Botanical Studies (2009) 50: 425-433.

PHYLOGENETICS

Phylogeny of Calocedrus (Cupressaceae), an eastern Asian and western North American disjunct gymnosperm genus, inferred from nuclear ribosomal nrITS sequences

Chih-Hui CHEN^1, ⁴, Jen-Pan HUANG², Chi-Chu TSAI³, and Shu-Miaw CHAW² *

¹Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan

²Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan

³ Crops Improvement Division, Kaohsiung District Agricultural Research and Extension Station, Council of Agriculture, Pingtung, Taiwan

⁴Division of Botany, Endemic Species Research Institute, Council of Agriculture, Nantou, Taiwan (Received March 18, 2009; Accepted April 20, 2009)

ABSTRACT. To resolve relationships between Taiwanese and other Asian species of Calocedrus and to infer the causes of the contemporary distribution of the genus, we analyzed 21 individuals sampled from Vietnam, Yunnan (China), Taiwan, and California (USA). Phylogenetic and phylogeographic analyses based on the nrITS sequence data suggested a deep split between Asian and American Calocedrus. In addition, our estimated dates of species divergence are consistent with fossil records and geohistorical evidence. Because, in addition to the distinctive morphology between C. formosana and C. macrolepis, the sequence divergence between them exceeds the interspecific level of divergence between species of Taxus, C. formosana could be regarded as a newly emerged distinct species. Speciation of the three species of Calocedrus studied was evidently shaped by geohistorical vicariant events, mainly allopatric fragmentations.

Keywords: Calocedrus; Disjunct distribution; Fossil records; Geohistorical events; nrITS; Phylogeography.

INTRODUCTION

Species of Calocedrus Kurz. (Cupressaceae), also known as incense cedars, are characterized by their flat branchlets with strongly decussate and dimorphic leaves, which are composed of two pairs, a small and median facial pair and a larger, narrowly triangular lateral one (Figure 1). The pollen bearing cones of incense cedars are solitary; the ovule bearing ones are solitary (or sometimes in a pair in C. formosana) ovoid-oblong with three pairs of scales, of which the upper pair is united in a flat plate between the outer two pairs. The lowermost (or outermost) pair is short, reduced and reflexed, and only the middle pair is fertile. The seeds have two distinctly unequal wings (Krussmann, 1985; Page, 1990). Four species have been recognized in Calocedrus. They have a typical eastern Asia-western North American disjunct pattern of distribution (Guo, 1999; Xiang et al., 1998; Xiang et al., 2001) (Figure 2). The three eastern Asian taxa are C. macrolepis Kurz., C. formosana (Florin) Florin, and C. rupestris Aver., H.T. Nguyen & L.K. Phan. Calocedrus

*Corresponding authors：E-mail:smchaw@sinica.edu.tw; Phone: 886-2-27871155; Fax: 886-2-27898711.

macrolepis is indigenous to southwestern China (including Hainan Island), northern Vietnam, and Myanmar; C. formosana is endemic to Taiwan (Krussmann, 1985; Page, 1990; Li and Keng, 1994); and C. rupestris is endemic to northern Vietnam (Averyanov et al., 2008). The fourth taxon, C. decurrens (Torr.) Florin, is native to the Cascade Mountains of Oregon and the Sierra Nevada of California and extends into Baja California (Krussmann, 1985; Page,

1990).

Fossil records indicate that in the Tertiary Calocedrus not only occurred in eastern and southeastern Europe, but also in more northern regions than it does today. For example: Kvacek and Hably (1998) and Kvacek (1999) recognized a fossil collection of C. suleticensis from the early Oligocene of the Czech Republic and Hungary and the early Miocene of Greece and another fossil species, C. pliocenica, from the Pliocene (5.3-1.8 million years ago[MYA]) of Poland. Wolfe (1972) described a leafy branch of the genus from the Oligocene-Miocene boundary (23.8 MYA) of Alaska. Kvacek (1999) also found C. schornii from the Oligocene of Oregon and C. masonii from the Miocene of Idaho. Additionally, Liu and Zheng (1995), Kvacek and Hably (1998), and Kvacek (1999) reported C. lantenoisii from the Miocene of Yunnan

426

Botanical Studies, Vol. 50, 2009

Province (China) and C. notoensis from Japan. No fossils of the genus have been recorded from western Europe or eastern North America.

Of the four incense cedars, the taxonomic status of C. formosana has been exceptionally controversial. It was first attributed to Libocedrus macrolepis when it was reported from Taiwan by Hayata (1908). Because of its smaller and obtuse lateral leaves (see Figure 1 in this study), 3-anthered stamens, and larger cones (pollen bearing cones 4-6 mm long and ovule bearing cones 12-15 mm long) and seeds (5 mm long), C. formosana was considered different from plants on the mainland by Florin (1930), thus it was recognized as L. formosana. Kudo (1 931 ), however, claimed that the characters used by Florin were variable and not sufficiently significant to recognize the Taiwanese plants as a distinct species. He therefore treated it as L. macrolepis var. formosana (Florin) Kudo. At that time Calocedrus was included in Libocedrus, which is now limited to species native to New Zealand and New Caledonia. After the recognition of Calocedrus by Li (1953), Florin (1956)

made the combination, C. formosana (Florin) Florin, for the Taiwanese incense cedar. Interestingly, western taxonomists preferred to treat C. formosana as a distinct species (e.g. Florin, 1956; Krussmann, 1985; Kvacek, 1999) while eastern taxonomists (e.g. Li and Keng, 1994) followed Kudo. Hence, a new approach to resolving these conflicting views is needed.

Sequence data from the internal transcribed spacers of nuclear ribosomal DNA (nrITS) have been utilized successfully to elucidate phylogenetic relationships at the generic and specific levels of both gymnosperms (e.g. Liston et al., 1996; Cheng et al., 2000) and angiosperms (e.g. Shi et al., 1998; Wen and Shi, 1999). Here we present our comparative analyses of nrITS from 21 individuals of three taxa of Calocedrus, C. decurrens, C. formosana, and C. macrolepis. Relationships within the genus were inferred and discussed based on the sequence variation and reconstructed phylogenetic trees. Furthermore, phylogeographic inferences and fossil records were used to determine the historical scenarios that might account for the present disjunct distribution of Calocedrus.

