雜交與多倍體化為植物界中常見的種化機制，由這兩種機制所產生的新物種會帶有更高的基因歧異度，並將物種帶往新的適應高峰。堇菜屬(Viola, Violaceae)植物是研究植物演化的重要材料之一，屬內物種在雜交及倍體數上的變化長期以來十分受到學者的關注。台灣產堇菜屬植物中，以蔓莖群(Diffusae)和合生托葉群(Adnatae)的染色體數目變化最引人注目。在蔓莖群中的四個物種其染色體為n=13 （茶匙黃）、24 （台北堇菜、普萊氏堇菜）、37 （心葉茶匙黃），合生托葉群內四個物種的染色體數則是n=12 （短毛堇菜）、24（小堇菜、紫花地丁）、36 （箭葉堇菜），兩群中染色體數皆呈倍數增加，雖然早有學者推論六倍體物種是由二倍體和四倍體物種雜交而來，卻遲遲未被證實。本研究利用母系遺傳的葉綠體DNA序列片段(trnL/trnF、rpl16)及核DNA序列片段(ITS)進行分析，加上鄰近地區樣本之序列，以最大簡約法與貝葉氏導出式分析重建台灣產堇菜屬蔓莖群與合生托葉群的演化歷史。
根據葉綠體DNA序列所建立的親緣關係樹，蔓莖群中的台北堇菜自成一支，與其他蔓莖群的物種關係較遠，合生托葉群的物種則是聚成支持度高的一群。在核DNA序列的部份，因多倍體物種多有同源基因拷貝，在親緣關係樹上呈現較複雜的表現，蔓莖群中的心葉茶匙黃分別與茶匙黃、台北堇菜和普萊氏堇菜、合生托葉群四倍體物種各自形成具三個具有支持度的分支；合生托葉群的箭葉堇菜則分別和短毛堇菜、小堇菜、紫花地丁各自形成多個具支持度的分支。
依據研究結果推論心葉茶匙黃是由茶匙黃或其近緣種、與台北堇菜和普萊氏堇菜共有的二倍體祖先、以及與合生托葉群共有的二倍體祖先等三個親本經兩次雜交及多倍體化而來。箭葉堇菜則可能是由短毛堇菜或其近緣種、長萼堇菜或其近緣種和與紫花地丁、早開堇菜共有的二倍體祖先雜交形成的。

Hybridization and polyploidization are common mechanisms in plant speciation. Via these mechanisms, the new lineages often can get higher genetic diversity than their parents and reach new adaptive peaks. Violets (Viola, Violaceae) are used as a material for plant evolution process, particularly focus on the ploidy level. In Taiwan, Viola group Diffusae and group Adnatae have very special cytological situation. The chromosome numbers in group Diffusae are n=13 (V. diffusa), 24 (V. nagasawai var. nagasawai and V. nagasawai var. pricei), 37 (V. tenuis), and the chromosome numbers in group Adnatae are n=12 (V. confusa), 24 (V. mandshuica and V. inconspicua subsp. nagasakiensis), 36 (V. betonicifolia), both of them increase as polyploidy. Early studies inferred that the hexaploid may be derived from hybridization and subsequent polyploidization between diploidy and tetraploidy ancestors which in the same group, but none of them be tested. This study used cpDNA (trnL/trnF, rpl16) and nrDNA (ITS) and be analyzed by maximum parsimony analysis (MP) and Bayesian inference analysis (BI) to reconstruct the relationship within these two polyploidy complex.
Based on the phylogeny tree of cpDNA, group Adnatae and group Diffusae were grouped separately in two well-supported clades but V. nagasawai var. nagasawai was not included. The nrDNA phylogeny tree showed that V. teunis nests in three different clades: V. diffusa, V. nagasawai var. nagasawai and V. nagasawai var. pricei, and the species of group Adnatae. Viola betonicifolia was separated to several different clades and gathered with V. mandshuica or V. inconspicua subsp. nagasakiensis.
The results indicated that V. teunis have three different parental species, one is V. diffusa or closely related species, another is a unknown diploid which shares a genome with V. nagasawai var. nagasawai and V. nagasawai var. pricei, and the last one is another unknown diploid which were shared with group Adnatae. Viola betonicifolia maybe a hybrid between V. confuse or closely related species, V. incospicua or closely related species and a diploid which were shared with V. mandshuica and V. prionantha.

