紫金牛葉瘤共生菌之鑑定及與宿主之共譜系分析

Abstract

細菌葉瘤共生現象只出現在少數被子植物。在紫金牛屬中，葉瘤為圓齒亞屬的主要特徵。過去研究發現，硃砂根頂芽腔內的細菌會感染幼葉，使葉緣形成葉瘤。此外，這些細菌也會感染開花側枝，並透過種子傳至下一代。目前葉瘤共生菌尚無法培養，且在共生關係中所扮演的角色仍然未知。為對葉瘤共生關係有進一步的了解，本研究利用分子方法，並輔以形態觀察及脈衝式電泳分析鑑定共生菌。另外也重建宿主植物譜系關係，並與共生菌比較，以瞭解其共演化關係。 電子顯微鏡觀察中，不同圓齒紫金牛物種之頂芽腔、胚珠表面或葉瘤中的共生菌形態均相當類似，皆為不具鞭毛及夾膜的桿菌，且有外膜，為典型格蘭氏陰性菌。根據 16S rDNA 序列，紫金牛葉瘤細菌與茜草科葉瘤細菌同樣屬於 β 變形桿菌亞綱的伯克霍德氏菌屬。脈衝式電泳顯示硃砂根及黑星紫金牛共生菌均具有兩條染色體，約 3.5 Mb 及 1.2 Mb，基因體較大多已知的伯克霍德氏菌小。在所取樣的物種中，共生菌的 rrn 操縱組可分為三型（第 1、2A 及 2B 型）。其中八個分類群都具有的第 1 型，與其他變形桿菌最常見的相同。其餘二型只出現在硃砂根及黑星紫金牛。第 2A 型在 16S-23S 區間的 trnI 及 trnA 基因與之間的區域有 163 bp 之缺失。第 2B 型則有完整之 16S-23S 區間，但在 rDNA 區域則有許多不同長短之缺失或插入，顯示此二型有功能分化的現象。 紫金牛屬譜系分析利用細胞核（nrITS、TM6 第二內插子）及葉綠體（trnL-trnF 、atpB-rbcL、psbA-trnH）片段，共納入 7 個亞屬的 23 個種、1 個亞種及 1 個變種。結果顯示葉瘤在紫金牛屬內應為單次起源。而圓齒亞屬外的其他三個亞洲亞屬（高木、鋸齒、腋序），本研究所分析的物種亦各自為支持度良好之單系群。整體來說，葉緣形態及毛被物為重要之屬內分類特徵，而生長型、胚珠數目及輪數則較不適合。此外，花藥及花絲之長度比在亞屬間及圓齒亞屬內有明顯之變異，顯示該特徵對紫金牛系統分類之潛在重要性。 共譜系分析顯示宿主與細菌譜系樹有顯著相關。此外，同種宿主但來源地不同之樣本形成姊妹群，亦支持循環式共生假說。在細菌譜系樹上，所取樣的圓齒亞屬中分為三個系群：廣泛分佈的硃砂根–黑星紫金牛系群、屯鹿紫金牛–百兩金–雪下紅–裡堇紫金牛系群及台灣特有的雨傘仔–玉山–阿里山–高士佛紫金牛系群。值得注意的是，阿里山與高士佛紫金牛有許多相似之處，而 TM6 第二內插子譜系樹亦顯示過去在該系群內可能有過雜交事件。

Bacterial leaf-nodule symbiosis is only found in a few angiosperm taxa. In Ardisia, leaf nodules are the distinctive feature of the subgenus Crispardisia. Previous anatomical studies of Ardisia crenata showed that a population of bacteria kept in the shoot tip inoculates the margins of young leaves, where leaf nodules later develop. The bacteria also infect floral primordia and are passed on to the next generation through seeds. To date, the bacteria are still unculturable and the role they play in the symbiotic relationship remains uncertain. To better our understanding of leaf-nodule symbiosis, a molecular approach, combined with morphological observation and PFGE analysis, was used to identify and characterize Crispardisia symbionts. A phylogeny of the host plants was also constructed and compared with that of the symbionts. Under electron microscopes, symbiotic bacteria from shoot apices, ovaries or leaf nodules of different host species are similar in morphology. They are rod-shaped, non-flagellated and non-capsulated and have an outer membrane, a typical Gram-negative structure. Analyses of 16S rDNA sequences suggest that the leaf-nodule symbionts of Ardisia belong to the genus Burkholderia in the β-subclass of Proteobacteria, as do the leaf-nodule bacteria of Rubiaceae. The PFGE patterns of A. crenata and A. polysticta symbionts indicate that they have two replicons of about 3.5 Mb and 1.2 Mb, which comprise a relatively small genome compared to other Burkholderia. One notable finding is that the symbionts have three types of rrn operons (Types 1, 2A and 2B). Eight sampled Crispardisia taxa have Type 1, the most common type in Proteobacteria. The other types are only found in A. crenata and A. polysticta. Type 2A is similar to Type 1 in the 16S and 23S regions, but has a 163-bp deletion in the trnI and trnA genes and their spacer within the 16S-23S spacer. In contrast, Type 2B has an intact spacer and rDNA regions with indels of various sizes, suggesting the two types are functionally complementary. For the phylogenetic analyses of Ardisia, 23 species, 1 subspecies and 1 variety from 7 subgenera were included in the data matrices of nuclear (nrITS, TM6 second intron) and chloroplast (trnL-trnF, atpB-rbcL, psbA-trnH) markers. The phylogenies suggest a single origin of leaf nodules within the genus. In addition to Crispardisia, the sampled taxa of the other three Asian subgenera (Tinus, Bladhia and Akosmos) also each form a well supported clade. In general, leaf-margin morphology and vestiture appear to be more important characters, rather than growth forms or numbers of ovules and ovule series. The ratios of anther and filament lengths were also found to be variable among the subgenera and within Crispardisia, suggesting its potential systematic value for Ardisia. Cophylogenetic analyses suggested that the host and symbiont phylogenies are significantly dependent. Furthermore, symbionts from hosts of the same species are grouped together irrespective of their geographic origins. These all supported the hypothesis of cyclic symbiosis. Within Crispardisia, three clades are recognized in the symbiont phylogeny: the widely distributed crenata-polysticta, brevicaulis-crispa-villosa-violacea and cornudentata-kusukuensis, a clade endemic to Taiwan. It should be noted that a number of similarities have been found between A. cornudentata ssp. morrisonensis var. stenosepala and A. kusukuensis, and the TM6 second intron phylogeny suggests possible hybridization events in the past.