Chinese Joumal of Oceanology and Limnology Vol. 23 No. 3, E 335-339, 2005

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)*

YOU Feng ( ~ ) , LIU Jing (~lJj~), ZHANG Peijun ( ~ : ~ ) , XIANG Jianhai ( ~ ) (Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China)

Received Oct. 12, 2003; revison accepted Apr. 10, 2005

Abstract A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematies of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P olivaceus clustered with K. bicoloratus, but P cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

Key words: mtDNA, 16s rRNA gene sequences, phylogeny, Pleuronectiformes

1 I N T R O D U C T I O N in molecular phylogeny.

The reconstruction of phylogenetic relation-

ships has increased dramatically since the advent of

modem molecular techniques. Mitochondrial DNA

markers successfully improved the systematics of

several fish groups at multiple taxonomic levels (He,

1999; Tinti and Piccinetti, 2000; Takehana et al.,

2005). A preliminary study was carried out on eight

species of three different flatfish families using

5'-portion of mtDNA control region (Tinti, 1999).

Other special sections of mtDNA, such as cyt b, ND,

16S rRNA and COl etc, were also sequenced to

study the relationships of different species among

levels of family, order or class.

This paper is a molecular study of several of

economically valued flatfish including Paralichthys

olivaceus, Pseudorhombus cinnamomeus, Kareius

bicoloratus and Psetta maxima in genus and family

by partial sequence analysis of mtDNA 16S rRNA

gene. Phylogenetic relationships among Pleuronec-

tiforms taxa were assessed by applying some effi-

cient methods at genetic level of differentiation for

comparison with other 6 published sequences in the

GenBank. The present study tries to use this mo-

lecular marker as a tool to study Pleuronectiformes

2 M A T E R I A L A N D M E T H O D S

All the specimens collected represent three

Pleuroneciform families in Shandong, China. Fresh

specimens of P maxima (Scophthalmidae, Psetta), P

cinnamomeus (Paralichthyidae, Pseudorhombus), K.

bicoloratus (Pleuronectidae, Kareius) were collected

in Nanshan and Shazikou free markets in Qingdao,

and live samples of P olivaceus (Paralichthyidae,

Paralichthys) were collected in the Institute of

Oceanology, the Chinese Academy of Sciences. To-

tal DNA in the individual muscle was extracted in

the method of Sambrook et al. 0989). DNA con-

centration was measured by spectrophotometer

(Beckman DU-600).

About 600 bp fragment of mitochondrial 16S

rRNA gene was amplified with the oligonucleoride

primers L (5' CCGGTCTGAACTCAGATCACGT

3') and H (5' CGCCTGTTTATCAAAAACAT 3')

(Kessing et al., 1989) supplied by Shangon Co. PCR

amplifications were carried out in 50 ~tl volumes.

* Supported by Natural Science Fund of China (No. 30271036) and Natural Science Fund of Shandong Province of China

3 3 6 C H I N . J. O C E A N O L . L I M N O L . , 23(3) , 2005 Vol.23

Reaction solutions contained 10xbuffer, 5 lal; dNTPs, 200 pxnoFL; Mg 2+, 2.5 mmoFL; primers, 0.2 lamol/L each; Taq DNAase, 1 unit; and total DNA, 300-400 ng, adjusted to 50 lal with dd H20. Reaction solu- tions were overlain with 15 ~tl of mineral oil to pre- vent condensation and evaporation. A negative con- trol was set up for each test. The amplifications were achieved in a Gene Cycler TM programmed for an initial denaturing at 94*(? for 5 minutes: 30 cy- cles, each at 94~ denaturing for 30 sec, 56~ an- nealing for 30 sec, 72~ extension for 1 min; and final extension at 72~ for another 10 min. The amplification products were analyzed by electro- phoresis in 1.5% agarose gels with 1 xTAE buffer, stained with ethidium bromide and photographed under UV light. A DNA marker was included for each gel. The amplified DNA was purified using the

Wizard PCR Preps DNA Purification System by Promega Co. Sequencing reactions were obtained using the ABI Prism Dye-Terminator kit (PE) with thermal cycling performed in a Thermlyne Amphil- tron I. Completed sequencing reactions were puri- fied by Centri Sep., and run on a MegaBACE auto- sequencer. Complementary strands for both primers were read.