Figure 1. ML phylogeny for Calocedrus (-lnL = 2379.00157; model: TrN + r). Branch support was calculated using MP bootstrapping (above branch before slash), ML bootstrapping (above branch after slash), Decay index (below branch before slash), and BPP (below branch after slash). Bayesian relative rate (BRR) test is shown next to individual codes; orange points indicate MLEs; and blue lines at both edges illustrate 95% BPPs. Estimated values overlap 95% BPPs, indicating a constant evolutionary rate. Left figures illustrate leaf branches for three species of Calocedrus (scale bar = 0.5 cm) and ovulate cones for C. formosana and C. macrolepis (scale bar = 1 cm); C. formosana and C. macrolepis (HAST accessions 65530 and 43827, respectively).

CHEN et al. ― NrITS phylogeny of Calocedrus

427

MATERIALS AND METHODS

and 43827 (C. macrolepis, from Yunnan, China).

Sampling

Nr ITS sequences from a total of 21 individuals of three species of Calocedrus were obtained (Table 1). All vouchers were deposited in the herbarium of Taiwan Endemic Species Research Institute (TAIE) except for the Chinese samples, which were gathered from cultivated individuals in the Atlanta Botanical Garden (The original collecting information for the Chinese species of Calocedrus is given in Table 1 ). GenBank nrITS sequences from Microbiota decussata, C. macrolepis and C. formosana were downloaded and included in our analysis. Based on the chloroplast rbcL gene (Brunsfeld et al., 1994) and combined molecular and morphological data (Gadek et al., 2000), Microbiotia decussata was selected as the outgroup because it was shown to be one of the closest sisters of Calocedrus and has a published nrITS sequence. Herbarium specimens examined are HAST

accessions 24005, 35634, 59941, 62772, 65530, 67179, 94644, 101265, 101266 (C. formosana, all from Taiwan),

DNA extraction and sequencing

Genomic DNA was extracted from fresh leaves or leaves dried in silica gel following the method of S hure et al. (1 983). We designed the PCR primers based on conserved regions of two published

GenBank accessions, U77962 and U77954 (both

Cupressus arizonica). The forward primer CUP1 (5' GGTATTCACGCCTGACTTGG3') is located at the 3' end of 18S rRNA gene, and the reverse one CUP2 (5'

ATAGGTGAACCTGCGGTAGG3') is at the beginning

sequence of the 26S rRNA gene. The PCR products were purified using GeneClean II (Bio 101, CA) and subcloned into a pGEM T-Easy vector (Promega, WI). Plasmid DNA was purified using a Qiaprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Sequencing was performed using an ABI377 automated sequencer with Bigdye^TM Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, CA). For each individual, at least two independent PCR clones were sequenced.

Table 1. Analyzed individuals and their corresponding accession numbers.


Species	Codes^a	Vouchers	Accessions	Sampling Localities
Microbiota decussata			AY380874	Little et al., 2004
C. decurrens	Cd1	Chen 3227	AY150679	Lloyd Meadows Basin, Tulare County, California, USA
	Cd2	Chen 3228	AY150680	Lloyd Meadows Basin, Tulare County, California, USA
	Cd3	Chen 3229	AY150681	Lloyd Meadows Basin, Tulare County, California, USA
	Cd4	Chen 3231	AY150682	Lloyd Meadows Basin, Tulare County, California, USA
	Cd5	Chen 3232	AY150683	Lloyd Meadows Basin, Tulare County, California, USA
	Cd6	Chen 3233	AY150684	Lloyd Meadows Basin, Tulare County, California, USA
	Cd7	Chen 3234	AY150685	Lloyd Meadows Basin, Tulare County, California, USA
	Cd8		AY380854	Little et al., 2004
C. macrolepis	CmVN^b1	Chen 4161	AY150686	Dalat, Vietnam
	CmVN^b2	Chen 4162	AY150687	Dalat, Vietnam
	CmCN^b1	ABG 98-0764	AF287249	Mekong Salween Divide, Yunnan, China; Cultivated in Atlanta Botanical Garden
	CmCN^b2	ABG 97-1416	AY150688	Kunming Inst. of Botany, Yunnan, China; Cultivated in Atlanta Botanical Garden
	CmCN^b3	ABG 97-1422	AY150689	An Fen Ying, Yunnan, China; Cultivated in Atlanta Botanical Garden
	CmCN^b4	ABG 97-1421	AY150690	An Fen Ying, Yunnan, China; Cultivated in Atlanta Botanical Garden
C.formosana	Cf1	Chen 3539	AY150691	Ching-Shui Village, Chung-Liao, Nantou, Taiwan
	Cf2	Chen 4163	AY150692	Chi-Chi Township, Nantou, Taiwan
	Cf3	Chen 4164	AY150693	Chi-Chi Township, Nantou, Taiwan
	Cf4	Chen 4159	AY150694	Fusing village, Taoyuan, Taiwan
	Cf5	Chen 4160	AY150695	Fusing village, Taoyuan, Taiwan
	Cf6	Chen 3540	AF287248	Ching-Shui Village, Chung-Liao, Nantou, Taiwan
	Cf7		AY380855	Little et al., 2004

428

Botanical Studies, Vol. 50, 2009

Sequence alignment and phylogenetic analyses

Sequences were aligned using the Clustal W implemented in the MegAlign program (DNASTAR, Inc.) with manual inspection. The best fit sequence evolution model was selected using both the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC)

by MODELTEST (version 3.7, Posada and Crandall,

1998). Phylogeny was reconstructed based on the selected molecular evolution model using the maximum likelihood (ML) method implemented in PAUP* (version 4.0b10, Swofford, 2001). An ML heuristic search was performed using a neighbor joining (NJ) starting tree and tree bisection and reconnection (TBR) swapping strategy.

