M OLECULAR TAXONO M Y OF CAMELLIA (THEACEAE) INFERRED

Transcription

1 American Journal of Botany 96(7): M OLECULAR TAXONO M Y OF CAMELLIA (THEACEAE) INFERRED FROM NRITS SEQUENCES 1 K UNJUPILLAI VIJAYAN, 2 W EN-JU ZHANG, 3 AND CHIH-HUA TSOU 2,4 2 Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan-115, ROC 3 Department of Ecology and Evolutionary Biology, Fudan University, Shanghai , China Camellia, comprising more than 200 species, is the type genus of the family Theaceae. Currently, the interspecies relationship of the economically important genus is still a matter of great debate and controversy. In an attempt to help settle this dispute using molecular phylogeny, we analyzed ITS sequences of 112 species of Camellia. The maximum parsimony and Bayesian trees grouped these species into eight major clades and four isolates. The current study supported the monophyly of sections Thea and Furfuracea, a merged section of Theopsis and Eriandra and the formation of section Oleifera by H, -t. Chang ( Flora of Reipulicae Popularis Sinicae. Tomus 49 (3), Science Press, China). The study suggested the polyphyletic nature of the sections Camellia, Paracamellia, Pseudocamellia, and Tuberculata and the paraphyletic nature of the section Chrysantha but did not support the sectional status of the three small sections, Archecamellia, Piquetia, and Sterocarpus. We also discuss the results in terms of morphology, geographic distribution and the results from an earlier molecular phylogeny analysis. Key words: Camellia ; ITS; molecular phylogeny; tea; Theaceae, Camellia is the type and the largest and economically most important genus in the family Theaceae. Species of Camellia are largely distributed in southeastern and eastern Asia and China, which, being the center of species diversity, possess more than 80% of the species ( Gao et al., 2005 ). The most economically important species in Camellia are C. sinensis and C. assamica, which are used to make nonalcoholic tea ( Eden, 1958 ; Kondo, 1975 ). Currently, more than 40 countries produce the tea commercially, and about 3.6 million tons of tea leaves are produced annually ( Antonios, 2005 ; Chen et al., 2007 ). Biochemically, tea leaves have more than 700 chemical constituents, of which catechins, flavonoids, amino acids, vitamins (C, E, K), caffeine, and polysaccharides are important for human health. The potential benefits of polyphenols and catechins in tea for the treatment of cancer, diabetes, obesity, and many cardiovascular ailments have been highlighted in many recent reports ( Khan and Mukhtar, 2007 ; Mukhtar and Ahmad, 2000 ; Murase et al., 2002 ). Vitamin C content in tea leaves is comparable to that in lemon ( Mondal et al., 2004 ). Camellia is also important in floriculture. Historically, the cultivation of Camellia as an ornamental started in China more than 1300 years ago ( Gao et al., 2005 ). Today, plants from C. japonica, C. reticulata, C. sasanqua, and a group of yellow-flowering species called golden camellias are grown as ornamental plants worldwide. Although Camellia is native to Asia, the cultivated species are well adapted in several other countries. For instance, plants of 1 Manuscript received 22 August 2008; revision accepted10 March The authors thank J. Y. Gao, H. G. Yeh, H. Wang, F. Lu, C. Wang, X. Gong, H. Peng, F. Maxwell, L. Li, and J. L. Huang for generous support in providing valuable samples. Thanks to B. G. Baldwin and Y. W. Yang for valuable advice, and to Y. L. Fu, M. H. Su and C. C. Wu for making several critically important field trips with them. They also thank the directors of the Hong Kong herbarium, K. L. Yip, and HAST herbarium, and C. I. Peng for invaluable help and L. Heraty for English editing. The authors received financial support for the study from the National Science Council, Taiwan, Republic of China (project NSC B MY2) and the Institute of Plant and Microbial Biology, Academia Sinica, R.O.C. 4 Author for correspondence ( chtsou@gate.sinica.edu.tw) doi: /ajb C. japonica are so popular in the southern United States that the flower of this species is recognized as the state flower of Alabama ( Gao et al., 2005 ). The third industrial use of Camellia is in the oil industry: seeds of several species belonging to the sections Oleifera, Paracamellia, Camellia, and Furfuracea are used for extracting edible oil, which is used extensively for cooking in China ( Ming, 2000 ; Gao et al., 2005 ; Zhang et al., 2007). In China, more than 3 million hectares are used for Camellia oil production, and nearly tons of seeds are harvested each year, which yield nearly tons of edible oil ( Ming, 2000 ; Gao et al., 2005 ). Camellia s.l. is considered a single genus encompassing all 17 genera described between 1753 and 1957 ( Prince, 2007 ). Morphologically, the monophyly of Camellia is supported by a rounded or rounded-cuneate seed with thick cotyledons ( Keng, 1962 ) and an Allium type of embryo sac formation ( Wu, 1962 ; Tsou, 1997 ). A molecular phylogenetic study of intron sequences of the RNA polymerase II ( RPB2 ) gene from nuclear DNA also confirmed the monophyletic nature of the genus Camellia ( Xiao and Parks, 2003 ). Although the generic circumscription is settled, the intrageneric classification continues to be a matter of great debate and confusion. Two recent classifications, which were proposed by Chang (1998) and Ming (2000) have many areas of disagreement, particularly for the circumscription of subgenera, sections, and species. Chang (1998) identified about 280 species and classified them into four subgenera and 21 sections. However, Ming (2000), with a broader species concept, recognized only 119 species and divided them into two subgenera and 14 sections, largely on the basis of a structural framework proposed by Sealy (1958). Xiao (2001) discussed a number of differences between these two major classifications, the most salient of which at the sectional levels are listed in Appendix S1, and those at the species level are listed in Appendix S2 (see Supplemental Data with the online version of this article). Controversies and contradictions among taxonomic classifications based on morphological characters in plants are not rare phenomena because the evolutionary dynamics behind all morphological character formations are not fully understood. Therefore, information from nonmorphological

