利用微隨體基因座探討軸孔珊瑚種間界線與族群遺傳之研究

Abstract

軸孔珊瑚是石珊瑚中種類最多的屬，總共包含113個種，大部分物種廣泛分布在印度太平洋海域，但其中有三個種只分布在加勒比海。因為種內變異過大加上可能發生種間雜交，而造成軸孔珊瑚分類上的困難，因此很多學者利用各種型態上、雜交試驗和分子標誌的方式，以釐清珊瑚的種間界線問題，但是往往沒有很好的成效；加上在族群遺傳研究中，利用的分子標誌缺乏高解析力，因此我們需要尋找更適合的分子標誌，藉以應用在軸孔珊瑚的種間界線及族群遺傳的相關研究中。微隨體基因座是一段以1-6 bp大小為重複單元所組成的DNA片段，因為其變異性高，且屬於共顯性與中性遺傳，這些特點使它們成為族群遺傳、遺傳疾病、親屬關係鑑定等研究的重要工具之ㄧ。因此，本研究欲探討微隨體基因座在軸孔珊瑚種間界線與族群遺傳的研究中的可用性。 目前在本實驗室中有五組由美麗軸孔珊瑚篩選出的GT重複序列片段，以及在文獻中有八組由麋角軸孔珊瑚篩選出的AAT重複序列片段，欲探討這些AAT重複序列片段是否在印度太平洋的軸孔珊瑚之同源性，利用在四種西太平洋的軸孔珊瑚和麋角軸孔珊瑚的PCR反應和序列定序後，共有五組AAT重複序列片段在印度太平洋物種是具有同源性的，加上有三組可用的GT重複序列片段，因此共有八組微隨體基因座可用於研究同域共存的軸孔珊瑚種間界線問題與族群遺傳之研究。 在研究同域共存的美麗軸孔珊瑚與指型軸孔珊瑚之種間界線研究結果可知：從單一基因座的探討，共有四組基因座的對偶基因頻度在兩種珊瑚間有顯著差異，而且經由八組基因座的複因子成分分析結果，兩種珊瑚間的微隨體基因型有明顯的分群，結果顯示多組微隨體基因座應可利用在軸孔珊瑚種間界線的研究上。 經由比較親代與子代的基因型，這些微隨體基因座都符合孟德爾遺傳。在族群遺傳研究中，利用其中七組微隨體基因座，初步觀測西太平洋的美麗軸孔珊瑚族群分化情形。在族群的哈溫平衡測試中，有半數的試驗不符合哈溫平衡，應為有效族群過小與族群內的自交情形嚴重所致；而在FST和Nei’s (1983)遺傳距離分析結果支持眺石和Togian Islands的遺傳關係比較近，和蛇頭山反而比較遠，而且根據Neighbor-Joining方法所做的樹型圖也支持蛇頭山的族群是一有別於眺石與Togian Islands的獨立類群，因此推測美麗軸孔珊瑚的族群分化情形可能是受到西太平洋洋流流向的影響。 從本研究可知，多組微隨體基因座有機會被利用在軸孔珊瑚種間界線的研究中，而且也可被用在觀測軸孔珊瑚的族群遺傳分化情形，因此，增加微隨體基因座與欲觀測的族群大小有機會更進一步釐清軸孔珊瑚種間界線與在印度太平洋族群遺傳之研究。

Acropora is by far one of the largest extent reef-building corals genus comprising 113 species. It is also one of the most widespread genera of corals, spanning the Indian and Pacific Oceans and Caribbean Sea. Due to great intraspecific variations and interspecific hybridization, species boundaries among Acropora were not clear. Hence, morphological, breeding, and molecular criteria were utilized to examine species boundaries among Acropora. But, many lower resoluble genetic markers were used for species boundaries among Acropora spp. Therefore, due to high polymorphism within populations, microsatellites, 1-6 bp tandemly repeats stretches, might possibly be utilized for species boundary and population genetics of Acropora. There were five GT repeats, isolated from A. muricata, and eight AAT repeats, isolated from A. palmata, tested on four Indo-Pacific Acropora species and Caribbean A. palmata. After PCR amplification and sequencing, there were three GT repeats amplified in Indo-Pacific species and five homologous AAT-repeat microsatellite loci applied for Indo-Pacific Acropora species. As a result, there were eight microsatellite markers, including three GT and five AAT repeats, utilized for species boundary between sympatric A. digitifera and A. muricata in Togian Islands, Indonesia. From the results at each locus, allelic frequency distributions of these two Acropora species were significantly different at four microsatellite loci (Acr53, Acr1-60, 180, 181), but the others were not. In multi-locus analyses, FCA (Factorial correspondence analysis) showed that two species were significantly divided into two groups. According to this result, it revealed multi-locus microsatellite markers would be possibly highly resoluble genetic markers for species boundary among Acropora spp. Due to unsuccessful PCR amplifications at the 181 locus, there were also seven microsatellite markers applied for population subdivisions of A. muricata in Shertoushan, Tiaoshi in Taiwan and Togian Islands, Indonesia. Based on pairwise FST and Nei’s genetic distance (1983), it revealed that the smaller genetic distance between Tiaoshi and Togian Islands compared with Shertoushan. However, the NJ tree, based on Nei’s genetic distance (1983) and the proportion of shared alleles, also revealed the similar patterns. Maybe the sea surface currents of the West Pacific Ocean possibly have significant impacts on the population subdivisions of A. muricata in these sites. In conclusion, multi-locus microsatellite loci could be applied for the species boundary Acropora spp, and the population subdivision of A. muricata in Indo-Pacific oceans. In the future studies, these highly polymorphic microsatellite loci will be available for further species boundary studies and population genetics of the genera Acropora.