【作者】 王敏晓；

【导师】 孙松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生态学， 2010， 博士

【摘要】 桡足类是海洋浮游动物中生物量最大的类群,堪称海洋中的昆虫。作为海洋生态系统次级生产力的主要组成部分,浮游桡足类处于海洋食物网的枢纽环节,在海洋物质能量通路中起到调节作用。其庞大的数量决定了桡足类在全球碳循环中的支撑作用。然而浮游桡足类的形态具有高度进化保守性,许多种类形态差别细微,因此基于形态学桡足类的种类鉴定、系统发育研究困难重重。分子标记具有直接反应生物自身遗传特征的优点,不同变异速率的分子标记可以解决种群结构划分、种类鉴定、高阶分类单元系统发育关系等各个水平的遗传分化的问题。为了更好的了解中国近海的浮游动物特别是浮游桡足类,我们使用线粒体细胞色素氧化酶第一亚基（COX1）,核核糖体RNA小亚基（18S rRNA）以及线粒体基因组研究桡足类不同尺度的遗传分化。本文主要分为四个部分,其主要内容如下:1.中华哲水蚤（Calanus sinicus）的线粒体基因组及桡足类的系统发育由于缺少合适的分子标记,桡足类的分子遗传研究尚处于起步阶段,从种群到高阶系统发育存在一系列问题亟待解决。线粒体基因组信息量大,除包含大量的序列变异外,还涵盖了包括基因排布、密码子使用等基因组层面的信息。中华哲水蚤是西北太平洋的陆架海区的关键种,处于该海域生态系统物质能量通路的枢纽环节。因此我们测定了中华哲水蚤的线粒体基因组并由此探讨了桡足类在节肢动物门中的系统发育位置及其内部各目之间的关系。中华哲水蚤线粒体基因组存在大量长片段的非编码序列,是已测甲壳生物线粒体中最长的,无处不在的基因重排使得中华哲水蚤线粒体基因组的基因排列组织结构完全不同于其他节肢动物线粒体基因组。线粒体的重组、非编码序列的产生及其相互作用可能是导致桡足类线粒体基因重排的主要原因。此外,中华哲水蚤线粒体基因组中还包括以下一系列特征,例如:偏低的AT含量,不同编码链间相似的核苷酸组成,以及相对其他桡足类较长的ATP酶编码基因。基于线粒体基因组蛋白氨基酸序列的系统发育结果显示,桡足类是单系发生的,哲水蚤目和Podoplea总目在桡足亚纲的基部分化。本研究构建的进化树中剑水蚤目和鞘口水蚤目聚在一起,显示了较近的亲缘关系;我们的结果支持Boxhall将剑水蚤目和鞘口水蚤目合并的建议。颚足纲则是多系发生的,可以分成三大类群。桡足类线粒体基因组结构独特,广泛使用的La PCR技术用于桡足类线粒体测序时却往往困难重重。本研究借助La PCR对中华哲水蚤线粒体测序时曾获得部分序列缺失的闭合环状DNA分子,后证实该序列为线粒体基因组内的嵌合序列。该现象与Lunt（1997）提出的线粒体重组的模型一致,提示该种线粒体可能发生重组,有待进一步研究。该结果警示使用La PCR进行线粒体测序要十分小心,需进一步验证所获序列的完整性。2.哲水蚤目桡足类的分子系统发育及分化时间估算哲水蚤目桡足类占海洋浮游动物生物总量的25%-80%,广泛分布于海洋生态系统的各类生境中。它们是浮游动物从洋底进入大洋水体,并成功占领大洋生态系统的最成功范例。哲水蚤目桡足类从底栖到大洋浮游生活方式的每一次变迁都在该类群进化过程中留下了深刻的印记。本研究首次通过分子手段构建哲水蚤目及其内部各主要类群间的系统发育关系,根据分子钟初步估算各主要类群的分化时间,进而探讨哲水蚤进化过程中推动该类群改变栖息环境和生活方式的可能因素,从分子遗传学的角度验证Bradford（2002）提出的若干假说。哲水蚤目18S rRNA二级结构相当保守,仅纺锤水蚤属43螺旋（Helix 43）的环部长度大幅延伸,折叠成稳定的茎环结构:Helix E43₁和Helix_E43₂。基于18S rRNA的系统发育结果中,哲水蚤目、管口虱目和怪水蚤目的单系性得到了很好的重现。鞘口水蚤目和剑水蚤目互为并系发生,共同组成一个单系群。本结果支持Boxhall将鞘口水蚤目并入剑水蚤目的建议。怪水蚤目落入猛水蚤内部,破坏了猛水蚤目的单系性。与18S rRNA贝叶斯树的结果一致,基于18S rRNA和COX1序列的系统发育分析中Eucalanoidea,Clausocalanoidea,Diaptomoidea和Arietelloidea总科的单系性得到了很好重现;虽然Calanoidea总科所在分支节点支持度不高,但约束Calanoidea为单系的系统树的似然概率最高,支持该总科的单系性。哲水蚤目总科间的系统发育关系为: （（Epacteriscioidea, Pseudocyclpoidea）, （Arietelloidea, （Diaptomoidea, （Calanoidea, （Eucalanoidea,Clausocalanoidea）））））。哲水蚤目起源于400.0百万年前（Mya）的泥盆纪的早期,营浮游生活的哲水蚤起源于398 Mya,此时大洋的高浊度促使了浮游类群的诞生,该时间节点与Bradford假设中的时间一致。Calanoidea-Clausocalanoidea以及Diaptomoidea所在分支起源于343百万年前的石炭纪早期,泥盆纪晚期的灭绝事件促使了桡足类的进一步分化以更好适应大洋水域的浮游生活。具髓鞘包被神经的高级类群起源于三叠纪早期（246.0Mya）,大洋浊度的下降、头足类的繁盛以及二叠纪三叠纪的大灭绝事件驱动了该类群的起源。3.中国近海浮游桡足类DNA Barcode分析桡足类在海洋生态系统中具有重要作用,其种类的准确鉴定是海洋生态系统的基础。由于桡足类形态进化中的保守性,隐存种在海洋浮游动物特别是桡足类中非常普遍。即便某些成体分类特征明确的种类,其幼体阶段依然难以甄别。因此有必要建立一套快速、准确、易于标准化的整合多种信息的中国近海浮游桡足类鉴定平台。DNA Barcode的兴起为桡足类种群鉴定提供了新的方法。本研究获得共90种浮游动物222条COX1序列。其中AT含量为61.1%,T和G出现的频率较高,转颠比为1.1。由于COX1序列尤其是摆动密码子具有显著的突变饱和,因此该序列不适于高阶系统发育分析。然而该标记可以准确区分近缘种,是种类鉴定的有效工具。基于COX1基因构建的进化树中,224种（149种来自GenBank）哲水蚤目桡足类大都聚成独自的单系群。种内遗传差异从0到26.8%不等,均值为1.9%;同属种间差异从0.3%到36.1%不等,均值为23.62%,两者差异显著（t=18.57,p<<0.01）。但部分哲水蚤目桡足类种内、种间遗传差异发生重叠,可能是由隐存种的分化以及形近种的鉴定错误所致,故该类群的多样性水平存在一定程度的低估。以上结果表明mtCOX1是哲水蚤目种类鉴定的有效工具。基于DNA Barcode,我们对中国近海的浮游哲水蚤组成有了进一步的了解,许多形近种得以区分。4.中国近海中华哲水蚤的种群遗传结构研究中华哲水蚤是西北太平洋陆架海域的关键种,弄清其种群遗传结构的组成对于理解该种的种群动力学演化及其同洋流、锋面等物理因素的关系有着重要意义。由于环境可塑性,传统的形态学方法很难筛选出鉴定种群的有效信息。分子标记则直接反映样品本身的内在差异,分辨率高,包括线粒体基因组调控序列在内的多种分子标记已广泛应用于种群的研究中。使用1060 bp的COX1序列和核ITS1-5.8S-ITS2序列,本文分析了采自东黄海鸭绿江口、黄海冷水团、长江口以及南海共计50只中华哲水蚤个体的遗传多样性,结果显示ITS序列在种内高度保守,没有发现任何变异位点。COX1基因变异位点十分有限,1060个碱基中可变位点仅15个,包含14个单倍型。各站位中华哲水蚤单倍型多态性水平中等（0.6132）,核苷酸多态性水平降低（0.000626）。不同站位均拥有多个特有单倍型,各海区存在一定程度的自我补充。H1单倍型以最高频率共享于所有站位中,不同站位间中华哲水蚤未发现显著遗传差异。使用最小生成网格和BEAST最大置信树分析不同单倍型的系统发育关系,所有单倍型分成两大分支,但未发现明显的地理分布格局。基于COX1序列的AMOVA分析表明中华哲水蚤没有显著的遗传结构分化,不同站位种群遗传差异不显著（FST=0.01389）。为了获得适合种群研究的高变分子标记,本研究筛选了来自鸭绿江口、长江口、黄海冷水团以及韩国北部沿海的11只中华哲水蚤分析其线粒体基因组序列,共发现401个可变位点,其中插入缺失突变全部出现于非编码区。整个基因组包含3个高变区,其中碱基11216到12260间的区域最为高变,包含两个微卫星位点,平均不到5个碱基就有发生一次变异。基于线粒体基因组序列,所有个体都拥有自己的单倍型,线粒体基因组表现出很强的基因型筛选能力。相对COX1基因,线粒体基因组特别是非编码区高变序列更适合该种的种群遗传学研究。更多还原

