Botanical Studies (2007) 48: 97-116.

*

Corresponding author: E-mail: chtsou@gate.sinica.edu.tw.

INTRODUCTION

Taiwan is situated at the junction of the Ryukyu and

the Luzon Arcs, and is separated from Fujian Province,

China by the 150 km wide Taiwan Strait (Figure 1). When

the overall flora of this region became explored in the

early 20

th

century, it became clear that the flora of Taiwan

mainly originated from continental China (Wilson, 1920;

Masamune, 1934; Kanehira, 1936; Li,

1957; Hosokawa,

1958) and the elements from the Philippines were limited

and restricted to the very south (Merrill, 1926; Li and

Keng, 1950; Li, 1953); while the flora of the Ryukyus (up

to the Amami group) was considered as an extension of the

flora of Taiwan,

separated from the flora of Japan (Kyushu

Island and to the north) (Masamune, 1934; Sonohara et

al., 1952; Hosokawa, 1958). These earlier viewpoints on

the floristic affinities have been supported by later floristic

analyses (Liu and Teruya, 1980; Hsieh et al., 1994; Shen,

1997; Hsieh, 2002). A well accepted explanation based

on geological/geographical information is that the Taiwan

Strait is mostly only 60-80 m deep and served as a bridge

connecting continental China and Taiwan several times

during the Pleistocene glacial episodes, whereas the Bashi

Strait between Taiwan and Luzon Island is deeper than

2,000 m and these islands have never been connected

(Shen, 1996). Our comparison on the native genera of

the Ryukyus listed in the "Flora of the Ryukyus, South of

Amami Island" (Hatusima and Amano, 1994) also reveals

that 96% (650 out of 672) of its native genera are also

native to Taiwan.

Phylogeny and taxonomy of Eurya (Ternstroemiaceae)

from Taiwan, as inferred from ITS sequence data

Chi-Chih WU, Zhi-Fu HSU, and Chih-Hua TSOU*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, R.O.C.

(Received May, 19 2006; Accepted August 24, 2006)

ABSTRACT. Eurya is the largest genus in the Ternstroemiaceae. The 13 Eurya species in Taiwan constitute

an important element in various habitats from low to high elevated forests in the island. In an attempt to

understand the interspecies relationships and geographical history of these 13 species, we sequenced 123

samples representing 32 Eurya species from Taiwan, the Ryukyus, continental China, and Southeast Asia

at the nuclear ITS region. Sixteen species of five other genera of Ternstroemiaceae were also incorporated.

Molecular phylogenetic trees show that the Eurya species studied form a monophyletic group, organized into

five clades with geographical correlations. All the 17 Eurya species from Taiwan and the Ryukyus studied,

except E. loquaiana, together constitute two coherent, but distantly related clades: one clade stands at a basal

and isolated position, with four out of its five species endemic to these two regions; they are probably Tertiary

elements of northern China or their direct descendants. The other clade is much more advanced and has strong

affinity to the Eurya in southeast China, which suggests that the members were probably originated from the

Quaternary flora of southeast China. Ecological partitioning is thought to be the major mechanism for the

speciation of four Taiwan-endemic species from E. chinensis.

Keywords: Eurya; ITS; Ternstroemiaceae; Phylogeny; Taiwan; The Ryukyus.

TAXONOMY

Figure 1. Map of SE Asia and E Asia.

98

Botanical Studies, Vol. 48, 2007

These phytogeographical studies of Taiwan were large-

scaled analyses of the entire floras or ligneous floras of

these regions. In recent years, adopting molecular tools

in phytogeographical studies on the plants of Taiwan has

become prevalent; however, the majority were focused

on the inter-polulation relationships within a single

species, e.g., Cycas taitungensis (Huang et al., 2001),

Cyclobalanopsis glauca (Huang et al., 2002); Kandelia

candel (Chiang et al., 2001), Michelia formosana (Lin,

2001; Lu et al., 2002), Myrica japonica (Cheng et al.,

2000), Trochodendron aralioides (Wu et al., 2001), and

Tritanotrichun oldhamii (Wang et al., 2004). Congeneric

species have rarely been investigated, e.g., Gentiana

(Chen, 2001).

The goal of our study is to approach the

phylogeography of a genus from Taiwan by analyzing it

together with congeneric species occurring in the Ryukyus

and Fujian Province, China. The genus Eurya was chosen

for three reasons. First, we have been working on a

revision of Eurya in Taiwan and have acquired an essential

species concept of the genus in this region. Second, the

total 19 Eurya species occurring in the Ryukyus and

Taiwan exhibit complex distribution patterns. Five are

endemic to the Ryukyus (E. osimensis, E. ryukyuensis,

E. sakishimensis, E. yaeyamensis, and E. zigzag), eight

to Taiwan (E. crenatifolia, E. glaberrima, E. hayatai, E.

leptophylla, E. nanjenshanensis, E. rengechinensis, E.

septata, and E. strigillosa). Three occur in both Taiwan

and SE China, but not in Japan/the Ryukyus (E. chinensis,

E. loquaiana, E. nitida). One is present in Taiwan and

probably in the Philippines as well (E. gnaphalocarpa)

(Ling, 1998), one is present in the Ryukyus (and Japan)

and China, but not in Taiwan (E. japonica), and one occurs

in all three regions (E. emarginata). Thirdly, the ratio of

endemic Eurya species is high in Taiwan (8/13=62%) and

the Ryukyus (5/7=71%); thus, Eurya appears to be a good

subject for phytogeographical research.

Eurya, comprising c. 130 species (Ling, 1998),

was the largest of the nine genera in the subfamily

Ternstroemioideae of Theaceae; but in the recent

angiosperm phylogeny system, Ternstroemioideae is

removed from Theaceae and combined with Pentaphyllax

(Pentaphyllacaceae) to form Ternstroemiaceae (Stevens,

2006). The genus Eurya distributes from SE Asia, E Asia,

to the Pacifics, with continental China as the center of

diversity (Ling, 1998). The species of Eurya are often

common or even dominant in various habitats in Taiwan,

such as riverbanks, coastal forests, windy mountain slops,

or cold and humid forests. In this study, for the taxonomic

treatment of Eurya, we followed the Manual of Taiwan

Vascular Plants (Liu et al., 1998) for the species in

Taiwan, with the addition of one new species, E. septata

(Wu et al., 2003). For Eurya in the Ryukyus, besides the

six species recognized in the Flora of Okinawa and the

Southern Ryukyu Islands (Walker, 1976) and the Check

List Vascular Floras of Ryukyus Islands (Shimabaku,

1997), we also accepted E. ryukyuensis as an independent

species. For Eurya in continental China, the Flora of

China (Ling, 1998) was followed.

MATERIALS AND METHODS

Sampling: All the 13 Eurya species distributed in

Taiwan, five out of the seven occurring in the Ryukyus,

and 12 out of the 18 occurring in Fujian, China were

collected in the field. (The 12 Fujian species were

collected from Fujian, Guangdong, and/or Hong Kong).

Eurya species from other areas and five other genera

in Ternstroemioideae, including Adinandra, Cleyera,

and Euryodendron of tribe Freziereae and Anneslea

and Ternstroemia of tribe Ternstroemieae, were also

incorporated (Table 1, Appendix 1). Leaf materials were

immediately put in silica gel after detachment in the

field; while voucher specimens were deposited in the

herbaria listed in Table 1. Sequencing for chloroplast

DNA regions psbA-trnH and trnL-trnF was carried out

in 24 and 4 species, respectively (Appendix 2); because

their respective sequence variations were too low for

interspecies comparison, no more sequencing was

continued (details provided in the Results). Sequencing

for the nuclear ITS (internal transcribed spacer) DNA

region was carried out in 123 samples of 32 Eurya species

and 23 samples of 14 species from the other five genera

(Table 1). The rather extensive sampling was intended

to have a preliminary survey on the ITS intraspecific

variation of Eurya species and to ascertain the suitability

of inferring ITS sequences in the present phylogenetic

study. The design of the sampling was that

for each Eurya

species occurring in Taiwan and the Ryukyus at least

three samples should be sequenced. When two or more

con-specific samples exhibited identical sequences, no

more sequencing for that species was required and that

sequence type would be chosen to represent the species

and registered in the GenBank; otherwise, more samples

should be sequenced until the requirement was fulfilled.

