哈氏狗脊蕨（Woodwardia harlandii Hook.）及細葉狗脊蕨（W. kempii Copel.）為烏毛蕨科（Blechnaceae）狗脊蕨屬（Woodwardia）植物，兩者除葉片形態特徵不同外，無其他顯著差異，而其生態習性雷同及地育地重疊性高等現象，使得此二物種的分類關係一直具有爭議。因此本研究首先針對地理分布、物候現象、細胞學資料與分子遺傳證據進行系統分類學研究，以釐清其系統演化關係。生物的配育系統會影響物種族群內與族群間的遺傳組成，甚至整個物種的演化，因此在進行族群遺傳多樣性與親緣地理學研究之前，先針對台灣產哈氏狗脊蕨與細葉狗脊蕨材料進行生殖生物學觀察與試驗，以明瞭其生殖行為與配育系統。最後以同功異構酶電泳分析法與植物葉綠體DNA進行族群遺傳多樣性與族群間遺傳變異分布結構之分析，重建種內族群間地理親緣關係與評估族群在地質歷史過程的遷移路線，並檢測不同的地理遷移模式假說。細胞學證據顯示，除了中國大陸廣東省新會地區之哈氏狗脊蕨族群為二倍體外，其餘檢測的哈氏狗脊蕨與細葉狗脊蕨族群皆為四倍體，而根據其核基因片段組成基因型的差異性，推測這些四倍體族群皆為異源四倍體（allotetraploid）。除了地理分布或生育環境都高度重疊，共域分布的四倍體哈氏狗脊蕨與細葉狗脊蕨更具有一致的物候現象。細葉狗脊蕨在葉綠體與細胞核基因片段均與共域分布的哈氏狗脊蕨有一致的單套基因型，顯示此二種外型不同的類群在遺傳組成上並無差異，而兩者除了葉片分裂型式差異外，其他證據都顯示其並非獨立的分類群。相對地，二倍體的哈氏狗脊蕨與其他四倍體哈氏狗脊蕨與細葉狗脊蕨之間在地理分布、染色體倍體性與分子證據都具有明顯區隔，顯示二倍體哈氏狗脊蕨是可與四倍體的哈氏狗脊蕨及細葉狗脊蕨區分的不同生物種。因此哈氏狗脊蕨複合種群實包括二倍體的哈氏狗脊蕨與四倍體的哈氏狗脊蕨及細葉狗脊蕨。台灣產四倍體哈氏狗脊蕨複合種群配子體發育類型屬於「三叉蕨型」，成熟配子體以單性配子體為主，雌配子體先熟，雌雄配子體發育成熟時間差距達3週，推測四倍體哈氏狗脊蕨複合種群之配子囊個體發生行為有利於異配子體交配，而雌配子體附近小型配子體短時間發育成雄配子體並具有大量藏精器，推測此複合種群具有促精素系統，藉此促進異配子體交配。同功異構酶電泳分析結果顯示二倍體哈氏狗脊蕨族群的基因座平均對偶基因數明顯高於四倍體類群，推測可能原因為大陸廣東新會一帶為上次冰期極盛時祺廣大族群退縮之避難所，因此保留較多的遺傳多樣性。此外，二倍體類群同質合子比例偏高，推測應是華倫得效應（the Wahlund effect）所造成。四倍體哈氏狗脊蕨複合種群對偶基因頻率多形成固定模式，推測應是此多倍體類群是異源四倍體，而多數基因座上固定之異質配對對偶基因則使得四倍體族群內異質合子比例偏高。利用族群間遺傳相似度進行群叢分析，四倍體之哈氏狗脊蕨與細葉狗脊蕨族群間I值高達0.919以上，遠高於蕨類植物種內族群間之最小I值，因此推論四倍體之哈氏狗脊蕨與細葉狗脊蕨應為同種，而同地區四倍體哈氏狗脊蕨與細葉狗脊蕨族群總是擁有近似的對偶基因頻率，亦支持此推論。哈氏狗脊蕨複合群中二倍體類群的葉綠體DNA核苷酸歧異度（π=0.00108）高於整體四倍體類群（π=0.00072），而二倍體類群單套型歧異度（h=0.797）亦顯著高於四倍體類群（h=0.294）。二倍體類群之9種基因型呈現星狀之譜系關係，且具有較高的單套基因型歧異度與較低的核苷酸歧異度，顯示大陸廣東省新會一帶之二倍體族群有近期族群快速成長與分布範圍擴張現象。以GST及NST檢測族群間遺傳分化，結果顯示哈氏狗脊蕨複合群、四倍體哈氏狗脊蕨複合群、四倍體哈氏狗脊蕨與二倍體哈氏狗脊蕨等四群之群內族群間都具有明顯的遺傳分化，且其Nst與Gst值都具有顯著差異，顯示各群內族群間都具有明顯的親緣地理結構。15種葉綠體單套基因型中大陸廣東新會一帶之二倍體族群具有9種單套基因型，其中8種更是侷限分布於此，推測此地區是二倍體類群長期之避難所。大陸南嶺及海南島地區族群同時都具有兩群序列差異頗大的基因型，其可能為最後一次冰期以來其他地區族群擴散之溶匯點（melting point），因此都具有較高之核苷酸歧異度。台灣烏來地區為另一可能之避難所，根據所擁有之2單套基因型與葉綠體非編碼區突變率推算，此地區族群可能在最近一次冰期開始前即已存在。本研究所使用之DNA序列證據並不支持Cranfill之異地種化再共域分布之族群遷徙假說，而核DNA單套基因型部分地區的地理變異則對本研究所主張之由北向南多路線族群遷徙假說提供部分的支持。

Woodwardia harlandii Hook. and W. kempii Copel. are two Woodwardia species of Blechnaceae. They have no distinct difference in morphology excepting the division of fronds. The systematic relationship between them is a controversial issue until now because of their similar ecology and sympatric distribution. We first implement the systematic study, by distribution pattern, phonological observation, cytological data and molecular evidences, to find their phylogenetic relationship. Mating systems would determine the infraspecial genetic contents and evolutionary process of a species. Therefore, the reproduction study of these two species was conducted, before the population genetic and phylogeographic studies proceeding, to have insight into the reproductive mode and mating system. Finally, genetic diversity and genetic structure of populations are studied by allozyme analysis and chloroplast DNA sequence to find the genetic relationship among populations, to infer the migration route in the geological history, and to test the different hypotheses of geographic distribution and transition of population. Cytological results show that all populations detected are tetraploid but the two ones, which are diploid, at Hsinhui in Guangtung Province of China. According to the distinction between the nuclear haplotypes, these polyploidy taxa are determined as allotetraploid. Addition to sympatric distribution and similar habitants, the phenology is identical for the tetraploidy W. harlandii and W. kempii living in the same habitat. Although having different division of fronds, these two species living sympatrically show the same genetic contents in both nuclear and chloroplast genes. All characters excepting morphology indicate that, without essential difference, they are not ‘good’ species. On the other hand, the results of distribution information, cytological data and molecular evidences indicate that