Maximum parsimony (MP) branch support was done using a non-parametric bootstrap (Felsenstein, 1985) with ten random sequence additions, TBR branch swapping, and 1000 replicates using PAUP*. ML branch support was carried out using 1000 replicates with the first iteration using an NJ starting tree (TBR branch swapping). The decay index (DI, Bremer, 1 988) was first calculated by TreeRot (version 3, Sorenson and Franzosa, 2007) using the obtained MP topologies to generate a PAUP* command file and then implemented in PAUP*. The Bayesian Posterior Probability (BPP) for branches was calculated using MrBayes (version 3.1.2, Huelsenbeck and Ronquist, 2001). The sequences obtained were partitioned into three regions, ITS1, 5.8S, and ITS2, using "charset," and each region was given the most complicated model (nst=6, rate=invgamma) using "applyto." Two independent runs were performed simultaneously, and each run contained four Markov chains. The Markov Chain Monte Carlo (MCMC) searches were run for 1x10⁶ generations, with chains being sampled every 100 generations. We calculated the Bayes factor between the two runs to investigate whether these two runs converged at the end of the analysis. The initial 1,000 trees were discarded as burn-in. The post-burn-in trees were imported into PAUP* to compute the 50% majority rule trees.

Rate constancy and estimation of divergence time

A Bayesian relative rate (BRR) test was conducted using Cadence (version 1.08beta, Wilcox et al., 2004). Microbiota decussata was selected as the outgroup for calculating the branch lengths from all ingroup individuals to the most recent common ancestor (MRCA). Significantly different evolutionary rates were identified if the 95% posterior distributions of calculated branch lengths did not overlap with each other.

Estimation of divergence times of nodes using the Bayesian network was carried out using PAML (version 4, Yang, 2007) and MULTIDIVTIME (version 9/25/03, Thorne and Kishino, 2002) with the F84 + r model (kept 1 x10⁵ samples, samples taken every 100 cycles, and the first 1 x 10⁶ cycles discarded as burnin). The root of Calocedrus was constrained between 33.7 and 23.8 MYA because fossil records of the genus first emerged

and became abundant during the Oligocene. One ML ultrametric tree which assumed a molecular clock was obtained by "multidivtime" and visualized using MEGA (version 4, Tamura et al., 2007).

Phylogeographic analysis

Nested clade analysis (NCA, Templeton et al., 1995; Templeton, 2008) was carried out first using TCS (version 1.21, Clement et al., 2000) to construct the most parsimonious network for those obtained sequences with a 95% confidence interval. This network was then nested using the rules of Templeton et al. (1992). GeoDis (version 2.5, Posada et al., 2000) was used to calculate the clade distance (D_c) and nested clade distance (D_n) for those designed nested clades. Once the D_c and/or D_n was/were detected as significant, the inference key (Templeton, 2004) was applied to discriminate effects of different types of historical events―restricted gene flows, past fragmentation, and range expansion―that may have resulted in correlation of sequence divergence and spatial and/or temporal factors.

RESULTS

Sequence variation

The amplified nrITS sequences comprised the ITS1, 5.8S rRNA, and ITS2 regions. Thirteen unique haplotypes were found within the 21 individuals of Calocedrus. The 19 newly-determined Calocedrus sequences were deposited in GenBank (Table 1 ). The lengths of the nrITS sequences were 1,067 bp in C. decurrens, 1,088 bp in the Asian mainland C. macrolepis, and 1,087 bp in the Taiwanese C. formosana before alignment. Gene boundaries were identified by comparison with other GenBank available sequences, e.g. Taiwania and Taxus (Cheng et al., 2000). The ITS1 regions have the highest length variations, which are due to a 34 bp deletion in the accessions from western North America, an 11 bp deletion in those from Asia, and one further deletion in those of C. formosana. The 5.8S regions are 145 bp long. The ITS2 regions are 219 bp long in C. decurrens and 218 bp in both C. macrolepis and C. formosana.

Phylogenetic analyses

TrN + r (-lnL = 2381.512) was selected as the best fit evolutionary model for the nrITS regions using

both AIC (4775.0239) and BIC (4805.1665). The ML

phylogeny (Figure 1) indicated that Asian and American Calocedrus were each monophyletic and that the sampled C. macrolepis was paraphyletic if we treated the C. formosana as a distinct species. The tree topologies also strongly suggest that the Taiwanese accessions comprise a monophyletic lineage with robust support (95/89/2/100) and is sister to the accessions of the Chinese C. macrolepis. Individuals of C. macrolepis from Vietnam occupied the basal position on the Asian Calocedrus lineage. Twice the deviation in the harmonic means was 1.8 between the

CHEN et al. ― NrITS phylogeny of Calocedrus

429

Figure 2. Bayesian estimations of divergence times for three species of Calocedrus using known fossil records for calibration. Numbers before nodes denote estimated divergence times; gray bars represent 95% CIs. Diagram below time scale denotes corresponding geological period; shaded boxes and scaled horizontal lines represent locations and geological time for fossils of Calocedrus. Circles show current distribution of Calocedrus.

two Bayesian runs, indicating that the difference between two runs is not statistically significant (Kass and Raftery,

1995).

Rate constancy and the estimation of divergence time

A BRR test (Figure 1) showed an overlapping 95% BPP for the analyzed data, which implies constant evolutionary rates within the nrITS regions of the samples of Calocedrus that were examined. Our estimated time of divergence between Asian and American Calocedrus

is 25.2 (28.9-23.8) MYA. The divergence time is 4.3

(11.8-0.6) MYA for the American individuals, 15.2

(24.4-4.6) MYA for Asian Calocedrus, 9.1 (19.4-1.7) MYA

for the Chinese C. macrolepis, and 5.4 (14.6-0.5) MYA for C. formosana (Figure 2).