2 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1349 characters such as nucleic acids and biochemical molecules is often needed to resolve taxonomic problems. During the last 10 years, efforts to resolve classification issues in Camellia have involved use of data on DNA sequences ( Thakor and Parks, 1997 ; Tang and Zhong, 2002 ; Orel et al., 2003 ; Xiao and Parks, 2003, Yang et al., 2006 ). However, most of these studies were small scale and focused primarily on species relationships within specific sections or groups ( Vijayan and Tsou, 2008 ). The only large-scale attempt was by Xiao and Parks (2003), who used introns and 23 of RPB2 gene. Although clades in the RPB 2 trees were poorly resolved and the sequences were not deposited in public databases, the study presented a view of the interspecies relationships of Camellia different from that of traditional treatments. For instance, the traditional circumscriptions of many sections were not supported by this study, and large sections such as Camellia were divided into subgroups showing geographic associations. These findings, coupled with the poor resolution of the clades, prompted us to undertake further investigation of the vexing taxonomic problems associated with the classification of the genus Camellia by using DNA sequences with better resolving power. Among the selectively less-constrained DNA sequences used for molecular phylogeny in plants, nuclear ribosomal internal transcribed spacer (nrits) sequences are considered the most powerful ( Rogers and Bendich, 1987 ; Baldwin, 1992 ; Baldwin et al., 1995 ; Alvarez and Wendel, 2003 ). In fact, use of nrits data for Camellia has been attempted, but success was reported in only a single study on the intrasectional relationships in the section Chrysantha ( Tang and Zhong, 2002 ). Technical difficulties associated with PCR amplification and subsequent sequencing of PCR products were cited as the major reasons for this lack of nrits data for Camellia ( Yang et al., 2006 ; Vijayan and Tsou, 2008 ). Therefore, in our initial attempt to resolve these technical problems associated with the sequencing of nrits in Camellia, in a report on the molecular phylogeny of Camellia (Vijayan and Tsou, 2008 ), we provided technical details about these problems and their solutions, along with the potential use of nrits sequences for resolving the complex species relationships in Camellia. In this second report, we discuss the phylogenetic relationships among the species of Camellia, with a focus on the sectional treatment, in comparison to the traditional classifications by Chang (1998) and Ming (2000). We also discuss conflicts in species delimitations in these previous classifications. MATERIALS AND METHODS Plant materials and data mining Young, healthy leaves of Camellia were collected from their natural habitats in Hong Kong, mainland China, northern Thailand, northern Vietnam, Taiwan, and the Ryukyus. A great number of samples were also collected with permission from the International Camellia Species Garden, Jinghua, Zhejiang Province, China, and other botanical gardens in Hong Kong and mainland China, where the plants are labeled and well maintained. Leaf samples, after collection, were immediately kept in a sealable packet containing silica gel and stored at 4 C. Voucher specimens of these collections are preserved in the HAST or CMU herbaria. These 109 leaf samples from 100 species covered 15 of 21 sections in the Chang (1998) classification and 11 of 14 sections in the Ming (2000) classification ( Table 1 ). An additional set of nrits sequences from 12 species of the golden camellias of section Chrysantha was also retrieved from GenBank (AF AF315496). Thus, the total number of species of Camellia assessed was 112. The sample list with relevant information is in Table 1. Outgroup sequences Five sequences from the genus Pyrenaria were used as outgroup taxa, because the genus is the closest to Camellia (Prince and Parks, 2001 ). We sequenced the sample of P. menglaensis (EU579798) and retrieved the other four sequences from GenBank (AF and AF ). DNA isolation, PCR amplification, cloning, sequencing, sequence alignment The protocols for DNA isolation, PCR amplification, cloning, and sequencing were as described ( Vijayan and Tsou, 2008 ). At least 10 clones were sequenced for each sample, and the sequences were aligned using the program PILEUP 8.1 ( Genetic Computer Group, 1994 ). The borders of ITS1, 5.8S and ITS2 regions were identified on the basis of earlier reports ( Baldwin, 1992 ; Tang and Zhong, 2002 ; Feldberg et al., 2004 ). Sequence analysis Gene tree Earlier efforts to sequence nritss in Camellia revealed sequencing errors, even with the use of pfu -DNA polymerase ( Vijayan and Tsou, 2008 ). Thus, both forward and reverse sequences of each clone were aligned to validate sequence integrity. The IUPAC ambiguity code was used when required. Our earlier study of nrits sequences in Camellia revealed that multiple sequences from one or more accessions of an individual species formed monophyletic groups ( Vijayan and Tsou, 2008 ). In this expanded study, we used the sequence from a single clone as the representative of a given species. To determine the representative sequence for each species, we constructed gene trees, which reflect the evolution of paralogous and orthologous copies of genes in a genome ( Wendel and Doyle, 1999 ; Small and Wendel, 2000 ). The limitations of current software for analysis (e.g., PAUP*; Swofford, 2001 ) and visualization (e.g., TreeView; Page, 1996 ) necessitated dividing the whole sequence data set into subsets, because a matrix of nearly 1000 sequences would have prevented effective analysis. The analysis was thus divided into six subsets: section(s) (1) Archecamellia + Brachyandra + Chrysantha + Longipedicellata + Piquetia + Stereocarpus, (2) Camellia, (3) Thea, (4) Eriandra + Theopsis, (5) Pseudocamellia + Tuberculata, and (6) Furfuracea + Oleifera + Paracamellia, following the classification proposed by Chang (1998). Gene trees were constructed with the use of maximum parsimony (MP) analyses in PAUP* version 4.0b8 ( Swofford, 2001 ). We conducted heuristic searches using stepwise addition of random sequences with 100 replicates, saving 1000 trees per replication and keeping tree-bisection-reconnection (TBR), branch swapping, multrees, collapse and steepest descent options. Gaps were treated as missing. Jackknife support ( Farris et al., 1996 ) for each branch was estimated by 100 replicates of heuristic searching with random addition of sequences. Trees were rooted with the five Pyrenaria species used as outgroups. Selection of sequences for species trees Sequences for species trees were selected from gene trees on the basis of a combination of tree reconciliation and mapping clade methods ( Page, 1994 ). In the tree reconciliation method, a species tree is constructed on the basis of a minimal number of duplications and lateral transfers, whereas in the mapping clade method, each cluster on the gene tree is mapped to the smallest cluster on the species tree that contains all the species from which the sequence was sampled. Species tree All 112 sequences, which included 