利用ITS和AFLP标记探讨中国、日本和澳大利亚3种珠母贝的亲缘关系

喻达辉, 朱嘉濠

喻达辉, 朱嘉濠. 利用ITS和AFLP标记探讨中国、日本和澳大利亚3种珠母贝的亲缘关系[J]. 南方水产科学, 2006, 2(5): 36-44.
引用本文: 喻达辉, 朱嘉濠. 利用ITS和AFLP标记探讨中国、日本和澳大利亚3种珠母贝的亲缘关系[J]. 南方水产科学, 2006, 2(5): 36-44.
YU Dahui, CHU Kahou. Species status of Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata using ITS and AFLP markers[J]. South China Fisheries Science, 2006, 2(5): 36-44.
Citation: YU Dahui, CHU Kahou. Species status of Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata using ITS and AFLP markers[J]. South China Fisheries Science, 2006, 2(5): 36-44.

利用ITS和AFLP标记探讨中国、日本和澳大利亚3种珠母贝的亲缘关系

基金项目: 

国家“863”高技术研究发展计划项目 2002AA603022

广东省科技计划项目 2002B2150101

广州省自然科学基金 037148

详细信息
    作者简介:

    喻达辉(1963-),男,博士,研究员,从事海洋生物技术研究。E-mail:pearlydh@pub.gz.gd.cn

  • 中图分类号: Q523+.8

Species status of Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata using ITS and AFLP markers

  • 摘要:

    我国合浦珠母贝Pinctada fucata的命名较混乱,与日本的P. fucata martensii和澳大利亚的P. imbricata的分类关系存在争议。用ITSs序列和AFLP标记对这3个种的遗传关系进行了分析。结果表明这3个地理种的遗传分化很低,种内和种间的遗传距离相互重叠,AFLP数据的聚类分析表明澳大利亚种群大部分个体单独聚合成一支,主成分分析结果相似。种间的遗传距离与分化程度与其地理距离呈正相关。分子方差结果也表明种间的差异较小,低于6%。这些结果表明这3个种实际上是同一物种。由于大西洋的P. mbricata与澳大利亚的不同,因此根据分类命名的优先原则,正确的种名应为P. fucata

    Abstract:

    Species status for Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata was investigated using ITSs and AFLP. The results indicated that genetic differentiation among populations of the three nominal species is low, and intraspecific and interspecific genetic distances are overlapping, as revealed by ITSs and AFLP markers. Based on AFLP data, phylogenetic analysis showed that most individuals from P. imbricata population are clustered together, consistent with the result from principal component analysis. It was found that the three nominal species are genetically isolated by geographic distance. AMOVA indicated that no more than 6% of the total variation is attributed to interspecific differences. These observations support the hypothesis that the three species are conspecific. According to the priority rule of nomination, the correct name of the species should be P. fucata because Atlantic P. imbricata is reported to be genetically different from Australian P. imbricata.

  • 合浦珠母贝(Pinctada fucata)是我国常见的海水育珠母贝,但其分类与命名存在一定的争议。其中文名称早期用马氏珠母贝,文革期间改为了合浦珠母贝[1],现在2个名称并存。其拉丁文学名也随之相应改变,早期用P. martensi,之后用P. fucata,现在则为P. martensiiP. fucataP. fucata martensii并存。如王祯瑞早期用合浦珠母贝P. martensi[2],现用P. fucata martensii[3]。日本的常见种早期主要用P. martensii[4],后来有的用P. fucata[5]。KURODA等[6]则将之分为P. fucata fucataP. fucata martensii 2个亚种。前者指南部亚热带如冲绳一带种群,后者指北部温带如三重县一带种群[7]。HYND[8]在对澳大利亚珠母贝种类的分类整理中将澳大利亚的常见小型珍珠贝定为P. fucata,并认为P. radiataP. fucata的同物异名。RANSON[9]则认为澳大利亚的P. fucataP. radiata的次同物异名。后来SHIRAI[10]则将P. fucata订为P. imbricata并被采用[11]。那么我国、日本和澳大利亚的这3种珠母贝是否为同种呢?