Phylogeographic analysis

A most parsimonious network was generated from the analyzed data (Figure 3). The Asian specie of Calocedrus are connected to one another using 95% CI, as are the American haplotypes. Nevertheless, the connection

between Asian and American lineages should be extended to 81 steps. D_c and/or D_n were detected to be statistically significant within most of the clade and nested clade levels. The geohistorical events inferred for the Asian Calocedrus clade and the total cladogram were allopatric fragmentations (Figure 3).

DISCUSSION

Two specific deletions in the aligned nrITS sequences of the Calocedrus accessions we examined could easily distinguish the Asian and North American taxa and also the two Asian ones. The estimated nrITS pairwise sequence divergence using Kimura's 2-parameter (K2P) distance (Kimura, 1980) among the species of Taxus was 0.18% to 1.87% (Li et al., 2001); the pairwise K2P distance between C. macrolepis and C. formosana, however, was 0.67-1.31%. Considering the similar evolutionary rates of the nrITS region between Taxus [ca. 5x10^-10 base/site/ year in Taxus (Li et al., 2001)] and Calocedrus (4.5x 10^-10-5.1x10^-10), the sequence diver gence between C. macrolepis and C. formosana exceeds the interspecific level of the species of Taxus. Hence, the recognition of

430

Botanical Studies, Vol. 50, 2009

Figure 3. The most parsimonious network of 13 haplotypes (21 individuals) estimated using TCS and the nested clade design. Individuals with same haplotype are combined in an oval box. Geographic coordinates obtained using GoogleEarth are shown next to clades. Dc and Dn and statistical significance are shown within clades (*^L: significantly large; *^S: significantly small). Allopatric fragmentations are inferred to be major geohistorical events that shaped current distribution of the Calocedrus.

C. formosana at specific rank, as proposed by Florin, based on differences in leaf size and shape and cone size appears justified. It is worthy to note that C. formosana is also easily distinguished from C. macrolepis by its thicker leaves (see also Krussmann, 1985) and cones with apparently shorter pedicels (Figure 1).

The comparatively long branches revealed by ML phylogeny support the monophylies and distinctiveness of the American and Asian clades (Figure 1), which clearly indicate that the Asian and American lineages have fully diverged. Some plant species are thought to be undifferentiated across continents during an isolation time of about 8 MY (Mayr, 2001). The phylogeographic study of Tsuga (Havill et al. , 2008) and our present study, however, suggest that the two continental clades have genetically as well as morphologically diverged after a long period of isolation. Since C. formosana was resolved to be monophyletic with strong support, it should be treated as distinct from C. macrolepis of the Asian mainland (Figure 1).

Despite the findings in a number of molecular studies that some disjunct congeneric species of seed plants between Taiwan, China, and the Ryukyu Islands are paraphyletic (Huang et al., 2001; Lu et al., 2001; Chiang

and Schaal, 2006; Chiang et al., 2006), our nrITS data

found Calocedrus on Taiwan to be monophyletic. The divergence pattern of in Calocedrus is similar to that in Amentotaxus argotaenia, as revealed by ISSR (Ge et al., 2005). Multiple introduction to Taiwan have been hypothesized for many plant species (Huang et al., 2001;

Lu et al., 2001; Chung et al., 2004; Chiang et al., 2006), and a sorting event could have resulted in discordant phylogenies if organelle and nuclear markers were used (Chiang et al., 2004). Given the relatively short geological history of Taiwan (Liu et al., 2000; Sibuet and Hsu, 2004; Huang et al., 2006), a nuclear marker other than nrITS used here, might better reflect the speciation processes of plants in Taiwan.

The time of divergence of the Asian and the Chinese species of Calocedrus is estimated to be during the Miocene, the period from which fossils are known in Yunnan (Figure 2). Previously, no fossils of Calocedrus have been reported in Taiwan, but the divergence time of the C. formosana is estimated to be around 5 MYA (Figure

2) , approximately just after the island of Taiwan emerged above sea level (Sibuet and Hsu, 2004). Fossils provide direct evidence of the existence of species at a given place in the past (Norell and Novacek, 1992; Ren, 1999; Wellman et al., 2003; Xiao et al., 2005), and geohistorical events are limited to species that have existed for a certain period of time in a particular place (Schindewolf et al., 1993; Mueller-Dombois and Fosberg, 1997). A reasonable estimate of divergence time reinforces the robustness of our Calocedrus phylogeny.

The contemporary genetic structure of Calocedrus was obviously shaped by allopatric fragmentation (Figure

3) . Recent studies have demonstrated the importance of selection over pure isolation on speciation (Wu, 2001; Osada and Wu, 2005; Stadler et al., 2008). Nevertheless, species of Calocedrus are forest trees with life spans

CHEN et al. ― NrITS phylogeny of Calocedrus

431

in excess of 20 years. Their highly diverged sequence structure, distinctive morphological characteristics, and discontinuous and localized distributions provide support for allopatric speciation (Hoskin et al., 2005; Nakazato et al., 2007). A more or less contiguous forest distribution throughout the northern Hemisphere was proposed during the Tertiary period (65-2 MYA) via the Bering land bridge (Tiffney, 1985a; 1985b). With the gradual fragmentation of habitats as the earth's climate fluctuated, plants and animals became isolated and diverged into distinct species

(Xiang et al., 1998; Xiang and Soltis, 2001; Von Dohlen

et al., 2002; Havill et al., 2008; Sanmartin et al., 2008). Speciation within formerly connected and widespread species of Calocedrus through fragmentations of the range of the genus could have been the result of global climate changes during that period.

CONCLUSION

The present study, with support from fossil and geohistorical evidence, clearly demonstrates the genetic uniqueness of the three species of Calocedrus. The North American and the Asian species of Calocedrus are each monophyletic. Geohistorical events have obviously impacted the diversification of these species. Calocedrus formosana was derived from an Asian ancestor and appears to be newly emerged.