After the primary sequences were read manu- ally with the help of Chromas software and were aligned by BioEdit, Genestar, Genetyx-win, and

Clustal W software, all of them with six flatfish par- tial 16S rRNA gene sequences from GeneBank were blast on NCBI. Each paired genetic distance of these ten species was calculated and MP (maximum par- simony), ML (maximum-likelihood) and NJ (neighbour-joining) trees were constructed by 500 bootstrap replications using PHYLIP Version 3.57c.

3 RESULTS

The 590 bp, 595 bp, 582 bp and 590 bp of par- tial 16S rRNA gene from these four species were amplified. All G+C level was less than 50%. After being blasted on NCBI, the 605 bp of the sequences was analyzed to examine the phylogenetic relation- ship for all ten species (including other six flatfish sequences from GenBank). These 605 bp sequences contained 153 variable sites, of which 108 were in- formative (Fig.l). The inter-species genetic dis- tances in their sequences ranged from 0.014 1 to 0.263 9 (Table I). But the intra-family genetic dis- tance between P cinnamomeus and P olivaceus was up to 0.216 9, which was much bigger than in- ter-family ones between P olivaceus and P. flesus

(0.093 1), and P olivaceus and K. bicoloratus

(0.090 4). MP, ML and NJ phylogenetic trees were gener-

ated as shown in Fig.2-Fig.4.

Table 1 Genetic distances among 10 flatfish based on partial sequence of 16S r R N A gene

Pl. f l Ka. bi Po l i Mi. va Pt. ma Ps. ci Mo. hi Sy. kl So.so So. se

e / . f l . . . .

Ka. bi 0.014 1 - - -

P oli 0.093 1 0.090 4 .. . .

Mi.va 0.1274 0.1274 0.1247

Pt. ma 0.1492 0.1445 0.1349

Ps. ci 0.201 0 0.194 4 0.216 9

Mo. hi 0.215 6 0.221 3 0.213 3

Sy. kl 0.203 2 0.208 8 0.199 3

So.so 0.213 3 0.216 1 0.199 2

So. se 0.1926 0.1953 0.1969

0.039 5 ....

0.220 4 0.216 6 .. . .

0.169 1 0.196 9 0.257 6 . . . .

0.1526 0.1734 0.2585 0.1275 ....

0.153 1 0.1798 0.261 7 0.1008 0.0529

0.146 1 0.178 5 0.263 9 0.096 8 0.057 5 0.027 4

Abbreviations: PL r , Platichthysflesus, AFII 3180, Tinti. F., Nadiani. M.and Tommasini, S.et al., 1998; Ka.bi, Kareius bicoloratus, this paper;

P. oil, Paralichthys olivaceus, this paper; MLva, Microchirus variegatus, AFl12851, Tinti, F., Nadiani, M. and Tommasini, S. et al., 1998; Pt.

raa, Psetta maxima, this paper;, Ps. Ci, Pseudorhombus cinnamomeus, this paper; Mo. Hi, Monochirus hispMus, AFl12852, Tinti, F., Nadiani,

M. and Tommasini, S. et al., 1998: Sy. KI, Synaptura ldeini, AF112847, Tinti, F., Nadiani, M. and Tommasini, S. et al., 1998; So. so, Solea solea, AF122825. Tinti, F., Nadiani, M. and Tommasini, S. et al., 1998; So. Se, Solea senegalensis, AF112846. Tinti, F.. Nadiani. M. and Tommasini, S. et al., 1998

N o . 3 3 3 7 Y O U e t a l . : S t u d y o n m i t o c h o n d r i a l 1 6 S r R N A g e n e s e q u e n c e s a n d p h y l o g e n y o f f l a t f i s h e s

1 20 4 0 60 80 100

P L f l . . . . . . . . . . ATCGTTGAACAAACGAACCC~TAATAGCGGCTGCACCATTAGGA~GTCCTGATCCAACATCGAGGTCG~AAACCCCCTTGTCGATA~GGG

Ka. b i AGGACT-TTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P. o l i AGGACT-TTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ f . V a . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P s , m a AGGACT TTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . .

P s . c i AGGACTATTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

Jto. h i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . .

S.v. k i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1". . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

S o . s o . . . . . . . . . �9 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1". . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

S o . s o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101 120 140 160 180 2 0 0

P L f l C~CTAAMtG(~;GATTGC~(~G~TA~CTAGGGT't~CTTGG~CGTTGATCGGTTTT-A~GGAT~AG~TT-GGTCAGAATT-~C~GC~TGT~AGAGCTG

K a . b i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . - . . . . . . . . . . - . . . . . . . . . . . . . . . . .