    COLGAN和PONDER[11]用等位酶(Allozyme)对澳大利亚的P. imbricata和日本的P. imbricata(即P. fucata martensii)进行了比较研究,发现两者遗传距离很近(Nei氏遗传距离 < 0.08),但与白珠母贝P. albina则相距较远(0.73~1.24),表明日本的P. fucata martensii与澳大利亚的P. imbricata是同种。ATSUMI等[12]通过等位酶和繁殖交配实验也认为中国的P. fucata与日本的P. fucata martensii应为同种。应用ITS1和ITS2(internal transcribed spacers)序列所作的系统发育研究也发现,中国、日本和澳大利亚3个种聚合在一起,且遗传距离非常近,种内和种间的遗传距离相互重叠,表明它们应为同种[13-14]。为了验证这一结果,本文测序了更多个体的ITS1和ITS2序列,并进行了AFLP(amplified fragment length polymorphism)实验分析,拟从种群遗传学的角度对3个种的遗传关系进行深入的分析,以探讨它们之间的遗传关系。

    本文共采集3个种群,即中国的P. fucata种群(采自海南三亚,32个个体),日本的P. fucata martensii种群(采自三重县24个个体),澳大利亚的P. imbricata种群(采自Port Stephens,30个个体)。采样点分布见图 1。样品取闭壳肌保存于95%的酒精中。DNA的提取用试剂盒(QIAamp DNA Mini Kit,QIAGEN)并按其说明操作。

    图  1  中国、日本和澳大利亚3个种群的采样点分布图
    CN. 中国海南;JP. 日本三重县;AUS. 澳大利亚Port Stephens
    Figure  1.  Sampling localities of the Chinese, Japanese and Australian populations
    CN. Hainan, China; JP. Mie Prefecture, Japan; AUS. Port Stephens, Australia

    ITS1(包括两侧的18S和5.8S基因的部分序列)和ITS2(包括两侧的5.8S和28S基因的部分序列)分别用相应的引物进行PCR扩增。ITS1的扩增引物为:SP-1-5(5′-AAC AAG GTT TCC GTA GGT G A-3′)和SP-1-3(5′-ATT TAG CTG CGG TCT TCA TC-3′)[15]。ITS2的扩增引物为:5.8S-F(5′-GCA GGA CAC ATT GAA CAT CG-3′)和28S-R (5′-CCA AGG ACG TTC TTA GCA GAA G-3′)[16]。ITS1和ITS2的扩增条件相同。PCR反应和测序方法见喻达辉等[16]

    AFLP实验参照VOS等[17]的方法稍作修改。模板DNA的量为100 ng,双酶切反应体积为20 μL,包含2.5 U EcoRI和2.5 U MseI (Biolabs,New England),37℃温浴3 h,70℃变性20 min。然后再加5 μL连接反应混合液,其中包含40 U T4 DNA连接酶(Biolabs,New England),EcoRI和MseI接头各0.25 μM,37℃温浴3 h。连接产物作为模板进行预扩增和选择性扩增。预扩增PCR反应总体积为20 μL,包含1 μL连接模板DNA,1(PCR缓冲液,1.5 mmol·L-1 MgCl2,dNTP各0.2 mmol·L-1EcoRI预扩增引物(37 μmol·L-1)和MseI预扩增引物(33 μmol·L-1)各0.17 μL,Taq DNA聚合酶(Promega)0.5 U。PCR反应程序为:94℃预变性2 min;94℃变性30 s,53℃退火30 s,72℃延伸1 min,20个循环。预扩增产物稀释10倍作为选择性扩增的模板。PCR反应条件与预扩增类似,不同的是模板DNA为5 μL,引物为选择性扩增引物。PCR反应程序为:94℃变性2 min,65℃退火30 s,在72℃延伸1 min;然后94℃变性30 s,65℃退火30 s,72℃延伸1 min,10个循环,每个循环退火温度降1℃至56℃;然后再运行28个循环。选择性扩增产物用6%聚丙烯酰胺胶分离、银染、统计扩增条带、有带的个体该位点记为1,无带的个体相应位点记为0。共用3对选择性引物:E-ACT/M-CAA、E-ACT/M-CTG和E-AGT/M-CTG,扩增结果合并分析。

