陳昭倫臺灣大學：海洋研究所郭美貞Kuo, Mei-JenMei-JenKuo2007-11-272018-06-282007-11-272018-06-282004http://ntur.lib.ntu.edu.tw//handle/246246/56606多齒圍沙蠶種群廣泛分佈在印度太平洋沿岸潮間帶地區。根據形態特徵的研究結果，多齒圍沙蠶種群至少包含十四個種。本研究欲闡明台灣和大陸東南沿海地區，多齒圍沙蠶種群在形態型質和分子遺傳標誌之變異。依據外部形態可區分出七個不同表型 (morph)，然而形態特徵在表型內及間皆有變異存在，並且表型間變異的重疊，常造成部分樣本難以確定屬於何種表型。根據兩組獨立分子遺傳標誌的分析結果，包括細胞核內組織蛋白2A2B次單元區間 (H2A-H2B histone gene spacer region) ，以及粒線體細胞色素氧化酶第一次單元 (cytochrome c oxidase subunit 1) 核苷酸序列分析，台灣和大陸東南沿海地區之多齒圍沙蠶種群，可分成五個顯著的系群 (clade)，而且系群間的分化程度已達到種的層級。系群一包含了表型一、表型四和表型五。而系群二至五則分別對應於表型二、三、六、七相對應。在本研究中，依據外部形態特徵和分子遺傳標誌的檢驗結果大致相符，確定台灣和中國大陸東南沿海的多齒圍沙蠶種群至少包含五個種。這個結果也顯示形態特徵結合分子遺傳標誌，將可提供多齒圍沙蠶種群種別界定更可信的依據。The Perinereis nuntia species group is widely-distributed in the intertidal areas of Indo-Pacific. Currently, there are at least fourteen recognized species in the group based on morphological characters. The objective of the present study is to elucidate morphological and molecular variations of the Perinereis nuntia species group in Taiwan and southeastern China. Seven morphs of the Perinereis nuntia species group were recognized based on morphological characters. However, these characters exhibited variations within and between clades. The overlap of characters made identification between these morphs difficult. Five distinct clades of the Perinereis nuntia species group were identified using molecular markers, H2A-H2B histone gene spacer region (H2A-H2B spacer) and cytochrome c oxidase subunit 1 (COI) DNA gene. In addition, genetic distances between clades of this species group showed an order of divergence magnitude similar to that between species in other marine invertebrates. Therefore, the divergence between clades of this species group reached the level of species. Clade I comprised morph #1, #4, and #5. While clade II, clade III, clade IV, and clade V was equivalent to morph #2, #3, #6, and #7 respectively. The identification of morphological characters and molecular markers for the P. nuntia species group were compatible and revealed that there were at least five species of the P. nuntia species group in Taiwan and the southeastern of China. The present study also indicated the utility of molecular tools in conjunction with morphological characters to delineate species boundaries in the P. nuntia species group.Contents 1. Introduction 1 1.1. Nereididae systematics: past and present 1 1.2. The Perinereis nuntia species group 2 1.3. The objective of present study 4 2. Materials and methods 5 2.1. Sampling, fixation, and preservation 5 2.2. Morphological analysis 5 2.3. Molecular analysis 7 2.3.1. DNA extraction 7 2.3.2. Polymerase chain reaction (PCR) amplification and DNA sequencing 7 2.4. Sequence alignment and phylogenetic analysis 8 2.4.1 Sequence alignment 8 2.4.2. Nucleotide statistics 8 2.4.3. Saturation analysis 8 2.4.4. Modeltest 9 2.4.5. Reconstructions of phylogenetic trees 9 3.1. Morphological analyses 11 3.1.1. The assessment of allometry 11 3.1.2 Descriptions of the Perinereis nuntia species group 11 3.1.2. Morphological variation 12 3.2. Base composition, substitution pattern, and tests for phylogenetic signal 14 3.2.1. H2A and H2B histone gene 14 3.1.2.Cytochrome c oxidase subunit I 15 3.2. Phylogenetics 16 3.2.1. H2A-H2B spacer 16 3.2.2. Cytochrome c oxidase subunit I 17 4. Discussion 18 4.1. Variability of morphology in the Perinereis nunita species group 18 4.2. Utilities of molecular markers 19 4.3. Comparisons of morphological variations and molecular phylogenies for the Perinereis nuntia species group 20 4.4. Species status and species identification using multi-loci approach 22 4.5. Speciation and evolution history of the Perinereis nunita species group 23 5. Conclusion 26 6. References 28 Tables Table 1. The sampled sites, habitats and numbers of individuals used in morphological and molecular analyses…………………….……..………..32 Table 2. Comparison of seven morphs in the Perinereis nuntia species group…...…34 Table 3. Comparison of paragnath counts and the length ratio of the dorsal cirrus to the dorsal notopodial lobe of six morphs of the Perinereis nuntia species group and P. mictodonta……………………………......…….………….....36 Table 4. Nucleotide statistics of H2A-H2B spacer gene…………………………….38 Table 5. Nucleotide statistics of COI gene…………………………………………..39 Table 6. Uncorrected genetic distance for COI and H2A-H2B spacer of the Perinereis nuntia species group………………………………………………...……...40 Table 7. Comparison of COI gene divergence……………………………………….41 Figures Figure 1. Diagrams of crucial morphological characters in the Perinereis nuntia species group…………………………….………………………………..42 Figure 2. The map of the collection sites………..…………………………………..43 Figure 3. Relationships between body width and (a) body length, and between body width and numbers of setiger (b)………………………………………….44 Figure 4. Relationship between body width and total numbers of paragnaths………45 Figure 5. Relationship between body width and the ratio of the dorsal cirrus to the dorsal notopodial lobe………………………………………………..……46 Figure 6. Seven morphs of the Perinereis nuntia species group from Taiwan and southeastern China………………………………………………………...47 Figure 7. Plots of paragnath counts for the six morphs of the Perinereis nuntia species group………………………………………………………………49 Figure 8. Plots of the length ratio of the dorsal cirrus to the dorsal notopodial lobe………………………………………………………………………...50 Figure 9. Alignment of H2A-H2B spacer of the Perinereis nuntia species group.....52 Figure 10. Skewness of tree length distributions…………………………...……….53 Figure 11. Proportions of transition and transversion to uncorrected pairwise distance plot of H2A-H2B spacer regions for the the Perinereis nuntia species group………………………………………………………………………54 Figure 12. Proportions of transition and transversion to uncorrected pairwise distance plot of COI for the Perinereis nuntia species group………………………55 Figure 13. Phylogenetic relationship of the Perinereis nuntia species group obtained from the H2A-H2B histone gene spacer regions …………………………56 Figure 14. Phylogenetic relationship of the Perinereis nuntia species group obtained from the COI gene………………………………………………………...59 Figure 15. The schematic synthesis of morphological and molecular analyses of the Perinereis nuntia species group in Taiwan and southeastern China……...62 Appendixes Appendix I. Key to differentiate the Perinereis nuntia species group………...…….63en-US多齒圍沙蠶種群第一次單元組織蛋白2A2B次單元區間粒線體細胞色素氧化酶cytochrome c oxidase subunit 1 geneH2A-H2B histone gene spacer regionThe Perinereis nuntia species group[SDGs]SDG14thesis