Abbott, R. J., M. J. Hegarty, S. J. Hiscock, and A. C. Brennan. 2010. Homoploid Hybrid Speciation in Action. TAXON 59:1375-1386.
Anderson, E. 1948. Hybridization of the Habitat. Evolution 2:1-9.
Ballard, H. E., K. J. Sytsma, and R. R. Kowal. 1998. Shrinking the Violets: Phylogenetic Relationships of Infrageneric Groups in Viola (Violaceae). Based on Internal Transcribed Spacer DNA Sequences. Systematic Botany 23:439-458.
Belaeva, V. A. and V. N. Siplivinsky. 1975. Chromosome Numbers and Taxonomy of Some Species of Baikal Flora. Botanicheskii Zhurnal 60:864-872.
Chen, C.-H. 2001. The Phylogenetic Study of Gentiana sect. Chondrophyllae Bunge (Gentianaceae). National Taiwan Normal University, Taipei.
Chen, Y. S. 2006. Toxonomic Revision of Viola L.(Violaceae) in China In stitue of Botany,Chinese Academy of Sciences, Beijing.
Choi, H. W., J. S. Kim, and J. W. Bang. 1996. Chromosome Numbers and DNA Polymorphism of Viola in Korea. Gene and Genomics 18:241-248.
Clausen, J. 1927. Chromosome Number and the Relationship of Species in the Genus Viola Annals of Botany 41:677-714.
Clausen, J. 1929. Chromosome Number and Relationship of Some North American Species of Viola Annals of Botany 43:741-764.
Comes, H. P. and R. J. Abbott. 2001. Molecular phylogeography, reticulation, and lineage sorting in Mediterranean Senecio sect. Senecio (Asteraceae). Evolution 55:1943-1962.
Duchoslav, M., L. Šafářová, and F. Krahulec. 2010. Complex Distribution Patterns, Ecology and Coexistence of Ploidy Levels of Allium oleraceum (Alliaceae) in the Czech Republic. Annals of Botany 105:719-735.
Edgar, R. C. 2004. MUSCLE: Multiple Sequence Alignment with High Accuracy and High Throughput. Nucleic Acids Research 32:1792-1797.
Erben, M. 1996. The Significance of Hybridization on the Forming of Species in the Genus Viola. Bocconea 5:113-118.
Fant, J. B., E. Kamau, and C. D. Preston. 2005. Chloroplast evidence for the multiple origins of the hybrid Potamogeton x fluitans. Aquatic Botany 83:154–160.
Feliner, G. N., J. F. Aguilar, and J. A. Rossello. 2001. Can Extensive Reticulation and Concerted Evolution Result in a Cladistically Structured Molecular Data Set? Cladistics 17:301-312.
Frajman, B., F. Eggens, and B. Oxelman. 2009. Hybrid Origins and Homoploid Reticulate Evolution within Heliosperma (Sileneae, Caryophyllaceae)-A Multigene Phylogenetic Approach with Relative Dating. Systematic Biology 58:328-345.
Goloboff, P., S. Farris, and K. Nixon. 2000. TNT (Tree Analysis Using New Technology) (BETA) ver. 1.0. Tucum Argentina.
Gong, Q., J. S. Zhou, Y. X. Zhang, G. X. Liang, H. F. Chen, and F. W. Xing. 2010. Molecular Systematics of Genus Viola L. in China. Journal of Tropical and Subtropical Botany 18:633-642.
Grant, V. 1981. Plant Speciation. 2 edition. Columbia University Press.
Hall, T. A. 1999. BioEdit: a User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95-98.
Harrison, R. G. 1990. Hybrid Zones: Windows on Evolutionary Process. Oxford Surveys in Evolutionary Biology 7:69-128.
Holder, M. T., J. A. Anderson, and A. K. Holloway. 2001. Difficulties in Detecting Hybridization. Systematic Biology 50:978-982.
Holstein, N. and S. S. Renner. 2011. A Dated Phylogeny and Collection Records Reveal Repeated Biome Shifts in the African Genus Coccinia (Cucurbitaceae). BMC Evolutionary Biology 11:28-43.
Huang, R. F., S. D. Shen, and X. F. Lu. 1996. Studies of the Chromosome Numbers and Polyploidy for Some Plant in the North-East Qinghai-Xizang Plateau. Acta Botanica Boreali-Occidentalia Sinica 16:310-318.