100 sequences from the species sampled in this study and the 12 sequences retrieved from GenBank, were aligned with the program PILEUP 8.1 ( Genetic Computer Group, 1994 ) and fine-adjusted manually with the program BioEdit ( Hall, 1999 ). Because gaps that formed in the sequence alignment as a result of insertion and deletion events (indel) are as informative as the nucleotides themselves in phylogenetic reconstruction ( Lloyd and Clader, 1991 ), gaps were coded into a binary matrix (presence/absence) following the simple indel coding methods of Simmons and Ochoterena (2000). We considered putatively homologous gaps ( de Pinna, 1991 ) as those with identical 5 and 3 termini. Gaps with different termini were not treated as homologous because at least one indel event must be postulated to transform one gap into another. The MP analysis was done with PAUP* 4.0b8 ( Swofford, 2001 ). To prevent the search stranding on an island of suboptimal trees and overflow of the tree buffer zone, heuristic searches were carried out with stepwise addition of random sequences with 100 replicates, saving trees per replication. Tree-bisection-reconnection (TBR) branch swapping, multrees, collapse, and steepest descent options and ACCTRAN character optimizations were in effect. Jackknife support ( Farris et al., 1996 ) was estimated with 1000 replicates of heuristic search with 10 random additions of sequences. Bayesian analysis was conducted with the program MrBayes 3.0b4 ( Huelsenbeck and Ronquist, 2001 ) as an alternative for likelihood analysis. Four Markov chain Monte Carlo (MCMC) runs were carried out for 1.5 million generations with four incrementally heated chains, starting from random trees and sampling 1 of every 100 generations. The evolutionary model that best fits the data set was determined as GTR+G by the program Modeltest 3.6 ( Posada and Crandall, 1998 ). The first generations were discarded as burn in on the basis of

3 1350 AMERICAN JOURNAL OF BOTANY [Vol. 96 log-likelihood plots. For estimating the posterior probability of the recovered branches, 50% majority-rule consensus trees were created from the remaining trees. Analyses were repeated three times to confirm the results. Trees were rooted with the five Pyrenaria species used as outgroups. RESULTS Length and sequence variations Alignment of multiple sequences from each leaf sample revealed varying degrees of intragenomic variability for most of the species. The intragenomic variability largely consisted of single substitutions and indels present in areas with mono-, di- or trinucleotide repeats. Gaps were common in the ITS1 and ITS2 regions, and therefore, their total lengths varied considerably, ranging from 626 bp in C. pachyandra to 699 bp in C. brevistyla. The GC content varied from 67.43% in C. azelea to 72.66% in C. latipetiolata in the ITS1 region and from 72.69% in C. ptilophylla to 78.66% in C. azalea in the ITS2 region. The 5.8S region was 164 bp for most species, but in members belonging to section Oleifera and several allied species had an insertion of A or AA after the first four (TAAA) conserved bases of 5.8S. The unusual length variability in the 5.8S region revealed the evolutionary dynamism of nrdna in Camellia. The GC content of the 5.8S region was much lower than that of ITS1 and ITS2; in most species, the content was 53.66% or 54.27%. Gene trees The most parsimonious gene tree of group 1 generated with 134 sequences from the 16 samples of 14 species from sections Archecamellia, Brachyandra, Chrysantha, Longipedicellata, Piquetia, and Stereocarpus, along with five outgroup sequences, had a tree length of 761 steps, consistency index of , and retention index of One of the most parsimonious trees with jackknife support values is shown in Fig. 1. The MP gene trees from the remaining five analyses for other sections are provided in online Appendices S3 S7 (see Supplemental Data with the online version of this article). The tree lengths, consistency indices, and retention indices of these five gene trees are, respectively, 934, , and for section Camellia (Appendix S3); 510, , and for section Thea (Appendix S4); 378, , and for sections Eriandra and Theopsis (Appendix S5); 379, , and for sections Pseudocamellia and Tuberculata (Appendix S6); and 1482, , and for sections Furfuracea, Oleifera, and Paracamellia (Appendix S7). In the gene trees, conspecific sequences were usually grouped together as a single major clade, as evidenced in Fig. 1 ; however, in some species one or a few sequences remained out of the main clade either as isolates or were grouped with sequences from other species. In just two or three instances, such as for C. japonica (Appendix S3) and C. rubituberculata and C. rhytidocarpa (Appendix S6), sequences from the same species were grouped into two or three small groups. From the major clade of each species, a representative sequence was chosen following the tree reconciliation and the mapping clade methods mentioned previously. The representative sequences selected from the gene trees are marked with boldfaced letters ( Fig. 1, Appendix S3 S7, see Supplemental Data with the online version of this article). For species that formed two or three clades, we selected the representative sequence from the clade with most of the sequences, keeping in mind the numerical dominance rule (La Jeunesse and Pinz ó n, 2007) because in a multiple-copy gene such as rdna, a variant copy can arise instantaneously in a single generation as a result of point mutations and has little probability of sweeping through the rdna to become the numerically dominant copy within a known time period. Thus, for the purpose of phylogenetic reconstructions, following the rule of numerical dominant seems logical (La Jeunesse and Pinz ó n, 2007). Species tree The MP tree with tree length of 584, consistency index of (excluding uninformative characters), and retention index of was largely congruent with the 50% majority-rule consensus tree from Bayesian trees ( Fig. 2 ). Both the MP tree and the Bayesian tree resolved the 112 ingroup species into eight major clades (clades A to H) and four isolates: C. gilbertii and C. pachyandra from section Brachyandra and C. amplexicaulis and C. longipedicellata from section Longipedicellata. The species included in these eight clades were exactly the same in both trees, and most of these eight clades were well supported in both trees. However, the interrelationships among these clades remained unresolved. The molecular phylogenetic scheme largely differed from the traditional ones, because only two clades (clades D and H) were consistent with the sectional composition of the Chang (1998) classification, whereas the other six clades accommodated species from two or more sections. Nevertheless, most of these