As for the species from other regions and of other genera,

the amount of samples was not very many, thus most of

the samples were sequenced. The number of con-specific

samples in this study ranged from one to 27. For those

Eurya species with wide distribution ranges, multiple

con-specific samples were usually chosen from different

countries/islands.

DNA extraction, amplification, and sequencing.

Total DNA was extracted from silica gel-dried or

herbarium material following the protocols described in

Struwe et al. (1998).

ITS.

Amplification of the internal transcribed spacer

(ITS) region which is composed of ITS1, the 5.8 gene, and

ITS2 was done by using primers ITSleu1 (Urbatsch et al.,

2000) and ITS4 (White et al., 1990). The PCR program

was set as 94¢XC for 5 min followed by 35 cycles of 94¢XC

for 1 min, 56¢XC for 1 min, 72¢XC 1 min, and a single cycle

of 72¢XC for 7 min. Sequencing was done by using ITSleu1

and ITS4 primers.

trnL-trnF.

Amplification of the trnL-trnF which

is composed of trnL intron, trnL3¡¦ gene and trnL-trnF

intergenic spacer was done by using primers TabC and

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

99

100

Botanical Studies, Vol. 48, 2007

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

101

102

Botanical Studies, Vol. 48, 2007

TabF (Taberlet et al., 1991). The PCR program was set as

94¢XC for 5 min followed by 30 cycles of 94¢XC for 1 min,

49¢XC for 1 min, 72¢XC min for 1 min 30 sec, and a single

cycle of 72¢XC for 7 min. Sequencing was done by using

primer TabC and TabF, when necessary, TabD and TabE

primers were used in addition (Taberlet et al., 1991).

trnH-psbA.

Amplification of the trnH-psbA

intergenetic spacer was done by using primers trnH (Tate

and Simpson, 2003) and psbA (Sang et al., 1997). The

PCR program was set as 96¢XC for 5 min followed by 35

cycles of 96¢XC for 50 sec, 53¢XC for 1 min, 72¢XC for 30

sec, and a single cycle of 72¢XC for 5 min. Sequencing was

done by using trnH and psbA primers.

PCR products were cleaned with the QIAquick PCR

purification kit (Qiagen, Valencia, California, USA).

Sequencing was done on an ABI 377 automated DNA

sequencer.

Sequence alignment.

Sequences were assembled

from both directions and ambiguous sites were checked

against the electrophenograms. All the sequence data

were aligned with the GCG program (Wisconsin Package

Version 10.3) and then adjusted manually by using Se-Al

(Rambaut, 1996).

Phylogenetic relationship analysis

Cladistic analysis was performed with Maximum

Parsimony by using PAUP* 4.0b10 (Swofford, 2002)

and Bayesian analysis by using MrBayes version 3.0b4

(Huelsenbeck and Ronquist, 2001). In the parsimony

analysis all characters were unordered and weighted

equally (Fitch, 1971). The data matrix was analyzed by

employing an heuristic tree search with 1000 replicates

with stepwise to create the initial trees, and asis sequence

addition and tree-bisection-reconnection (TBR) branch

swapping were set. The MaxTrees was set to 10000.

Relative support was estimated with the bootstrap option

in PAUP* employing a heuristic search with 1000

replicates. In the Bayesian analysis, MrModel test version

3.6 (Posada and Crandall, 1998) was adopted to estimate

the parameters, the Markov chain Monte Carlo algorithm

was set as four simultaneous chains, and a tree was saved

every 5000 generations. Two million generations were

performed in each analysis. Trees from the burn-in period

were discarded, and a 50% majority rule consensus tree

was constructed from the remaining trees.

RESULTS

Chloroplast DNA sequences

The psbA-trnH sequences of the 20 Eurya species

examined were uniformly 416 bp long and 16 of them

showed exactly the same sequence (Table 2); as for the

other four species, E. acuminata and E. groffii had two

single substitution mutations each, and E. glaberrima

and E. distichophylla one substitution mutation (Table

2). The total six variations were singletons. Sequencing

of the trnL-trnF region was carried out in four distantly

distributed Eurya species (Table 2). Three of them showed

identical trnL-trnF sequences. As for the fourth species,

it had a deletion of 27 bp. Both psbA-trnH and trnL-trnF

regions seemed unsuitable for the phylogenetic analysis of

Eurya, this part of sequencing work was thus intermitted.

Nuclear ITS sequences

The length of ITS in the six genera of Ternstroemiaceae

ranged from 632 to 648 bases before alignment and was

673 after alignment. Within Eurya the length was fairly

uniform, with 637 bases long in 27 species, 638 bases in

three species, and 632 in one; this segment included the

ITS1 247-248 bases long, 5.8S 164 bases long, and ITS2

221-227.

ITS sequence variation

The number of con-specific samples of Eurya species

varied from one to

27. For the species with two or more

samples, limited intraspecific variation did exist in most

of them; nevertheless, the representative sequence of

each species, which was generated from at least two con-

specific samples, usually emerged within a small sample

size, and the other con-specific sequence types differed

from the representative at only one to three sites (Table 1).

Most of these intraspecific variations were independent,

single substitution mutations within the intron and a small

percentage were insertions or deletions. The exceptions

were found in E. crenatifolia and E. leoptophylla where

their ITS sequences were much variable. It is worthy

to mention that Eurya chinensis was much extensively

surveyed (20 samples) because its morphological

variations were great and many individuals with different

features were sampled (Table 1); but it turned out that ITS

sequence was highly conserved among the con-specific

samples of E. chinensis that seven out of the 10 samples

from Taiwan exhibited the same type (E. chinensis-T)

and all nine samples from Hong Kong and Guangdong,

China showed the same type (E. chinensis-C). These

two dominant sequence types, E. chinensis-T and E.

chinensis-C differ at only one site that the former has a

C whereas the latter has a T at base number 200 in the

aligned matrix (Table 2). It is important to note that a C

at base 200 characterizes all the samples of Taiwanese E.

chinensis, E. emarginata, and E. septata and the Ryukyu¡¦s

E. sakishimensis; whereas a T is found in the remaining

species. Further discussions on this point will be given

in Discussion. In the combined data matrix including the

representative sequences of the 32 Eurya species, the total

variation site is 62 bases in the aligned matrix (Table 2).

ITS distance within Eurya

The distance between any two of the 32 Eurya species

studied ranges from 0 to 16 bases, under the condition

that the representative sequences are compared (Tables

1, 2). Identical ITS sequences were found in two pairs of

species, E. chinensis-T and E. septata, and E. chinensis-C

and E. nanjenshanensis. These two pairs actually differ

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

103

Table 2. ITS sequence variations among the 32 Eurya species, the numbers of the sites are based on the aligned matrix.

at a single site, site 200 in the matrix (Table 2), as just

mentioned. Minor ITS variation is commonly found

between species occurring in the same geographical

region. Among the 13 Eurya species from Taiwan, 10 of

them form two coherent groups. Group one including E.

glaberrima, E. gnaphalocarpa, E. rengechiensis, and E.

strigillosa is mainly distributed in

the Central Mountain

Range of Taiwan, from 650 m to 2,000 m elevation.

With E. glaberrima as the center, the other three differ

from it at only one or two sites, and the total variation in

this group is three sites (Table 2). Group B includes six

species, viz., E. chinensis, E. crenatifolia, E. emarginata,

E. leptophylla, E. nanjenshanensis, and E. septata, with

interspecies ITS distance from zero to two sites except that

E. leptophylla differs from E. chinensis-T and E. septata

at three sites (Table 2). Seven species from SE China form

a coherent group as well and with E. amplexifolia as the

center, E. hebeclados, E. loquaiana, E. macartneyi, E.

muricata, E. subintegra, and E. weissiae differ from the

center at only one or two sites (Table 2). Although minor

ITS variation is mostly found between species occurring

in the same geographical area; the reserve is not true.

Eurya loquaiana, E. glaberrima, and E. hayatai co-occur

in many mid-elevated forests in Taiwan, but their pairwise

distance is 12 (E.l. vs. E.g.), 10 (E.g. vs. E.h.), and 13

(E.l. vs. E.h) bases, respectively. Eurya yaeyamensis and

E. ryukyuensis can be found in the same locality in the

Iriomote island, Japan; but their ITS sequences differ at 10

sites (Table 2).