Huelsenbeck, J. P., F. Ronquist, R. Nielsen, and J. P. Bollback. 2001. Bayesian Inference of Phylogeny and Its Impact on Evolutionary Biology. Science 294:2310-2314.
Joly, S., P. A. McLenachan, and P. J. Lockhart. 2009. A Statistical Approach for Distinguishing Hybridization and Incomplete Lineage Sorting. The American Naturalist 174:E54-E70.
Joly, S., J. R. Starr, W. H. Lewis, and A. Bruneau. 2006. Polyploid and Hybrid Evolution in Roses East of the Rocky Mountains. American Journal of Botany 93:412-425.
Jordan, W. C., M. W. Courtney, and J. E. Neigel. 1996. Low Levels of Intraspecific Genetic Variation at a Rapidly Evolving Chloroplast DNA Locus in North American Duckweeds (Lemnaceae). American Journal of Botany 83:430-439.
Juslén, A., H. Väre, and N. Wikström. 2011. Relationships and Evolutionary Origins of Polyploid Dryopteris (Dryopteridaceae) from Europe Inferred Using Nuclear pgiC and Plastid trnL-F Sequence Data. TAXON 60:1284-1294.
Kim, S.-T., S. E. Sultan, and M. J. Donoghue. 2008. Allopolyploid Speciation in Persicaria (Polygonaceae): Insights from a Low-Copy Nuclear Region. Proceedings of the National Academy of Sciences 105:12370-11237.
Koch, M. A., C. Dobes, and T. Mitchell-Olds. 2003. Multiple Hybrid Formation in Natural Populations: Concerted Evolution of the Internal Transcribed Spacer of Nuclear Ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae). Molecular Biology and Evolution 20:338-350.
Kubatko, L. S. 2009. Identifying Hybridization Events in the Presence of Coalescence via Model Selection. Systematic Biology 58:478-488.
Laureto, P. J. and T. J. Barkman. 2011. Nuclear and Chloroplast DNA Suggest a Complex Single Origin for the Threatened Allopolyploid Solidago houghtonii (Asteraceae) Involving Reticulate Evolution and Introgression. Systematic Botany 36 209-226.
Lee, Y. N. 1967. Chromosome Numbers of Flowering Plants in Korea. Journal of Korean Research Institute Ewha Women's University 11:455-478.
Liang, G. X. and F. W. Xing. 2010. Infrageneric Phylogeny of the Genus Viola (Violaceae) Based on trnL-trnF, psbA-trnH, rpl 16, ITS Sequences Cytological and Morphological Data. Acta Botanica Yunnanica 32:477-488.
Mallet, J. 2007. Hybrid Speciation. Nature 446:279-283.
Marcussen, T., K. S. Jakobsen, J. Danihelka, H. E. Ballard, K. Blaxland, A. K. Brysting, and B. Oxelman. 2012. Inferring Species Networks from Gene Trees in High-Polyploid North American and Hawaiian Violets (Viola, Violaceae). Systematic Biology 61:107-126.
Marhold, K. and G. N. Feliner. 2008. IAPT/IOPB Chromosome Data 6. TAXON 57:1267-1273.
Morgan, D. R. and B. Holland. 2012. Systematics of Symphyotrichinae (Asteraceae: Astereae): Disagreements between Two Nuclear Regions Suggest a Complex Evolutionary History. Systematic Botany 37:818-832.
Popp, M. and B. Oxelman. 2007. Origin and Evolution of North American Polyploid Silene (Caryophyllaceae). American Journal of Botany 94:330-349.
Posada, D. 2008. jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution 25:1253-1256.
Rentsch, J. D. and J. Leebens-Mack. 2012. Homoploid Hybrid Origin of Yucca gloriosa: Intersectional Hybrid Speciation in Yucca (Agavoideae, Asparagaceae). Ecology and Evolution 2:2213-2222.
Rieseberg, L. H. 1997. Hybrid Origins of Plant Species. Annual review of Ecology and Systematics 28:359-389.
Rieseberg, L. H. and N. C. Ellstrand. 1993. What Can Molecular and Morphological Markers Tell Us about Plant Hybridization? Critical Reviews in Plant Sciences 12:213-241.