【Abstract】 In terms of their size, diversity and abundance, copepods can be regarded as the insects of the seas. Marine copepods are the most abundant and conspicuous component of a plankton sample. They are by far the most important primary consumers in the marine ecosystem and play a vital role by linking primary production to higher trophic levels. Furthermore, copepods are abundant and thus represent an ecologically important source of carbon flux. However, researches on copepods are usually hampered by the difficulties in species identification based on morphology in isolate owning to the subtle differences between species. The conservative trends of morphological evolution in copepods also confuse the phylogenetic researches in copepods. Molecular markers can offer us alternative tools for phylogenetic analysis at broad range of levels from population to inter-order relationships. Here we investigate copepods at different phylogenetic levels via separate molecular markers including COX1, 18S rRNA and complete mitochondrial genome. The thesis paper was divided into 4 independent issues:1. The mitochondrial genome of C. sinicus with multiple long noncoding regions and reshuffled gene order, useful molecular markers for phylogenetic analysis.With greatest diversity and richest abundance, copepods exhibit extensive ecological radiation in the marine ecosystem. Calanus sinicus dominates continental shelf waters in the Northwest Pacific Ocean and plays an important role in local ecosystem by linking primary production to higher trophic levels. Lacking effective molecular markers, several phylogenetic issues on copepods are still elusive from population to higher levels such as their phylogenetic position within pancrustacea. Being genome-level informative, mitochondrial DNA sequences are used as markers not only for population genetic studies but also for phylogenetic studies. Concurrence of multiple noncoding regions and reshuffled gene arrangement makes the mitochondrial genome of C. sinicus remarkably distinctive from other arthropods, including copepods. Furthermore, there are another suit of particularities found in mitogenome of C. sinicus, such as low AT content and symmetrical nucleotide composition between strands, abbreviated stop codons for some PCGs, as well as extended length of ATP6 and ATP8 relative to other copepods. Monophyly of copepods and basal split between Calanoida and Podoplea were well resolved while Maxillopoda were resolved polyphyletic. The closest affinity between Cyclopoida and Poecilostomatoida supports Boxshall in reassigning the latter subordinate to the former. The occurrence of mosaic sub-genomic fragments （mosaic cycler sequence） during laboratory work calls attention to the application of LA PCR to mitogenome sequencing in copepods, and may give an additional evidence of mitochondrial recombination. The accelerated