台灣植物親緣地理研究-台東蘇鐵與琉球蘇鐵之親緣地理研究

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(1)行政院國家科學委員會補助專題研究計畫成果報告台灣植物親緣地理研究台東蘇鐵與琉球蘇鐵之親緣地理研究. 計畫類別：個別型計畫計畫編號：NSC 執行期間：90 年. 整合型計畫. 90－2311－B－006－00108 月. 01 日至. 90 年. 07 月. 31 日. 計畫主持人：蔣鎮宇共同主持人：計畫參與人員：. 本成果報告包括以下應繳交之附件：赴國外出差或研習心得報告一份赴大陸地區出差或研習心得報告一份出席國際學術會議心得報告及發表之論文各一份國際合作研究計畫國外研究報告書一份. 執行單位：國立成功大學生物系. 中. 華. 民. 國. 91 年. 1. 10 月. 29 日.

(2) 行政院國家科學委員會專題研究計畫成果報告台灣植物親緣地理研究-台東蘇鐵與琉球蘇鐵之親緣地理研究計畫編號：NSC 90-2311-B-006-001執行期限：90 年 08 月 01 日至 91 年 07 月 31 日主持人：蔣鎮宇行機構及單位名稱:國立成功大學生物系一、中文摘要:. model, which describes a migratory pattern with colonists recruited from a random sample of earlier existing populations. Phylogenies obtained from cpDNA and mtDNA were discordant according to neighbour-joining analyses. In total four chlorotypes (clades I-IV) and five mitotypes (clades A-E) were identified based on minimum spanning networks of each locus. Significant linkage disequilibrium in mitotype-chlorotype associations excluded the possibility of the recurrent homoplasious mutations as the major force causing phylogenetic inconsistency. The most abundant chlorotype I was associated with all mitotypes and the most abundant mitotype C with all chlorotypes; no combinations of rare mitotypes with rare chlorotypes were found. According to nested clade analyses, such nonrandom associations may be ascribed to relative ages among alleles associated with the geological history through which cycads evolved. Nested in networks as interior nodes coupled with wide geographical distribution, the most dominant cytotypes of CI and EI may represent ancestral haplotypes of C. taitungensis with a possible long existence prior to the Pleistocene glacial maximum. In contrast, rare chlorotypes and mitotypes with restricted and patchy distribution may have relatively recent origins. Newly evolved genetic elements of mtDNA, with a low frequency, were likely to be associated with the dominant chlorotype, and vice versa, resulting in the nonrandom mitotype-chlorotype associations. Paraphyly of CI and EI cytotypes, leading to the low level of genetic differentiation between cycad populations, indicated a short period for isolation, which allowed low possibilities of the attainment of coalescence at polymorphic ancestral alleles.. 本研究利用葉綠體 DNA atpB-rbcL noncoding spacer 以及粒線體 DNA ITS 來重建台東蘇鐵親緣關係，顯示高頻蠹蟲組事件使兩分子標誌物皆具有高度遺傳變異，族群結構上顯示低度分化，兩者所重建之親緣樹狀圖並不一致，由於連鎖不平衡造成兩樹狀圖所得之結果不一致，葉綠體 DNA 中頻度高的單型 I 個體包含所有出現粒線體 DNA 的單型(A-E)，粒線體 DNA 中單型 C 個體包含所有出現葉綠體 DNA 的單型(I-IV)，根據親緣網狀圖顯示此現象主要來自兩標誌物相對年紀的不同以及地質歷史所致，其中 C-I 以及 E-I 出現於網狀圖中心顯示兩者最為古老，反之稀少的單型皆位於網狀圖末端，為近代起源。族群分化程度低主要因隔離時間尚短，族群並沒有各自達到單一起起源。關鍵詞：葉綠體、粒線體、親緣樹狀圖 Abstr act: The phylogegraphic pattern of Cycas taitungensis, an endemic species with two remaining populations in Taiwan, was investigated based on genetic variability and phylogeny of the atpB-rbcL noncoding spacer of chloroplast DNA (cpDNA) and the ribosomal DNA (rDNA) internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA). High levels of genetic variation at both organelle loci, due to frequent intramolecular recombination, and low levels of genetic differentiation were detected in the relict gymnosperm. The apportionment of genetic variation within and between populations agreed with a migrant-pool 2.

