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鳜类及其两种牛首科寄生虫的系统发育研究
Alternative TitleMolecular Phylogeny of sinipercid fishes (Perciformes: Sinipercidae) and their two bucephalid digeneans (Digenea: Bucephalidae)
陈达丽
Subtype博士
Thesis Advisor聂品
2007-06-11
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword鳜类 鳜属 少鳞鳜属 细胞色素b基因 S7核糖体蛋白基因 内含子 16s Rrna 细胞色素c氧化酶亚基1基因 Viperin基因 核糖体转录间隔区 系统发育 单系 物种鉴定 范尼道弗吸虫 合肥道弗吸虫
Abstract鳜类(sinipercids)鱼类是一群淡水鲈形目鱼类,而隶属于牛首吸虫科的(Bucephalidae)范尼道弗吸虫(Dollfustrema vaneyi)和合肥道弗吸虫(Dollfustrema hefeiensis),是鳜类鱼类多数成员肠道寄生虫中的常见种。尽管目前已有一些基于形态和分子水平的系统方面的研究,但对鳜类系统发育及其进化地位尚有较大争议。另一方面,范尼道弗吸虫和合肥道弗吸虫在地理分布、宿主分布以及形态特征均有较高相似性。因此,我们对鳜类和这二种近缘的牛首科吸虫进行了系统发育分析,并对鳜类与这二种寄生虫的协同进化关系进行了初步探讨。 首先,以线粒体细胞色素b(cyt b)基因全序列为标记,分析了鳜类中的10种以及11种与鳜类相近并代表不同的科的外类群的系统发育关系。采用邻接法、最大简约法、最大似然法和贝叶斯法分别进行系统发育重建,采用Shimodaira-Hasegawa test (SH test)、approximately test (AU test)和paramatric bootstrap test检验相互竞争的拓朴树或系统发育假设,并运用Bayesian松散分子钟方法,估算鳜类鱼类的分化时间。系统发育分析表明:作为外类群的9个物种,即分别代表鮨科(Serranidae)、锯盖鱼科(Centropomidae)、发光鲷科(Acropomatidae)、谐鱼科(Emmelichtyidae)、篮子鱼科(Siganidae)、海鲈科(Moronidae)和太阳鱼科(Centrarchidae)的种类,均插入鳜类的鳜属(Siniperca)和少鳞鳜属(Coreoperca)之间,这为鳜类的非单系起源提供了明显的证据。少鳞鳜属似乎与代表太阳鱼科的Centrachus属亲缘关系更近,而在少鳞鳜属,日本少鳞鳜(C. kawamebari)位于系统树基部,中国少鳞鳜(C. whiteheadi)和朝鲜少鳞鳜(C. herzi)形成姐妹种。在鳜属中,长身鳜(S. roulei)位于基部,麻鳜(S. fortis)次之,鳜(S. chuatsi)和大眼鳜(S. kneri)是最分化的,并与暗鳜(S. obscura)形成姐妹群。而斑鳜(S. scherzeri)和波纹鳜(S. undulata)的系统发育位置不确定。 然而,不同的系统发育重建方法得到的系统发育树拓扑结构并不完全一致,且只有paramatric bootstrap检验能显著拒绝鳜类的单系性(P < 0.01)假设,支持鳜类的并系起源。鳜科的非单系起源可能表明,传统分类上建立鳜科的“共同离征”,比如说圆鳞,可能是至少两次独立起源产生的。基于最优的系统发育假设,采用贝叶斯松散分子钟,首次为鳜类鱼类的进化提供了分子钟尺度。鳜属大约起源于22.71±4.84 Mya,从渐新世到中新世之交辐射到更新世,在中新世伴有几次快速的成种事件;而少鳞鳜属最有可能起源于约22.90±3.78 Mya。 核基因的内含子被普遍用作系统发育标记,但是当产生系统发育假设时,关于排序策略和插入/缺失的处理方法的一系列问题并没有明确的阐述。通过设置不同的比对参数,采用Clustal x和POY 3.0.11对核基因S7核糖体蛋白(rpS7)基因内含子2序列进行比对,进而探讨鳜类的系统发育。比对产生的空位分别处理成缺失性状(missing data),用GapCoder软件对空位进行编码并将这些编码附加在原有序列之后,以及将不能排定的位点去掉。处理好的序列矩阵采用最大简约法、最大似然法和贝叶斯法分别构建分子系统发育树。用一致节点的总数目(MP:BP ≥ 75%;ML:BP ≥ 75%;BI:PP ≥ 0.95)来评价分析策略。发现,POY软件(gap cost分别为1和2)比对得到的数据建树一致性节点数均比Clustal x比对建树的一致性节点数高。采用了gapcoder编码空位(gap)的数据建树普遍比其它两种方法好。同时,将不能排定的位点剔除会使系统发育信息减少,对系统发育分析是不利的。基于POY软件(gap cost = 2)比对得到的数据构建的系统发育树中,鳜属和少鳞鳜属的单系性得到支持,但鳜类的单系性由于N. spinosus的插入被破坏。在少鳞鳜属中,朝鲜少鳞鳜(C. herzi)位于基部,中国少鳞鳜(C. whiteheadi)和日本少鳞鳜(C. kawamebari)形成姐妹种。在鳜属中,麻鳜(S. fortis)和波纹鳜(S. undulata)聚成一枝,其它种类形成另一枝:斑鳜1(S. scherzeri 1)位于基部,长身鳜(S. roulei)和斑鳜2(S. scherzeri 2)次之,鳜(S. chuatsi)和大眼鳜(S. kneri)是最分化的,并与暗鳜(S. obscura)形成姐妹群。 我们选用一种免疫分子,即viperin基因来重建鳜类的系统发育。为了评价viperin基因的系统发育意义及它与其它基因的一致性,采用单独以及联合的方法分析7307bp的分子数据,这些数据包括cyt b、16S rRNA和细胞色素c氧化酶亚基1(CO1)这三个线粒体基因,以及核基因viperin基因和S7核糖体蛋白基因(rpS7)内含子1和2。各基因片段的系统发育意义和系统发育一致性通过一致性检验、一致节点数和PBS(Partitioned Bremer support)来确定。尽管检测到线粒体基因和核基因之间的系统发育信号的显著异质性,通过AU检验(approximately unbiased test)和SH检验(Shimodaira-Hasegawa test)都支持基于viperin基因构建的贝叶斯树是最佳拓扑结构树,其拓扑结构为((中国少鳞鳜,(日本少鳞鳜,朝鲜少鳞鳜)),((暗鳜,斑鳜),((波纹鳜,麻鳜),(长身鳜,(大眼鳜,鳜)))));而基于其它基因片段和联合数据构建的贝叶斯树都被显著拒绝。六个基因片段的拓扑评估表明,在现有的采样水平上,viperin基因比本研究中所选用的其它基因的系统发育适用性更好,将该基因用于其它鱼群的系统发育分析可能会很有趣。 从采自不同地点、寄主和寄生部位的范尼道弗吸虫(Dollfustrema vaneyi)和合肥道弗吸虫(D. hefeiensis)共60个个体中扩增并克隆得到完整的核糖体转录间隔区(ITS1-5.8S-ITS2)序列。