The greatest ITS distance, 16 variation sites, was found

in three species pairs, i.e., E. laotica vs. E. sakishimensis;

E. acuminata var. acuminata vs. E. ryukyuensis; and

E. acuminata var. acuminata vs. E. sakishimensis.

Coincidentally, these three pairs have the greatest

geographical distance that E. laotica and E. acuminata are

SE Asian whereas E. ryukyuensi and E. sakishimensis are

Ryukyu endemics. The next level, 15 variation sites, was

found in species pairs between these two SE Asian species

and many Taiwan-Ryukyus endemic species (Table 2).

Gene tree and species tree

In order to evaluate the level of paralogy of the ITS

intraspecific variation revealed in this study, all the

sequence types, 92 types generated from 146 samples of

the 46 species from six genera (Table 1) and three other

sequences from GenBank, were analyzed to produce

the gene tree (Figure 2). The most parsimony (MP) was

adopted for analysis with Anneslea and Ternstroemia

from the tribe Ternstroemieae as the outgroups. In the

gene tree, the con-specific sequence types are usually

grouped in a clade of their own, such as in cases of E.

yaeyamensis, E. distichophylla, E. muricata, E. loquaiana,

E. acuminata, E. japonica, E. hayatai, E. nitida, E .

ryukyuensis, E. sakishimensis, and E. emarginata. In many

other species they form polytomous branches in a larger

clade, such as those of E. rengenchiensis, E. strigillosa,

E. glaberrima, E. gnaphalocarpa, E. hebeclados, E.

macartneyi, E. nanjeshanensis, and E. septata. Only those

of E. crenatifolia, E. chinensis, and E. leptophylla spread

somewhat widely in the lowest clade and are occasionally

mixed with some other species to form a smaller clade

(Figure 2). On the basis of the gene tree (Figure 2),

the intraspecific sequence variation of most species

shows shallow paralogy and the selected representative

sequences do group with their con-species samples except

that sequences of E. crenatifolia, E. chinensis, an d E.

leptophylla are widely spread in the lowest clade. Thus, we

consider that the propriety of the representative sequence

104

Botanical Studies, Vol. 48, 2007

Figure 2. The gene tree based on 92 ITS sequence types produced from 49 species of six genera. This is the MajRule tree generated

from the parsimony analysis, with total characters=673, informative characters=148, tree length=347, CI=0.6945, RI=0.9132, and

10000 trees retained. Numbers showing on the branches are percentage of the presence of the clade among the 10000 trees (numerator)

and bootstrap values larger than 50 (%) (denominator). The bootstrap values were based on 800 replicates.

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

105

is supported. The species trees (Figures 3, 4) were then

produced based on the 52 representative sequences which

representing 49 species (Table 1) by using the most

parsimony and Bayesian analyses. In general, the MajRule

gene tree and the two species trees are highly congruent

in their topologies (Figures 2, 3, 4). Their common and

important points revealed in these trees are:

1. Among the ingroups, Adinandra and Cleyera together

form a clade with 100% bootstrap support, this clade

is sister to Euryodendron and Eurya. The three species

of Cleyera are embedded within the seven Adinandra

species, further consideration on the unification/

separation of these two genera is necessary. The two

Adinandra species endemic to Taiwan, A. formosana

and A. lasiostyla, differing at 19 sites, are not the

closest to each other. The lowland A. formosana forms

a clade with the SE China distributed A. millettii,

with only one site in difference; the highland A.

lasiostyla forms a clade with the Ryukyu endemic A.

yaeyamensis, with six sites different, but sharing seven

substitution mutations of their own.

2. Euryodendron, a monotypic genus, is sister to and the

closest to the Eurya clade. The status of Euryodendron

as a monotypic genus is strongly supported in this

study.

3. Eurya, with 32 species studied, appears as a

monophyletic genus. The 32 species unanimously form

five clades and the allocation of these species in five

clades is exactly the same in all these MajRule trees

(Figures 2, 3, 4). The relationships of these five clades

are better revealed in MP trees than in Bayesian trees

since the five clades are arranged in three levels in the

former, but are parallel in the latter. These five clades

possess strong geographical constraints and are thus

defined:

(1)

Ryukyu-Taiwan (RT) endemic group ¡X this clade

is sister to the other four clades in MP MajRule

trees (Figures 3, 4). Five species are included, with

Eurya yaeyamensis endemic to the Ryukyus, and

E. reneichiensis, E. strigillosa, and E. glaberrima

endemic to Taiwan. Eurya gnaphalocarpa is

reported as also occurring in the Philippines and

Taiwan (Ling, 1998). Among these five species, the

former two are sister to the latter three.

(2)

SE Asia-SW China group ¡X four species are

included, E. acuminata, E. laotica, and E. groffii

with a distribution range in SE Asia and E.

quinquelocularis in SW China. It is interesting

that E. acuminata wallichiana shows intermediate

sequencing between E. groffii and E. acuminata

(Table 2).

(3)

S China grou p ¡X E. acuminatissima, E. disticha

and E. distichophylla form a small clade, they

mainly distributed in the southern provinces of

China.

(4)

SE China group ¡X eight species, viz., E .

amplexifolia, E. herbeclados, E. impressinervis,

E. loquaiana, E. marcartneyi, E. muricata, E.

subintegrifolia, and E. weissiae form a well

supported clade. All these species are confined to

SE China except that E. loquaiana also extends to

Taiwan.

(5)

Ryukyu-Taiwan (RT) dominant group ¡X twelve

species are included in this clade. The most basal

one, E. rubiginosa, is only present in Guangdong,

China. At the next level is E. nitida, occurring

in continental China and Taiwan. Among the

remaining 10 species, a clade consisting of E.

hayatai and E. japonica is sister to the other eight

species. Eurya japonica occurs in continental China

and Japan and E. hayatai is endemic to Taiwan.

The remaining eight species are endemic to either

Taiwan or the Ryukyus except for the widely

distributed E. chinensis. Since E. chinensis stands

as the center of these eight species, as the sequences

are concerned, these eight are collectively termed as

an E. chinensis branch. This E. chinensis branch is

successfully established in Taiwan and the Ryukyus.

The sequence variations among them are so low

that the resolution of this branch is very poor. It is

worthy to note that in this ladder-like arrangement

of this big clade, species distribution range shifts

stepwise from SE China (E. rubiginosa) to China

+ Taiwan (E. nitida), to E China-Taiwan-Japan

(E. chinensis, E. hayatai, E. japonica), and then

to Taiwan-Ryukyu endemism (E. crenatifolia, E.

leptophylla, E. nanjenshanensis, E. ryukyuensis, E.

sakishimensis, and E. septata).

In summary, the 32 Eurya species studied form five

well supported clades (Figures 2, 3, 4). Seventeen out of

the 18 Eurya species occurring in Taiwan and the Ryukyus

are confined to two clades, the R-T endemic and the R-T

dominant clades; these two clades are situated respectively

at the most basal and the most derived positions in the MP

MajRule trees. The remaining Taiwanese E. loquaiana is

grouped with seven SE China distributed species. The 12

Eurya species occurring in Fujian, on the other hand, are

spread in four out of the five clades.

DISCUSSION

The nuclear ITS gene (18S-5.8S-26S) has hundreds

to thousands copies arrayed as tandem repeats on the

chromosomes. Its phylogenetic inference has been

extensively employed by plant taxonomists (Alvarez

and Wendel, 2003), but more questions on the impact

of its sequence polymorphism on the phylogenetic

reliability have been raised (Alvarez and Wendel, 2003;

Andreasen and Baldwin, 2003; Bailey et al., 2003). In

order to ascertain the suitability of ITS being used in

the present study, a lot of efforts were made to collect

and sequence multiple samples for each Eurya species

in Taiwan and neighboring regions. We sequenced 3-27

106

Botanical Studies, Vol. 48, 2007

Figure 3. Species tree one based on 52 ITS sequence types representing the total 49 species. This is the Majrule tree generated from

the parsimony analysis, with total characters=673, informative characters=141, tree length=309, CI=0.6958, RI=0.9035, and 121 trees

retained. Bootstrap supports higher than 50% are shown beneath the branches. Numbers showing on the branches are percentage of the

presence of the clade among the 121 trees (numerator) and bootstrap values larger than 50 (%) (denominator). The bootstrap values

were based on 1000 replicates. The five clades of the Eurya species are defined, and the Arabic numerals 1, 2, and 3 are designated to

the distribution range covering Fujian, Taiwan, and the Ryukyus, respectively.