clades were well defined by a set of morphological characters ( Table 2 ). Clade A, with jackknife value (JV) of 81% and posterior probability (PP) of 100%, is a large assemblage of species belonging to six sections of the Chang classification (1998). The 28 species included all 18 species from the section Chrysantha, five from the section Tuberculata, two from the section Pseudocamellia (C. chungkingensis and C. szechuanensis), one from the section Piquetia (C. piquetiana ), and one each from sections Archecamellia (C. petelotii ) and Stereocarpa (C. yunnanensis ). In the MP tree, seven species from the section Chrysantha and C. petelotii from Archecamellia formed the basalmost group of clade A, which was followed by another group of five species from the section Tuberculata, along with C. chungkingensis from the section Pseudocamellia. In Chang s system (1998), C. chungkingensis was in section Pseudocamellia, but in Ming s system (2000) it was included in section Tuberculata. Thereafter, the inner large group had 11 species of Chrysantha forming a series of small subclades and the single species each of sections Piquetia and Stereocarpus positioned as singletons. In the Bayesian tree, four parallel subclades and eight singletons joined together with strong branch support of 100%. In this case also, the major groupings formed by species from the sections Chrysantha and Tuberculata remained the same as those observed in the MP tree, and the species from sections Piquetia and Stereocarpus remained as singletons. Clade B (JV = 87% and PP = 95%) was a mixture of 13 species from three sections: one from section Archecamellia (C. granthamiana ), nine from section Camellia, and three from section Paracamellia. In both Bayesian and MP trees, two species from the section Paracamellia and one species from the section Camellia ( C. subintegra ) formed the basal subclade. The species C. granthamiana joined with this subclade as a sister taxon in the MP tree and as the basalmost taxon of the entire clade in the Bayesian tree. The remaining eight species of the section Camellia, along with C. weiningensis (section Paracamellia ), formed two subclades. Of note, in the Ming system (2000), C. weiningensis was treated under section Camellia as a synonym of C. saluenesis. Clade C (JV = 89% and PP = 100%) consisted of all 18 species sampled from the sections Eriandra and Theopsis, which formed three subclades and several singletons in both trees, and the members in the subclades remained almost the same in both

4 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1351 T ABLE 1. List of samples, section classification according to Chang (1998), voucher information, and Gen Bank accession numbers for sequences determined in this study. Taxon (Sample no.) Section Place of collection Voucher (source origin) Accession a C. acuticalyx Chang (T1356) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. acutiperulata Chang et Ye ex Chang (T1355) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. amplexicaulis (Pit.) Cohen-Stuart (T1226) Longipedicellata Chang Zhejiang, China Zhang, JH , Golden Cam. Gard. (cult.) EU579676, FJ FJ C. angustifolia Chang (T1341) Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. assamica (Mast) Chang (S455) Thea (L.) Dyer Taipei, Taiwan Su, MH 670; Tea Improv. Inst. (cult.) FJ C. assamica (T561) Thailand Maxwell (CMU) C. atrothea Chang et Wang (T1002) Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. azalea Wei (T650)Camellia (L.) Dyer Guangdong, China Tsou, CH 1977 (HAST) EU C. bailinshanica Chang et Xiong (T1003) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. brachygyna Chang (T1373) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. brevistyla (Hayata) Cohen- Stuart (T1043) Paracamellia Sealy Taitung, Taiwan Su, MH 664 (HAST) EU C. caudata Wall (T666)Eriandra Coh. St. Guangdong, China Tsou, CH 1993 (HAST) EU C. chekiangoleosa Hu (T860) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. chekiangoleosa Hu (T875) Camellia (L.) Dyer Hunan, China Zhang, WJ EU C. chrysantha (Hu) Tuyama (T865) Chrysantha Chang Yunnan, China Kunming Bot. Inst. (cult.) EU579687, FJ FJ C. chungkingensis Chang (T847) Pseudocamellia Seally Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. cordifolia (Metcalf) Nakai (T327) Eriandra Coh. St. Guangdong, China South China Bot. Inst. (cult.) EU C. costei Lev. (T866) Theopsis Coh.St Yunnan, China Kunming Bot. Inst. (cult.) EU C. crapnelliana Tutcher (T1001)Furfuraceae Chang Guangxi, China Zhang, WJ GX0506 EU C. cuspidata (Kochs) Wright (T711) Theopsis Coh.St Fijian, China Tsou, CH 2050 (HAST) EU C. delicata Li (T1005) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. edithae Hance (T871) Camellia (L.) Dyer Hunan, China Zhang, WJ HN EU C. euphlebia Merr. ex Sealy (T804) Chrysantha Chang Yunnan, China Trop. Bot. Gard., Xishuangbanna (cult.) EU579697, FJ FJ C. euryoides Lindl. (T622) Theopsis Coh.St Guangdong, China Tsou, CH 1949 (HAST) FJ C. fl ava (Pit.) Sealy (T850) Chrysantha Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU579699, FJ FJ C. fl uviatilis Hand. et Mazz. (T621) Paracamellia Sealy Guangdong, China Tsou, CH 1948 (HAST) FJ C. forestii (Diels) Cohen-Stuart (T878) Theopsis Coh.St Yunnan, China Zhang, WJ EU C. formosensis Su, Hsieh, et Tsou (S318) Thea (L.) Dyer Taiwan Su, MH 497 (HAST) EF C. formosensis (T827) Taiwan Tsou, CH 2139 (HAST) C. fraterna Hance (T1208) Theopsis Coh.St Yunnan, China Kunming Bot. Gard. (cult.) EU C. furfuracea (Merr.) Cohen-Stuart (T1044) Furfuraceae Chang Taiwan Su, MH 606 (HAST) EU C. gauchowensis Chang (T1008) Oleifera Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. gilbertii (Chev.) Sealy (T1230) Brachyandra Chang Tamdao Natl. Park, Vietnam Zhang, WJ VN001 EU579709, FJ FJ C. granthamiana Sealy (T1009) C. granthamiana (T1299) Archecamellia Sealy Zhejiang, China Hong Kong Intern. Cam. Sp. Gard. (cult.) Tsou, CH 2377 (HAST) EU579710, FJ FJ C. grijsii Hance (T207) Paracamellia Sealy Yunnan, China Tsou, CH 2349, Kunming Bot. EU Gard. (cult.) C. hengchunensis Chang (C169) Pingtung, Taiwan Su, MH 442 (HAST) FJ C. hengchunensis (T1042) Pingtung, Taiwan Tsou, CH (HAST) C. hongkongensis Seem (T1089) Camellia (L.) Dyer Taiping Mt., HK Tsou, CH 2242, EU C. hongkongensis (T1093) Taiping Mt., HK Tsou, CH 2246 (HAST) C. hunanica Chang et Liang (T1010) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. hupehensis Chang (T1011) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. impressinervis Chang et Liang (T1212) Yunnan, China Kunming Bot. Gard. (cult.) EU579721, FJ FJ C. japonica L. (T1235) Camellia (L.) Dyer Keelung, Taiwan Su, MH 638 (HAST) EU C. jinshajiangica Chang et Lee (T1366) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. jinyunshanica Chang et Qi (T1013) Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. kissi Wall (T851) Paracamellia Sealy Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. kwangsiensis Chang (T1332) Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. lapidea Wu (T1014) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. latipetiolata Chi (T846H) Furfuraceae Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. lawii Sealy (T1015) Eriandra Coh. St. Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. leptophylla Liang (T852) Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. longipedicellata (Hu) Chang (T1016) Longipedicellata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU579734, FJ FJ C. longtousanica (T1347)Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. lungshenensis Chang (T1018) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. mairei (Levl.) Melchior (1369) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. meiocarpa Hu (T20) Taipei, Taiwan Taipei Bot. Gard. (cult.) EU C. microphylla (Merr.) Chun (T853) Paracamellia Sealy Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EF C. multiperulata Chang (T647) Camellia (L.) Dyer Guangdong, China Tsou, CH 1974 (HAST) FJ432109

5 1352 AMERICAN JOURNAL OF BOTANY [Vol. 96 TABLE 1. Continued. Taxon (Sample no.) Section Place of collection Voucher (source origin) Accession a C. nitidissima Chi (T1000) Chrysantha Chang Guangxi, China Zhang, WJ GX05 04 EU579740, FJ FJ C. nokoensis Hayata (T17) Theopsis Coh.St Taipei, Taiwan Taipei Bot. Gard. (cult.) EU C. obovatifolia Chang (1359) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. octopetala Hu (T727) Furfuraceae Chang Fujian, China Tsou, CH 2066 (HAST) EU C. oleifera Abel (T898)Oleifera Chang Zhejiang, China Tsou, CH 2149 (HAST) FJ C. omeiensis Chang (T1021) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. pachyandra Hu (T989) Brachyandra Chang Yunnan, China Zhang, WJ YN05-36 EU579748, FJ FJ C. parafurfuracea Liang et Chang (T854) Furfuraceae Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. parvilimba Merr. et Metcalf (T1022) Theopsis Coh.St Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. parvimuricata Chang (T1361) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. paucipetala Chang (T1023) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. petelotii (Merr.) Sealy (T707) Archecamellia Sealy Guangdong, China South China Bot. Gard. (cult.) EU579753, FJ FJ C. phellocapsa Chang et Lee (T1025) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. phaeoclada Chang (T1024) Paracamellia Sealy Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ EU579757, FJ FJ C. piquetiana (Pierre) Sealy (T1224) Piquetia Chang Zhejiang, China Zhang, JH Golden Cam. Gard. (cult.) C. pitardii Chang (T992) Camellia (L.) Dyer Yunnan, China Zhang, WJ YN05-39 FJ C. polyodonta How ex Hu (T998) Camellia (L.) Dyer Guangxi, China Zhang, WJ GX05-02 EU C. ptilophylla Chang (1343) Thea (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. pubifurfuracea Zhong (T855) Furfuraceae Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. rhytidocarpa Chang et Liang (T1028) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. rhytidophylla Li et Yang Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. rubituberculata Chang (T1030) Tuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. rusticana Honda (T1380) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. salicifolia Champ. ex Benth. (T1046) Eriandra Coh. St. Pingtung, Taiwan Su, MH 649 (HAST) EU C. salicifolia Champ. ex Benth. (T725) Eriandra Coh. St. Fujian, China Tsou, CH 2064 (HAST) EU C. saluensis Stapf ex Bean (T1213) Camellia (L.) Dyer Yunnan, China Kunming Bot. Gard. (cult.) EU C. saluensis (T886) Yunnan, China DYJ 0502 C. sasanqua Sims (T988) Oleifera Chang Taipei, Taiwan Su, MH 409 (HAST) FJ C. semiserrata Chi (T680) Camellia (L.) Dyer Guangdong, China Tsou, CH 2007 (HAST) EU C. shensiensis Chang (T937) Paracamellia Sealy Sichuan, China Zhang, WJ & Lu, F EU C. sinensis (L.) Kuntze (T1236) C. sinensis (T894) Thea (L.) Dyer Taipei, Taiwan West Tien-Mu Mt., Zhejiang, China Yang-ming Mt. (cult.) Tsou, CH 2145 (HAST) FJ C. subacutissima Chang (T1034) Theopsis Coh.St Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. subintegra Huang et Chang (T1065) Camellia (L.) Dyer Jiangxi, China Lu, F. & Wang, C. EU C. szechuanensis Chi (T928) Pseudocamellia Seally Sichuan, China Zhang, WJ & Lu, F EU C. taliensis (Smith) Melch. (T864) Thea (L.) Dyer Yunnan, China Wang, C FJ C. tenuifl ora (Hayata) Cohen-Stuart (S163) Paracamellia Sealy Taipei, Taiwan Su, MH 128 (HAST) FJ C. transarisanensis (Hayata) Cohen-Stuart (S333) Theopsis Coh.St Taichung, Taiwan Su, MH 515 (HAST) EU C. transnokoensis Hayata (S305) Theopsis Coh.St Nantou, Taiwan Su, MH 476 (HAST) EU C. tsaii Hu (T1035) Theopsis Coh.St Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. tuberculata (T858) ChienTuberculata Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. tunghinensis Chang (T1036) Chrysantha Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU579785, FJ FJ C. uraku (Makino) Kitamura (T1378) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. vietnamensis Huang (T856) Oleifera Chang Zhejiang, China Intern. Cam. Sp. Gard. (cult.) FJ C. waldeniae Hu (T1131) Thea (L.) Dyer Tatung Mt., HK Tsou, CH 2284 (HAST) EU C. waldeniae (T1150) Tatung Mt., HK Tsou, CH 2303 (HAST) C. weiningensis Li ex Chang (T857) Paracamellia Sealy Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. wenshanensis Hu (T1037) Eriandra Coh. St. Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. xifongensis Li ex Chen et Zheng (T1038) Camellia (L.) Dyer Zhejiang, China Intern. Cam. Sp. Gard. (cult.) EU C. yuhsienensis Hu (T1206) Paracamellia Sealy Yunnan, China Kunming Bot. Gard. (cult.) EU C. yunnanensis (Pit. ex Diels) Cohen-Stuart (T1218) Sterocarpus (Pierre) Yunnan, China Kunming Bot. Inst. (cult.) C. yunnanensis (T1222) Sealy Yunnan, China Kunming Bot. Inst. (cult.) Pyrenaria menglaensis Tao (T995) Yunnan, China Zhang, WJ YN05-44 EU EU579796, FJ FJ Notes: Bot. = Botanical, HK = Hong Kong, Inst. = Institute; Intern. Cam. Sp. Gard. = International Camellia Species Garden. a Sequences retrieved from GenBank are not included. Single accession numbers are the representative sequences used in the species trees ( Fig. 2 ). The serial accession numbers provided for 14 species are the cloned sequences used in the gene tree ( Fig. 1 ). Fig. 1. Gene tree of the sections Archecamellia, Brachyandra, Chrysantha, Longipedicellata, Piquetia, and Stereocarpus of the genus Camellia. Analysis of 134 ingroup nrits sequences from 16 leaf samples of 14 species from these sections showing one of the most parsimonious gene trees with tree length of 761 steps, consistency of index , and retention index of ; numbers on the branches are jackknife values.