mutational rates within copepods as well as interaction of non-coding regions with recombination in C. sinicus mitogenome give rise to present highly rearranged mitochondrial genome.2. Molecular phylogeny and divergence estimation of calanoid copepods based on 18S rRNA and mtCOX1 genes.Calanoid copepods, which are major component in all plankton samples, exhibit extensive radiation in the marine ecosystem. They are by far the most successful colonizers of pelagic realm. Their successful colonization is closely, if not completely, associated with the evolutionary history they have encountered. Here we tested the hypothesis regarding phylogenetic relationships of Calanoid copepods with molecular evidences.Secondary structures for 18S rRNA of calanoid copepods were constructed based on their consensus sequences. Owing to functional constraints, the structures are almost unchangeable among calanoid copepods with some exceptions in the genus of Acartia. Acartia species expanded loop regions in the helix 43, which was refolded into new helixes, being Helix E43₁ and Helix E43₂. The structure has been proved useful to assist in aligning 18S rRNAs and in giving more reasonalbe evolutionary models. Our phylogenetic analysis based on 18S rRNAs confirmed monophyly of Calanoida, Misophrioida and Siphonostomatoida. Cyclopoida and Poecilostomatoida which were resolved reciprocal paraphyletic coalescent into a monophyletic clade, supporting Boxhall in subordinating Poecilostomatoida to Cyclopoida. Misophrioida penetrated into Harpacticoida and destroyed the monophyly of the latter. In accordance with the results from 18S rRNA dataset, monophyly of Eucalanoidea, Clausocalanoidea, Diaptomoidea and Arietelloidea were well resolved by the combined dataset of both 18S rRNA and mtCOX1 gene. Though ambiguously resolved with low node confidence support, monophyly of the superfamily Calanoidea was confirmed by the best likelihood score for the Calanoidea-monophyletic constraint tree during the hypothesis testing. Based on 18S rRNA, divergence time for the order Calanoida was estimated at 400 Mya while the pelagic forms originated 398 Mya, which is in congruent with Bradford’s hypothesis. The high turbidity during early Devonian may trigger the explosion of pelagic animals, as supported by fossil evidence from ostracods. About 343 million years before, the clade comprising of Calanoidea-Clausocalanoidea and Diaptomoidea diversified. Extinction event at late Denovian should have accelerated this divergence which gave rise to the lineage that fits the pelagic realms better. Divergence dating of advanced groups （calanoids from species in Clanoidea-Clausoclanoidea with Myelin-like sheath covered nerve fibers） was estimated at 246 Mya, when the turbidity decrease to a minimum level and the abundance of predator retained a high level. The extinction event at the boundary of Permian and Triassic should have driven the divergence process.3. DNA barcoding the calanoid copepods in the Chinese coastal regionsCopepods play an important role in the marine ecosystem. Accurate copepod species identification is thus necessary for understanding the conditions of local marine ecosystem. However, even the most skillful experts may get confused since the copepods resemble each other with subtle