Acta Oceanologica Sinica 2007，V01．26，No．6，P．83～90

http：／／www．oceanpress．COB．cn

E—mail：hyxbe@263．net

on the Phylogenetic analysis of Pectinidae()based ribosomal DNA internal transcribed spacer region

Zhenminl+ HUANG Xiaotin91，BI Kel，HU Jingjiel，HU Xiaolil，BAO

of of Marine Life of China， 1．Laboratory of Molecular Genetics and Breeding Mollusk，College Science，Ocean University

Qingdao 266003，China

Received 14 September 2006；accepted 2 1 March 2007

Abstract

and The ribOSOmal DNA internal transcribed spacer(ITS)region is auseful genomic region for understanding evolutionary genetic

molecular of using relationships．In the current study，the phylogenetie analysis Pectinidae(：Bivalvia)was performed nine of this were obtained from the the nucleotide sequences of the nuclear ITS region in species family．The sequences sca]lop

and nobilis，and compared with the species Argopecten irradians，，Amusium pleuronectes Mimachlamys

an species published sequences of Aequipecten opercularis，Chlamys知rreri，C．distorta，肛varia，Pecten maximu$，and outgroup

&黝viridis．The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods·Phylo。 the nor— their combination trees of similar results suppofl genetic analysis based on ITSl，ITS2，or always yielded topology．The

with classification of two and Pectininae) Dhological classificatioas of bivalve and are nearly consistent subfamilies(Chlamydinae made of that fbmulated by Waller．However，A．irrad／ans，together with A．opercularis up genera Amusium，evidences they may of Wailer who them in belong to the subfamily Pectinidae．The data are incompatible with the conclusion placed Chlamydinae by

and con。 morDhological characteristics．These results provide new insights into the evolutionary relationships among species

of classification tribute to the improvement existing systems．

Key words：bivalve，Pectinidae，ribosomal DNA，ITS，phylogeny

1 Introduction veniles．He divided the family Pectinidae into three subfamilies，Camptonectinae，Chlamydinae，and

Pectininae．He further divided into the The family Pectinidae(Mollusca：Bivalvia)in— Chlamydinae tribes cludes many well．known marine invertebrates that Chlamydini，Crassadomini，Mimachlamydini， and Aequipectinini．and Pectininae into Palliolini， play important roles in aquaculture．The phylogenet— Pectinini．Molecular phyloge— ic relationships among these species are still an issue Decatopectinini，and netic studies of the Pectinidae have of debate．Among the classification systems devised family developed

over the last decades(Barucca et a1．，2004； for the family Pectinidae，Waller(1993，1991) rapidly shell Insua et a1．，2003；Canapa et a1．，2000；Matsumo— proposed a system based on microsculptural to and et a1．，1999；Steiner features and the morphological characteristics of ju一 Hayami，2000；Canapa and Mtiller，1996；Littlewood，1994；Gjetvaj et

}Corresponding author．E—mail：zmbao@OUC．edu．cn

HUANG Xiaoting et a1．Acta Oceanologica Sinica 2007，V01．26，No．6，P．83—90

a1．，1992)． of the systematic and phylogenetic relationships of

Ribosomal DNA is a multiple··copy cluster of re·· several groups of bivalves，especially at the genus peat units；each unit contains the 18S，5．8S and level．We cloned and sequenced the ITS region of

28S rRNA genes，and the internal transcribed spacer four (Mimachlamys nobilis，Mizuhopecten between the 18S and 5．8S rRNA genes(ITSl)as yessoensis，Am潞ium pleuronectes and A．irradians) well as between the 5．8S and 28S rRNA genes to obtain the basic characteristics of these se—

(ITS2)．ITS sequences are of an appropriate size to quences．Moreover，the ITS sequences，both indi— be amplified easily with a pair of primers designed vidually 7 ITSl or ITS2) and collectively according to the 3 7end of 18S rDNA and the 5 7end of (ITSt+ITS2)were used to determine the phyloge—