P. o J i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . CGT. -G . . . . . . . . . . . . - . . . . . . . . . . - . . . . . . GA . . . . . . . . .

J/f . va . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . CG.. -G . . . . . . . . T . . . T . . . . . A . . G. - . T . . T. AA.. T . . . G. .

P s . m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . C . . . -G . . . . . . . . T . . . T . . . . . A . . G . - . . . . T. AA.. G . . . G. .

P s . c i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . A . . . . CC . . . . . . CA. ACG . . . . . . . T A . . . - . . . . . . . . . . A . . . . . . AA.. G . . A . . .

Mo. h i . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . CA. AGGC . . . . . . . A. . AA . . . . . . . . G . . . . . . . A . . . . . . . G. .

S y . k l . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . --TAAG . . . . . . . . T . . AA.. C . . . . . G. - . . . . T. AA . . . . . . G. .

So. s o . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CT . . . . . . C- AGG . . . . . . . . T . . A A . . . . . . . . G . - . . . . T. AA . . . . . . G. .

So. se . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C - AAG . . . . . . . . T . . A A . . . . . . . . G . - . . . . T . A . . . . . . . G . .

201 220 240 260 280 300 PL f l TTGCTCTGGCTTGCGGG~GAAGAAGTAACTTAGGGGT---GTGCTCCC~TTTCCACGT(~GGGTTTTGTATT-CCCCATGGTCGCCCCAACCGAAGACAT

Ka. b i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . .

P. o l i . C . . G . . A . T . . . T A . . . . G . . . G . - . . T G . . . . . . . . . A . . . . . - . . . . . . . . . . . . . . . . . . . . G . . - . . . . . . . . . . . . . . . . . . . . . . . . . . .

M L v a . G . . . . A. T . . . GA . . . . CNNNNNNNNNNNT . . . . ACGG . . . . . . . T-A . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . .

Ps. ma . G . . . . C A T . . . GA . . . . T T A . G. - . . . . . . . . G - - - . . . . . . . T - A . . . . . . A . . . . . . . . . . . . . . . - . . . . . G . . . . . . . . . . . . . . . . . . . . .

Ps. cd . C . . . . . A T . . . AA . . . . TG. T T . . . . AA. T. CTT . A - - C . . . . . . TG . . . . . . . . . . A . . . . . . . . . G . . - . . T . . . . . . . . . . . . . . . . . . . . . . . .

tr h i C C C . . . A. T . . T T A A . . . C . . T G . . I 'GAG. - . . . . , ~ G A . . . . . . T T - A C . . . . . A . . . . . . . . . . . . . . . T . . . . GC . . . . . . . . . . . . . . . . . . . . .

Sv. k l . CC . . . . . T . . C T A . . . . T G . . G A G . . AG- - - . . . . . --. . . . . . . . T. - A . . . . . . A . . . . . . . . . . . . . . . 1 " . . . . GC . . . . . . . . . . . . . . . . . . . . .

So. so . C C . . . A. T . . C T A . . . . C . . . G A G . . A A - - . . . . . T A . . . . . . . T T -A . . . . . . A . . . . . . . . . . . . . . . T . . . . GC . . . . . . . . . . . . . . . . . . . . .

So . se . C C . . . A. T . . T T A . . . . C . . . . A . . . A G - - . . . . . T - . . . . . . . T T - A . . . . . . A . . . . . . . . . . . . . . . T . . . . GC . . . . . . . . . . . . . . . . . . . . .

3 0 1 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0

P ] . f l CAAGGCAGGTTCCATTTAGTTC-AGGCCCTTAGCTGGGGGT---ACTTGAC -ATGGTCCACCT(;TGTGTCTtuAAGCTCCATAGGGTCTTCTCGTCTTAT

Ka. b i . . . . . . . . . C . . . . . . . . . . . . - G . A . . . . . . . . . . . . . . . - - - . . . . . . . - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . .

P. o l i . . G . . . T . . . . T . . . . . . . . . . AG . . . . . . . . . . . . . . T . . - - . T . . . . . - - . . . A . . T G . . C T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

g i . va T . . . . . . . . AG . . . . . . . . . . T - - . . T . . GGT. TG . . . . . . - - - G T . . A . . . . . . . . T G . . C T A T C . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ps. m a T. GA . . . . . GGT . . . . . . . . . T - - . . T . . GGT. AG . . . . . . - - - . . . . A . . - - . . . . G. T G . . C T A T C . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ps. c i . . . A C G . . . G . . . . C. G . . C. T - - . TAA. C. G. T A . . . . . . CTT . G . . A. C - - . . . . G T . . AT . T. A . A . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . .

J/o. h i T T . . C . . . . G G . . G . . . . . . G T - - . AT . T . . "FAT. T A A . A. - - - - T . . A. AGG. C . . G. TGG . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sy. k l T . . T A . . . . G G . . G . . A . . . G T - G C T T T . . . . T T . . . A A . . - - - - T . . . G G - - . C . . G. T G T A . T G T . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . .

So. so T . . "FA . . . . G G . . G . . A . . . ( ; T - - . T T . G . . T . T T . A. AA. - - - - T . . . G G - - . C . . G. TGAA. T G T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

<9o. so T . . T A . . . . G G . . G . . A . . . G T - - . T T . G . . T A T C T . . AA . - . . . . . , . GGG . C . . G. TG. A. T G T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

401 420 440 460 480 500 PL s G(;TTTTATCCCC(;•TTCTGCACGGGGAGATCAATTTCATTGA•CAGGGGAAGGAGACAGTTAAGCCCTCGTTAT•C•ATTCATACAGGTCTTCATTTAAA

Ka. bi . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . TG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P. o l i AGG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TG . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . .

Y i . va TAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . TT. A. AG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P s . ma TGAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . TT. A. AG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

Ps. c i OFAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . T . . . . G . . . . . . . . . . . . . . . . . . . . . C T A . . . . . . . . . . . G . . . . . . T . . . . . . .

go. hJ . A A , ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . AAAG . . . . . . . . . . . . . . . . . . . . . CT . . . . . . . . . . . . G . . . . . . . . . . . . . .

Sy . k l . GAG . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . T . . ATAG . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . G . . . . . . . . . . . . . .

So. so . "rGG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TG. ATAG . . . . . . . . . . . . : . . . . . . . . . T . . . . . . . . . . . . G . . . . . . . . . . . . . .

So. so . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TG. ATAG . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . G . . . . . . . . . . . . . .

501 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 6 0 5

P L f_l A G A C A A G T G A Y ~ A C G C T A C C T T T ~ C A C G G T C A A A A T A C C ~ C G ~ C C G T T G A A C ~ A A G T T G T C A C T G G G C A G G C G G G A C C T C T T A T A C T G T G ( ; T ~ A ~ G C A A . . . . . .

Ka. b i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GAGGCG

P. o l i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TGT.. C T C . . . . GAGGCG

Mi. va . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G - . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y. A . . . --. . . . . - . . . . .

Ps . m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T C A . . . T . . . . . GAGGCG

Ps. c i . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . T . . . . . . . . . . . . . . . . . . . . . - . T T A C . . . . GGC . . . . . . . A . . . . . . . . . . . . T . A C AG. - . . . . . GAGGCG

Jfo. h i . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . T . . . . . . . . A . . . . . . . . T . . . . T - A . A . . . . . . . . . . . . . . A . . . . . . . . . . . . . . A G . . G G - - T . . . - . . . . .

Sy. kJ . . . . . . . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T - - G . . . . . . . . . . . . . . . A . . . . . . . . . . . - - . T. T . . . G G . . . . - . . . . .

So. so . . . . . . . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . T - - G . . . . . . . . . . . . . . . A . . . . . . . . . . . - - . T. T. G. G G T . . . - . . . .

So. so . . . . . . . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T - - G . . . . . . . . . . . . . . . A . . . . . . . . . . . - - . T. TA . GGG. C . . - . . . . .

F i g . 1 V a r i a t i o n s i t e s i n 1 0 f l a t f i s h 6 0 5 b p s e q u e n c e s o f 1 6 S r R N A g e n e

338 CHIN. J. OCEANOL. LIMNOL., 23(3), 2005 Vol.23

t 82.1 i l l

100

78.1 99.2

Ps.ci

So .~

95.6 ~ So.so

~oo [ sy.kt

Mo.hi

Mi. va

l~ma

Eoli

Ka.bi

I P1.fl

Fig.2 Maximum parsimony phylogenetic tree based on mi- tochondrial 16S rRNA gene sequences (The numbers at the forks indicate the number on the tree and correspond to the percentages of 500 bootstrap replicates where the clades were founded)