6 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1353

7 1354 AMERICAN JOURNAL OF BOTANY [Vol. 96 trees. However, both trees showed no clear demarcation between species of Eriandra and Theopsis. Clade D (JV = 80% and PP = 100%) included all 12 species sampled from the section Thea. A subclade with three species C. longtousanica, C. kwangsiensis, and C. taliensis received high support and was sister to the remaining nine species, of which seven were singletons and two (i.e., C. sinensis and C. jinyunshanica ) joined together. Clade E, with weak support values, comprised 13 species: eight from section Camellia and five from section Paracamellia. The Bayesian and MP trees have identical topology. The eight species of section Camellia were split into two subclades and three singletons arranged in a stepwise manner, whereas the five species of the section Paracemellia formed a strongly supported subclade embedded well inside clade E. Clade F, with strong support (JV = 97% and PP = 100%), was an assemblage of 13 species, including all five species from section Oleifera, four from section Tuberculata, two from section Paracamellia, one from section Camellia (C. lungshenensis ), and one Taiwan-endemic species ( C. hengchunensis ), which was not treated by Chang (1998) or Ming (2000). Again, Bayesian and MP trees had highly similar topologies. Three species from the section Tuberculata formed a basal subclade with 100% support values. As well, the section Oleifera and its allied species formed another well-supported subclade. Clade G, with both JV and PP of 100%, contained all six species from the section Furfuracea, along with a single species from section Camellia (C. hongkongensis ). With C. pubifurfuracea at the base, the remaining six species formed two subclades. Both trees had identical topology with similar support values for most of the internal nodes. Clade H is a small clade with strong support (JV = 96% and PP = 100%) comprising six species from the section Camellia. Isolates In both the Bayesian and MP trees, C. pachyandra was the basalmost taxon, sister to all other species of the genus Camellia. However, C. gilbertii, C. amplexicaulis, and C. longipedicellata joined at the base of a branch containing clades A and B and one containing clades B and C. In addition to very weak support values, these three species are morphologically distinct from other species of these clades and are thus treated as isolates. DISCUSSION Intragenomic sequence variability Considerable intragenomic sequence variability existed in the ITS1 and ITS2 regions of Camellia in this study of nrits sequences and was mainly due to single-nucleotide substitutions in one or two clone sequences and indels largely located before tandem repeats of mono, di-, tri- or tetranucleotides. In most cases, the indels started with G, C, or A but not T. The high GC content, coupled with the occurrence of indels in spacer regions could explain why earlier attempts to sequence PCR products of the nrits region in Camellia, without cloning, were unsuccessful ( Thakor and Parks, 1997 ; Yang et al., 2006 ; S. H. Suh, Sun Yatsen Univ., Guangzhou, China; personnel communication). Slippage of DNA polymerase during DNA replication may result in the addition or deletion of short spans of sequences that repeat on one side of the region flanking the indel ( Levinson and Gutman, 1987 ). Many other mechanisms may cause varia- tions among the tandem repeats ( Kelchner and Clark, 1997 ; Benson and Dong, 1999 ; Zhu et al., 2000 ). If the rate of concerted evolution is not sufficient to homogenize the ITS sequences within individuals and species, accumulation of intragenomic variation may occur ( Appels and Honeycutt, 1986 ; Rogers and Bendich, 1987: Hillis and Dixon, 1991). Interspecies hybridization could also contribute to intragenomic variability in Camellia because interspecies hybridization events have been reported in some species ( Wickremasinghe, 1979; Banerjee, 1988 ; Tanaka et al., 2003 ). In hybrid species, divergent ribosomal DNA copies may coexist due to different evolutionary trajectories before they merge into a single genome through recombination and concerted evolution ( Wendel, 2000 ). Nonetheless, in our trees ( Fig. 1, Appendix S3 S7), for most species, the multiple conspecific sequences grouped together into compact and distinct clades, which clearly suggested that those intragenomic substitutional changes were inconsequential and were not shared by sequences of another species. The changes could result from shallow paralogy or PCR/sequencing errors because leaves of Camellia contain a large amount of PCR-interfering phenolic compounds (Singh et al., 1999; Vijayan and Tsou, 2008 ). Phylogeny based on morphology and DNA sequencing data Camellia is a large genus with more than 200 species. The current study included 112 species, which covered nearly 50% of the total species and more than 70% of the sections (15 of 21 sections in the Chang [1998] system or 11 of 14 in the Ming [2000] system); thus, the phylogenetic scheme generated in this nrits study is important in understanding the interspecies relationships within this genus. In the present analysis, the 112 species were resolved into eight major clades, which were largely strongly supported. The interspecies relationships exhibited by these eight clades and those revealed by an earlier analysis with RPB2 sequences ( Xiao and Parks, 2003 ) differed greatly from those revealed by traditional classifications ( Sealy, 1958 ; Chang, 1998 ; Ming, 2000 ). Nevertheless, a number of morphological criteria used for defining sections in traditional systems are still useful in defining the clades of this study. Morphology is the basis of taxonomy, and the molecular scheme requires support from morphological data. We found four floral characters and two fruit characters fairly compatible with our molecular cladograms and providing good support for many of our clades ( Table 2 ). These characters include the persistency of bracteoles and of sepals in young fruit, the level of fusion among the filaments and that among the styles, the sculpture of the pericarp, and the number of seeds in the fruit. The first two characters highly supported two important results we obtained. In the section Tuberculata, of the eight species sampled, five (Tub-1 group) were associated with golden camellias in clade A, and the remaining three species (Tub-2 group) are associated with the section Oleifera in clade F. These two groups of Tuberculata were not separable morphologically ( Chang, 1998 ; Ming, 2000 ); however, the line drawings of the Tuberculata species in the monograph by Ming (2000) show sepals persisting on young fruits in the Tub-1 group (congruent with golden camellias), whereas the sepals drop off after flowering in the Tub-2 group (congruent with the Oleifera group). In another instance, the 25 species from section Camellia were arranged in three clades (B, E, H), and species that grouped in clade E were clearly distinct from the remaining species of section Camellia by their persistent sepals. The characters of

8 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1355 filamental fusion and stylar fusion were highly congruent with our clades and deserve special mention. The level of filamental fusion and that of stylar fusion varied considerably among groups; nonetheless, if styles are completely or nearly free, filaments are usually free or fused only at the very base. On the contrary, with high filamental fusion (e.g., half the length), the stylar fusion is also high. An exception to this rule was found in clade D ( Thea clade), in which filamental fusion was restricted to the base, but stylar fusion was at a higher level. Useful fruit characters are few in number, but they are good taxonomic criteria. Among the eight major clades, four were homogeneous in morphology and were compatible with the architecture of existing sectional divisions. These clades were strongly supported in both MP and Bayesian analyses. Thus, they deserved to be accepted and treated as monophyletic sections or groups. Eriandra-Theopsis clade (clade C) All 16 species from sections Eriandra and Theopsis sampled were joined and intermixed in this clade. These two sections have long been considered closely related, and their taxonomic distinction was mainly based on the nature of the ovary, because in Eriandra the ovary is pubescent, whereas in Theopsis it is glabrous ( Sealy, 1958 ; Chang, 1998 ; Ming, 2000 ). Xiao and Parks (2003) showed this intermixing of species in these two