28 S rDNA．Furthermore，ITS sequences are more netic relationships among ten Pectinidae species． diverse than ribosomal RNA genes f Hillis and Dix— on，1991)，and they have been employed to resolve 2 Materials and methods phylogenetic classification problems in the lower tax— onomical levels such as genera and species(Morgen 2．1 Sample collection and DNA extraction and Blair，1998；Navajas et a1．，1998；Perera et M．nobilis and were collected a1．．1998)．In bivalve，ITS has been studied in A．pleuronectes from the wild of the East China Sea and several species．The size and restriction pattern of population the South China A．irradin瑚 the ITS region have been employed to distinguish Sea．respectively．The collected in this was introduced from Mytilus mussels(Toro，1998；Heath et a1．， study originally 1996)，Veneridae clams(Ferntindez et a1．，2001)， the USA and reared in the Huanghai Sea，and

was from and reared in the Bo— certain pectinid scallops(Wang et a1．，2006； M．yessoensis Japan hai Sea．Only adult specimens were used for DNA L6pez—Pifi6n et a1．，2002)，and populations of the was extracted from the adductor giant clam Tridacna crocea(Yu et a1．，2000)．The preparation．DNA

muscle a traditional method complete sequence of ITS l or ITS2 has been used in using phenol／chloroform

as described Sambrook and the phylogenetic analysis of freshwater bivalves Las— by Russell(2001)．

migona(King et a1．，1999)，Brazilian Biomphalar— 2．2 PCR amplification，cloning and sequencing ia species(Vidigal et a1．，2000)，Mytilus species The ITS was (Riginos et a1．，2002)，and certain Pectinidae region prepared by polymerase chain reaction(PCR)．The forward primer(5’一 scallops(Insua et a1．，2003)．

reverse Pectinidae scallops have a wide distribution in GT／TCTGTAGGTGAACCTGC-3’)and primer

the world．Waller(1993，1991)produced a de— (5'-CTCGTCTGATCTGAGGTCG一3 7)were designed

based on the nucleotide retrieved from tailed morphological taxonomy of Pectinidae．Barue— sequences GenBank(AF245687 and AF245688；Yu et al，， ca(2004)constructed phylogenetic trees of Pectini-

was set in a volume dae using 16S and 12S ribosomal rRNA 2001)．PCR up 50／xL containing 10 of of each genes．However，there is a disagreement between ng templates DNA．0．1扯mol／dm3 of each morphological and molecular classifications about the primer，0．2 mmol／dm3 dNTP，with 1．0 U

phylogeny of bay scallop，Argopecten irradians(La— 如DNA polymerase(Promega)and 1×buffer．

o marck，1819)．In the present study，we focused on The amplification conditions were as follows：94 C，

the ribosomal DNA internal transcribed spacer region 60 s(denaturation)；56。c，60 s(anneal)；72。C，

because it has proved to be very useful for the study 90 s(elongation)for 30 cycles．Amplified fragments

HUANG Xiaoting et a1．Acta Oeeanologica Sinica 2007，V01．26，No．6，P．83～90 85

were checked by electrophoresis with l％agarose gel 2．3 Sequence alignment and phylogenetie analysis and visualized by ethidium bromide．

The PCR．．amplified fragments of three individu．． The identity of the sequences obtained was deter-

als from each species were ligated into a pMDl8一T mined using the BLAST program(Altschul et a1．，

Vector(TaKaRa)and transferred into the competent 1 997)，Beside the sequences of OUF clones from坛

E．col；DI-IS∞The recombinants were identified nobilis，M yessoensis，A．irradians and A．pleuronect—

retrieved also those of through blue or white selection(Sambrook and Rus— es，we Chlamys扣rreri，C．dis—

sell．2001)．Sequence reactions were carried out with torta，胍varia，Aequipecten opercularis，Pecten maxi— mlA$and Perna viridis from GenBank．The species uti— an ABI PRISM 3730xL DNA sequencer． A1l se—

lized in this comparative study are listed in Table 1． quenees were deposited into GenBank with the acces． All were sion number listed in Table 1． sequences aligned using CLUSTALX(Thomp- son et a1．，1997)．The base composition，the ratio of Table used for the 1．Species phylogenetic analysis and transition to transversion and Kimura 2-parameter dis- Genebank accession number tance were calculated using the MEGA version 3，l

