177 resultados para Mitochondrial-dna
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本工作用线粒体细胞色素b基因全序列探讨了白腹鼠属(Niviventer)分子系统发育关系,利用线粒体控制区全序列研究小家鼠(Mus musculus)在中国的分子系统地理学。 在白腹鼠属的分子系统发育研究中,本研究首次对来自青藏高原东南部和台湾的9种(安氏白腹鼠、川西白腹鼠、社鼠、刺毛鼠、梵鼠、灰腹鼠、褐尾鼠、台湾白腹鼠和台湾社鼠)32个个体的细胞色素b全序列进行了测定和分析。用最大简约法、最大似然法和贝叶斯法三种构树方法构出的进化树一致表明,9种分为3个线粒体DNA系:系A包括社鼠、台湾社鼠、台湾白腹鼠、刺毛鼠、梵鼠和灰腹鼠6种;系B包括安氏白腹鼠和川西白腹鼠2种;系C仅包括褐尾鼠一种。应用DNA分类的分析方法表明台湾社鼠不应是社鼠的亚种,而应是一个独立的种。此外,以来自更新世中晚期(1.2-0.13百万年)的社鼠化石为依据推算该属的分歧时间。结果表明该属的起源于1.64百万年前,系A和系B分化发生在1.46百万年以前,该属的其余物种分化时间为1.29-0.67百万年前。这些分化时间和青藏高原的最后一次隆起、昆黄运动以及第四纪大冰期时间相一致,提示地理隔离和冰期作用可能对该属的形成演化起到重要的作用。 在小家鼠的分子系统地理学研究中,通过测定来自中国12个采集地的184只小家鼠的控制区全序列,并结合了Genbank里的单倍型,首次研究小家鼠在中国的分子系统地理格局、种群历史和生物地理过程。184个样品定义了66个单倍型,单倍型多样性为0.95,核苷酸多样性为0.012。用66个单倍型及Genbank里来自周边国家的62个小家鼠单倍型进行分子系统发育分析,邻接法和贝叶斯法构得的分子系统发育树基本一致。结果表明小家鼠在中国分为两个线粒体DNA系(南方系和北方系)。分子变异等级分析结果表明主要的分子变异发生在两个线粒体DNA系之间(84.03%)。根据研究结果推断长江是两个线粒体DNA系之间基因交流受限的主要地理障碍。单倍型进化网络关系表明两个线粒体DNA是异域扩散。两个系的单倍型歧点分布图呈钟型分布,说明经历了快速的种群扩散。经推算小家鼠在中国北方系的种群扩散时间为16 600(22 000-11 000)年前,而南方系的种群扩散时间早于北方系,扩散时间为20 000(40 000-6 700)年前。依据单倍型和核苷酸的多样性、单倍型进化网络关系以及单倍型歧点分布图综合推断小家鼠在中国的两个系在末冰期时经历了快速的种群扩张导致今天的地理分布格局。根据系统发育树的结果推断,北方系种群的大体扩散方向是从北往南,而南方系是从南往北迁移。小家鼠以长江为界分为南方系和北方系的系统地理模式、扩散方向和种群的扩散时间与人以长江为界分为南方人和北方人、扩散方向、扩散时间(60 000-16 000)基本一致,这可能暗示人类活动对小家鼠的迁移产生的重要影响。
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本研究用线粒体细胞色素b 基因全序列和ND4 基因序列探讨了哀牢髭蟾 (Vibrissaphora ailaonica)分子亲缘地理学和保护遗传学,利用线粒体细胞色素b、 控制区全序列和部分12SrRNA 基因序列研究了分布于中国的红瘰疣螈 (Tylototriton verrucosus)分子亲缘地理学和保护遗传学。 哀牢髭蟾为生活于高海拔生境(大约2000-2600m)的濒危蛙类。各种致危因 素引起了关于对小的、隔离种群长期存活的关注,然而迄今没有关于该物种遗传 多样性的报道。本论文首次研究了分布于中国云南省的哀牢髭蟾的亲缘地理学和 保护遗传学。对采自于9 个种群81 个个体,我们应用线粒体mtDNA cyt b 和 ND4 基因共计1990bp, 获得了51 个单倍型。系统发育分析显示三个分化较深且互为单 系的族群,大致相应于分别被元江和藤条江河谷分开的三个地理单元(元江以东、 金平和藤条江以西地理单元),暗示了长期的地理隔离分化。分子变异等级分析 (AMOVA)显示遗传变异主要为三个地理单元之间(81.68%)和种群内的个体之 间(11.26%)。三个地理单元显著的地理分布暗示在空间和时间上的隔离,这与中 国西南地区在晚中新世到上新世的哀牢山的造山运动和隆升所引起的重要的气候 和古地质变化时间相一致。我们推测低海拔干热河谷可能是三个地理单元之间基 因交流受限的主要生态障碍。根据上述研究结果,我们建议对这三个遗传分化显 著的地理单元,元江以东地区、金平地区、藤条江以西地区作为独立的管理单元 分别加以保护。 