    ITS1和ITS2经双向序列验证(测序)后用Clustal X 1.83[18]进行比对分析并作简单手工调整。同时通过NCBI的GenBank数据库进行Blastn同源分析[19]。根据比对分析结果和同源分析结果,确定ITS1和ITS2两端的边界。并根据碱基突变和缺失突变确定个体的等位基因,所有等位基因的序列均已在GenBank注册。

    (1) 聚类分析。ITS1和ITS2数据用MEGA 3[20]进行邻结法(NJ)[21]聚类分析,构建NJ系统树,并按种群分组计算组间平均K2P(Kimura 2-parameter)遗传距离。考虑到bootstrapping在种内水平上的检测能力有限[22],NJ树的可信度用interior branch test[23]检测,1 000个重复。AFLP数据用Phyltools软件计算距离矩阵[24],然后用MEGA 3构建NJ树。

    (2) 遗传分化分析。ITS1和ITS2数据用Dna SP 4.0[25]进行卡方(χ2)和KST*检验分析,其中χ2基于等位基因频率信息,其余基于DNA序列变异信息[26]。AFLP数据用Arlequin 2.0[27]FST进行了基于等位基因信息的精确检验(global exact test)[28]和基于位点变异信息的随机组合检验(random permutation test),10 000次重复。ITS和AFLP数据的AMOVA[29]都用Arlequin 2.0软件计算。此外还用Network 4.1(www.fluxus-engineering.com)[30-31]分析了ITSs数据的网络结构,用MVSP V3.1[32]对AFLP数据进行了主成份分析(principal component analysis,PCA)。

    获得ITS1序列44条,其中:CN、JP和AUS 3个种群分别获得18、12和14条序列。ITS1序列长401~405 bp,比对长度406 bp,包含19个变异位点,由11个简约信息位点(parsimony informative sites)和8个缺失位点组成。44条序列可分成29个等位基因,3个种群的等位基因和共享情况见表 1

    表  1  ITS1的等位基因及其在各种群中的分布
    Table  1.  Alleles of ITS1 and their distributions in populations of each species
    种群
    population
    h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15
    CN 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1
    JP 3 3 1
    AUS 1
    种群
    population
    h16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26 h27 h28 h29 sum
    CN 1 18
    JP 1 1 1 1 12
    AUS 1 3 1 1 1 3 1 1 1 14
    注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note: h01, h02, …: allele;CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV 
    | 显示表格

    获得ITS2序列34条,CN、JP和AUS种群分别获得16、8和10条。ITS2序列长229~237 bp,比对长度237 bp,包含20个为变异位点,由8个简约信息位点和12个缺失位点组成。34条序列可分成15个等位基因。3个种群的等位基因和共享情况见表 2

    表  2  ITS2的等位基因及其在种群中的分布
    Table  2.  Alleles of ITS2 and their distributions in populations of each species
    种群
    population
    h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 sum
    CN 2 2 1 2 1 1 3 1 1 1 1 16
    JP 1 3 1 2 1 8
    AUS 3 1 3 1 2 10
    注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note:h01, h02, …:allele;CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV 
    | 显示表格

    ITS1等位基因序列在GenBank的注册号为:AY877514-5,AY877517,AY877524-5,AY877527,AY877533-5,AY877537,AY877543-5,AY877547,AY877557-8,AY877563-72,AY877573-80。ITS2等位基因序列在GenBank的注册号为:AY877582,AY877585,AY877588-90,AY877592-4,AY877597-8,AY877600-1,AY877606-11,AY877612-6。