Accession No． Species (Kumar et a1．，2004)． Chlamys fa册一 AF245687 Phylogenetic trees were produced using the neigh— (Jones et Preston，1904) A蹦5688 bor-joining(NJ)(Saitou and Net，1 987)and maxi— Chlamys distorta A『428409 mum parasimony(MP)methods with MEGA 3．1 pro— (da Costa，t778) gram．Neighbor—joining trees were constructed emplo— Mizuhopecten yessoemis AY690600 ying the Kimura 2-parameter distance．Maximum-par- (Jay，1856) DQnl7585 simony trees were produced using branch·-and--bound DQ417586 search by attributing equal mass to transitions and Mimachlamys nobilis AY690599 transversions．In all analyses，the deletion (Reeve，1852) DQ417587 complete was selected for values DQ444298 option handling gaps．Bootstrap (Felsenstein，1 985)，indicating robustness of nodes in Mimachlamys vart。a AJ428408

(Linnaeus，1758) neighbor．joining and maximum—parsimony trees， refer

to 1 000 Argopecten irradianz AY695802 replications，

(Lamarek，1819) DQ417588

DQ417589 3 Results

Amt“dum plelrronectes DQ417590

(Linnaeus，1758) DQ4t7591 3．1 PCR amplification and data analysis DQ417592 The entire ITS region was amplified from at least Aequipecten opercularis AJ428407

30 individuals of each scallop species．In all cases． (Lamarck，1758) the PCR yielded a single band of approximately 750 bp Pecten maximus Aj428410 in length．Tahle 2 lists the ITS lengths and the nueleo— (Linnaeus，1758) tide composition percentage of three individuals from Perna viridis AF353098 each species．The total length of the ITS region ranged (Linnaeus，1758) from 520 bp(旭nobilis)to 558 bp(A．irradians) and the GC content from 44．4％(舱yessoensis)to 50．1％(A．pleuronectes)．

86 HUANG Xiaoting et a1．Acta Oceanologica Sinica 2007，V01．26，No．6，P．83～90

Table 2．Nucleotide composition(GC)and length

17ISl ITS2

5pecms G+C(％)length／bp G+C(％)length／bp

月．irmdioml 41．9 277 46．9 281

，4．i，mdin，u2 41．9 277 47．3 281

A．imzdi∞53 41．9 277 47．7 281

———————————————————————————————————————————————————————————————————————————————————————————一一一M．nobillsI 48．9 254 45．9 266

M．nobilis2 48．9 254 46．2 266

M．nobilis3 48．9 254 45．9 266

244 46．3 279 M。yessoensisl 42．6

45．9 281 M．yessoensis2 43．2 243

M．vessoens／s3 43．2 243 45．6 281

A．D2eMro，wc把s1 48．4 271 51．9 272

272 A．Dleun)，}ec￡e52 48，2 272 51．5

A．Dleur0M以∞3 47．8 272 51．4 272

were 1．0 for for ITS2 P．viridis，which belongs to Mytilidae was used as values of those ratios ITSl，1．2 and 1． 1 for their combination． Genetic distance value the outgroup．Sequences of the ten species were

0．10 to 0．37 in to 0．44 in aligned to produce 730 unambiguously aligned nueleo— ranged from ITSl，0．13

0．1 2 to 0．37 in combination．The mean tide．positions，including 3 1 5 parsimony-informative ITS2，and distance of 0．22±0．09 in ITSl was sites．The 5．8S rRNA gene displayed 13 variable Kimura genetic that sites，three of which were parsimony—informative．By significantly smaller than inⅡS2(0．28±0．12)． The distances of the P．viridis and other contrast．ITSl displayed a total of 224 variable sites and outgroup spe-

ITS2 displayed 253．The number of parsimony—informa— eies varied significantly．

tive sites was 145 in ITSl and 170 in ITS2．In the case 3．2 Phylogenetic analysis of M。yessoensis．three individuals had two nucleotide

substitutions and three indels in ITSl，with two nucleo· The construction of molecular phylogenetic trees

tide substitutions and two indels in ITS2．Those of was carried out using ITSl，or ITS2，or ITSl+ITS2，

M．nobilis displayed one nucleotide substitution in ITS2． with P．viridis as the outgroup．NJ trees and MP trees differences in Those of A．pleuronects displayed two nucleotide substi— exhibited a similar topology with sli曲t

tutions and one indel in ITSl．with three nucleotide branch len甜h when constructed based on ITS2 or

substitutions in ITS2：and those of A．irradians two nu— ITSl+ITS2(Figs lc and d)，and a slightly different

cleotide substitutions in ITSl and two in ITS2． topology when constructed using ITSl alone(Figs l a

trees were in Considering the 5．8S rRNA gene highly con— and b)．Moreover，MP perfect correspon— dence with the trees．The served．the sequences of ITSl alone，ITS2 alone，and NJ corresponding phylogenetic

in the members of Pectinidae were con． a combination of the two(ITSl+ITS2)were used trees indicated that

to transversion be- stituted two well-differentiated our analysis．The ratio of transition by clades．C．fa删矗．

tween clones from different species without the out— C．distorta。M．nobilis，M．yessoensis and M．varia be-

in to 1．7 in to one group ranged from 0．5 to 2．0 ITSl．0．6 longed clade，and A．irradians，A．opercularis，