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

107

Figure 4. Species tree two with the same dataset as species tree two. This is the MajRule tree generated from the Bayesian analysis.

The posterior probabilities higher than 50% are shown beneath the branches. Asterisks (*) indicate the presence of anther septation,

two species are unknown in this regard and marked with a ".", the remaining species have no anther septa.

108

Botanical Studies, Vol. 48, 2007

samples for every Eurya species in Taiwan and most

species in the Ryukyus; in general, for most of these

species a specific and dominant sequence type was found

after a small sampling. Identical ITS sequences are

commonly obtained from con-specific samples collected

from different countries/islands (e.g., E. chinensis, E.

emarginata, E. japonica, E. macartneyi, E. nitida, E.

ryukyuensis, and Cleyera japonica) or from localities

hundreds kilometers away (e.g., E. glaberrima, E.

loquaiana, E. septata, E. strigillosa, and Adinandra

formosana) (Table 1, Appendix 1). As for con-specific

samples collected from the same population, their ITS

sequences are even more homogeneous. The tendency of

retaining a specific ITS sequence type within a species

resulting from the concerted evolution has been reported

in many plant groups (Wendel et al., 1995; Sang et al.,

1995); we believe that concerted evolution is affecting

Eurya as well. In addition, in our integrated analysis,

all the 92 sequence types generated in this study were

incorporated to produce the gene tree (Figure 2), the

intraspecific sequence variations of most species represent

shallow paralogy which indicates that they would not

adversely affect species tree reconstruction (Figures 2, 3,

4). In conclusion, we believe that ITS sequence is reliable

for this phylogenetic study of Eurya. In the E. chinensis

lineage the interspecific sequence variations are usually

low or even none; whereas the intraspecific variations of

E. chinensis, E. crenatifolia, and E. leptophylla are greater

than the interspecific variations; thus the sequence types

of these three species may mix with a few other species

in several small clades. The much greater intraspecific

sequence variation of two Taiwan-endemic species, E.

crenatifolia and E. leptophylla, might be due to their

slower concerted evolution relative to mutation rates and

incomplete lineage sorting, etc. (Andreasen and Baldwin,

2003). It is noteworthy to point out that the dominant

sequence type of the species with multiple samples is

also the consensus sequence for each species. They were

chosen to be registered in the GenBank and used for

interspecies interpretations (Table 1).

Taxonomic implications

The genus Eurya has a rather large number of species,

c. 130; and criteria used for discriminating species are

poor and often microscopical. The present sequencing

work shows that the ITS sequences of the 32 Eurya

species differ at 62 sites (Table 2) and the greatest distance

between two species is 16 mutations, which is potentially

useful for taxonomic treatment of Eurya at theoretical

and practical levels. During the course of this study, a

few taxonomic problems were resolved by using the ITS

sequencing data:

1. Taxonomic treatments of E. chinensis in Hong

Kong. Eurya chinensis and E. nitida are the most

common Eurya species in Hong Kong and sympatric

in numerous sites. Usually they can be easily

distinguished by the thick pubescence and the smaller

leaves in E. chinensis and the lack of pubescence

and slightly larger leaves in E. nitida; however, in

many windward mountainous sites, abundant typical

(hairy) E. chinensis individuals are mixed with a small

percentage of glabrous individuals and the latter are

not readily distinguishable from E. chinensis or E.

nitida. Sometimes, there are hairy individuals, but with

much smaller (c. 1/2 of that of E. chinensis) and thicker

leaves, which are similar to the Taiwan-endemic E.

crenatifolia in gross morphology. Local taxonomists

use the name, E. chinensis var. glabra to the glabrous

individuals and E. chinensis var. chinensis to the small-

leaved ones (Lai et al., 2004). Owing to the lack of

any further studies, we sequenced eight individuals

of E. chinensis from Hong Kong including the three

phenotypes (typical, glabrous, and small-leaved).

Interestingly, their ITS sequences came out the same

as the E. chinensis-C type collected from Guangdong,

China (Table 1). The results strongly support the

taxonomic treatment of these phenotypes under E.

chinensis. In addition, this preliminary study suggests

that E. chinensis possesses great morphological

plasticity and may well sustain severe environments

where other Eurya species can not. The populations

surviving in the harsh conditions may express greater

morphological diversity.

2. D o E. acuminata and E. japonica occur in Taiwan.

In the most recent treatments of Eurya in Taiwan,

i.e., Flora of Taiwan (Hsieh et al., 1996), Manual of

Taiwan Vascular Plants (Liu et al., 1998), and Flora of

China (Ling, 1998), the recognition of E. acuminata

and E. japonica is controversial. The distribution of

Eurya acuminata in Taiwan is reported in the first and

the third treatments; and that of E. japonica in the

third. Nonetheless, our samples of E. acuminata var.

acuminata collected from Singapore and E. acuminata

var. wallichiana from N Thailand are well separated

from Taiwanese Eurya in both morphological and

ITS sequence aspects (Table 2). Plants in Taiwan

previously determined as E. acuminata were mostly of

E. loquaiana, probably due to their similar acuminate

leaf apices. For clarifying the issue of Eurya japonica,

samples were collected from Zhe-Jiang Province,

China and Okinawa Island, Japan and they showed the

same morphological characteristics and identical ITS

sequences (Table 1, Appendix 1); but in the sequence

aspect, E. japonica differs from those Eurya in Taiwan

at five sites (E. hayatai and E. nitida) or more. Among

the Taiwanese Eurya, the endemic E. hayatai is often

misidentified as E. japonica because they are similar

morphologically; with the major distinction on the leaf

lower surface where the venation is clear in E. japonica,

but nearly invisible in E. hayatai. These two species are

so far closely related as suggested by the species trees

(Figures 3, 4). In this study, ITS sequencing work helps

to delete the presence of E. acuminata and E. japonica

in the current flora of Taiwan.

3. Taxonomic status of Eurya ryukyuensis, E .

nanjenshanensis, a nd Cleyera morii. Eurya

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

109

ryukyuensis, a Ryukyu-endemic taxon, was first

published as an independent species by Masamune in

1935, but was transferred to a variety of E. emarginata

by Hatushima in 1956 which was followed in all the

important floras of that area, such as Flora of Okinawa

and the Southern Ryukyu Islands (Walker, 1976) and

Check List of the Vascular Flora of the Ryukyu Islands

(Shimabuku, 1997). ITS sequencing data shows that

E. ryukyuensis is different from all the other Eurya

species studied. The ITS distance from E. ryukyuensis

to E. emarginata is four sites and the shortest

distance is found in E. chinensis (sequence type of E.

chinensis-C), with two different bases (Table 2). The

treatment of E. emarginata var. ryukyuensis is thus

not supported. Eurya nanjenshanensis was published

as E. nitida var. nanjenshanensis in Flora of Taiwan

(Hsieh et al., 1996), mainly due to the glabrous feature;

but was then recognized as a distinct species in the

Manual of Taiwan Vascular Plants (Liu et al., 1998).

The ITS sequence of E. nanjenshanensis differs from

that of E. nitida at four sites, but is the same as the E.

chinensis-C sequence type. Again, the treatment of E.

nitida var. nanjenshanensis is not supported. Cleyera

morii, a Taiwan endemic, was first published as a

variety of Eurya, Eurya ochnacea Szyzy. var. morii

Yama. by Yamamoto in 1927. It was then transferred

to Cleyera japonica Thunb. var. morii (Yama.) Masa.

by Masamune (1935) and then changed to C. morii

(Yama.) Masa. in 1939. Nonetheless, C. japonica var.

morii was preferred by Kobuski (1938) and adopted in

later important treatments, such as Flora of Taiwan, the

First Edition (Li, 1976) and the Second Edition (Hsieh

et al., 1996), the Manual of Taiwan Vascular Plants (Liu

et al., 1998), and Flora of China (Ling, 1998). Our three

samples of C. japonica collected from the Ryukyus

and Taiwan have identical ITS sequence and the two

samples of C. morii collected from two populations in

Taiwan have identical sequences as well. These two

species differ at five sites, which strongly supports an

independent species status of C. morii, in addition to

their morphological distinctions (Hsieh et al., 1996).