本研究通过测定中国横断山区的红瘰疣螈(Tylototriton verrucosus)17个采集 点的123个红瘰疣螈标本的线粒体mtDNA Cyt b、D-loop和12SrRNA三个基因片段 (2347 bp)序列,首次研究了红瘰疣螈在横断山区的群体遗传结构和分子系统地理格 局,并讨论了T. shanjing的物种有效性。基于123个样品定义的49个单倍型的贝叶 斯和NJ系统发育分析表明:(1) T. verrucosus和T. shanjing均未各自构成单系,而是 共同构成一个单系群;(2) 横断山区的红瘰疣螈分为3个线粒体DNA地理单元,即滇 东南、滇中-滇西和片马地理单元,并且地理单元间不存在共享单倍型,说明红瘰 疣螈具有明显的系统地理分布格局。AMOVA分析同样表明3个地理单元之间存在 显著差异,并且分子变异主要发生在3个地理单元间(62.4%)。T. shanjing与T.verrucosus的mtDNA cyt b 序列差异平均值仅为1.1%,明显小于它们与两外群(贵 州疣螈和大凉疣螈)及外群间的遗传距离(6.5-9.9%)。因此,根据T. verrucosus和 T.shanjing的遗传差异以及系统发育分析结果都不支持T. shanjing的物种地位,T. shanjing为T. verrucosus的同物异名,并建议恢复T. verrucosus的中文名红瘰疣螈。 基于上述结果,我们建议将分布于滇东南、滇中-滇西、片马地区的红瘰疣螈作 为三个独立的管理单元分别加以保护。
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依据线粒体上ND2和CO1两个变异较大的基因序列分析了香港地区香港湍蛙7种群、华南湍蛙1种群,以及大陆其他地区华南湍蛙7种群,戴云湍蛙1种群,武夷湍蛙1种群的系统发育关系,进而探讨香港湍蛙的遗传多样性、香港湍蛙特有性、如何确定香港湍蛙最佳保护单元以及这四种湍蛙的物种分类地位。
1. 香港湍蛙保护遗传学研究
香港湍蛙核苷酸传多样性较低,从其遗传多样性信息、单倍型网络分析、中性检验值以及岐点分布结果一致显示香港湍蛙很可能经历了瓶颈后的扩张,种群正在由一个较小的有效种群大小迅速增长, 有足够的时间通过变异用于积累单倍型的多态性, 而对于提高核苷酸多样化而言, 时间尚短(Nei M et al,1975,Avise J C,2000;李明等,2003)。
分子变异分析结果显示香港湍蛙种群间存在较多的基因交流,且系统发育树上各种群间交叉在一起,没有形成与地理单元相关的分支,而从其单倍型网络看,他们源于共同的祖先,是一个单系群,与地理单元间没有形成显著的遗传分化。因此应作为一个进化显著单元(ESU)。结合其与其他湍蛙发育关系及遗传距离以及野外采集信息认为香港湍蛙只在香港地区有分布,属于香港特有种。该物种内遗传多样性较低,又属于世界自然保护联盟红皮书中的近危种,同时也是《野生动物保护条例》中的受保护野生动物,且由于香港城市建设等使得其栖息环境受到威胁,因此在香港特别行政区应该受到重点保护。
从单倍型分布和核苷酸多样性可以看出大榄涌种群和城门种群具有较高的单倍型多样性和核苷酸多样性,应该作为保护的重点区域。
2. 华南湍蛙东、南沿海种群间系统关系
华南湍蛙分布广,各种群存在着丰富的遗传多样性信息且中部种群广西龙胜和湖南张家界种群核苷酸多样性明显高于其他边缘种群华南湍蛙。种群间几乎没有基因交流,且各种群间无共享单倍型,可见已形成了显著的遗传分化。各种群间遗传距离都较远,其中广东南昆山种群以及福建三港种群与其他种群距离最远,因此可以推测其他种群(广东深圳、香港大屿山、广西龙胜和防城以及湖南张家界种群)可能为独立进化的种群。但是否是一新种或一隐存种,还需要结合形态学进行更深入的研究。
本研究中无论从系统关系看还是从遗传距离看,大屿山种群与深圳种群最近,支持陈坚峰等将其定为华南湍蛙,即华南湍蛙新增一个分布点:香港大屿山。
系统树上广西防城种群(支B)与龙胜和湖南种群(支A)形成姐妹群。香港大屿山种群与深圳种群先形成姐妹群(支C),但却没有与其距离很近的广东南岭及南昆山种群(支D)形成姐妹群,可能粤北和粤中的环境及气候较复杂因此与粤南其他种群形成了明显的隔离。同时可以看出华南湍蛙种群遗传分化与地理距离没有显著的相关性。
3. 四种湍蛙间的系统关系
根据线粒体CO1基因建立四种湍蛙间的系统关系及其遗传距离,很清楚地看到,香港湍蛙与戴云湍蛙关系很近,而华南湍蛙则与武夷湍蛙较近。然而,戴云湍蛙同一个种群内部共有两个单倍型DY1和DY2,且两个单倍型间遗传距离大于DY1与香港湍蛙间遗传距离,更远远大于香港湍蛙种群内部的距离,即戴云湍蛙内部两个单倍型间遗传距离达到了种级水平,同样在系统发育树上这两个单倍型与香港湍蛙形成并系。但是,戴云湍蛙种内在形态上差异不显著。因此,其是否属于萌芽物种分化形成(budding speciation)或已经完全分化为两个不同的种值得进一步研究?