    AFLP实验共有64个个体获得清晰条带,其中CN种群30个,JP种群19个,AUS种群15个。3对引物共扩增184个位点,其中E-ACT/M-CAA扩增58个位点,E-ACT/M-CTG 70个位点,E-AGT/M-CTG 56个位点。3个种群享有共同的单态带,但没有种群特异的鉴别条带(图 2)。

    图  2  3个种各8个体的AFLP扩增结果
    CN. 中国群体;JP. 日本群体;AUS. 澳大利亚群体
    Figure  2.  Amplified fragment length polymorphisms in eight individuals of each species
    CN. Chinesse P. fucata; JP. Japanese P. fucata martensii; AUS. Australian P. imbricata

    种内和种间的平均K2P遗传距离几乎相等(表 3)。ITS1显示CN种群的种内遗传距离较大,而ITS2则表明JP种群的种内遗传距离较大。但ITS1和ITS2都显示CN种群与JP种群之间的遗传距离最大,与地理分布距离呈反相关。但AFLP数据计算的CN种群与JP种群之间的遗传距离最近,AUS种群与CN种群和JP种群相距最远,与它们的地理分布格局具有高度的一致性。

    表  3  ITS1和ITS2的种内和种间的平均K2P遗传距离及AFLP的Nei & Li平均遗传距离
    Table  3.  Average intra- and interspecific K2P genetic distances from ITS1 & 2, and Nei and Li′s (1979) interspecific genetic distances from AFLP data
    种群
    population
    种内平均遗传距离/%
    intraspecific genetic distance
    种间遗传距离/%(下三角: ITS 1,上三角: ITS2)
    interspecific genetic distance (below diagonal: ITS1, above diagonal: ITS2)
    AFLP的Nei&Li′s
    遗传距离
    Nei&Li′s distance for AFLP
    ITS 1 ITS 2 CN JP AUS CN JP
    CN 0.87
    (0~2.00)
    0.77
    (0~1.80)
    - 1.25
    (0~3.10)
    0.87
    (0~2.20)
    JP 0.69
    (0~1.50)
    1.20
    (0~1.80)
    0.90
    (0~2.00)
    - 1.11
    (0~2.60)
    0.74
    AUS 0.54
    (0~1.20)
    0.79
    (0~1.80)
    0.84
    (0~2.00)
    0.67
    (0~1.30)
    - 2.39 2.49
    注:CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note: CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV 
    | 显示表格

    ITSs的NJ系统树显示各个种的个体不聚合成单系群(monophyly),而是相互交错(图 3-a图 3-b)。当种间遗传距离很小时,一般的系统发育分析容易受到同质性(homoplasy)的影响,我们采用网络法构建系统发育关系,结果与NJ法一致,不同种群仍未聚合成单系群(数据未给出)。但AFLP的NJ树中AUS种群15个体有11个聚合成单支(图 3-c),表明AUS相对比较自成一体,而CN与JP种群的个体仍混合在一起。

    图  3  基于(a) ITS1、(b) ITS2序列和(c) AFLP的NJ系统发育树
    Figure  3.  Neighbor joining trees of the three species based on (a) ITS1, (b) ITS2 and (c) AFLP

    遗传分化的显著性检验表明,当基于基因型或表型频率时,ITSs和AFLP数据都没有显著性分化(数据未给出),尤其是AFLP数据,每个个体都表现为不同的表型,不存在分化的问题。而当基于序列位点突变(ITSs)或不同位点等位基因频率的差异(AFLP)时,3个种之间的遗传分化达到显著水平但分化程度很低,CN和JP种群分化尤其低(表 4)。