In summary, the present sequencing work supports the

independent species status of E. ryukyuensis and E.

nanjenshanensis. Also, Cleyera morii, differing from

C. japonica at five sites, also deserves the status of a

species.

4. Taxonomic value of anther septation in Eurya. Large

scaled taxonomic studies on Eurya have been poor; the

most recent is by Ling (1998) in the Flora of China.

Since his earlier revision (1966) on Eurya, Ling has

strongly stressed the importance of anther septa and

even classified the genus as two subgenera based on this

single criterion. This work, though the sampling covers

only one fourth of the total number of species, shows

that anther septa could have evolved from nonseptate

ancestors in different lineages independently (Figure 4).

We assume that the evolution of anther septa in Eurya

is possibly associated with pollination advantages, i.e.,

retaining pollen grains in the anthers in the up-side-

down floral orientation. Taxonomic applications of this

character in Eurya classification are better restricted to

species discrimination.

Phylogeography of

Eurya of Taiwan

Our study included all the 13 species of Eurya

occurring in Taiwan, five of the seven (71%) occurring in

the Ryukyus, and 12 of the 18 (67%) in Fujian Province,

China, and several others from SW China and SE Asia.

Increasing the number of species and the regions of

sampling will certainly give better resolution of the

interspecies relationships. Nonetheless, the relationships

of the species occurring in Taiwan and the Ryukyus that

they constitute two coherent but distantly related groups

plus one isolated species (E. loquaiana) is strongly

indicated. First, the intimacy among the five species in

the RT endemic clade and that among the eight in the RT

dominant clade are obvious by the high ITS sequence

similarity and the possession of several synapomorphic

sites within each clade, which is also reflected in the high

bootstrap value. Second, the distant relationship between

these two clades as shown in Table 2 and Figures 3 and 4

can not be refuted because members of these two groups

constantly have 10 or more different sites at the ITS

region. Thirdly, E. loquaiana is closely associated with a

few members of SE China clade with a strong support of

92% bootstrap value (Figure 4). Its difference from other

Eurya species in Taiwan ranges from 10 to 15 sites (Table

2) and its isolated position within the Eurya in Taiwan

is definite. Phylogenetically, Eurya species of Taiwan

and the Ryukyus form two coherent, but distantly related

groups, plus one isolated species, is distinct and unlikely

to be altered even when more samples are analyzed.

On the basis of the species relationships of Eurya,

which we here revealed, a few phylogeographical points

can be deduced:

1. Among the seven Eurya species occurring in the

Ryukyus, five are here studied. They are either

embedded in the RT endemic clade (e.g., E .

yaeyamensis) or in the RT dominant clade (e.g.,

E. emarginata, E. japonica, E. ryukyuensis, and E.

sakishimensis). The remaining two Eurya species

yet studied are rare and endemic E. osimensis and E.

zigzag. Morphologically, E. osimensis is very similar

to the Taiwan-endemic E. strigillosa in general features

and especially in possessing yellowish, long (c. 0.6-0.8

mm) hairs on terminal buds and branches; it has even

been treated as a variety of the latter by Masamune

(1955). Eurya zigzag is very similar to another

Ryukyu-endemic E. yaeyamensis which is embedded

in RT endemic clade grouped with four species from

Taiwan. Generally speaking, the majority of Eurya in

the Ryukyus show close affiliations with the Eurya

in Taiwan. But there are other facts to be considered.

First, the endemic rate of the Eurya in the Ryukyus is

very high (5/7); second, E. japonica distributes in E.

110

Botanical Studies, Vol. 48, 2007

China, Japan, and Korea, but not in Taiwan. Thirdly,

the only Eurya species shared by the Ryukyus and

Taiwan, i.e., E. emarginata, is probably of avian seed

dispersal because it is distributed in the costal regions

of many islands in SE China, Taiwan and the Ryukyus;

thus its coexistence in these two regions does not imply

any pre-existing land bridges between the two regions.

And fourthly, no Eurya species is currently distributed

to the Ryukyus and Taiwan only. In conclusion, our

preliminary analysis suggests that the rather close

linkage between the Eurya of the Ryukyus and Taiwan

was based on ancient interactions; the prolonged

isolation of the Ryukyus probably since the middle of

Pleistocene (Kimura, 2000) results in the very high

endemism of Eurya. No recent interactions through

land bridges between the Ryukyus and Taiwan can be

identified in the case of Eurya.

2. The phylogeography of the Eurya in Taiwan is a little

more complicate. The four species in RT-endemic

clade show comparatively close associations with

the Ryukyu members, which is probably caused by

ancient interactions. Some species (e.g., E. hayatai, E.

loquaiana, E. nitida) show affiliations with extant SE

China or E China members. And, the remaining six

species, including four endemic species, the SE China-

Taiwan distributed E. chinensis, an d E. emarginata,

form an intimate lineage, with E. chinensis as the

center (the E. chinensis lineage) (Figures 3, 4). These

nine species, as a whole, are connected with the current

continental progenitors. In this connection, the origin

of the four endemic species in the E. chinensis lineage

can be discussed. Eurya crenatifolia, E. leptophylla,

E. nanjenshanensis, and E. septata possess identical

ITS sequences to that of E. chinensis, either to the

representative of SE China (E. chinensis-C) or Taiwan

(E. chinensis-T). As we suggested in the Results, E.

chinensis has great ability in living in diverse habitats

and developing great morphological diversities. It is

possible that during the several Pleistocene glacial

periods when Taiwan was connected with Fujian

(Zhao, 1982; Shi et al., 1986; Hsieh and Shen, 1994;

Voris, 2000), E. chinensis as a member of SE China

flora could have migrated to Taiwan several times.

Speciation originated from E. chinensis might have

taken place in different ecological habitats and at

different geological times, and then gave rise to

these four Taiwan-endemic taxa. Eurya crenatifolia,

characterized by tiny and thick leaves, is adapted to

windy and humid environment, mainly in northern

Taiwan; E. leptophylla, characterized by small and

thin leaves, is restricted to higher elevations; E .

nanjenshanensis, with glabrous and slightly obovate

leaves, is found in the tropical southern lowlands;

and E. septata, differing from E. chinensis in having

septate anthers and slightly larger leaves, can well

survive in disturbed habitats (Wu et al., 2003). As for

the other three Taiwan-endemic species, E. glaberrima,

E. hayatai, an d E. strigillosa, since their closest

relatives are not yet evident, no interpretations on their

endemism can be provided.

In summary, this study shows that the current

composition of the Eurya in Taiwan is derived from

various sources, which may well reflect the complicate

phytogeographical history of Taiwan. It has long been

noticed that the highlands of Taiwan not only possess

much higher rate of endemic species (Hsieh, 2002), but

also retain a great number of Tertiary relics from N China

(Li, 1957; Liu, 1988; Shen, 1996, 1997), whereas the

lowlands of Taiwan have strong affinity with the lowlands

of SE China (Hsieh et al., 1994). The rationale is that

during the Tertiary the climate of N China was warm

and humid. By the end of Tertiary and the beginning of

Quaternary when global temperature decreased drastically

and the cycles of glacial/interglacial episodes proceeded,

the Tertiary flora of N China migrated southwards (Liu,

1988). A considerable percentage of this flora completely

vanished in N China and SE China during the middle

Pleistocene, but a great number of species were retained

in the highlands in Taiwan and Japan to the east, and

Yunnan, Guanxie, Sichuan Provinces of China to the

west (Yang and Hsu, 1980; Liu, 1988, Shen, 1996). Such

floristic similarity between SW China and the highlands

of Taiwan was noticed as early as 1920 by Wilson. In our

study, the RT endemic group of Eurya stands at the basal

most position and is distant from the remaining Eurya in

Taiwan, and it is not close to Eurya species from other

regions, either. This group is likely to be an isolated

and ancient group based on the information available.