Acknowledgements. Sincere thanks are due to R. Determann of the Atlanta Botanical Garden and J. Shevock of National Park Service, Department of the Interior, USA for their help in providing plant materials. This study was

supported by a grant, 90AS-1.3.2-EI-eW2(9), from the

Council of Agriculture, Executive Yuan, Taiwan, to CHC and in part by an Academia Sinica grant to SMC. We also thank M. Ito, H. Hasegawa, two anonymous reviewers, and Dr. David Boufford for constructive comments on an earlier draft of the manuscript.

LITERATURE CITED

Averyanov, L.V., T.H. Nguyen, K.L. Phan, and V.T. Pham. 2008. The genus Calocedrus (Cupressaceae) in the Flora of Vietnam. Taiwania 53: 11-12.

Bremer, K. 1988. The limits of amino acid sequence data in angiosperm phylogenetic reconstruction. Evolution 42:

795-803.

Brunsfeld, S.J., P.S. Soltis, D.E. Soltis, P.A. Gadek, C.J. Quinn, D.D. Strenge, and T. A. Ranker. 1 994. Phylogenetic relationships among the genera of Taxodiaceae and Cupressaceae: evidence from rbcL sequences. Syst. Bot. 19: 253-262.

Cheng, Y.C., R.G. Nicolson, K. Tripp, and S.M. Chaw. 2000.

Phylogeny of Taxaceae and Cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA

nrITS region. Mol. Phylogenet. Evol. 14: 353-365.

Chiang, T.Y., K.H. Hung, T.W. Hsu, and W.L. Wu. 2004. Lineage sorting and phylogeography in Lithocarpus formosanus and L. dodonaeifolius (Fagaceae) from Taiwan. Ann. Missouri

Bot. Gard. 91: 207-222.

Chiang, T.Y. and B.A. Schaal. 2006. Phylogeography of plants in Taiwan and the Ryukyu Archipelago. Taxon 55: 31-41.

Chiang, Y.C., K.H. Hung, B.A. Schaal, X.J. Ge, T.W. Hsu, and

T.Y. Chiang. 2006. Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchuensis

complex. Mol. Ecol. 15: 765-779. Chung, J.D., T.P. Lin, Y.C. Tan, M.Y. Lin, and S.Y. Hwang.

2004. Genetic diversity and biogeography of Cunninghamia konishii (Cupressaceae), an island species in Taiwan: a comparison with Cunninghamia lanceolata, a mainland species in China. Mol. Phylogenet. Evol. 33: 791-801.

Clement, M., D. Posada, and K.A. Crandall. 2000. TCS: a

computer program to estimate gene genealogies. Mol. Ecol.

9: 1657-1659.

Florin, J. 1930. Die Koniferengattung Libocedrus Endl. Svensk Botanisk Tidskrift 24: 126. f. 2, pl. 2.

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

Florin, R. 1956. Nomenclatural notes on genera of living gymnosperms. Taxon 5: 186-192.

Gadek, P.A., D丄.Alpers, M.M. Heslewood, and C.J. Quinn. 2000. Relationships within Cupressaceae sensu lato: a combined morphological and molecular approach. Am. J. Bot. 87: 1044-1057.

Ge, X.J., X.L. Zhou, Z.C. Li, T.W. Hsu, B.A. Schaal, and

T.Y. Chiang. 2005 Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting. J. Plant

Res. 118: 415-422.

Gou, Q. 1999. Ecological comparisons between Eastern Asia and North America: historical and geographical perspectives. J. Biogeogr. 26: 199-206.

Havill, N.P., C.S. Campbell, T.F. Vining, B. LePage, R.J. Bayer,

and M.J. Donoghue. 2008. Phylogeny and biogeography of Tsuga (Pinaceae) inferred from nuclear ribosomal ITS and

chloroplast DNA sequence data. Syst. Bot. 33: 478-489.

Hayata, B. 1908. Flora montana Formosae. An enumeration of the plants found on Mt. Morrison, the Central Chain, and other mountainous regions of Formosa at altitudes of

3,000-13,000 feet. J. College Sci., Imperial University,

Tokyo, Japan 25: 1-260.

Hoskin, C.J., M. Higgie, K.R. McDonald, and C. Moritz. 2005.

Reinforcement drives rapid allopatric speciation. Nature

437: 1353-1356.

Huang, C.Y., P.B. Yuan, and S.J. Tsao. 2006. Temporal and spatial records of active arc-continent collision in Taiwan: a

synthesis. GSA Bulletin 118: 274-288. Huang, S., Y.C. Chiang, B.A. Schaal, C.H. Chou, and T.Y.

Chiang. 2001. Organelle DNAs phylogeography of Cycas taitungensis, a relict species in Taiwan. Mol. Ecol. 10:

432

Botanical Studies, Vol. 50, 2009

2669-2681.

Huelsenbeck, J.P. and F. Ronquist. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:

754-755.

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.

Krussmann, G. 1985. Manual of Cultivated Conifer, Timber Press, Hong Kong, pp. 60-61.

Kvacek, Z. and L. Hably. 1998. New plant elements in the Tard Clay Formation from Eger-Kiseged. Acta Palaeobotanica

38: 5-23.

Kvacek, Z. 1999. An ancient Calocedrus (Cupressaceae) from the European Tertiary. Flora: Morphologie, Geobotanik.

Oekophysiologie 194: 237-248. Li, J.H., C.C. Davis, P. del Tredici, and M.J. Donoghue. 2001.

Phylogeny and biogeography of Taxus (Taxaceae) inferred from sequences of the internal transcribed spacer region of nuclear ribosomal DNA. Harvard Pap. Bot. 6: 267-274.

Li, H. L. 1 953. A reclassification of Libocedrus and Cupressaceae. J. Arn. Arb. 34: 17-36.

Li, H. L. and H. Keng. 1 994. Flora of Taiwan, Volume 1 , Editorial Committee of the Flora of Taiwan, Taipei, Taiwan,

pp. 586-595.

Liston, A., W.A. Robinson, J.M. Oliphant, and E.R. Alvarez-Buylla. 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering

seed plants. Syst. Bot. 21:109-120.