ITS2，and 0．7 to 3．0 in their combination．Average A．pleuronectes and P．mlzximus to the other．

HUANG Xiaoting et a1．Acta Oceanologica Sinica 2007，V01．26，No．6，P．83～90 87

M yessoensis 3

M yessoensts I 脱，'essoensis 2 C．mr胛ri

M．”obili S3

M．nolfili Sl 骈．nobili娩

l：．distorta

M．ilalia

以．pleurottef·tes 2

以．pleuronectes 1

“．pleunmFf，P^3

，’．maximus A．()口P，℃?t如ris

A．irradians3

A．irradiansl A．irradifuls2

M．nobilis I

M．D(jbilis2 M．，，obilis3

C．^￡rMri

M．yessoen．sis 2 M．vessoensis 3 肼ye 9SOeltsis 1

C distorta

M．varia

A．pleurone(·tes 2

A．pleuronectes 3

A．pleuronecles I

t’．maximus

A．OlM!FCltlaris

A．irradiansl

A．irradiam3

A．irradian．_2

P．viridis

Fig．1．Pectinidae phylogenetic relationships derived from ITS sequence．a．Neighbor-joining tree inferred from ITSl，b．

maximum trees inferred from parsimony ITSl．e．ITS2 or ITSl+ITS2 and d．ITS2 or ITSl+ITS2．The numbers represent the of 1 percentage 000 bootstrap replications in which a given node appeared．Numbers above a branch in C and D indicate a boot—

value in the ITS2 below a a strap tree；numbers branch indicate bootstrap value in the ITSl+ITS2 tree．The bar representsge—

netic distance．

The size of ITS varies considerably in different species

(i．e，1 060～1 370 in Veneridae：650 in Hall— 4 Discussion bp bp otis discus hanna；600 bp in fungi；and 2 400 bp in

humans)．The total ITS len@h of the ten species in our Previous studies of other organisms have suggested study varied from 520 to 558 bp，which was consistent that ITS region is highly variable SO it is suitable for with that of other bivalve species(470～1 010 bp)．In phylogenetic analysis of closely related species and addition，the lengths of ITSl(243～277 bp)showed a and populations(Morgen Blair，1 998；Navajas et a1．， similar size with those of ITS2(266～281 bp)．It

1998；Perera et a1．，1998；Hillis and Dixon，1991)． should be noted that the ITSl and ITS2 of SCallops dis．

88 HUANG Xiaoting et a1．Acta Oceanologica Sinica 2007，V01．26，No．6，P．83～90

played a similar size as other bivalyes do(Insua et indicated an excess of transvertions and some saturatmn a1．。2003)，but it dose not seem to be the nolqXl， in these species．As discussed by Waller(1991)，the since in a few bivalves I鸭l can be twice or more than subfamilies Chlamydinae and Pectininae(including the

ITS2(Ferntindez et a1．，2001；Yu et a1．，2000)． nine species in our study)were already in place well

of 5．8S rRNA in the ten before the end of the Mesozoic is The lengths the gene species era，Therefore，it were all 1 57 bp，which were deduced by comparing likely that the speciation of Pectinidae occurred an— data from certain bivalve(Insua et a1．，2003)，but ciently． difierent from three clam species．in which 5．8S rRNA In terms of the phylogenetic trees，the NJ trees

and MP trees identical construc— gene contains 158 bp(Fermindez et a1．，2001)， the displayed topology the ITS 1+ITS2， The GC content of ITSl(41。9％～48．9％)and tion based on sequences ITS2 or

ITS2(45．6％一51．9％)showed little variation among However，the NJ trees and the MP trees did exhibit a the scallop species，which is consistent with previous small difference based on ITSl．Insua(2003)sugges— results，i．e．，between 45％and 66％in ITSI and be- ted ITSl alone should offer significant phylogenetic res— tween45％and 68％in ITS2(1nsua et a1．，2003)． olution since the bootstrap values on its phylogenetic