与戴云湍蛙香港湍蛙关系类似,从系统树上看华南湍蛙不形成单系,而是分成两个大支,与武夷湍蛙形成并系,且福建和南昆山的华南湍蛙与武夷湍蛙遗传距离远大于武夷湍蛙种内福建种群与浙江种群的遗传距离,达到了种级分化水平。由此,可以推断武夷湍蛙是有效种。系统树上广东深圳、香港大屿山、广西防城和龙胜以及湖南张家界种群与华南湍蛙福建及南昆山各种群间遗传距离已超出了种内各种群间的遗传距离,但是至于这一支是否应为另外一个种,有必要扩大采样,并结合核基因及形态信息进行进一步研究。
MtDNA of ND2 and CO1 gene were used to investigate genetic diversity of Amolops in Hongkong .We collected seven populations of A. hongkongensis,,one population of A.ricketti from Hong Kong and other seven populations of A.ricketti from East and South of Chinese mainland. As well as one population of A. daiyunensis and one population of A.wuyiensis Phylogenetic relationship were analyzed of four species. Discussed whether A.hongkongensis is an endemic species and how can we make the conservation and management decisions.
1. Conservation Genetics of A. hongkongensis
A. hongkongensis has a low nucleotide diversity, the results of genetic diversity, haplotype network, neutrality test and the mismatch distributions indicate that A. hongkongensis experienced a recent expansion after a bottle neck. They had enough time to accumulated haplotype diversity, but it’s too short to have a high nucleotide diversity(Nei M et al,1975,Avise J C,2000;Li et al,2003).
The result of AMOVA reveals that it has much gene exchange among the populations of A. hongkongensis. The clades of the phylogenetic tree were mixed together, no significant genetic differentiation among 8 populations and they share the same ancestor from the network analysis, these indicate that they are monophyly and should be protected as one ESU. Combined with the information of relationships of interspecies, genetic distance and distribution investigate, We conclude that A. hongkongensis is an endemic species of Hong Kong. Considering on the status of low genetic diversity in A.hongkongensis, and this species was listed in the IUCN red list as near threatened, as well as listed in the
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Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.
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Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global theta (ST) (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise theta (ST) comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.
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A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.