84.4

100

P.oli

- - Ka.bi

- - Pl.fl

t _

76.2 I L Mo.hi

199.8[ ~].ma

- - Ps.ci

- - So.se

- - So.so

- - Sy.kl

Fig.3 Maximum likelihood phylogenetic tree based on mito- chondrial 16S rRNA gene based on mitochondrial 16S rRNA gene sequences (The numbers at the forks indicate the num- ber on the tree and correspond to the percentages of 500 bootstrap replicates where the clades were founded)

t

68.8 [

100

90.4 100 [ _ _

88.6 Mo.hi

85.2 [ So.so

So.se

sy.kt

Ps.ci

96.4 ~

- - P.oli

Mi. va

Pt.ma

Kn.bi

PLfl

Fig.4 Neighbor-joining phylogram tree based on 16S rRNA gene sequences (The numbers at the forks indicate the num- ber on the tree corresponding to the percentages of 500 boot- strap replicates where the clades were founded)

4 D I S C U S S I O N S A N D CONCLUSIONS

Partial sequences of mtDNA 16S rRNA gene of the four flatfish were blasted search in the GenBank. We found that these parts of 16S rRNA gene in P. olivaceus and K. bicoloratus were similar to the se- quences of mitochondrial genome of P. olivaceus

(Accession No. NC_002386) and K. bicoloratus

(Accession No. NC_003176) studied by Miya et al. (2001). The identities were up to 98% (590 bp) and 99% (590 bp), respectively. The sequence of P. maxima was also similar to the result of Cunha et al. (Accession No. AY157323), and the identity was up to 99%. The partial sequence of P. cinnamomeus

was not found in the GenBank, but the identity with P. olivaceus was more than 90%. Then all four se- quences could be confirmed to be homologous se- quence of mtDNA 16S rRNA gene.

The results showed that the mtDNA 16S rRNA gene sequences in these ten flatfish could present in genetic differentiation level clearly. The cross- gen- era genetic distances ranged from 0.052 9~).100 8 in the Family Soleadae, and the genetic distances between Soleidae and Paralichthyidae, Soleidae and Pleuronectidae ranged from 0.090 4-0.221 3, which showed the variation of inter-species, -genera and -families, respectively. The same-family genetic distance between P. cinnamomeus and P. olivaceus

was 0.216 9. These family-level differences were higher than that of cross-family ones such as be- tween Paralichthyidae and Pleuronectidae. The rea- son of the data being different from the results of traditional morphological data has not been found because no relevant sequences of allied species in the GenBank were available. Therefore, new study aiming to find the reason is needed.

There are many flatfish phylogenic trees re- ported based on either morphological data or isozyme data. Li and Wang (1995) drew a phyloge- netic tree of the Pleuronectiform families and sub- families according to their plesiomorph and apo- morph which showed that Family Paralichthyidae and Family Pleuronectidae clustered; and that Fam- ily Bothedae and Family Paralichthyidae have the same source. Hensley and Ahlstrom (1984) believed that the relationship between Family Paralichthidae and Family Pleuronectidae was closer than that be- tween Family Paralichthidae and Family Scophthal-

No.3 YOU et al.: Study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes 339

midae. Vemeau et al. (1994) set up a phylogenetic

tree of Pleuronectidae in terms of isozyme electro- phoresis showing that the systematical level of Fam-

ily Pleuronectidae paralleled to that of Family

Bothedae. In the cluster in genetic distance of isozyme (Fujino, 1986) 1, P. olivaceus clustered with

C1eisthnes herzensteini and then with K. bicoloratus

accidentally (the later two are of Family Pleuronec-

tidae). However, the tree of molecular marker is different from that of morphological marker. Our

results from three trees (ME ML and NJ trees) were not very different. Among them, MP tree (Fig.2) and ML tree (Fig.3) were visually similar. The NJ tree

(Fig.4) was little different from the above two trees, but the general topotaxis is similar. The location and

relation of P. olivaceus, K. bicoloratus and P. maxima were the same in many papers. Most closest

relation was between P. olivaceus and K. bicoloratus,

and between P maxima and Family Soleadea. However, P. cinnamomeus of Family Paralichthidae

clustered outside to P olivaceus and K. bicoloratus.

Nonagreement of cladograms in molecular and

morphological datasets is very common for some mitochondrial genes or other genes of different spe-

cies. The reason can be analyzed by very different methods such as Lineage sorting, Gene duplica-

tion/extinction and Horizontal transfer (Avise et al., 1983, Goodman et al., 1979, Kidwell, 1993). So,

careful study must be carried out on molecular

characters of species with the reference to their morphological features and use different mitochon-

drial markers such as cyt b or COl genes. Otherwise, longer sequences or combination with other nuclear

genes should be applied to decrease the statistical

deviation.

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