Liu, Y.S. and Y.H. Zheng. 1995. The flora of late Tertiary. In X.X. Li (ed.), Fossil Flora of China Through the Geological Ages. Guangdong Science and Technology Press, Guangzhou,

China, pp. 383-416. Liu, T.K., Y.G. Chen, W.S. Chen, and S.H. Jiang. 2000.

Rates of cooling and denudation of the early Penglai Orogeny, Taiwan, as assessed by fission-track constraints. Tectonophysics 320: 69-82.

Lu, S.Y., C.I. Peng, Y.P. Cheng, K.H. Hong, and T.Y. Chiang.

2001. Chloroplast DNA phylogeography of Cunninghamia konishii (Taxodiaceae), an endemic conifer of Taiwan.

Genome 46: 797-807.

Mayr, E. 2001. Wu's genic view of speciation. J. Evol. Biol. 14:

866-867.

Mueller-Dombois, D. and F.R. Rosberg. 1997. Vegetation of the Tropical Pacific Islands, Springer Verlag, Berlin, Germany,

pp. 4-17.

Nakazato, T., M.K. Jung, E.A. Housworth, L.H. Rieseberg, and G.J. Gastony. 2007. A genomewide study of reproductive barriers between allopatric populations of a homosporous fern, Ceratopteris richardii. Genetics 177: 1141-1150.

Norell, M.A. and M.J. Novacek. 1992. The fossil record and evolution: comparing cladistic and paleontologic evidence for vertebrate history. Science 255: 1690-1693.

Osada, N. and C.I. Wu. 2005. Inferring the mode of speciation from genomic data: a study of the great apes. Genetics 169:

259-264.

Page, C.N. 1990. Cupressaceae, In K.U. Kramer and P.S. Green (eds.), The Families and Genera of Vascular Plants. Volume 1, Pteridophytes and Gymnosperms. Springer Verlag, Berlin, Germany, pp. 302-316.

Posada, D. and K.A. Crandall. 1998. MODELTEST: testing the

model of DNA substitution. Bioinformatics 14: 817-818.

Posada, D., K.A. Crandall, and A.R. Templeton. 2000. GeoDis: a

program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. Mol. Ecol. 9: 487-488.

Ren, D. 1999. Flower-associated Brachycera flies as fossil evidence for Jurassic angiosperm origins. Science 280:

85-88.

Sanmartin, I., H. Enghoff, and F. Ronquist. 2008. Patterns of animal dispersal, vicariance and diversification in the

Holarctic. Biol. J. Linn. Soc. 73: 345-390.

Schindewolf, O.H., W.E. Reif, J. Schaefer, and S.J. Gould. 1993. Basic Questions in Paleontology: Geologic Time, Organic Evolution, and Biological Systematics, University of Chicago Press, Illinois, USA, pp. 4.

Shure, M,, S. Wessler, and N. Fedoroff. 1983. Molecular identification and isolation of the waxy locus in maize. Cell

35: 225-233.

Shi, S.H., H.T. Chang, Y.Q. Chen, L.H. Qu, and J. Wen. 1998. Phylogeny of the Hamamelidaceae based on the nrITS sequences of nuclear ribosomal DNA. Biochem. Syst. Ecol.

26: 55-69.

Sibuet, J.C. and S.K. Hsu. 2004. How was Taiwan created?

Tectonophysics 379: 159-181.

Sorenson, M.D. and E.A. Franzosa. 2007. TreeRot, version 3. Boston University, Boston, Massachusetts, USA.

Stadler, T., U. Arunyawat, and W. Stephan. 2008. Population genetics of speciation in two closely related wild tomatoes (Solanum section Lycopersicon). Genetics 178: 339-350.

Swofford, D.L. 2001. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts, USA.

Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4:

molecular evolutionary genetics analysis (MEGA) software

version 4.0. Mol. Biol. Evol. 24: 1596-1599. Templeton, A.R., K.A. Crandall, and C.F. Sing. 1992. A cladistic

analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132: 619-633.

Templeton, A.R., E. Routman, and C.A. Phillips. 1995.

Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140: 767-782.

Templeton, A.R. 2004. Statistical phylogeography: methods of evaluating and minimizing inference errors. Mol. Ecol. 13:

789-809.

Templeton, A.R. 2008. Nested clade analysis: an extensively validated method for strong phylogeographic inference.

CHEN et al. ― NrITS phylogeny of Calocedrus

433

Mol. Ecol. 17: 1877-1880.

Tiffney, B.H. 1985a. Perspectives on the origin of the floristic similarity between eastern Asia and eastern North America.

J. Arn. Arb. 66: 73-94.

Tiffney, B.H. 1985b. The Eocene North Atlantic land bridge: Its importance in Tertiary and modern phytogeography of the Northern Hemisphere. J. Arn. Arb. 66: 243-273.

Thorne, J. L. and H. Kishino. 2002. Divergence time and evolutionary rate estimation with multilocus data. Syst.

Biol. 51: 689-702. Von Dohlen, C.D., U. Kurosu, and S. Aoki. 2002. Phylogenetics

and evolution of the eastern Asian-eastern North American disjunct aphid tribe, Hormaphidini (Hemiptera: Aphididae).

Mol. Phylogenet. Evol. 23: 257-267. Wellman, C.H., P.L. Osterloff, and U. Mohiuddin. 2003.

Fragments of the earliest land plants. Nature 425: 282-285.

Wen, J. and S.H. Shi. 1999. A phylogenetic and biogeographic study of Hamamelis (Hamamelidaceae), an eastern Asian and eastern North American disjunct genus. Biochem. Syst.

Ecol. 27: 55-66.

Wilcox, T.P., F.J. Garcia de Leon, D.A. Hendrickson, and D.M. Hillis. 2004. Convergence among cave catfishes: long-

branch attraction and a Bayesian relative rates test. Mol.

Phylogenet. Evol. 31: 1101-1113.

