臺灣梅花鹿類緣關係與族群之分子遺傳結構研究

• Main Authors: 江慶華, Chiang, Ching-Hua
• Other Authors: 臺灣大學: 動物科學技術學研究所, 朱有田; 姜延年
• Format: Thesis
• Language: Chinese; English
• Published: 2011
• Subjects: 臺灣梅花鹿;粒線體DNA;微衛星DNA;類緣關係;遺傳結構; Cervus nippon taiouanus;mitochondrial DNA;microsatellite DNA;phylogenetic relationship;genetic structure; Green Island; Siberia
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/253832; http://ntur.lib.ntu.edu.tw/bitstream/246246/253832/1/ntu-100-R98626006-1.pdf

梅花鹿（Cervus nippon）是遍布於亞洲地區的鹿科物種，自日本、中國、臺灣至越南均可見其蹤跡。臺灣梅花鹿（C. n. taiouanus）屬於臺灣特有亞種，曾因過度獵捕與棲地減少造成野生族群數量銳減，於西元1969年的調查中，臺灣梅花鹿已於野外絕跡。遂於1984年起，在墾丁社頂地區展開梅花鹿復育工作，自臺北圓山動物園引進22頭梅花鹿作為復育核心鹿群，歷經26年籌備與努力，目前已有復育鹿群於野外生活，達成復育目標。期間金門畜試所亦有保育臺北圓山動物園梅花鹿，綠島則有野放之圈養族群。惟復育鹿群之族群遺傳結構尚未完全清楚，種原來源問題亦常受質疑。本研究之目的在於探討臺灣梅花鹿之遺傳特徵與族群遺傳結構，藉以釐清種原來源疑慮，瞭解其族群間及與其他梅花鹿亞種之遺傳關係。 粒線體DNA序列之多型性可用以研究物種間或物種內母系類緣關係及基因交流情形。另一方面，細胞核內微衛星DNA標記多型性可探討族群或個體間遺傳關係，本研究同時以這兩種遺傳標記探討上述問題。 本研究分析164頭臺灣梅花鹿樣本之粒線體DNA序列，包含84個來自墾丁復育鹿群、35個來自綠島、16個來自金門及29個來自畜養鹿群之樣本，結果發現共有2種細胞色素b及5種D-loop之基因單套型。復育族群墾丁鹿群共有1種細胞色素b單套型及3種D-loop單套型；金門鹿群皆只有一種基因單套型。另自NCBI取得28條其他亞洲梅花鹿亞種之粒線體DNA序列，用以探討梅花鹿之類緣關係。所建構之類緣關係樹圖顯示臺灣梅花鹿與中國梅花鹿亞種關係較近，與日本亞種關係較遠。此外，自淇武蘭遺址收集距今約450年前之古老樣本進行遺傳分析，結果成功增幅及定序出591 鹼基長度之粒線體DNA D-loop序列片段，將其定義為古代梅花鹿粒線體DNA序列，此序列與現生梅花鹿D-loop第一型單套型序列相同，顯現其於母系遺傳之時間連續性。 本研究以22組微衛星標記之多型性分析臺灣梅花鹿復育族群及畜養族群之遺傳結構與類緣關係，共分析126個臺灣梅花鹿樣本，84個來自墾丁復育鹿群、16個來自金門及26個來自養殖鹿場。結果顯示，本研究所用微衛星標記之平均多型性指數為0.466，平均有效對偶基因數為2.2，顯示這22組微衛星標記於臺灣梅花鹿族群中具有多型性。估算族群遺傳結構及以遺傳距離建構之類緣關係，顯示臺灣梅花鹿可分為復育族群及畜養族群兩大類群。為瞭解其族群間分化程度，估算FST值，墾丁復育鹿群與臺南畜養鹿群、臺東畜養鹿群及金門鹿群間之FST值分別為0.110、0.104及0.088（p < 0.05），顯示復育族群與畜養族群間分化程度較高。 綜合上述結果，臺灣梅花鹿古代DNA研究可定義臺灣梅花鹿之遺傳特徵，協助釐清復育鹿群種原問題與演化歷程，未來若能獲取更多臺灣梅花鹿古代DNA序列之遺傳資訊，即可作深入且較完整之探討。此外，缺乏粒線體單套型多型性，證實遺傳管理之迫切性，而透過對臺灣梅花鹿遺傳結構與分化情形之瞭解，顯示復育鹿群與畜養族群遺傳特徵具有差異，可作為長期實行遺傳監控與管理之基礎，維持遺傳多樣性，使研究與保育工作得以永續發展。 Sika deer (Cervus nippon) are widespread throughout Asia, from mainland China in the west to Japan, and from Siberia in the north Russia to south China, Taiwan and Vietnam. The Formosan sika deer (C. n. taiouanus) had ever been an endemic subspecies in the wild of Taiwan. Because of the destruction of their habitats and suffered from strong hunting pressure, the sika deer was extincted in the wild in 1969. Since 1984, a recovery program has been executed, in which the original population included 22 deer obtained from Yuanshan Zoo was conserved in Kenting National Park (KNP). The conserved deer had reintroduced into the wild till 1994. In the meantime, several sika deer populations were reared in different sites in Taiwan, including Kinmen County Livestock Research Institute (KCLR) and Green Island (GI). However, these population genetic structure is not defined completely, and the wild origin of these conserved deer populations has been queried frequently. The purpose of present study is to determine genetic characteristics and structures among Formosan sika deer