Sexually derived larvae, intracolonial genotypic variations, and heterozygosity excess of a brooded coral (Pocillopora damicornis) in Taiwan revealed by microsatellite markers

本論文探討一種廣泛分佈的孵育型珊瑚-細枝鹿角珊瑚 (Pocillopora damicornis) 在精細尺度上的無性繁殖系遺傳結構。本研究共利用七組微隨體基因標誌 (microsatellite markers)，其中四組 (PD1、PD2、PD3、PD5)在本實驗中獨立發展而得，分析採自台灣南部南灣海域後壁湖地區的三個獨立鹿角珊瑚族群。為了測試此種珊瑚可能的精子散佈距離，其中二個族群 (HB1和HB2) 採用窮盡式採集法– 我們採集了樣區內的每一個珊瑚個體，包括一個位置中心的珊瑚母群體 (mother colony) (n = 41 x 2)；第三個族群 (HBc)則因地理環境和珊瑚群體密度較低，僅隨機採集了25株珊瑚群體當作比較族群。除此之外，我們也從兩個獨立的珊瑚母群體各隨機取樣100隻幼生來作基因型分析。從200隻幼生的多基因座基因型 (multilocus genotypes)分析中，發現有高比例的無性繁殖系基因型 (82.5%)，顯示此地區的鹿角珊瑚以無性生殖為主要繁殖策略；其中，僅有12隻基因型相異的幼生 (n = 35) 可成功追溯其父源珊瑚體 (father colony)。在珊瑚群體的層級上，也發現有高比例的無性繁殖系多基因座基因型 (74.8%)，而且無性繁殖系基因型之間存在著遺傳變異 (intracolonial genotypic variations)。為了客觀定義出此變異的界線，以便於與有性生殖造成的遺傳變異做區隔，我們先用基因型距離矩陣 (genotypic distance matrix) 將所有珊瑚群體歸類成10個無性繁殖系群 (clonal lineage, C1-C10)，再從基因型頻率推算出各無性繁殖系群為有性生殖產物的機率 (Psex)，最後將此機率和電腦模擬出的機率下限 (critical-Psex) 做比較，以統計學的觀點驗證某無性繁殖系群是否僅為隨機產生；C1為此地區最大的無性繁殖系群，佔了所有樣本的53%。最後，不論在幼生層級或是群體層級，我們發現這三個族群都有異型合子過量 (heterozygosity excess) 的現象，這種現象在固著型海洋無脊椎動物 (sessile marine invertebrates) 中很少見，可能的原因包括：高度的無性生殖、異型合子優勢 (heterosis)、以及生殖上的自體不相容 (self-incompatibility)。The thesis described a research conducted on the fine-scale clonal genetic structure of a brooded coral, Pocillopora damicornis, which is widely distributed throughout the world’s coral reefs. Seven simple tandem repeat (STR) loci, or microsatellites, were utilized to characterize coral colonies sampled in the HoBihoo area, Nanwan Bay, southern Taiwan; among them, four microsatellite loci (PD1, PD2, PD3, and PD5) were independently developed by us in this study. In order to elucidate the potential sperm dispersal distance of this coral species, two populations (HB1 and HB2) were sampled exhaustively, i.e. all observed coral colonies were sampled within a demarcated area that was centered by a mother colony (n=41x2). A third population (HBc) was sampled randomly for index comparisons only (n=25) due to its distinct geographic environment and lower colony density. Besides, we also randomly collected and genotyped 100 larvae from each mother colony during planulations. A high proportion of the 200 larvae genotyped multilocusly were found to be clonal (82.5%), indicating asexual reproduction was the main reproductive strategy for P. damicornis within this area; among these larvae, only 12 genotypically distinct ones (total n=35) could be successfully traced down to their father colony. At the colony-level, a high proportion of the clonal genotypes was also found (74.8%); what is more, intracolonial genotypic variations from the identified clonal genotypes were observed as well. In order to objectively define the line drawn between genotypic variations caused by sexual recombinations and somatic mutations (the assumptive cause of intracolonial genotypic variations), 10 clonal lineages were tentatively grouped (C1-C10) with a threshold value inferred from the genotypic distance matrix. Then the probability that a given clonal lineage thus defined was only the coincidental product by chance alone (Psex) was calculated from the observed allele frequencies for each clonal lineage. At last, we compared these probabilities with the statistical boundaries set at the 5% level (critical-Psex) obtained from computer simulations. In this way, we could therefore verify these clonal lineages with a statistical viewpoint; C1 dominated the area of study by 53% in number and was the biggest clonal lineage. Last but not least, heterozygosity excess, a phenomenon rarely seen in sessile marine invertebrates, was observed at both the colony- and larva-level. Possible interpretations included widespread asexual reproductions, selection for heterozygotes (heterosis), and reproductive self-incompatibility.