通过计算P-distance遗传距离和系统发育重建,以探讨ITS在物种鉴定和系统发育重建中的意义。在范尼道弗吸虫的38个个体中,发现44个多态位点及21个单倍型;而在合肥道弗吸虫的22个个体中,发现43个多态位点和16个单倍型。两种寄生虫之间没有共享的单倍型。两种寄生虫之间的单倍型多样度较核苷酸多样度更为相似。所有用于分析的样本的ITS1种间遗传距离变化范围在0.0441—0.0744,平均为0.0536,种内遗传距离在0—0.0421之间,平均为0.00948;ITS2种间遗传距离变化范围在0.0513—0.0736,平均为0.0576,种内遗传距离在0—0.0435之间,平均为0.00525。种间遗传距离远远大于种内距离;表明以ITS1和ITS2为遗传标记,可以在物种水平上对这二个种相似种进行准确的鉴定。系统发育分析(包括邻接法,简约法,最大似然法和贝叶斯法)都得到两个支持率很高的分枝,分别对应于范尼道弗吸虫和合肥道弗吸虫。然而,两个物种的内部进化关系并不一致,尽管它们均未显示与地理和寄主相关联的特异性结构,但能说明这两个种含有复杂的进化历史。
Other AbstractSinipercids are a group of freshwater percoid fish endemic to East Asia. Dollfustrema vaneyi and D. hefeiensis are two common digenean parasites in the intestines of sinipercid fish and even other non-sinipercid fish. Despite several taxonomical attempts and preliminary mtDNA-based molecular phylogeny among the sinipercids, their phylogenetic relationship and systematic position remained unsolved. D. vaneyi and D. hefeiensis are regarded as sibling species, which share morphological similarities. Thus, the molecular phylogeny of sinipercids and the two digeneans were investigated in the present study, with a preliminary discussion on the co-evolutionary relationship between the two digeneans and siniprcids. The complete cytochrome b gene sequences from nine sinipercid species and four non-sinipercid fish species were cloned, and a total of 12 complete cyt b gene sequences from 10 species of sinipercids and 11 sequences from 10 species of non-sinipercid fish also in Perciformes were included in the phylogenetic analysis including neighbor-joining, maximum parsimony, maximum likelihood and Bayesian analyses. The competing sinipercid topologies were tested by using the Shimodaria-Hasegawa test (SH test), approximately unbiased test (AU test) and parametric bootstrap test. As expected, the two genera Siniperca and Coreoperca within sinipercids are recovered as monophyletic. However, nine species representing Moronidae, Serranidae, Centropomidae, Acropomatidae, Emmelichtyidae, Siganidae and Centrarchidae included in the present study are all nested between Coreoperca and Siniperca, which provides marked evidence for a nonmonophyly of sinipercid fishes. Coreoperca appears to be closest to Centrachus representing the family Centrarchidae. Coreoperca whiteheadi and C. herzi are sibling species, which together are closely related to C. kawamebari. In the Siniperca, the node between S. roulei and the remaining species is the most ancestral, followed by that of S. fortis. S. chuatsi and S. kneri are sibling species, sister to S. obscura. However, the sinipercids do not seem to have a very clear phylogenetic history, for different methods of phylogenetic reconstruction result in different tree topologies, and the only conclusive result in favor of a paraphyletic origin of the two sinipercid genera is the parametric bootstrap test. The paraphyly of Sinipercidae may suggest that the ‘‘synapomorphs’’ such as cycloid scales, upon which this family is based, were independently derived