瓶鼻海豚主要組織相容性複合體DQB與DRB基因變異與演化

Abstract

主要組織相容性複合體（MHC）對於一個動物族群的演化與環境適應十分重要 ，因此適合作為研究演化生態學與保育學上的議題。本研究首先針對兩種來自西太 平洋的瓶鼻海豚之DQB與DRB基因之全長序列與其表現之胺基酸進行分析。這是鯨 類的第一個MHC全長序列研究。此外，這兩種瓶鼻海豚可能是一個可用來研究不同 棲息環境下哺乳動物MHC如何受到影響的動物模式。來自台灣與日本的兩頭Tursiops truncatus 與來自台灣與印尼的 兩頭T. aduncus 之血液分別被用於RACEPCR。 以結 果推測此屬動物應有一個DQB基因座 與兩個DRB基因座。Tutr-DQB、Tuad-DRB、 與Tutr-DRB在免疫功能上應有功能性存在。 但Tuad-DQB分子因不符合典型的胺基酸 結合角色所以還需更多樣本的進一步研究。譜系樹分析結果推測此兩種海豚因分別 棲息於不同環境（T. truncatus 遠洋；T. aduncus 沿岸) 因此序列會分成兩群。 此部 份的結果提供了研究瓶鼻海豚MHC基因多樣性與疾病生 態學的基礎資料。 為了進一步的了解瓶鼻海豚的免疫多樣性，自台灣海域擱淺或誤捕收集之62隻 瓶鼻海豚樣本 (T. truncatus 42隻 T. aduncus 20隻) 被用作研究DQB基因座第二外顯子 之多樣性。其增幅產物為172bp。每隻動物得到的等位基因數目不超過兩個。其高 比例的不同義核苷酸取代與中等程度的變異量顯示此基因座受到正向選汰壓影響。 此結果也不支持前人研究指出海洋環境選汰壓較低的假設。譜系樹分析顯示兩種瓶 鼻海豚與其他海豚科動物並不符合分類學結果，暗示有跨物種演化形式的存在。此 外，兩種瓶鼻海豚呈現明顯分群，暗示他們的確身處不同的環境，而不同的選汰壓 已影響其MHC class II 基因演化，目前所見的MHC等位基因對該物種應有適應上的價 值。 最後是鯨偶蹄目之MHC演化關係與分化時間研究。結果指出鯨類與偶蹄類於六 千萬年前左右分家，而病原驅動之指向性選汰應是其分化的原動力。此外，兩種瓶 鼻海豚於兩千四百萬年前左右分家，此結果比最早的海豚 (一千一百萬年前左右) 還要早出許多。這些結果指出平衡選汰將這些等位基因族系維持相當長的一段時間 ，以致他們能累積許多取代使得彼此差異越來越大。此研究對於MHC與病原的共演 化過程提供了新的理解，並且也是鯨偶蹄目MHC class II基因的第一個演化歷史研究 。

Variability of the major histocompatibility complex is very suitable for investigating a wide range of open questions in evolutionary ecology and conservation because it reflects evolutionarily relevant and adaptive processes within and between populations. First, the 1st full-length expressed DQB and DRB gene products in the order Cetacea from 2 species of bottlenose dolphins (genus Tursiops, family Delphinidae) of the western Pacific, which differ in their diet, microflora, and habitat, was presented. These species might represent a natural model through which we can examine the immunogenetic consequences of different pathogenic and environmental influences on the MHC of mammals. Blood samples from 2 T. aduncus (from Taiwan and Indonesia) and 2 T. truncatus (from Taiwan and Japan) were used for RACE-PCR. The presence of 1 DQB locus and 2 DRB loci in this genus was revealed. The presence of Tutr-DQB, Tuad-DRB, and Tutr-DRB suggests a functional role for these molecules in pathogen-specific immune responses. However, several features do not support a traditional role for Tuad-DQB molecules in peptide binding, and further study is needed. The phylogenetic analysis revealed that the sequence divergence of these 2 species might reflect different selective pressures between pathogens in oceanic (T. truncatus) and coastal (T. aduncus) waters. This study provides an essential foundation for analyzing variations in MHC genes and studying infectious disease ecology in bottlenose dolphins. For a better understanding of the immunologic diversity in bottlenose dolphins, the sequence variation of the exon 2 region of MHC DQB locus was analyzed in 62 bottlenose dolphins (42 T. truncatus and 20 T. aduncus), collected from strandings and fishery bycatch in Taiwanese waters. The 172 bp sequences amplified showed no more than two alleles in each individual. The high proportion of nonsynonymous nucleotide substitutions and the moderate amount of variation suggest positive selection pressure on this locus, arguing against a reduction in the marine environment selection pressure. The phylogenetic relationship among DQB exon 2 sequences of bottlenose dolphins and other delphinids did not coincide with taxonomic relationship, indicating a trans-species evolutionary pattern. Moreover, there is no mixture but a clear division between T. truncatus and T. aduncus, indicating that different selective pressures from pathogens exist in oceanic and coastal waters because shallow waters along the coast may influence by the terrestrial runoff and the pathogen diversity and abundance in the coastal waters may be different from that in the oceanic area. The habitat-specific alleles may have adaptive value for certain species and can be discriminately selected. Lastly, the evolutionary relationship and estimates of divergence time of cetartiodactyls by using MHC class II genes was reported. The results suggest that cetaceans (T. truncatus and T. aduncus) diverged from artiodactyls (pig, hippo, and ruminants) about 60 Mya or slightly earlier and the pathogen-driven directional selection is very likely the driving force of the sequence divergence of MHC genes of the cetaceans and the terrestrial relatives. Furthermore, the results show that T. truncatus and T. aduncus diverged about 24 Mya, which predates the emergence of oldest dolphin (approximately 11 Mya) quite much. It implies that these allelic lineages were maintained for a long evolutionary period through speciation events by balancing selection and the long persistence time could allow the accumulation of multiple substitutions between allelic lineages. This research provides new insights into the dynamics of MHC-pathogen coevolution and is the first study on the evolutionary history of cetartiodactyls using the MHC class II genes.