    表  4  基于DNA序列信息或等位基因频率的遗传分化显著性检验
    Table  4.  Significant test on genetic differentiation among species based on sequence information (K*ST) or allelic frequencies (FST)
    KST* FST
    ITS1 ITS2 AFLP
    种群
    population
    CN AUS CN AUS CN AUS
    AUS 0.057*** 0.004ns 0.084*
    JP 0.034* 0.034* 0.095*** 0.113* 0.021* 0.092*
    注:ns. 不显著;*. 0.01 < P < 0.05;***. P < 0.001;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note:ns. not significant;*. 0.01 < P < 0.05;***.P < 0.001;CN. Chinesse P. fucata;JP. Japanese P. fucata martensii;AUS. Australian P. imbricata
    下载: 导出CSV 
    | 显示表格

    AFLP数据的主成份分析也表明,CN和JP种群的个体重叠在同一个区域,而AUS的大部分个体则有所分开(图 4),与AFLP的NJ树一致。ITSs和AFLP数据的AMOVA分析都表明,主要变异来源于种内(94%~96%),种间的差异较小(4%~6%)。

    图  4  AFLP的主成份分析
    CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Figure  4.  Principal component analysis on AFLP data
    CN. Chinesse Pinctada fucata; JP. Japanese P. fucata martensii; AUS. Australian P. imbricata

    中国的P. fucata和日本的P. fucata martensii是重要的育珠母贝[12, 33],澳大利亚的P. imbricata也具有潜在的育珠价值[34],经济价值高,分布广[10]。但它们之间的分类地位存在一定争议。ITSs由于协同进化(concerted evolution),导致种内的差异被迅速同化而种间差异则快速积累[35],因而在种类鉴定方面特别有用,已成功用于珊瑚、贝类、多毛类等亲缘关系近、形态相似的种的区分[36-41]。在珊瑚和多毛类的ITSs序列研究中表明,种间的序列差异显著高于种内的序列差异[36, 39]。2种牡蛎(Saccostrea commercialisS. glomerata)的ITS1序列分析亦表明它们是同种[42]。本研究表明CN、JP和AUS这3个种群的ITS1和ITS2的种间遗传距离非常小,与种内遗传距离相互重叠,而系统发育分析也表明不同种的个体相互混聚在一起。此外,种间的遗传分化也很低。这些证据都支持它们是同种的结论[13-14]

    由于ITSs是多拷贝基因,可能存在个体内变异[43]。为此,我们增加了AFLP标记的应用。AFLP结合了RAPD和RFLP的优点,具有稳定可靠、重复性好的特点,且不需要研究对象的任何前提条件,可用于所有生物[44]。AFLP指纹技术可产生大量位点,这些位点比较均匀地分布于整个基因组,因而在种类鉴定上比微卫星还要优越得多[45]。目前AFLP已广泛用于物种区分和系统发育研究[46-48]。本研究表明,AFLP标记所揭示的3个种之间的遗传关系,包括遗传分化程度和遗传距离都与地理隔离的远近呈正相关,即地理相隔远的种群之间遗传距离较大,分化程度也较高。NJ聚类分析和PCA分析结果也完全吻合,均揭示出澳大利亚种群相隔较远。但3个种群都没有各自特异的AFLP标记,我们在珠母贝属其他种间的AFLP分析中可发现不同种的特征条带非常明确(未发表资料)。在虹鳟(Oncorhynchus mykiss irideus)、褐鳟(O. clarki clarki)及其杂种的AFLP分析中,133个多态标记中就有23个鉴别标记(diagnostic marker)将3者区分开[49]。本研究中AFLP的结果和ITSs也是非常一致的,而且更加灵敏,能够把AUS种群有所区分出来。这些结果与蛋白质等位酶的结果相同[11-12]

    本研究表明,中国的P. fucata、日本P. fucata martensii和澳大利亚的P. imbricata 3个地理种群是同一物种。按照分类命名优先法则应该用P. imbricata一名。但由于大西洋的P. imbricata与太平洋的P. imbricataP. fucataP. fucata martensii不同[50],因此这3个种群正确的种名应为P. fucata

  • 图  1   中国、日本和澳大利亚3个种群的采样点分布图

    CN. 中国海南;JP. 日本三重县;AUS. 澳大利亚Port Stephens

    Figure  1.   Sampling localities of the Chinese, Japanese and Australian populations