(3) In this study, we are interested in looking into the genetic variability of chloroplast and mitochondrial loci and the phylogenetic consistency between organelle lineages in Taiwan's cycads.. Keywor ds: phylogegraphy, cpDNA, neighbour-joining analyses 二、緣由與目的: Cycads represent an ancient lineage, whose origin can be dated to the lower Permian (Zhifeng & Thomas 1989), unique from 'conifers'. Most extant species of Cycas, a Laurasian genus, are restricted to local flora with relictual distributions, a result of limited seed dispersal. According to palynological evidence, ancient cycads were widespread in Taiwan, a continental island 150 km east of mainland Asia (Fig. 1), before the extant genus became dominant and recognizable during the early Tertiary (Johnson & Wilson 1990). Cycas taitungensis Shen et al., however, is the only extant and endemic species with two endangered populations along the eastern coast of the island (Osborne et al. 1999). Phylogenetically, C. taitungensis and C. revoluta , a species distributed in Ryukyu and the Chinese mainland, constitute a unique lineage, Section Asiorientales, sister to other cycads (Hill 1999).. 三. 結果: At both loci, no within-individual variation was detected. The rITS region of mtDNA in Cycas taitungensis was amplified and sequenced with the length varying from 482 bp (isolate RL 81) to 536 bp (isolate RL 141). The mtDNA sequences were aligned with a consensus length of 538 bp. Of these, 299 bp (55.6%) were variable, including indels of 115 bp. A+T (56.4%) was rich in the DNA fragment, which agreed with the nucleotide composition of most noncoding regions (cf. Li 1997). The length of the atpB-rbcL spacer of cpDNA varied from 762 bp (isolate RL159) to 830 bp (isolate RL64). In total, 838 bp of the consensus length were aligned. There were 372 (44.4%) polymorphic sites, including indels of 108 bp. A+T (63.6%) was also rich in this noncoding spacer. Ninety-seven haplotypes and 55 haplotypes were determined in cpDNA and mtDNA of C. taitungensis, respectively. In the larger population (RL), nucleotide diversity at both loci was higher than that of the smaller population (C).. Like most cycads, dioecious plants of C. taitungensis are insect-pollinated, primarily by beetles (cf. Donaldson et al. 1995), as opposed to the ancestral wind-pollination mechanism of conifers and Ginkgo (Owens et al. 1998). The fleshy outer seed coat of cycads attracts rodents, which serve as dispersal agents (Nostorg & Nicholls 1997). The level of gene flow via seed and pollen is thus constrained by the migratory capabilities of pollinators and seed carriers. Gene flow distance between members of local populations was estimated to be 2-7 km in cycads (Yang & Meerow 1996). Gene exchange between distantly separated populations would be less possible than that between neighbouring demes. Hence, high genetic differentiation between populations is expected and has been detected in many cycads (e.g. Ellstrand et al. 1990; Walters & Decker-Walters 1991; Yang & Meerow 1996).. An NJ tree and a spanning network were recovered based on the nucleotide variation of the ribosomal ITS region of mtDNA. Three major clades (mitotypes) were identified: clade A [RL106 (RL151, RL132)] (bootstrap value = 100%), clade B (RL81, RL143) (bootstrap value = 100%) and clade C (bootstrap value = 100%). According to the links suggested by MIN-SP-NET analysis, two additional types were identified, i.e. D and E. Mitotype C was nested in the network as an interior node, which happened to be most dominant in number (89.21%) over other mitotypes (A, 2.94%; B, 1.96%; D, 1.96%; and E, 3.92%). Seventy substitutions and a 53-bp deletion characterized the mitotype D; whereas 18 substitutions 3.

(4) characterized the mitotype E. Likewise, 66 substitutions and a 44-bp deletion distinguished mitotype A from B. Within mitotype C, all sequences were linked to a most dominant haplotype (n = 12), which was nested in the group as the most interior node. All mitotypes were restricted to the larger populations, except for types C and E.. 1999). Instead, they are likely to represent historical migration events (cf. Lu et al. 2001). Low genetic differentiation due to shared dominant alleles and heterogeneous composition of organelle DNAs within each population (i.e. CI and EI cytotypes), and the high deduced Nm between populations agreed with a migrant-pool model (Wade & McCauley 1988), which describes a migratory pattern with colonists recruited from a random sample of previously existing populations. This unusual model is usually associated with glaciation or vicariance events.. Low consistency was found between NJ trees of cpDNA and mtDNA. Two of the major clades, A and B, identified in the mtDNA tree did not correspond to any clade of the cpDNA tree. The topology of the haplotypes within the most dominant type C of the mtDNA was not congruent with the dominant clade I of the cpDNA either. Individuals of the same cpDNA haplotype usually had different mtDNA sequences. For example, within clade I of cpDNA, RL135, RL108, C23, and C36 had identical cpDNA sequences, whereas their mtDNA sequences differed from each other by 1-24 bp. Furthermore, individuals of the same clade of mtDNA may belong to different clades of cpDNA, and vice versa, e.g. RL81 and RL106 of chlorotype I belonging to clades B and A of mtDNA, respectively.. According to geological evidence (Kizaki & Oshiro 1977), Taiwan had been long linked to the Asiatic mainland via a land bridge until today's Taiwan Strait became a geographical barrier for seed dispersal some 20 000 years BP. During the early Pleistocene, ice ages occurred at regular intervals of 100 000 years followed by a 20 000-year warm period (Milankovitch cycles) (cf. Bennett 1990). Like other gymnosperms, such as Cunninghamia (Lu et al. 2001) and Pinus (Strauss et al. 1993), and angiosperms (e.g. oaks, Dumolin-Lapégue et al. 1997; Petit et al. 1997; Ferris et al. 1998; beech, Koike et al. 1998; beets, Desplanque et al. 2000), C. taitungensis should have survived glacial cycles (Chiang & Peng 1998).. 四討論: In this study, unexpectedly high Nm values between RL and C populations of C. taitungensis were deduced from the nucleotide sequences of cpDNA and mtDNA markers, indicating that a migratory mode deviated from the regular stepping-stone, Wright's island, or isolation-by-distance models (cf. Hamrick & Nason 1996). According to ecological observations (Hsieh 1999), dispersal of cycad seeds across a wide geographical range, in this case 30-40 km, in modern habitats is most unlikely due to the discontinuity of vegetation and the constraint of migratory capabilities of the seed-carrying animals. Under such migration model, the high Nm values between cycad populations deduced from F ST were invalid for the use of estimating current population structure and ongoing gene flow (Whitlock & McCauley. With a likely tropical origin, the ancestral populations of Taiwan's cycads may have entered the island and become dominant during the de-glaciation period (Shaw & Huang 1995), being forced to migrate into refugia during the subsequent glacial maximum. According to palynological evidence, such back and forth migration of cycad populations may have occurred since the Cretaceous. During the last glacial period, 100 000 years BP, due to dramatic temperature oscillation, most cycad populations probably went extinct (cf. Bennett 1990). The remaining populations along the eastern coast of the island thus represent relics for Taiwan's cycads. According to the literature, long-distance seed dispersal during the glacial maximum, 4.

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