Wolfe, J.A. 1972. An interpretation of Alaskan Tertiary floras, In A. Graham (ed.), Floristics and paleofloristics of Asia and eastern North America. Elsevier, Amsterdam, The

Netherland, pp. 201-233.

Wu, C.I. 2001. The genic view of the process of speciation. J.

Evol. Biol. 14: 851-865.

Xiang, Q.Y. and D.E. Soltis. 2001. Dispersal-vicariant analyses of intercontinental disjunctions: Historical biogeographical implications for angiosperms in the Northern Hemisphere.

Int. J. Plant Sci. 162: S29-S39. Xiang, Q.Y., D.E. Soltis, and P.S. Soltis. 1998. The eastern

Asian and eastern and western North American floristic disjunction: Congruent phylogenetic patterns in seven diverse genera. Mol. Phylogenet. Evol. 10: 178-190.

Xiao, S., B. Shen, C. Zhou, G. Xie, and X. Yuan. 2005. A uniquely preserved Ediacaran fossil with direct evidence for a quilted bodyplan. Proc. Natl. Acad. Sci. USA 102: 10227-10232.

Yang, Z. 2007. PAML 4: Phylogenetic analysis by maximum

likelihood. Mol. Biol. Evol. 24: 1586-1591.

根據核醣體基因間片段重建分佈於亞洲東部及北美西部肖楠屬

的親緣關係

陳志輝^1,4 黃仁磐² 蔡奇助³ 趙淑妙²

¹國立成功大學生命科學系

²中央研究院生物多樣性中心

³行政院農業委員會高雄區農業改良場作物改良課

⁴行政院農業委員會特有生物研究保育中心植物組

我們分析21個來自越南、雲南、台灣及加州的肖楠個體之核醣體基因間片段，探討分布在台灣及亞洲大陸個體之間的關係，並推論導致本屬植物目前分佈型式的原因。我們的分析結果都顯示產於亞洲與美洲的肖楠彼此之間分化相當明顯。除此之外估算出來的物種分化時間皆與化石出現時間或地理事件發生時間一致。台灣肖楠與亞洲大陸產之翠柏之間除了外觀上明顯的形態差異之外，兩者間分子層面的差異亦超出另一個屬（紅豆杉屬）所估算出的物種間差異，故台灣肖楠應被視為一個新形成的獨立物種。而歷史上的不連續分佈的地理事件為造成本研究所選取的三個物種異域種化的機制。

關鍵詞：肖楠；不連續分佈；化石紀錄；nrITS ；親緣地理；地理事件。



	432	Botanical Studies, Vol. 50, 2009

	2669-2681. Huelsenbeck, J.P. and F. Ronquist. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120. Krussmann, G. 1985. Manual of Cultivated Conifer, Timber Press, Hong Kong, pp. 60-61. Kvacek, Z. and L. Hably. 1998. New plant elements in the Tard Clay Formation from Eger-Kiseged. Acta Palaeobotanica 38: 5-23. Kvacek, Z. 1999. An ancient Calocedrus (Cupressaceae) from the European Tertiary. Flora: Morphologie, Geobotanik. Oekophysiologie 194: 237-248. Li, J.H., C.C. Davis, P. del Tredici, and M.J. Donoghue. 2001. Phylogeny and biogeography of Taxus (Taxaceae) inferred from sequences of the internal transcribed spacer region of nuclear ribosomal DNA. Harvard Pap. Bot. 6: 267-274. Li, H. L. 1 953. A reclassification of Libocedrus and Cupressaceae. J. Arn. Arb. 34: 17-36. Li, H. L. and H. Keng. 1 994. Flora of Taiwan, Volume 1 , Editorial Committee of the Flora of Taiwan, Taipei, Taiwan, pp. 586-595. Liston, A., W.A. Robinson, J.M. Oliphant, and E.R. Alvarez-Buylla. 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering seed plants. Syst. Bot. 21:109-120. Liu, Y.S. and Y.H. Zheng. 1995. The flora of late Tertiary. In X.X. Li (ed.), Fossil Flora of China Through the Geological Ages. Guangdong Science and Technology Press, Guangzhou, China, pp. 383-416. Liu, T.K., Y.G. Chen, W.S. Chen, and S.H. Jiang. 2000. Rates of cooling and denudation of the early Penglai Orogeny, Taiwan, as assessed by fission-track constraints. Tectonophysics 320: 69-82. Lu, S.Y., C.I. Peng, Y.P. Cheng, K.H. Hong, and T.Y. Chiang. 2001. Chloroplast DNA phylogeography of Cunninghamia konishii (Taxodiaceae), an endemic conifer of Taiwan. Genome 46: 797-807. Mayr, E. 2001. Wu's genic view of speciation. J. Evol. Biol. 14: 866-867. Mueller-Dombois, D. and F.R. Rosberg. 1997. Vegetation of the Tropical Pacific Islands, Springer Verlag, Berlin, Germany, pp. 4-17. Nakazato, T., M.K. Jung, E.A. Housworth, L.H. Rieseberg, and G.J. Gastony. 2007. A genomewide study of reproductive barriers between allopatric populations of a homosporous fern, Ceratopteris richardii. Genetics 177: 1141-1150. Norell, M.A. and M.J. Novacek. 1992. The fossil record and evolution: comparing cladistic and paleontologic evidence for vertebrate history. Science 255: 1690-1693. Osada, N. and C.I. Wu. 2005. Inferring the mode of speciation from genomic data: a study of the great apes. Genetics 169:	259-264. Page, C.N. 1990. Cupressaceae, In K.U. Kramer and P.S. Green (eds.), The Families and Genera of Vascular Plants. Volume 1, Pteridophytes and Gymnosperms. Springer Verlag, Berlin, Germany, pp. 302-316. Posada, D. and K.A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817-818. Posada, D., K.A. Crandall, and A.R. Templeton. 2000. GeoDis: a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. Mol. Ecol. 9: 487-488. Ren, D. 1999. Flower-associated Brachycera flies as fossil evidence for Jurassic angiosperm origins. Science 280: 85-88. Sanmartin, I., H. Enghoff, and F. Ronquist. 2008. Patterns of animal dispersal, vicariance and diversification in the Holarctic. Biol. J. Linn. Soc. 73: 345-390. Schindewolf, O.H., W.E. Reif, J. Schaefer, and S.J. Gould. 1993. Basic Questions in Paleontology: Geologic Time, Organic Evolution, and Biological Systematics, University of Chicago Press, Illinois, USA, pp. 4. Shure, M,, S. Wessler, and N. Fedoroff. 1983. Molecular identification and isolation of the waxy locus in maize. Cell 35: 225-233. Shi, S.H., H.T. Chang, Y.Q. Chen, L.H. Qu, and J. Wen. 1998. Phylogeny of the Hamamelidaceae based on the nrITS sequences of nuclear ribosomal DNA. Biochem. Syst. Ecol. 26: 55-69. Sibuet, J.C. and S.K. Hsu. 2004. How was Taiwan created? Tectonophysics 379: 159-181. Sorenson, M.D. and E.A. Franzosa. 2007. TreeRot, version 3. Boston University, Boston, Massachusetts, USA. Stadler, T., U. Arunyawat, and W. Stephan. 2008. Population genetics of speciation in two closely related wild tomatoes (Solanum section Lycopersicon). Genetics 178: 339-350. Swofford, D.L. 2001. PAUP: Phylogenetic Analysis Using Parsimony (and Other Methods), Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts, USA. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. Templeton, A.R., K.A. Crandall, and C.F. Sing. 1992. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132: 619-633. Templeton, A.R., E. Routman, and C.A. Phillips. 1995. Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140: 767-782. Templeton, A.R. 2004. Statistical phylogeography: methods of evaluating and minimizing inference errors. Mol. Ecol. 13: 789-809. Templeton, A.R. 2008. Nested clade analysis: an extensively validated method for strong phylogeographic inference.



