現生裸子植物的葉綠體基因組演化與親緣研究

Abstract

現生的種子植物可分為五大群，蘇鐵植物(cycads)、銀杏(Ginkgo)、針葉樹植物(conifers)、買麻藤植物(gnetophytes)及被子植物(angiosperms)。然而，此五群植物間的親緣關係仍未有定論。為重新檢驗此一長久的爭議，本研究定序了台東蘇鐵(Cycas taitungensis)和小葉買麻藤（Gnetum parvifolium）的完整葉綠體基因組(chloroplast genome)。台東蘇鐵的葉綠體基因組為163,403 bp的圓形分子，其結構具有兩段25,074 bp的反向重複序列 (inverted repeat)。 本研究整理已知的37種陸生植物所共有的56個葉綠體基因組蛋白基因，以三種不同演算法重建親緣演化樹。所獲得之演化樹的樹形(topology)具一致性－皆支持現生所有種子植物、裸子植物、被子植物各為單系群(monophyly)。裸子植物單系群內，可再分成兩個次群組(subclade)，即蘇鐵–銀杏群組及買麻藤–松群組。以上結果與gnetifer及gnepines兩假說一致。此外，本研究亦提出cpDNA結構上突變的證據來支持上述的親緣分析結果。重建的演化樹中，買麻藤植物的分支(branch)明顯長於其他裸子植物，表示買麻藤植物有相對快速的演化速率。以相對速率檢驗法（relative rate test）分析，顯示買麻藤植物的快速演化速率主要發生在密碼子(codon)的第三位置，以及密碼子的第一與第二位置的transversion核苷酸位點。此外，蘇鐵的葉綠體基因組仍保存部份的tufA基因序列，稱為假tufA基因，此假基因亦存在於Anthoceros及銀杏的葉綠體基因組。以此假基因建構的演化樹，顯示tufA基因可能在種子植物的共同祖先就已遺失，推算此遺失事件應發生在距今約3億年前。本研究提出tRNAPro-GGG在被子植物的共同祖先(存在時間距今約1.5億年前)時已遺失的假說。另外，對松科植物如何遺失一段反向重複序列亦提出新的看法。本研究雖同時支持gnetifer及gnepines假說，但無法判定何者較正確。因此，需要分析更多非松科的針葉樹(non-Pinaceae conifers)葉綠體基因組的完整序列，以解決裸子植物的親緣演化。 為進一步探討導致買麻藤植物相對快速演化速率的因子，本研究另外定序了四種裸子植物之完整葉綠體基因組：三種買麻藤植物－Ephedra equisetina (木賊麻黃：109,518 bp)，Gnetum parvifolium (小葉買麻藤: 114,914 bp)，和Welwitschia mirabilis (千歲蘭: 118,919 bp)，以及一對照組植物：松科的台灣油杉（Keteleeria davidiana; 117,720 bp)。千歲蘭的葉綠體基因組在2008年已被完整定序，並為陸生光合作用植物中，最小且最緊密 (compact)的物種。然而，買麻藤綱之其他兩科－麻黃科 (Ephedrales) 及買麻藤科 (Gnetales) 的葉綠體基因組仍未被研究過，吾人對買麻藤綱植物的葉綠體基因組如何縮小(reduction)及緊密化(compaction)的機制仍未了解。 本研究發現Ephedra及Gnetum的葉綠體基因組比Welwitschia的更小且更緊密。買麻藤綱植物的葉綠體的共同特徵是: (1)遺失了18個在其他種子植物仍保留的基因；(2)基因間的序列 (spacer) 及基因的內顯子 (intron) 有明顯的序列刪除現象(sequence deletion)；又後者在操作組間 (inter-operon) 比在操作組內 (intra-operon) 明顯，且偏好刪除一整段長序列，而不是單一核苷酸。由此可推論在買麻藤綱植物的葉綠體演化過程中，有一選擇壓力 (selection) 迫使其葉綠體基因組縮小及緊密化。此外，買麻藤綱植物的快速演化速率與葉綠體基因組含高比率的腺嘌呤 (adenine: A) 及胸腺嘧啶 (thymine: T)有關，並在統計上具有顯著意義。綜合上述的發現，本研究推論買麻藤綱植物之葉綠體基因組的縮小及緊密化是一種減少資源消耗的演化策略 (a lower-cost strategy);其目的為: (1)對抗其惡劣的生存環境與(2)提高本身的競爭力來抗衡同生育地周遭的被子植物。此一演化策略導致買麻藤綱植物具有較快的演化速率。前人的研究已發現，買麻藤綱植物的核基因組為裸子植物中最小的，此一特性對本研究所作的演化推論，提供了一有力的證據。

Phylogenetic relationships among the five groups of extant seed plants are presently unsettled. To re-examine this longstanding debate, we determined the complete chloroplast genomes (cpDNAs) of Cycas taitungensis and Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with two typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms, respectively. Within the modern gymnosperms are 2 highly supported sister clades: Cycas–Ginkgo and Gnetum–Pinus. This result agrees with both the ‘‘gnetifer’’ and ‘‘gnepines’’ hypotheses. The sister relationships in Cycas–Ginkgo and Gnetum–Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas–Ginkgo and Gnetum are consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A pseudo tufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the pseudo tufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms. To comprehend the mechanisms driving the rapid evolutionary rates of gnetophytes, four additional cpDNAs, including one from each of the three gnetophyte orders, Ephedra equisetina, Gnetum parvifolium, and W. mirabilis, and one from the non-Pinus Pinaceae, Keteleeria davidiana were determined. The cpDNA of Welwitschia mirabilis (the only species of Welwitschiales) was recently reported to be the most reduced and compact among photosynthetic land plants. However, cpDNAs of the other two gnetophyte lineages (viz. Ephedrales and Gnetales) have not yet been studied. It remains unclear what underlining mechanisms have downsized the cpDNA. To pin down major factors for cpDNA reduction and compaction in gnetophytes, we have determined the cpDNAs of E. equisetina (109,518 bp) and G. parvifolium (114,914 bp). They are not only smaller but more compact than that of W. mirabilis (118,919 bp). The gnetophyte cpDNAs have commonly lost at least 18 genes that are retained in other seed plants. Furthermore, they have significantly biased usages of AT-rich codons and shorter introns and intergenic spaces, which are largely due to more deletions at inter-operon than intra-operon spaces and removal of segment sequences rather than single-nucleotides. We showed that the reduced gnetophyte cpDNAs clearly resulted from selection for economy by deletions of genes and non-coding sequences, which then led to the compactness and the accelerated substitution rates. The smallest C-values in gnetophyte nuclear DNAs and the competitive or resource-poor situations encountered by gnetophytes further suggest a critical need for an economic strategy.