The four Taiwanese species in the RT endemic clade are

restricted to the middle and upper elevated forests in the

Central Mountain Range where many well known Tertiary

elements are accommodated, for examples Amentotaxus,

Chamaecyparis, Fagus, Hedyderia, Juglans, Kalopanax,

Keteleria, Taiwania, Taxus, etc. (Shen, 1996). The other

group of Eurya, the RT dominant clade, shows high

affinities with Eurya species currently present in SE China

(Fujian Province), and most of them are distributed in

lowlands. Therefore, we propose that the five species of

RT endemic clade are most likely Tertiary elements from

N China or their direct descendants which evolved in

Taiwan. The remaining nine Eurya species in Taiwan are

mostly members or derivatives of the Quaternary flora of

the SE China. Along with the uplifting of Taiwan during

the late Tertiary and Quaterany, earlier immigrants tend to

be found in higher elevated areas in Taiwan. Ecological

partitioning is suggested as an important mechanism for

the speciation of several Taiwan-endemic species from

different populations of E. chinensis at probably different

geological times.

Acknowledgements. Great thanks are extended to the

following directors: Tokushiro Takaso of Iriomote Station,

the Ryukyus, Kwok-leung Yip of HK Herbarium, and

Ruth Kiew of Herbarium of Singapore Botanical Garden

for the help with local collecting and the use of herbaria.

We also appreciate the following people providing us

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

111

valuable materials for sequencing: Hua-Gu Ye (South

China Institute of Botany, Academy of Science, China),

Wen-Ju Zhang (Fudan University, China), Tetsuo Denda

(University of the Ryukyus), Jenn-Che Wang (Normal

Taiwan University, Taiwan), Mong-Huai Su and Chun-

Neng Wang (National Taiwan University, Taiwan),

Yu-pin Cheng (Forestry Research Institute, Taiwan),

David Johnson (Ohio Wesleyan Univ., USA), Yin-Wei

Lin (HK Herbarium) and Suei-Ya Liu (Herbarium of

Academia Sinica, Taiwan). And, valuable comments

from Lucia Kawasaki, J. F. Maxwell, and Susan Renner

on the manuscript were much appreciated. The project

was partially supported by National Science Council

(NSC 88-2311-B-001-037) and the Institute of Plant and

Microbial Biology, Academia Sinica, Taiwan, Republic of

China.

LITERATURE CITED

Alvarez, I. and J.F. Wendel. 2003. Ribosomal ITS sequences and

plant phylogenetic inference. Mol. Phy. Evol. 29: 417-434.

Andreas en, K. and B.G. Baldwin. 2003. Nuclear ribos omal

DNA sequence polymorphism and hybridization in checker

mallows (Sidalcea, Ma lvac eae). Mol. P hy. Evol . 29:

563-581.

Bailey, C.D., T.G. Carr, S.A. Harris, and C.E. Hughes. 2003.

Characterization of angiosperm nrDNA polymorphis m,

paralogy, and pseudogene. Mol. Phy. Evol. 29: 435-455.

Chen, C.H. 2001. The phylogenetic study of Gentiana sect.

Chondrophyl lae B unge (Gent iana cea e). P h D t hes is ,

National Taiwan Normal Univerity, Taipei, Taiwan, R.O.C.

(in Chinese, with English abstract)

Cheng, Y.P., C.T. Chien, and T.P. Lin. 2000. Population genetics

of ge ographica lly re strict e and wi de spread s pe cies of

Myrica (Myricaceae). J. Hered. 91: 61-66.

Chiang, T.Y., Y.C. Chiang, Y.J. Chen, C.H. Chou, S. Havanond,

T.N. Hong, a nd S . Huang. 200 1. Ph yloge ograph y of

Kandeli a c andel in E as t As iati c ma ngrove s ba se d on

nuc leotide variation of chloropla st and m itochondrial

DNAs. Mol. Ecol. 10: 2697-2710.

Fitch, W.M. 1971. Toward defining the cours e of evolution:

minimal change for a specific tree topology. Syst. Zool. 20:

406-416.

Hatusima, S. and T. Amano. 1994. Flora of the Ryukyus, South

of Amami Island. Second Edition. The Biological Society

of Okinawa.

Hsieh, C.F. 2002. Composition, endemism and

phytogeographical affinities of the Taiwan Flora. Taiwania

47: 298-310.

Hsieh, C.F. and C.F. Shen. 1994. Introduction to the flora of

Taiwan, 1: geography, geology, climate, and soils. In T.C.

Huang et al. (eds.), Flora of Taiwan, Second Edition, Vol. 1,

pp. 1-3.

Hsieh, C.F., C.F. Shen, and K.C. Yang. 1994. Introduction to

the flora of Taiwan, 3: floristics , phytogeogra phy, and

vegetation. In T.C. Huang et al. (eds.), Flora of Taiwan,

Second Edition, Vol. 1, pp. 7-18.

Hsieh, C.F., L.K. Ling, K.C. Yang, S.Z. Yang, and C.H. Tsou.

1996. Thea cea e. In T.C. Hua ng et al . (e ds.), F lora of

Taiwan, Second Edition, Vol. II., pp. 662-693.

Hosokawa, T. 1958. On the synchorological and floristic trends

and discontinuities in regard to the Japan-Liukiu-Formosa

area. Vegetatio 8: 65-92.

Huang, S ., Y.C. Chiang, B.A. Schaal, C.H. Chou, and T.Y.

Chiang. 2001. Organelle DNA phylogeography of Cycas

taitungensis, a relic species in Taiwan. Mol. Ecol. 10:

2669-2681.

Huang, S.S.F., S.Y. Hwang, and T.P. Lin. 2002. Spatial pattern

of chloroplast DNA variation of Cyclobalanopsis glauca in

Taiwan and East Asia. Mol. Ecol. 11: 2349-2358.

Heulsenbeck, J.P. and F. Ronquist. 2001. MRBAYES: Bayesian

inference of phylogenetic trees. Bioinformatics 17:

754-755.

Kanehira, R. 1936. Formosan Trees. Department of F orestry,

Government Research Institute, F ormosa, pp. 5-20. (in

Japanese)

Kimura, M. 2000. Pale ogeography of t he Ryukyu Is lands.

Tropics 10: 5-24.

Kobuski, C.E. 1938. Studies in the Theaceae. III. Eurya

subgenera Eur yodes and Penteurya. Ann. Missouri Bot.

Gard. 25: 99-359.

Lai, C.C., Y.W. Lam, P.S. So, K.Y. Tam, Y.M. Wan, and K.L.

Yip. 2004. Checklist of Hong Kong Plants. Hong Kong

Herba rium , Agricu lture , F i she ries and C onse rvat ion

Department, Hong Kong.

Li, H.L. 1953. Floristic interchanges between Formosa and the

Philippines. Pacific Sci. 2: 179-186.

Li, H.L. 1957. The genetic affinities of the Formosa flora. Eighth

Pacific Science Congress, pp. 189-195.

Li, H.L. 1976. F lora of Taiwan, 1

st

Ed. Epoch Press , Taipei,

Taiwan.

Li, H.L. and H. Keng. 1950. Phytogeographical affinities of

southern Taiwan. Taiwania 1: 103-128.

Lin, T.P. 2001. Allozyme variations in Michelia formos ana

(Kanehira) Masamune (Magnoliaceae), and the inference

of a glacial refugium in Taiwan. Theor. Appl. Genet. 102:

450-457.

Ling, L.K. 1966. A revis ion of genus Eurya in China. Acta

Phytotaxonomica Sin. 11: 263-342. (in Chinese)

Ling, L.K. 1998. Theaceae, Ternstroemioideae. In Flora

Reipublicae Popularis Sinicae, edited by Delectis Florae

Reipublicae Popularis Sinicae Agendae Academiae Sinicae

Edita, Tomus 50 (1), Science Press. (in Chinese)

Liu, H.Y., Y.P. Yang, S.Y. Lu, and B.L. Shih. 1998. Manual of

Taiwan Vascular Plants. Vol. III. The Council of Agriculture,

Excutive Yuan, Taipei, Taiwan, R. O. C. (in Chinese)

Liu, K.B. 1988. Quaternary history of the temperate forests of

China. Quat. Sci. Rev. 7: 1-20.