at least twice within sinipercid fishes, and further study should be carried out to include the other two Siniperca species and to incorporate other genes. By using a Bayesian relaxed molecular clock approach for inferring dating information from molecular phylogeny, the first molecular clock timescale was presented for sinipercids evolution. The genus Siniperca appears to have emerged approximately 22.71 ± 4.84 Mya, and radiated from Oligocene–Miocene boundary to Pleistocene, along with several rapid speciation events in Miocene; while genus Coreoperca have emerged most possibly approximately 22.90 ± 3.78 Mya. Nuclear introns are commonly used as phylogenetic markers. Based on different alignment parameters, the rpS7 intron 1 sequences are aligned with Clustal x(GO = 20, 10, 5, 2.5 and 1, respectively; GE = 10, 5, 1 and 0.5, respectively)and POY 3.0.11 (gap costs = 1, 2, 4 and 8, respectively). The gaps generated in the process of alignment are treated as missing data, generating a “simple indel coding” with the program GapCoder, and excluding the areas of ambiguous alignment. The matrixes generated after the gap treatment were analyzed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian (BI) methods. The different analytical strategies are assessed based on the totality of congruent nodes (MP: BP≥75%; ML: BP≥ 75%; BI: PP ≥ 0.95). It is suggested that there are more congruent nodes of phylogenetic trees reconstructed on the matrix generated from POY (gap cost = 1 and 2, respectively) than from Clustal x. The topology of the trees based on the matrix containing new information generated from GapCoder are prevalently better than the other two-gap treatments. It is also shown that excluding the areas of ambiguous alignment will decrease the phylogenetic information. In the tree reconstructed on the matrix aligned by POY (gap cost = 2), the monophyly of sinipercids was disputed by the injection of N. spinosus. The two genera Siniperca and Coreoperca were recovered with high posterior probability. In the Coreoperca, C. whiteheadi formed a sister species with C. kawamebari, and C. herzi was a basal species. The Siniperca consisted of two main clades. One was formed by S. fortis and S. undulata, and the other clade was represented by the rest members of the Siniperca. S. scherzeri 1 branched basal to this clade, and (S. roulei, S. scherzeri 2) was the secondly basal species. S. chuatsi and S. kneri was the most derived species, and formed a sister species that was sister to S. obscura. An innate immunity gene, the virus-induced protein (viperin) gene was for the first time employed in the present study to illustrate the phylogeny of sinipercids. The A total of 7307 bp data from three mitochondrial partitions (cytochrome b, CO 1 and 16S rRNA) and viperin gene plus the first two introns of nuclear S7 ribosomal protein gene was analyzed in order to estimate the relationship among sinipercids and to assess the phylogenetic utility of viperin gene. The phylogenetic utility and homogeneity of all partitions