The difference of the GC content between I髑1 and trees were slightly higher than those of ITS2 and very

ITS2 was less than 7％．This resuh indicates coevolu- sinlilar to those of the combined data set．Neverthe．

trees based on tion of the two spacers at the level of base composition 1ess，with our result，the phylogenetic

(L6pez—Pifi6n et a1．，2002)．Interspecific diver- the sequences ITS2 and ITS 1+ITS2 were identical but

the ten was much than in‘ different from those of ITSl．After these gence among scallops higher comparing with et traspecific polymorphism，and ITS2 divergence is sig。 classification other molecular(Barucca a1．， nifieantly higher than that of ITSl between species． 2004)and morphological taxonomy(Waller，1 993)，

This has also been reported in other researches(Insua we found only the results based on the sequences ITSl

to the et a1．，2003；L6pez—Pifi6n et a1．，2002)．There was were identical with those results，especially clas— of the tribes especially the case for M．nobilis．where clones of four sification Chlamydini，Crassadomini，and individuals were sequenced and only two nucleotide Mimachlamydini．As a resuh，we suggest that only substitutions were detected． ITSl should be used in the phylogenetic analysis since

be too variable in se- Molecular phylogenetic analysis relies on the sub— the ITS2 region may primary

se‘ stitution pattern of pairwise comparisons among quences· of Pectinidae has quences．In general，transitional changes(among pu’ The phylogenetic relationship more than been both and fines and among purimidines)are frequent investigated previously morphologically

transversional changes(between purines and pyrimi— molecularly．The present molecular results are consist— dines)，although theoretically，twice as many transver‘ ent with Waller’s system．which is based on an i1"1110．

sional changes are possible．As a consequence of this vative use of non．adaptive features．such as shell mi． divergence in substitution rate，transitions are likely to erosculptures and the morphological characteristics of become saturated sooner than transversion．The ratio of juvenilistice forms，which bear few signs of the sLlbse— transitions to transversions generally showed a value of quent divergence in lifestyle．According to Waller’s exceeding 1，indicating an incomplete saturation of (1993，1991)studies，the scallop species in our transition(while values close to 1 or below reflect satu- study were representatives of the tribes Chlamydini ration；DeSalle et a1．，1987)，In the present study， (C。如础^and胍yessoensis)，Crassadomini(c．dis— and the average values of ratios of transition to transversion torta)，Mimachlamydini(M．varia M．nobilis)， for ITSl and ITS2 were 1．0 and 1．2 respectively．This Aequipectinini(A．irradians and A．opercularis)，and

HUANG Xiaoting et a1．Acts Oceanologica Sinica 2007，V01．26，No．6，P．83—90 89

Pectinini(A．plearonectes and P n2aximll．$)．Further- more，the tribes Chlamydini， Crassadomini， Reference

Mimachlamydini and Aequipectinini belong to the sub- families Chlamydinae and Pectinini to the subfamily Ahschul S F，Madden T I。，Sch'affer A A，et a1．1997．Gapped

and new of data—r Pectininae．In terms of the classification of the tribe， BLAST PSI—-BLAST：a generation protein

base search programs。Nucleic Acids Research，25：3 389～ our results for Chlamydini，Aequipectinini and Pectini— 3 402 ni were identical with those of Waller’s；however， Barucca M，Olmo E，Schiaparelli S，et a1．2004．Molecular there were ambiguous findings for Crassadomini and phylogeny of the family Peetinidae(Mollusca：Bivalvia)based Mimachlamydini．According to Waller(1993)，M． on mitochondrial 16S and 12S rRNA genes，Molecular Phylo— varia and M．nobilis belong to the tribe Mimachlamydi· genetics and Evolution，31：89～95 ni；C．distorta belongs to the tribe Crassadomini．Nev— Canapa A，Barucca M，Marinelli A，et a1．2000．Molecular da—

our and舱uaria con- ertheless，in study，C．distorta ta from the 16S rRNA gene for the phylogeny of Pectinidae stituted one branch which differentiated obviously from (Mollusca：Bivalvia)．Journal of Molecular Evolution，50： the branch constituted by the species M。nobiliz．On 93～97 the other hand，in the classification of the subfamilies， Canapa A，Marota I，Rollo F，et a1．1999．The small—subunit

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