L iu, T.S . and Z. Teruya. 1980. The phytogeography of the

112

Botanical Studies, Vol. 48, 2007

Ryukyus and Taiwan from viewpoint of woody plants. Ann.

Taiwan Mus. 23: 1-65. (in Chinese, with Japanese abstract)

L u, S .Y., K.H. Hong, S .L. Liu, Y.P. Che ng, W.L. Wu, and

T.Y. Chi ang. 2002. Geneti c vari ation a nd populat ion

differentiation of Michelia formosana (Magnoliac eae)

ba se d o n cp DNA va ria ti on a nd R APD fing erpr int s :

relevance to post-Pleistocene recolonization. J. Plant Res.

115: 203-216.

Masamune, G. 1934. P hytogeographic pos ition of Form os a

when her indigenous genera are concerned. Trans. Nat.

Hist. Soc. Formosa 22: 164-194. (in Japanese, with English

abstract)

Masamune, G. 1935. Beitrage zur kenntnisse der Flora von Sud-

japan V. Trans. Nat. Hist. Soc. Formosa 25: 248-252.

Masamune, G. 1939. Miscellaneous notes on the flora of the

eas tern Asi a XIV. Trans. Nat. Hist. S oc. Formos a 29:

339-345.

Masamune, G. 1955. tracheophytarum Ryukyu Insularum. Sci.

Rep. Kanazawa Univ. (VII) 4: 45-134.

Merrill, E.D. 1926. Enumeration of the Philippine flowering

plants. The Government of the Philippine Islands, Manila.

Nylander, J.A.A. 2004. MrModeltest v2. Program distributed

by the aut hor. E volutionary Biol ogy Ce ntre, Upps ala

University.

Rambaut, A. 1996. Se-Al: Sequence Alignment Editor. Available

at

http://evolve.zoo.ox.ac.uk/

.

Posada, D. and K.A. Cradal. 1998. MODELTES T: testing the

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

Sang, T., D.J. Crawford, and T.F. Stuessy. 1995. Documentation

of reticulate evolution in peonies (Paeonia) using internal

transcribed spacer sequences of nuclear ribosomal DNA:

Implications for biogeography and concerted evolution.

Proc. Natl. Acad. Sci. USA 92: 6813-6817.

Sang, T., D.J. Crawford, and T.F. Stuessy. 1997. Chloroplast

DNA phylogeny, reticulate evolution, and biogeography of

Paeonia (Paeoniaceae). Amer. J. Bot. 84: 1120-1136.

Shen, C.F. 1996. The biogeography of Taiwan: 1. Background.

Ann Taiwan Mus 39: 387-428. (in Chinese, with English

abstract)

Shen, C.F. 1997. The biogeography of Taiwan: 2. Some

preliminary thoughts and studies. Ann. Taiwan Mus. 40:

361-450. (in Chinese, with English abstract)

Shi , Y.F., B.H. Ren, J .T. Wang, and E . De rbys hire. 1986.

Quaternary glaciation in China. Quat. Sci. Rev. 5: 503-507.

S himabuku, K.I. 1997. Check lis t of Vas cular flora of t he

Ryukyu islands, Revised Edition. Univ Kyusyu Publishing,

Japan.

Sonohara, S., S. Tawada, and T. Amano. 1952. Flora of Okinawa.

United States Civil Administrations of the Ryukyu Islands.

Stevens, P.F. 2006. Angiosperm P hylogeny Website. Version

7, May 2006. http://www.mobot.org/MOBOT/research/

APweb/.

Struwe, L., M. Thiv, J. Kadereit, T. Motley, A.S.R. Pepper,

J . Rova, K. Potgieter, P. W hite, and V.A. Albert. 1998.

Saccifolium (S accifoliaceae), an endemic of Sirra de la

Neblina on the Brazilian-Venezuelan frontier, is related to a

temperate-alpine lineage of Gentianaceae. Harvard Papers

Bot. 3: 199-214.

Swofford, D.L. 2002. PAUP *: phylogenetic analys is using

p ar s im o ny (* a nd ot he r m et h ods ) , v e r 4 . S i na u er,

Sunderland, Massachusetts, USA.

Taberlet, P., L. Gielly, G. Pautou, and J. Bouvet. 1991. Universal

primers for amplification of three non-coding regions of

chloroplast DNA. Pl. Mol. Biol. 17: 1105-1109.

Tate, J.A. and B.B. Simpson. 2003. Paraphyly of Tarasa

(Malvaceae) and diverse origins of the polyploid species.

Syst. Bot. 28: 723-737.

Urba tsc h, L .E ., B.G. Ba ldwin, and M.J. Donoghue. 2000.

P hyl ogeny of coneflowers a nd relat ive s (He liantheae:

Asteraceae) based on nuclear rDNA internal transcribed

spacer (ITS) sequences and chloroplast DNA restriction site

data. Syst. Bot. 25: 539-565.

Voris, H.K. 2000. Maps of Pleistocene sea levels in southeast

As ia: s horelines , ri ve r sys tems and tim e dura tions. J .

Biogeography 27: 1153-1167.

Walker, E.H. 1976. Flora of Okinawa and the southern Ryukyu

Islands. Smithsonian Instuitute P ress, Washington DC.

U.S.A.

Wang, C.N., M. Moller, and Q.C.B. Cronk. 2004. Polupation

genetic structure of Titanotrichum oldhamii (Gesneriaceae),

a subtropical bulbiliferous plant mixed sexual and asexual

reproduction. Ann. Bot. 93: 201-209.

Webb, T. and P.J. Bartlein. 1992. Global changes during the last

3 million years: climate controls and biotic responses. Ann.

Rev. Ecol. Syst. 23: 141-173.

Wendel, J.F., A. Schnabel, and T. Seelanan. 1995. Bidirectional

interlocus concerted evolution following allopolyploid

speciation in cotton (Gossypium). Proc. Natl. Acad. Sci.

USA 92: 280-284.

White, T.J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In M.A. Innis et al. (eds.), PCR protocols,

Academic Press, San Diego, California, USA, pp. 315-322.

Wilson, E.H. 1920. A phytogeographical sketch of the ligneous

flora of Formosa. J. Arn. Arbor. 2: 25-41.

Wu, J.E., S. Huang, J.C. Wang, and W.F. Tong. 2001. Allozyme

va riat ion and the gene tic s truc ture of popul ati ons of

Trochodendron aralioided, a mon otypi c and narrow

geographic genus. J. Plant Res. 114: 45-57.

Wu, C.C., Z.F. Hsu, and C.H. Tsou. 2003. S tudies of Eurya

(Ternstroemiaceae) in Taiwan (1), a new endemic species,

Eurya septata. Bot. Bull. Acad. Sin. 44: 67-72.

Yang, H.R. and X. Hs u. 1980. Qua ternary e nvironment al

changes in eastern China. J Nanjing Univ (Natr Sci Series)

1: 121-144. (in Chinese, with English abstract)

Zhao, Z.B. 1982. A preliminary study on the evolution of Taiwan

Strait. Taiwan Strait 1: 20-24. (in Chinese, with English

abstract).

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

113

Appendix 1. Information of collection site of the 146 vouchers listed in Table 1.

Species name

Collection site

Eurya acuminata DC. var. acuminata

Singapore: Tsou 1538*.

Eurya acuminata var. wallichiana Dyer

N. Thailand: Maxwell 2004-233*; Maxwell & Euder 1.

Eurya acuminatissima Merr. & Chun

Hong Kong: Tsou et al. 1813*, 1814.

Eurya amplexifolia Dunn

China - Guangdong: Tsou 1578*.

Eurya chinensis R. Br. var. chinensis

E. chinensis-T

Taiwan - Taipei: Tsou et al. 1269, 1327, 1330*, 1378, 1379;

Nantou: Tsou et al. 1258; Pingdong: Tsou et al. 1250, 1423;

Yilan, Turtle Isl.: Tsou et al. 1720.

Ryukyu - Iriomote Isl.: Tsou et al. 1425.

E. chinensis-C

China ¡V Guangdong: Tsou et al. 1583*; Hong Kong: Tsou et al.

1792, 1794, 1800, 1836, 1837, 1891.

Eurya chinensis var. glabra Hu et L. K. Ling

Taiwan - Yilan, Turtle Isl., Chen, C.C. 7403; Hong Kong: Tsou

et al. 1802*, 1892.