diploidy W. harlandii and tetraploidy W. harlandii and W. kempii belong to two different biological species. Therefore, the W. harlandii species complex comprises diploidy W. harlandii and tetraploidy W. harlandii and W. kempii. The prothallial development is Aspidium type. Almost all the gametophytes are unisexual, in which the female ones reach mature 3-week earlier than the males. The sexual progression of the gametophytes promotes the intergametophytic mating in tetraploidy W. harlandii complex. The observation that lots of small-size gametophytes nearby the female one become male and bear numerous antheridia in a short period indicates this complex producing antherridiogen to favor cross-fertilization. The results of allozyme analysis show that the diploidy W. harlandii complex has much higher mean number of alleles per locus than the tetraploids. The interpretation for this high value is that the population maintains significantly high genetic diversity because it was a refugium at Hsinhui in the last glacial period. The population there, however, has relatively high proportion of homozygotes. It might be the outcome of the Wahlund effect. Allele frequencies for the populations of tetraploid W. harlandii complex usually become fixed pattern. These taxa should be allotetraploids and have high proportion of heterozygotes within the populations because high percentage of fixed- and heterozygous-allele loci. The genetic identity of I is less than 0.919 between the populations of the tetraploidy W. harlandii and W. kempii, which is much higher than the mean genetic identity among the populations of the same species. The result of this UPGMA analysis consists with the argument that the tetraploidy W. harlandii and W. kempii should be the same species. Additionally, the fact that sympatric populations of these two tetraploids always have similar allele frequencies at most loci also supports the above argument. In W. harlandii species complex, diploidy taxon have higher nucleotide and haplotype diversities (π=0.00108, h=0.979) of chloroplast DNA than those of tetraploidy taxa (π=0.00072, h=0.294). In the diploidy taxon, the lineage relationship of its nine haplotypes shows a star-like pattern. In addition, this taxon has high haplotype diversity but low nucleotide diversity. These results indicate the populations at Hsinhui grow rapidly and experience a recent range expansion. The GST and NST indices of genetic differentiation indicate the populations of four groups of diploiy W. harlandii, tetraploidy W. harlandii, tetraploidy W. harlandii complex, and W. harlandii complex all show significantly genetic differentiation. The GST significantly higher than NST in all four groups indicates the populations of these four groups all show obviously phylogeographic structure. The diploidy populations at Hsinhui have nine ones, in which eight are endemic, of total 15 cpDNA haplotypes. Therefore, it is proposed that Hsinhui is a long-term refugium for diploidy taxon. The cpDNA haplotypes of the populations in Nanling Mountain Ridge and Hainan Island both include two groups of highly variant sequences. It may be the effect of melting point after the last maximum glacial period that there are high nucleotide diversities in both areas. According to the mutation rate at the noncoding region of cpDNA, Urai in Taiwan might be another candidate for refugium. The Urai populations having two haplotypes might exist before the last maximum glacial period. The hypothesis of Cranfill about migration pattern of historical populations is rejected by the molecular evidences in this study. However, the hypothesis of multiple migration routes directly from the north to the south proposed in this study is partially supported by nuclear DNA.

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