	CHEN et al. ― NrITS phylogeny of Calocedrus	433

	Mol. Ecol. 17: 1877-1880. Tiffney, B.H. 1985a. Perspectives on the origin of the floristic similarity between eastern Asia and eastern North America. J. Arn. Arb. 66: 73-94. Tiffney, B.H. 1985b. The Eocene North Atlantic land bridge: Its importance in Tertiary and modern phytogeography of the Northern Hemisphere. J. Arn. Arb. 66: 243-273. Thorne, J. L. and H. Kishino. 2002. Divergence time and evolutionary rate estimation with multilocus data. Syst. Biol. 51: 689-702. Von Dohlen, C.D., U. Kurosu, and S. Aoki. 2002. Phylogenetics and evolution of the eastern Asian-eastern North American disjunct aphid tribe, Hormaphidini (Hemiptera: Aphididae). Mol. Phylogenet. Evol. 23: 257-267. Wellman, C.H., P.L. Osterloff, and U. Mohiuddin. 2003. Fragments of the earliest land plants. Nature 425: 282-285. Wen, J. and S.H. Shi. 1999. A phylogenetic and biogeographic study of Hamamelis (Hamamelidaceae), an eastern Asian and eastern North American disjunct genus. Biochem. Syst. Ecol. 27: 55-66. Wilcox, T.P., F.J. Garcia de Leon, D.A. Hendrickson, and D.M. Hillis. 2004. Convergence among cave catfishes: long-	branch attraction and a Bayesian relative rates test. Mol. Phylogenet. Evol. 31: 1101-1113. Wolfe, J.A. 1972. An interpretation of Alaskan Tertiary floras, In A. Graham (ed.), Floristics and paleofloristics of Asia and eastern North America. Elsevier, Amsterdam, The Netherland, pp. 201-233. Wu, C.I. 2001. The genic view of the process of speciation. J. Evol. Biol. 14: 851-865. Xiang, Q.Y. and D.E. Soltis. 2001. Dispersal-vicariant analyses of intercontinental disjunctions: Historical biogeographical implications for angiosperms in the Northern Hemisphere. Int. J. Plant Sci. 162: S29-S39. Xiang, Q.Y., D.E. Soltis, and P.S. Soltis. 1998. The eastern Asian and eastern and western North American floristic disjunction: Congruent phylogenetic patterns in seven diverse genera. Mol. Phylogenet. Evol. 10: 178-190. Xiao, S., B. Shen, C. Zhou, G. Xie, and X. Yuan. 2005. A uniquely preserved Ediacaran fossil with direct evidence for a quilted bodyplan. Proc. Natl. Acad. Sci. USA 102: 10227-10232. Yang, Z. 2007. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24: 1586-1591.

	根據核醣體基因間片段重建分佈於亞洲東部及北美西部肖楠屬的親緣關係陳志輝^1,4 黃仁磐² 蔡奇助³ 趙淑妙² ¹國立成功大學生命科學系 ²中央研究院生物多樣性中心 ³行政院農業委員會高雄區農業改良場作物改良課 ⁴行政院農業委員會特有生物研究保育中心植物組我們分析21個來自越南、雲南、台灣及加州的肖楠個體之核醣體基因間片段，探討分布在台灣及亞洲大陸個體之間的關係，並推論導致本屬植物目前分佈型式的原因。我們的分析結果都顯示產於亞洲與美洲的肖楠彼此之間分化相當明顯。除此之外估算出來的物種分化時間皆與化石出現時間或地理事件發生時間一致。台灣肖楠與亞洲大陸產之翠柏之間除了外觀上明顯的形態差異之外，兩者間分子層面的差異亦超出另一個屬（紅豆杉屬）所估算出的物種間差異，故台灣肖楠應被視為一個新形成的獨立物種。而歷史上的不連續分佈的地理事件為造成本研究所選取的三個物種異域種化的機制。

	關鍵詞：肖楠；不連續分佈；化石紀錄；nrITS ；親緣地理；地理事件。