sections, but in their results, these species formed two separate clades along with some species from the section Camellia. Because the nrits sequence data strongly suggested the homogenous nature of this clade, these two sections should be combined into a single monophyletic group. Species of the Eriandra-Theopsis clade have rather distinct morphological features: they have the smallest leaves and flowers in the genus and have short pedicels, persistent bracteoles and sepals until the fruiting stage, white petals, two whorls of stamens with the filaments of the outer whorl fused to one half to two thirds to form a long corona tube, a long stylar tube with three short free ends, and, more importantly, a small and one-seeded fruit, which is not found elsewhere in this genus (Table 2 ). Thea clade (clade D) The monophyly of the section Thea was widely accepted by Sealy (1958), Chang (1998), and Ming (2000) and was recently supported by DNA analysis with RPB2 intron sequences ( Xiao and Parks, 2003 ). Here, nrits data once again confirmed this finding: all the 12 sampled species from section Thea grouped under a single clade with strong support values. Further, the 12 species formed two major subclades, one mainly with 3-carpellate species and the other with 5-carpellate members, which agrees with the infrasectional treatment by Chang (1998) and Ming (2000). Morphologically, species in Thea are characterized by short and stout pedicels, early falling of bracteoles, persistent sepals, white petals, filaments fused at the base, a long stylar tube with short free ends, fruits with a central column and 3 5 loculed, and a single seed per locule ( Table 2 ). The basal filamental fusion and medium height of stylar fusion for species of this section form an exceptional combination of characters in the genus Camellia. Furfuracea clade (clade G) This clade included all six species sampled from the section Furfuracea, along with C. hongkongensis from the section Camellia. Inclusion of C. honkongensis in this clade is strongly supported by both the MP tree (JV = 100%) and Bayesian tree (PP = 90%). The species C. hongkongensis is an odd member in the section Camellia. Although both Chang (1998) and Ming (2000) placed C. hongkongensis in the section Camellia, they noted its free styles and furfuraceous pericarp, which distinguishes this species from other members of the section Camellia. Inclusion of C. hongkongensis with species of Furfuracea was also found in the RPB2 analysis ( Xiao and Parks, 2003 ). The taxonomic treatment of the section Furfuracea is a matter of great debate because Sealy (1958) placed a number of species that were difficult to classify in the section Heterogenea. However, Chang (1981) considered the section Heterogenea as an artificial group and dissolved it by dispersing the species into four different sections, including Furfuracea. Chang (1981) created the section Furfuracea for species with free styles and spongy and furfuraceous pericarps. But later, Ming (2000) restored the section Heterogenea and considered it a natural group of species with free styles. Our nrits data, however, supported the monophyly of the section Furfuracea by Chang (1998), which should also include C. hongkongensis. Morphologically, the Furfuracea clade is well distinguishable by features such as the absence of a distinct pedicel, sepals persistent up to young fruiting stage, filaments fused at the base, free styles, spongy and furfuraceous pericarps, and all locules in the fruit containing at least one seed. Camellia-3 clade (clade H) This clade is another small and homogeneous one formed by six species from the section Camellia. Geographically, these six species are restricted to southwestern China and are found largely in medium high to high mountains. Morphologically, species of this clade have no pedicels, bracteoles and sepals falling after flowering, petals white or red, filaments fused to halfway, a long style with short free ends, large fruits, and all three locules setting seeds. As mentioned previously, the 25 sampled species of section Camellia are divided into three clades: Camellia -1 group in clade B, Camellia-2 group in clade E, and Camellia -3 group in clade H. Because of the persistence of bracteoles and sepals up to the fruiting stage, species of the Camellia -2 are well separated from species in the other two groups. Although, morphologically, species of the Camellia -3 group in clade H cannot be well distinguished from species of the Camellia -1 group in clade B, species in Camellia-3 are largely restricted to higher elevated regions in Sichuan and Guizhou provinces in China, whereas species of the Camellia-1 group are widely distributed in southern and central China, from low- to high-elevation regions, but largely in lowlands. Other clades The remaining four clades (i.e., clade A: species mainly from Chrysantha ; Tuberculata-1, Piquetia and Stereocarpus ; clade B: species from Camellia -1 and Paracamellia-1; clade E: species from Camellia -2 and Paracamellia-2; and clade F: species from Oleifera and Tuberculata -2) are basically heterogeneous groups containing species from two larger sections with or without species from smaller sections. Although these clades have many characteristic morphological features, as shown in Table 2, when more species from these sections are sampled, more distinct patterns may emerge from these clades. Therefore, intense sampling and investigation are suggested for these clades. Isolates Four species were treated as isolates in this study. Camellia pachyandra and C. gilbertii, both from section Brachyandra, appeared as the basalmost species sister to all other species of the genus, but they are not closely linked. These two species no doubt belong to the genus Camellia because they possess all the sequence synapomorphies of the genus in the nrits region but none of those of Pyrenaria. Species of the

9 1356 AMERICAN JOURNAL OF BOTANY [Vol. 96 Fig. 2. (A B) Species tree of the genus Camellia. Analysis of nrits sequences of 112 species of Camellia showing (I) the most parsimonious tree with tree length of 584 steps, consistency index (excluding uninformative characters) of , and retention index of and (II) the Bayesian tree from Markov Chain Monte Carlo of 1.5 million generations; numbers on the branches represent jackknife values in the parsimonious tree and posterior probability values in the Bayesian tree.

10 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1357 Fig. 2. Continued

11 1358 AMERICAN JOURNAL OF BOTANY [Vol. 96 T ABLE 2. Salient morphological characters of the eight major clades generated from ITS1-5.8S-ITS2 sequences. Clade Persistency after flowering Fusion Bracteole Sepal Filamental Stylar No. locules/fruit; no. seeds/locule Pericarp sculpture Eriandra-Theopsis (clade C) until young fruit until young fruit 1/2 - > 2/3 long with 1; 1 smooth short free ends Thea (clade D) deciduous until young fruit at base long with 3 5; 1 smooth short free ends Furfuracea (clade G) deciduous deciduous at base styles free 3 5; 1 2 furfuraceous Camellia -3 (clade H) deciduous deciduous 1/2 long with 3; 1 2 smooth short free ends Chrysantha, Tuberculata-1 (clade A) until young fruit until young fruit at base styles free (1 )3( 5); 1 2 with knobs in Tuberculata Oleifera, Tuberculata-2 (clade F) Camellia-1 & Paracamellia-1 (clade B) Camellia-2, Paracamellia (clade E) deciduous deciduous free or at base styles free or fused at base deciduous or until young fruit deciduous or until young fruit deciduous or till young fruit deciduous or till young fruit group 3 5; 1 3 with knobs in Tuberculata group 1/5 1/2 short long 3; 1 3 smooth 1/2 or at base long with short free ends or short (2 )3( 5); 1 3 smooth section Brachyandra of Chang (1998) have small flowers, short filaments, and free styles and represent a morphologically distinct group. Indeed, the species are well separated from other sections of the genus in our cladograms. Twelve species exist in the section Brachyandra ; thus, investigation of more species from this section might help in understanding this archaic group of the genus. Regarding the other two isolates, C. amplexicaulis and C. longipedicellata, Chang (1998) placed both under the section Longipedicellata, another morphologically distinct group. These two species have pedicels not shorter than 2 cm, sepals persistent until fruiting stage, white petals, filaments fused at the base, three free styles, and fruits with seeds in three locules. This group also deserves more attention. Comparison between the two molecular