以粒線體、微衛星與核基因序列探討觀霧山椒魚的族群分化

Abstract

觀霧山椒魚(Hynobius fuca)為臺灣產山椒魚中，海拔分佈最低的物種，主要分佈於雪山山脈的西北部地區，因全球暖化以及棲地開發等壓力，使觀霧山椒魚生存面臨危機。本研究藉由遺傳分析推估觀霧山椒魚在過去歷史中族群變動情形，進而了解其分類地位及分化歷史，利於未來保育工作上的實施。本研究使用觀霧遊憩區、北插天山、拉拉山與棲蘭山的觀霧山椒魚樣本。應用粒線體序列、核基因序列及簡化基因組序列(ddRADseq)進行族群遺傳分析。首先，利用次世代定序所得轉錄體(transcriptome)序列開發出2類型核基因標記，第一種為微衛星(microsatellites)：具有演化快速且易累積變異，利於探討族群遺傳結構；第二種為核基因內含子序列(nuclear gene introns)：因無功能上的限制，所以可累積更多的突變。另外，又再應用次世代定序技術獲得ddRADseq並以SNPs來進行分析。粒線體細胞色素b (mitochondrial cytochrome b, cyt-b)分析結果，把觀霧山椒魚分成3大系群，分別為觀霧系群、北橫系群與棲蘭山系群，其中觀霧群與北橫群較為接近，而棲蘭山群為獨立的支系。而觀霧地區與棲蘭山族群間遺傳距離為0.0442–0.0503，接近種間遺傳差異(0.0364–0.750)。觀霧遊憩區4個採樣點間無明顯分化，可視為一個族群。微衛星演化樹也可將觀霧山椒魚分成3大支系，其中北橫群與棲蘭山群較為接近。內含子演化樹顯示觀霧山椒魚在各個族群間無顯著遺傳分化，視為一大族群。ddRADseq分析所得演化樹與微衛星演化樹大致相同。親緣關係樹分群結果在粒線體與核基因標記有不一致的現象，其原因可能是演化速度為粒線體最快，微衛星次之，核基因序列最慢，故猜測觀霧山椒魚族群正處於不完全譜系排序(Incomplete lineage sorting)的狀態。所以棲蘭山族群雖未是一個獨立種，但為保留觀霧山椒魚的遺傳多樣性，應對該棲蘭山地區進行更全面性的調查以達保育目的。

Hynobius is a member of Hynobiidae, Caudata, Amphibia. Taiwan is the southernmost habitat of this genus. Because of global warming and excess of habitat utilization, Hynobius fuca are critically endangered. Through genetic study, the author tried to investigate genetic variation, construct phylogeography, and estimate population demography of H. fuca. It aims to provide the reference for conservation design. Samples of H. fuca were collected from Guanwu, Lalashan and North Chatianshan (combined as North-cross group), and Qilanshan. MtDNA was more often utilized to proceed the genetic variance analysis in the past studies; however, it only represents partial history of population differentiation. To offer overall genetic information, it is able to be reinforced to adopt the nuclear markers in the meantime. Based on transcriptome data, I developed two genetic markers, microsatellites and nuclear introns. Both of them are less constrained and are used as neutral markers. In addition, SNPs (Single Nucleotide Polymorphism) obtained from ddRADseq (double digest Restriction Associated DNA) were used for analysis. The result of mitochondrial cytochrome b subunit I analysis showed that samples from four spots around Guanwu area have limited difference and can be considered as a single population. The genetic distance between Guanwu and Qilanshan is 0.0452–0.051 which is close to the differences between species ranging is 0.0361–0.727. H. fuca can be divided into three groups Guanwu, North-Cross, and Qilanshan groups. Guanwu and North-Cross groups are close to each other with Qilanshan group as their sister group. The phylogenetic tree of microsatellite can be divided into three monophyletic groups as well. North-Cross and Qilanshan groups are close to each other with Guanwu group being their sister group. The nuclear introns show no sign of population differentiation among groups. From the phylogenetic tree of ddRADseq is similar to that of microsatellites. In consistent results derived from different markers may suggest that H. fuca are experiencing the stage of incomplete lineage sorting among different populations.