morphological differences. Even fewer clues are available for larval identifications. DNA barcodes provide an alternative approach for species identification.222 sequences covering 90 zooplanktons were identified in our study, AT content of which is 61.1%. Notable substitution saturation is apparent in all codons and particularly in the wobble ones, which preclude their utility in higher-level phylogenetic studies. Intraspecific variation of 224 calanoid copepods ranged from 0 to 26.8% with the means of 1.9% while congeneric intraspecific variation ranged from 0.3% to 36.1% with the means of 23.62%. Significant barcode gap was observed （t=18.574, p<<0.01）. Most individuals belonging to the lineages whose intra- and inter-specific variation overlapped have been proved to encounter cryptic speciations. The majority of morphological species were clustered together in a monophyletic clade. These findings suggest that mtCOX1 is an appropriate barcoding tool for calanoid copepods and the diversity of calanoid copepods have been overlooked based on morphology. Using this molecular approach, several marine copepods in China were revised here.4. Population genetics of Calanus sinicus in the Chinese coastal regions Calanus sinicus dominates continental shelf waters in the Northwest Pacific Ocean and plays an important role in local ecosystem by linking primary production to higher trophic levels. Lacking effective molecular markers, phylogenetic relationships among populations of the species are still elusive.For the preliminary study, COX1 genes of 1060 bp in length and complete ribosomal internal transcriptional spacers （ITS1-5.8S-ITS2） were adopted for population genetic analysis in 50 individuals of C. sinicus sampled from Yalujiang estuary, Changjiang estuary, the South China sea and the Cold Water Mass in the Yellow Sea. ITS sequences were revealed highly conservative with no variable sites in all sequences analyzed. Though more variable, only 15 variable sites were detected in COX1 genes, representing 14 haplotypes. Specific haplotypes can be found in separate sampling stations, indicating that self-recruitment may exist in local areas. However, H1 haplotype was shared by all stations with high frequencies, which illustrate the homological status between populations. As was confirmed by AMOVA analysis, no genetic structure could be resolved in C. sinicus with available markers, suggesting that high dispersal potential may lead to a strong gene flow among different regions. However, consensus is far away from being reached. Since more variable markers are needed.11 nearly complete C. sinicus mitochondrial genomes from Yalujiang estuary, Changjiang estuary, Cold Water Mass and Korea nearshore were screened for hyper-variable markers for population studies. Within the 16,670 bp alignment, there are a total of 401 variable sites. Indel variations all present in non-coding regions and transitions dominate the SNPs. Three“hot-spots”, especially the hyper-variable microsatellite locus in LNRs provide rich polymorphism for population studies. By the means of 3 hyper-variable regions, all individuals could be assigned to a unique haplotype. Compared to the COX1 gene, more information could be obtained from mitochondrial genome for the population studies.更多还原