    CN. Hainan, China; JP. Mie Prefecture, Japan; AUS. Port Stephens, Australia

    图  2   3个种各8个体的AFLP扩增结果

    CN. 中国群体;JP. 日本群体;AUS. 澳大利亚群体

    Figure  2.   Amplified fragment length polymorphisms in eight individuals of each species

    CN. Chinesse P. fucata; JP. Japanese P. fucata martensii; AUS. Australian P. imbricata

    图  3   基于(a) ITS1、(b) ITS2序列和(c) AFLP的NJ系统发育树

    Figure  3.   Neighbor joining trees of the three species based on (a) ITS1, (b) ITS2 and (c) AFLP

    图  4   AFLP的主成份分析

    CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群

    Figure  4.   Principal component analysis on AFLP data

    CN. Chinesse Pinctada fucata; JP. Japanese P. fucata martensii; AUS. Australian P. imbricata

    表  1   ITS1的等位基因及其在各种群中的分布

    Table  1   Alleles of ITS1 and their distributions in populations of each species

    种群
    population
    h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15
    CN 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1
    JP 3 3 1
    AUS 1
    种群
    population
    h16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26 h27 h28 h29 sum
    CN 1 18
    JP 1 1 1 1 12
    AUS 1 3 1 1 1 3 1 1 1 14
    注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note: h01, h02, …: allele;CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV

    表  2   ITS2的等位基因及其在种群中的分布

    Table  2   Alleles of ITS2 and their distributions in populations of each species

    种群
    population
    h01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 sum
    CN 2 2 1 2 1 1 3 1 1 1 1 16
    JP 1 3 1 2 1 8
    AUS 3 1 3 1 2 10
    注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note:h01, h02, …:allele;CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV

    表  3   ITS1和ITS2的种内和种间的平均K2P遗传距离及AFLP的Nei & Li平均遗传距离

    Table  3   Average intra- and interspecific K2P genetic distances from ITS1 & 2, and Nei and Li′s (1979) interspecific genetic distances from AFLP data

    种群
    population
    种内平均遗传距离/%
    intraspecific genetic distance
    种间遗传距离/%(下三角: ITS 1,上三角: ITS2)
    interspecific genetic distance (below diagonal: ITS1, above diagonal: ITS2)
    AFLP的Nei&Li′s
    遗传距离
    Nei&Li′s distance for AFLP
    ITS 1 ITS 2 CN JP AUS CN JP
    CN 0.87
    (0~2.00)
    0.77
    (0~1.80)
    - 1.25
    (0~3.10)
    0.87
    (0~2.20)
    JP 0.69
    (0~1.50)
    1.20
    (0~1.80)
    0.90
    (0~2.00)
    - 1.11
    (0~2.60)
    0.74
    AUS 0.54
    (0~1.20)
    0.79
    (0~1.80)
    0.84
    (0~2.00)
    0.67
    (0~1.30)
    - 2.39 2.49
    注:CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note: CN. Chinese population;JP. Japanese population;AUS. Australian population
    下载: 导出CSV

    表  4   基于DNA序列信息或等位基因频率的遗传分化显著性检验

    Table  4   Significant test on genetic differentiation among species based on sequence information (K*ST) or allelic frequencies (FST)

    KST* FST
    ITS1 ITS2 AFLP
    种群
    population
    CN AUS CN AUS CN AUS
    AUS 0.057*** 0.004ns 0.084*
    JP 0.034* 0.034* 0.095*** 0.113* 0.021* 0.092*
    注:ns. 不显著;*. 0.01 < P < 0.05;***. P < 0.001;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
    Note:ns. not significant;*. 0.01 < P < 0.05;***.P < 0.001;CN. Chinesse P. fucata;JP. Japanese P. fucata martensii;AUS. Australian P. imbricata
    下载: 导出CSV
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  • 收稿日期:  2006-05-18
  • 修回日期:  2006-06-03
  • 刊出日期:  2006-10-19

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