口試委員會審定書………………………………………………………………………... i Acknowledgement………...……………………………………………………………….. ii 中文摘要…………………………………………………………………………………... iii 英文摘要…………………………………………………………………………………... iv Prologue…………………………………………………………………………................. 1 Chapter 1 1.1. Introduction…………………………………………………………………………… 2 1.2. Materials and methods……………………………………………………................... 4 1.2.1. Sampling scheme………………………………………………………………. 4 1.2.2. DNA extraction and PCR………………………………………………………. 5 1.2.3. Data analysis……………………………………………………………………. 6 1.3. Results………………………………………………………………………………… 7 1.3.1. Marker applicability…………………………………………………................. 7 1.3.2. Characterizations of adult colonies and larvae………………………………...... 8 1.3.3. Paternal assignments…………………………………………………................. 8 1.4. Discussions……………………………………………………………………………. 9 1.4.1. Mixed mode of reproduction……………………………………………..……... 9 1.4.2. Sexually derived larvae…………………………………………………………. 10 Chapter 2 2.1. Introduction…………………………………………………………………………… 14 2.2. Materials and methods………………………………………………………………… 16 2.2.1. Sampling scheme……………………………………………………….............. 16 2.2.2. Genotype characterizations……………………………………………...……… 17 2.2.3. Data analysis…………………………………………………………….............. 17 2.2.4. Tentative grouping of clonal lineages…………………………………..………. 18 2.2.5. Verification with probabilities……………………………………………...…… 19 2.2.6. Tests based on the grouping…………………………………………………….. 19 2.3. Results………………………………………………………………………………… 20 2.3.1. Colony assignments…………………………………………………….………. 20 2.3.2. Linkage disequilibrium…………………………………………….……………. 23 2.3.3. Heterozygosity excess……………………………………………………...…… 23 2.4. Discussions……………………………………………………………………….…… 23 2.4.1. Clonal identity…………………………………………………………………... 23 2.4.2. Intracolonial genotypic variations………………………………………………. 25 2.4.3. Heterozygosity excess and self-incompatibility………………………………… 27 3. References………………………………………………………………………………. 30 4.1. Tables & Figures (Chapter 1)………………………….……………………………… 35 Table 1.1………………………………………………………..……………………… 35 Table 1.2……….………………………………………………………………….…… 36 Table 1.3…………………………………………………………………………..…… 37 Table 1.4………..……………………………………………………………………… 38 Table 1.5…………………………………………………………………………..…… 39 Fig 1.1………………………………………………………………………………….. 40 4.2. Table & Figures (Chapter 2)………………………………………………………...... 41 Table 2.1……………………………………………………………………………….. 41 Table 2.2………………………………………………………………………….……. 42 Table 2.3……………………………………………………………………………….. 43 Table 2.4……………………………………………………………………………….. 44 Table 2.5……………………………………………………………………………….. 45 Table 2.6……………………………………………………………………………….. 46 Fig 2.1………………………………………………………………………………….. 47 5. Appendix………………………………………………………………………………... 48 5.1. All samples in HB1……………………………………………………………….. 48 5.2. All samples in HB2……………………………………………………….………. 52 5.3. All samples in HBc……………………………………………………...……….... 5