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为了进一步研究青蟹属系统进化的科学问题,并揭示我国东南沿海青蟹群体遗传结构和群体进化细节信息,本论文主要开展了以下两个方面的研究:(1)基于线粒体12S rRNA、16S rRNA和COI三种基因序列探讨中国东南沿海青蟹的种类归属与青蟹属的系统进化;(2)利用线粒体COI基因标记分析中国东南沿海拟穴青蟹的群体遗传结构。序列特征、遗传距离和系统进化分析结果都表明本文研究的青蟹均为S. paramamosain。NJ、BAYES和ML系统进化树显示S. paramamosain与S. tranquebarica互为姐妹种,S. olivecea应该是4种青蟹中最早分化出来的种类。10个地理群体130只拟穴青蟹的线粒体DNA(mitochondrial DNA,mtDNA)细胞色素氧化酶亚基I(COI)基因序列Mantel检验结果显示群体间的遗传分化程度与地理距离没有显著的相关性。分子进化中性检验结果表明自然选择在分子进化过程中起了重要作用,并暗示该物种在最近经历了一个快速的群体爆发及扩张事件。
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The jinjiang oyster Crassostrea rivularis [Gould, 1861. Descriptions of Shells collected in the North Pacific Exploring Expedition under Captains Ringgold and Rodgers. Proc. Boston Soc. Nat. Hist. 8 (April) 33-40] is one of the most important and best-known oysters in China. Based on the color of its flesh, two forms of C rivularis are recognized and referred to as the "white meat" and 11 red meat" oysters. The classification of white and red forms of this species has been a subject of confusion and debate in China. To clarify the taxonomic status of the two forms of C. rivularis, we collected and analyzed oysters from five locations along China's coast using both morphological characters and DNA sequences from mitochondrial 16S rRNA and cytochrome oxidase 1, and the nuclear 28S rRNA genes. Oysters were classified as white or red forms according to their morphological characteristics and then subjected to DNA sequencing. Both morphological and DNA sequence data suggest that the red and white oysters are two separate species. Phylogenetic analysis of DNA sequences obtained in this study and existing sequences of reference species show that the red oyster is the same species as C. ariakensis Wakiya [1929. Japanese food oysters. Jpn. J. Zool. 2, 359-367.], albeit the red oysters from north and south China are genetically distinctive. The white oyster is the same species as a newly described species from Hong Kong, C. hongkongensis Lam and Morton [2003. Mitochondrial DNA and identification of a new species of Crassostrea (Bivalvia: Ostreidae) cultured for centuries in the Pearl River Delta, Hong Kong, China. Aqua. 228, 1-13]. Although the name C. rivularis has seniority over C. ariakensis and C. hongkongensis, the original description of Ostrea rivularis by Gould [1861] does not fit shell characteristics of either the red or the white oysters. We propose that the name of C. rivularis Gould [1861] should be suspended, the red oyster should take the name C. ariakensis, and the white oyster should take the name C. hongkongensis. (C) 2004 Elsevier B.V. All rights reserved.
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How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed-dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3-4) and paternally inherited, pollen- and seed-dispersed chloroplast DNA (cpDNA) (rpl16 and trnS-trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G(ST) = 0.738, N-ST = 0.771) indicating low levels of seed-based gene flow and significant phylogeographical structure (N-ST > G(ST), P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north-central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north-central part of the species' range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north-central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among-population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.
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We sequenced partial mitochondrial 16S ribosomal DNA (16S rDNA) of 18 firefly species from Southwest of China. Combined with homologous sequences previously reported, phylogenetic trees including Japanese, Korean and Chinese species were reconstructed by
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The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1 rhodopsin exon 1, and tyrosinase exon 1) genes from 57
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从9种科鱼类的福尔马林标本中获得了333bp的细胞色素b基因片段的序列。这9个种分别代表科鱼类的8个属。333bp的DNA序列经MUST软件排序后,有101个变异位点,其中有39个信息位点。序列在成对物种间的距离为8~48。平均遗传距离为24%~144%。简约分析产生了最大简约系统树,其步长是162(CI=0735,RI=0494)。在该系统树上,Bagarius是最原始的属,并与所有其他的物种形成姊妹群。其余8个属形成一个单系类群并分为二个姊妹群。尽管在形态上具有13个离征,但在分子系统树上
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The population genetic structure of fish parasitic nematode, Camallanus cotti, collected from the Yangtze River, Pearl River and Minjiang River in China was investigated. From these parasites, the similar to 730 bp of the first internal transcribed spacer of ribosomal DNA (ITS1 rDNA) and the 428 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene were sequenced. For the ITS1 rDNA data set, highly significant Fst values and low rates of migration were detected between the Pearl River group and both the Yangtze River (Fst = 0.70, P < 0.00001; Nm = 0.21) and Minjiang River (Fst = 0.73, P < 0.00001; Nm = 0.18) groups, while low Fst value (Fst = 0.018, P > 0.05) and high rate of migration (Nm = 28.42) were found between the Minjiang and the Yangtze rivers. When different host/locality populations (subpopulations) within each river were considered, subpopulations between the Yangtze River and Minjiang River had low Fst values (<= 0.12) and high Nm values (>3.72), while Pearl River subpopulations were significantly different from the Yangtze River and Minjiang River subpopulations (Fst >= 0.59; Nm < 1). The COI gene data set revealed a similar genetic structure. Both phylogenetic analyses and a statistical parsimony network grouped the Pearl River haplotypes into one phylogroup, while the Yangtze River and Minjiang River haplotypes formed a second group. These results suggested that the Yangtze River and Minjiang River subpopulations constituted a single reproductive pool that was distinct from the Pearl River subpopulations. In addition, the present study did not find host-related genetic differentiation occurring in the same drainage. (C) 2009 Published by Elsevier B.V.