taxonomic studies The two major molecular phylogenetic investigations of the genus Camellia (the present one with nrits sequences and the other with ndna RPB2 sequences by Xiao and Parks (2003) ) have provided considerable insight into the interspecies relationships of Camellia, which could not be provided by many previous attempts with use of cpdna sequences ( Thakor and Parks, 1997 ; Orel et al., 2003 ; Yang et al., 2006 ). Thus, we again underscore the importance of nuclear gene sequences in molecular phylogenetic study of tree species such as Camellia. Furthermore, these two molecular phylogenetic investigations share many important findings. Both studies revealed the need to revise the existing classifications, both supported the monophyly of sections Thea and Furfuracea and that the species C. hongkongensis should be shifted from section Camellia to section Furfuracea, and both revealed that sections Eriandra and Theopsis were closely related and not separable and that species of sections Tuberculata and Chrysantha, as well as C. szechuanensis from section Pseudocamellia, were closely related. Finally, results of both studies equally supported the section Camellia as polyphyletic. The species from the section Camellia formed groupings based on geographical origin and distribution, and species in this section distributed in southeastern and eastern China, Korea, and Japan are well separated from those in southern and southwestern China. Nevertheless, both studies disagreed on many points. The most notable disagreement was the monophyly of the section Paracamellia defined by Ming (2000) and Sealy (1958), which was supported by Xiao and Parks (2003), but our study showed a bifurcation of the section Paracamellia and supported Chang s (1981) creation of the section Oleifera from the section Paracamellia. A recent study of leaf anatomical characters also supported the separation of Oleifera from the section Paracamellia ( Lin et al., 2008 ). Other important differences are, first, species of the section Eriandra and Theopsis formed a monophyletic clade in our tree but mixed together with species from the section Camellia and divided into two well-separated clades in the study by Xiao and Parks (2003). Second, the positions of some species in small sections and isolates differed; for example, C. amplexicaulis of section Longipedicellata was isolated and was a sister to the clade of Eriandra and Theopsis in our trees but was associated with clades of species in sections Camellia, Oleifera, and Paracamellia in the Xiao and Parks (2003) trees. Also, C. yunnanensis of section Stereocarpus was embedded in the clade consisting of sections Chrysantha and Tuberculata in our trees but was allied to section Furfuracea in the Xiao and Parks (2003) trees. These types of conflicts in results are not uncommon in molecular phylogeny and can arise from both analytical and biological factors ( Rokas et al., 2003a ). Analytical factors that generally affect phylogenetic reconstruction are choice of optimality criterion ( Huelsenbeck, 1995 ), data availability (Cummings et al., 1995 ), taxon sampling ( Graybeal, 1998 ), and specific assumptions in the modeling of sequence evolution ( Yang et al., 1994 ). The major biological factor that affects phylogenetic reconstruction is the evolutionary dynamics that may cause the history of the genes under analysis to obscure the history of the taxa ( Rokas et al., 2003b ). Reexamination of the classifications of Chang (1998) and Ming (2000) The present phylogenetic study included samples representing 15 of the 21 sections of the classification by Chang (1998) and 11 of 14 of that by Ming (2000). For the Chang circumscriptions, our study supported the monophyletic nature of the sections Thea and Furfuracea (with C. hongkongensis included), a monophyletic section Theopsis merged with section Eriandra, and the creation of the section Oleifera but advocating expansion of it with a few species from sections Camellia and Paracamellia. The sections Brachyandra and Longipedicellata of Chang (1998) are distinct from other major

12 July 2009] VIJAYAN ET AL. MOLECULAR TAXONOMY OF CAMELLIA 1359 groups, although their sectional status cannot be settled because of lack of samples, their isolated and basal positions deserve further study. Regarding the remaining eight sections, we revealed the polyphyletic nature of large sections such as Camellia, Paracamellia, Pseudocamellia, and Tuberculata, which warrants a thorough revision. The section Chrysantha was found to be paraphyletic because all the golden camellias were together in a single clade, but species from sections Piquetia, Pseudocamellia, Stereocarpus, and Tuberculata were nested within this large clade. Species of three small sections (i.e., Archecamellia, Piquetia, and Stereocarpus ) did not appear as distinct groups; hence, their independent sectional status could not be supported. Regarding the classification by Ming (2000), the monophyly of section Thea and a combined group of Eriandra and Theopsis were supported by our results. However, other large sections such as Archecamellia, Camellia, Heterogenea, Paracamellia, and Tuberculata appeared to be polyphyletic or artificial. The small section Piquetia is merged within a big clade of golden camellias, whereas the other two, section Corallina represented by C. gilbertii and section Longipedicellata represented by C. longipedicellata, remained independent in our trees. In addition to sectional treatments of Camellia, we examined species circumscription because of the great discrepancy in the species treatments by Chang (1998) and Ming (2000). As mentioned previously, Chang recognized 280 species, whereas Ming recognized only 119 species (Appendix 1). We tried to reexamine the reductions made by Ming (2000) using information from nrits phylogeny and Kimura distances wherever required. We encountered and reexamined more than 30 cases of Ming s reduction from species to synonyms or to varieties in this study. In general, Ming (2000) carefully identified important characters, which was evident from his placement of C. chungkingensis in section Tuberculata, C. weiningensis in section Camellia, and C. petelotii with golden camellias as supported by our data. We also found that in a number of Ming s propositions for reduction, the species involved were indeed closely related; however, most of these reductions could not be supported by our study because, often, the species involved were not sister species, but the sister species as appeared in the cladograms were still recognized as independent species. Therefore, further investigation with more samples and more approaches from both molecular and morphological aspects are required to settle these conflicting issues of species circumscription. Conclusions The present molecular phylogenetic study of the interspecies relationships of the economically important genus Camellia with use of nrits sequencing strongly supported the need for revising the present classifications. The 112 species analyzed were grouped into eight major clades and four isolates. We found species belonging to different subgenera of the classification by Chang (1998) or Ming (2000) mixed in a single clade in our study, which thus does not support the subgeneric divisions by these two experts. Our nrits study confirmed the earlier RPB 2 study of Xiao and Parks (2003) in the monophyly of sections Thea and Furfuracea, the polyphyly of section Camellia and the inclusion of C. hongkongensis in the section Furfuracea. Our work further advances our understanding of Camellia phylogeny by providing new insights into the monophyly of a combined section of Theopsis and Erinadra ; the polyphyly of sections Paracamellia, Pseudocamellia, and Tuberculata ; the paraphyletic nature of section Chrysantha ; and the failure to show independent sections for three small sections (i.e., Archecamellia, Piquetia, and Sterocarpus ). The three distinct groups originating from the section Camellia can be differentiated on the basis of geographic distribution. Likewise, the two divergent groups in section Tuberculata were based on the persistence of bracteoles and sepals in young fruits. Detailed studies focusing on section Brachyandra are needed to shed light on the early evolution of the genus. Further study of sections Camellia, Paracamellia, and Tuberculata, however, are crucial for providing a better picture of the intrageneric lineage of the genus Camellia. LITERATURE CITED Á LVAREZ, I., AND J. F. 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