摘要
细首科(Cephalothricidae)隶属纽形动物门古纽目(Palaeonemertea)。由于其有限的形态学特征及形态学鉴定本身的复杂性，而成为分类学中较为混乱的类群之一。有关线细首科系统发育的研究较少，尤其从分子水平上来研究线细首科纽虫系统发育关系目前还未见报道。本研究将以细首科纽虫为研究对象，验证统计简约网络法进行物种界定的可行性；拟基于COI，16SrDNA，28SrDNA基因序列探讨细首科纽虫的系统发育关系，并且利用三种纽虫线粒体基因组基因排列信息及蛋白质编码基因，探讨纽虫在后生动物中的分类地位。
主要研究成果如下：
1．扩增了线细首科165个个体的线粒体COI基因，依据统计简约法原理，用TCS软件构建单倍型网络图。探讨此方法在线细首科纽虫中进行物种界定的可行性。同时在MEGA4．0软件中进行遗传距离分析，在Phyml和MrBayes软件中构建系统发育树。结果表明，同一种的个体明显地聚在同一个单倍型网络图中。种间K2p遗传距离显著地大于种内遗传距离，得到的最大似然树和贝叶斯树显示同一物种的不同个体均形成高支持率的单系。因此得出结论，基于线粒体COI基因的统计简约单倍型网络图方法能够对细首科物种进行有效地物种界定。
2．以核基因(28SrDNA)和线粒体基因(16SrDNA和COI)为遗传标记，对线细首科的系统发育关系进行了探讨。以针纽目Amphiporus angulatus，Amphiporus lactifloreus为外类群，采用最大似然法(ML)和贝叶斯法(BI)分别构建系统发育树。系统发育树的拓扑结构显示，细首科并不是单系。
3．首次测定了纽形动物门三种纽虫，Cephalothrix simula，Cephalothrix sp．和Paranemetes peregrine的线粒体基因组，基因组大小分别为16296bp，15800bp(部分非编码区没有测定)和14560bp。三者均包含后生动物线粒体基因组典型的基因组成(13个蛋白质编码基因、22个转运RNA和2个核糖体RNA)。C．simula与C．sp．基因排列完全一致。与这两种纽虫相比，Paranemetesperegrine的基因组中有11个基因发生了易位包括4个蛋白质编码基因(nad6，cox1，cox2，atp6)和7个tRNA(trnY，trnE，trnG，trnF trnQ，trnW，trnC)。
基于基因排列，蛋白质编码基因氨基酸及氨基酸数据的系统发育分析结果均支持纽虫与有体腔的冠轮动物有较近的亲缘关系，而不是无体腔的扁形动物。基于基因排列及蛋白质编码基因核苷酸数据系统发育研究结果表明纽虫与内肛动物近缘。基于蛋白质编码基因氨基酸序列的系统发育分析结果支持纽虫与软体动物近缘。
Cephalothricidae is one of the classes within the order Palaeonemertea of thephylum Nemertea. Taxonomy among this group remains confused due to their fewmorphological characters and the complication of morphology methods based on thehistological sectioning. So far, few reports have been published on the phylogeny ofCephalothricidae, especially at the molecular level. In the present study, we tried totest the potential use of statistical parsimony network analysis based on COI sequenceas a tool to delimit species in Cephalothricidae species, and to estimate thephylogenetic relationship among this group based on CO1, 16SrDNA and 28SrDNA.In addition, we used gene arrangement and the protein-coding genes to assess thephylogenetie position of Nemertea within the lophotrochozoans.
The main work and conclusion of the present study were as the following:
(1) To test the potential use of statistical parsimony network analysis as a tool todelimit species in Cephalothricidae species. COI gene sequences from 165Cephalothricidae specimens were sequenced and performed a statistical parsimonyanalysis in the program TCS. Genetic distance was calculated in MEGA3.0 software,and phylogenetic trees were constructed with Phyml and MrBayes software. COI genesequences from single species typically stick together in a single haplotype network.In class Cephalothricidae distance within species is obviously lower than that betweenspecies. And in the phylogenetic trees, all individuals of each species formed a clade.From these results, we can draw a conclusion that statistical parsimony networkanalysis based on the mitochondrial CO1 gene can effectively support the speciesdelimitation in Cephalothricidae.
(2) Three combined genes (COI, 16SrDNA and 28SrDNA) were employed toestimate the relationship of the Cephalothricidae. Two hoplonemerteans Amphiporus lactifloreus and Amphiporus angulatus were chosen as outgroup. Maximum likelihood (ML)and Bayesian inference (BI) methods were adopted to construct the phylogenetic trees.All these analyses consistently showed that the family Cephalothricidae is notmonophyletic, which is not consistent with the results from morphology study.
(3) The mitochondrial genomes from Cephalothrix simula, Paranemetes peregrineand Cephalothrix sp. were sequenced, which is first report of complete mitochondrialgenome in phylum Nemertea. The mitochondrial genomes of them is 16,296 bp,14,560 bp, and more than 15,800 bp in length (partial non-coding region was notdetermined), respectively.
As other metazoans, the genomes of C. simula, P. peregrine and C. sp. contain astandard set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNAgenes. The gene arrangements of C. simula and C. sp. mitochondrial genomes areconsistent. Compared to this two, transloeations of 4 protein-coding genes (nad6,cox1, cox2, atp6) and 7 tRNA (trnY, trnE, trnG, trnF, trnQ, trnW, trnC)were found inP. peregrine.
All analyses based on gene rearrangements, nucleotide and amino acid datasupport that the nemertean is close to the coelomate lophotrochozoans, rather than theacoelomate platyhelminths. Both the pattern of gene rearrangements andphylogenomic analyses using concatenated nucleotide sequences of the 13mitochondrial protein-coding genes support the close relationship between nemerteanand the phoronid. While the phylogenomic analyses based on the concatenated aminoacid sequences showed the sister relationship between Nemertea and Mollusca.
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