populations, and to understand the genetic relationship among different sika deer subspecies. Polymorphism of mitochondrial DNA (mtDNA) sequences provides reliable information to study the phylogeny and the gene flow among species or inter-populations. One hundred and sixty four Formosan sika deer mtDNAs including 84 from KNP, 35 from GI, 16 from KCLR, and 29 from 2 farms were obtained. There are 2 cytochrome b haplotypes and 5 D-loop haplotypes obtained. To understand the phylogeny and gene flow, 28 sequences from eastern Asian sika deer subspecies were obtained from NCBI GenBank. A Bayesian phylogenetic tree revealed Formosan sika deer are genetically closer to the sika deer from south China than deer from Japan. In the other way, an ancient specimen from Ki Wu Lan archaeological site (450 years before present) was collected and used to define the mtDNA characteristic of ancient Formosan sika deer. A portion of D-loop sequence (591 base pairs) obtained successfully, and it was totally identical to one of modern Formosan sika deer haplotypes. The result indicated the continuity among the maternal linkage of ancient and modern Formosan sika deer. Besides, the allelic diversity of microsatellite loci is commonly used for understanding the genetic relationship and differentiation among intra-population or individuals. Twenty two microsatellite markers were applied to study the genetic structures and phylogenetic relationship among conserved and cultivated populations in Taiwan. One hundred and twenty six of Formosan sika deer nuclear DNA including 84 from KNP, 16 from KCLR, and 26 from farms were analyzed. The polymorphism index content (PIC = 0.466) and effective alleles (mean = 2.2) were estimated, indicating the 22 microsatellite markers were informative. According to the results of Bayesian clustering analysis and phylogenetic trees, conserved and cultivated deer are divided as different populations. The F-statistic value for population differentiation (FST) of KNP versus Tainan, versus Taitung, and versus KCLR populations were 0.110, 0.104, and 0.088 respectively (p < 0.05), and showed that the higher population differentiation in KNP versus cultivated populations. In conclusion, the genetic characteristics of Formosan sika deer were defined which provide insights into the phylogenetic relationship and evolutionary history. Ancient DNA study may also help to clarify the queries of the origin of conserved Formosan sika deer, but more ancient sequences should be included to strengthen the evidence. Moreover, understanding the genetic structures and differentiation of Formosan sika deer populations was valuable to find genetic characteristics of different populations. A valid genetic management program based on these results for further conservation and maintaining biodiversity can be proposed properly.