Rosselló, G. N. F. J. A. 2007. Better the Devil You Know? Guidelines for Insightful Utilization of nrDNA ITS in Species-Level Evolutionary Studies in Plants. Molecular Phylogenetics and Evolution 44:911-919.
Sessa, E. B., E. A. Zimmer, and T. J. Givnish. 2012. Reticulate Evolution on a Global Scale: A Nuclear Phylogeny for New World Dryopteris (Dryopteridaceae). Molecular Phylogenetics and Evolution 64:563-581.
Soltis, D., P. Soltis, J. Pires, A. Kovarik, J. Tate, and E. Mavrodiev. 2004. Recent and Recurrent Polyploidy in Tragopogon (Asteraceae): Cytogenetic, Genomic and Genetic Comparisons. Biological Journal of the Linnean Society 82: 485-501.
Soltis, D. E., P. S. Soltis, and J.A.Tate. 2003. Advances in the Study of Polyploidy Since Plant Speciation. New Phytologist 161:173-191.
Soltis, P. S. and D. E. Soltis. 2000. The Role of Genetic and Genomic Attributes in the Success of Polyploids. Proceedings of the National Academy of Sciences:7051-7057.
Song, K., P. Lu, K. Tang, and T. C. Osborn. 1995. Rapid Genome Change in Synthetic Polyploids of Brassica and Its Implications for Polyploid Evolution. Proceedings of the National Academy of Sciences:7719-7723.
Stepanov, N. V. and E. N. Muratova. 1995. Chromosome Numbers of Some Taxa of Higher Plants of Krasnoyarsk Territory. Botanichekii Zhurnal 80:114-116.
Suda, J. and P. Trávníček. 2006. Estimation of Relative Nuclear DNA Content in Dehydrated Plant Tissues by Flow Cytometry.in J. P. Robinson, Z. Darzynkiewicz, J. Dobrucki, W. Hyun, J. Nolan, A. Orfao, and P. Rabinovitch, editors. Current Protocols in Cytometry. Wiley, New York, USA.
Sun, K. and Q. R. Wang. 1996. Cytological Studies on 6 Species of Viola from North West China. Chinese Bulletin of Botany 13:46-47.
Taberlet, P., L. Gielly, G. Pautou, and J. Bouvet. 1991. Universal Primers for Amplification of Three Non-Coding Regions of Chloroplast DNA. Plant Molecular Biology 17:1105-1109.
Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28:2731-2739.
Wang, C. J. 1991. Viola L. Pages 8-129 in Anonymous, editor. Flora Reipublicae Popularis Sinicae. Science Press, Beijing.
Wang, J. C. 1989. A Taxonomic study of Viola L. (Violaceae) of Taiwan Taiwan University, Taipei.
Wendel, J. and J. Doyle. 2005. Polyploidy and Evolution in Plants. Pages 97-117 in R. J. Henry, editor. Plant Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. Cromwell Press Trowbridge, UK.
Wendel, J. F. 2000. Genome Evolution in Polyploids. Plant Molecular Biology 42:225-249.
Wendel, J. F. and J. J. Doyle. 1998. Phylogenetic Incongruence: Window into Genome History and Molecular Evolution. Pages 265-296 in D. Soltis, P. Soltis, and J. Doyle, editors. Molecular Systematics of Plants, II: DNA Sequencing. Kluwer Academic Publishers, Boston, MA.
Wu, L. L., X. K. Cui, R. I. Milne, Y. S. Sun, and J. Q. Liu. 2010. Multiple Autopolyploidizations and Range Expansion of Allium przewalskianum Regel. (Alliaceae) in the Qinghai-Tibetan Plateau. Molecular Ecology 19:1691-1704.
Yang, H. A. 2011. Hybridization and Polyploidization of Taiwanese Begonias. National Taiwan University, Taipei, Taiwan.
Yoo, K. O. and S. K. Jang. 2010. Infrageneric Relationships of Korean Viola based on Eight Chloroplast Markers. Journal of Systematics and Evolution 48:474-481.
Yoshioka, H. and R. Tanaka. 1984. On the Occurrence of Accessory Chromosomes in Viola ovato-oblonga. Chromosome Information Service 37:28-29.
Zhou, J.-S., Q. Gong, and F.-W. Xing. 2008. Viola nanlingensis (Violaceae), a New Species from Guangdong, Southern China. Annales Botanici Fennici 45:233-236.