were estimated via a combination of homogeneity partition tests, congruent nodes, and partitioned Bremer support. Despite the detection of significant heterogeneity of phylogenetic signal between the mitochondrial and nuclear partitions, the Bayesian tree by viperin gene represented the best-supported topology of all the data by using the approximately unbiased test and the Shimodaira-Hasegawa test, with the following clades: ((C. whiteheadi, (C. kawamebari, C. herzi)), ((S. obscura, S. scherzeri), ((S. undulata, S. fortis), (S. roulei, (S. kneri, S. chuatsi))))). While all Bayesian tree from other individual and simultaneous data were significantly rejected. Topological appraisals of the six gene partitions suggested that the viperin gene is a better estimator of phylogenetic relationship of sinipercids than the cyt b, COI, 16S rRNA partitions and the first two introns of rpS7 at the taxonomic levels under consideration, and it would be interesting to employ this gene for the phylogenetic analysis of other fish groups. The complete internal transcribed spacer 1(ITS1), 5.8S rDNA, and ITS2 region of the ribosomal DNA from 60 specimens belonging to two closely-related bucephalid digeneans (Dollfustrema vaneyi and Dollfustrema hefeiensis) from different localities, hosts and microhabitat sites were cloned to examine the level of sequence variation and the taxonomic levels to show utility in species identification and phylogeny estimation. It is obvious that these molecular markers may enable the discrimination of the two species. A total of 21 haplotypes was found with 44 polymorphic positions in 38 individuals of D. vaneyi, and 16 haplotypes found with 43 polymorphic positions in 22 individuals of D. hefeiensis. There is no shared haplotypes between the two species. Haplotype rather than nucleotide diversity is similar between the two species. For ITS1, the largest intraspecific genetic distance is 0.0421, much lower than the average interspecific distance, which has a value of 0.0536, and still much lower than the shortest interspecific distance, 0.0441. For ITS2, the largest intraspecific genetic distance is 0.0435, much lower than the average interspecific distance, which has a value of 0.0576, and still much lower than the shortest interspecific distance, 0.0513. It is thus suggested that ITS1 and ITS2 are useful genetic markers for the identification of bucephalid digeneans. Phylogenetic analyses revealed two robustly supported clades, one corresponding to D. vaneyi and the another to D. hefeiensis. However, the population structures between the two species seem to be incongruent, and show no geographic and host-specific structure among them, further indicating that the two species may have had a much more complex evolutionary history than expected.
Pages147
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12106
Collection学位论文
Recommended Citation
GB/T 7714
陈达丽. 鳜类及其两种牛首科寄生虫的系统发育研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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