Eurya crenatifolia (Yamamoto) Kobuski

Taiwan - Taipei: Tsou et al. 1253*, 1319, 1381, 1512; Hsinchu:

Tsou et al. 1485, 1487; Taichung: Tsou et al. 1333; Yilan: Tsou

et al. 1604, 1614.

Eurya disticha Chun

China - Guangdong: Tsou et al. 1973*, 1975.

Eurya distichophylla Hemsl

China - Guangdong: Tsou et al. 1577*, 2016; Fujian: Tsou et al.

2113.

Eurya emarginata Makino

Taiwan - Kinmen Isl.: Tsou et al. 1399, 1400*; Taipei: Tsou et al.

1252, 1495.

Eurya glaberrima Hayata

Taiwan - Taoyuan: Tsou et al. 1370, 1374, 1376; Taichung: Tsou

et al. 1414*; Kaohsiung: Tsou et al. 1652; Yilan: Tsou et al.

1601, 1603.

Eurya gnaphalocarpa Hayata

Taiwan - Taichung: Tsou et al. 1331*; Nantou: Tsou et al. 1260,

1864; Yilan: Tsou et al. 1314, 1764.

Eurya groffii Merr.

Hong Kong: Tsou et al. 1890*, 1894.

Eurya hayatai Yamamota

Taiwan - Hsinchu: Su, Mong Huai 227, 282; Taichung: Tsou et

al. 1626*, 1627; Kaohsiung: Su, Mong Huai 109.

Eurya hebeclados Ling

China - Guangdong: Tsou et al. 1944*; Zhejiang: Zhang, Wen Ju,

2000-12-18-c.

Eurya impressinervis Kobuski

China - Guangdong: Ye, Hua Gu 2003-5-30-a*.

Eurya japonica Thunb.

Japan - Nago: Tetsuo Denda 2000-12-6-a*, 2000-12-6-b; China -

Shanghai: Zhang, Wen Ju 2001-9-19-a, 2001-9-19-b.

Eurya laotica Gagnep.

Vietnam - Sapa: Tsou et al. 1620*.

Eurya leptophylla Hayata

Taiwan - Hsinchu: Tsou et al. 1355*, 1356; Nantou: Lin, Chia

Hua 523; Tsou et al. 1456, 1634; Kaohsiung: Tsou 1647;

Taidong: Tsou 1503.

Eurya loquaiana Dunn

Taiwan - Hsinchu: Tsou et al. 1352; Nantou: Tsou et al. 1341;

Yilan: Tsou et al. 1316*.

Eurya macartneyi Charmp.

China - Guangdong: Tsou et al. 1579*; Hong Kong: Tsou et al.

1831, 1839.

114

Botanical Studies, Vol. 48, 2007

Species name

Collection site

Eurya muricata Dunn

China - Zhejiang: Zhang, Wen Ju 2000-12-18-a*, 2000-12-18-b.

Eurya nanjenshanensis (Hsieh, Ling, & Yang) Yang & Lu Taiwan - Pingdong: Tsou et al., 1419*, 1421, 1422.

Eurya nitida Korthals

Taiwan - Taipei: Tsou et al. 1279, 1328.

China - Guangdong: Tsou et al. 1582.

Hong Kong: Tsou et al. 1798, 1806, 1826*, 1868.

Eurya quinquelocularis Kobuski

China - Guangdong: Ye, Hua Gu 2002-6-29-b*, 2002-6-29-e.

Eurya rengechiensis Yamamota

Taiwan - Nantou: Tsou et al. 1342*, 1367; Su, Mong Huai 241,

242.

Eurya rubiginosa Chang

China - Guangdong: Tsou et al. 1580*.

Eurya ryukyuensis Masamune

Ryukyu - Iriomote Isl.: Tsou et al. 1436*; Wang, Chun Neng

1520; Wang, Jenn Che 2003-10-6; Okinawa: Wang, Jenn Che

2003-10-5.

Eurya sakishimensis Hatusima

Ryukyu - Iriomote Isl.: Tsou et al. 1301*, 1442; Wang, Chun

Neng, s.n.

Eurya septata Wu, Hsu, & Tsou

Taiwan - Taipei: Tsou et al. 1470, 1471; Nantou: Tsou et al.

1476, 1477*; Taidong: Tsou et al. 1499.

Eurya strigillosa Hayata

Taiwan - Nantou: Tsou et al. 1335*; Kaohsiung: Tsou et al. 1650,

1623.

Eurya subintegra Kobuski

China - Guangdong: Ye, Hua Gu 2002-6-29-c*; Tsou et al. 1987.

Eurya weissiae Chun

China - Guangdong: Ye, Hua Gu 2002-6-26-b*, Ye, Hua Gu s.n.

Eurya yaeyamensis Masamune

Ryukyu - Iriomote Isl.: Tsou et al. 1282, 1439, 1540*.

Adinandra dumosa Jack

Singapore: Tsou 1539*.

Adinandra elegans How & Ko ex Chang

China - Guangdong: Tsou 1584*.

Adinandra formosana Hayata

Taiwan - Hsinchu: Tsou et al. 1493; Taipei: Tsou 827

(AF089713), Tsou 1546*.

Adinandra lasiostyla Hayata

Taiwan - Nantou: Tsou et al. 1334, 1346*.

Adinanda millettii (Hook. & Arn.) Benth. & Hook. Ex Hance Hong Kong: Tsou et al. 1799*, 1889.

Adinandra yaeyamensis Ohwi

Ryukyu - Iriomote Isl.: Tsou et al. 1303*.

Anneslea fragrance Wall. var. lanceolata Hayata

Taiwan - Pingdong: Tsou et al. 1099*.

Cleyera japonica Thunb.

Ryukyu - Iriomote Isl.: Tsou et al. 1296*, 1446.

Taiwan - Kaohsiung: Tsou et al. 1631.

Cleyera morii Masamune

Taiwan - Taipei: Tsou et al. 858*, 1272.

Euryodendron exclesum Chang

China - Guangdong: Chung-Shan Univ., Tsou et al. 1590*.

Ternstroemia gymnanthera (Wight et Arn.) Sprague

Taiwan - Taipei: Tsou et al. 1098*, 1304.

Ternstroemia kwangtungensis Merr.

China - Guangdong: Tsou et al. 1585*.

Ternstroemia luteoflora Ling

China - Guangdong: Tsou et al. 1587*; Hong Kong: Tsou et al.

1842.

Ternstroemia microphylla Merr.

China - Guangdong: Tsou et al. 1586*.

Appendix 1. (Continued)

WU et al. ¡X Phylogeny and taxonomy of

Eurya

from Taiwan

115

Appendix 2. List of psbA-trnH and trnL-trnF samples and their accession numbers. Vouchers are deposited at HAST (Herabrium,

Academian Sinica, Taiwan)

Species

Accession

number Voucher information

psbA-trnH

Eurya acuminata DC. var. acuminata

AY943244 Tsou 1538, Singapore

Eurya acuminata var. wallichiana Dyer

AY943245 Maxwell 2003-6-13, N Thailand

Eurya acuminatissima Merr. & Chun

AY943246 Tsou et al. 1813, Hong Kong

Eurya amplexifolia Dunn

AY943247 Tsou 1578, Guangdong, China

Eurya disticha Chun

AY943248 Ye, Hua Gu 2002-6-26, Guangdong, China

Eurya distichophylla Hemsl

AY943249 Tsou et al. 1577, Guangdong, China

Eurya emarginata Makino

AY943250 Tsou et al. 1400, Kinmen Isl., Taiwan

Eurya glaberrima Hayata

AY943251 Tsou et al. 1414, Taichung, Taiwan

Eurya gnaphalocarpa Hayata

AY943252 Tsou et al. 1331, Taichung, Taiwan

Eurya groffii Merr

AY943253 Tsou et al. 1890, Hong Kong

Eurya hayatai Yamamota

AY943254 Tsou et al. 1626, Taichung, Taiwan

Eurya hebeclados Ling

AY943261 Zhang, Wen Ju, 2000-12-18-c, Zhejiang, China

Eurya impressinervis Kobuski

AY943255 Ye, Hua Gu 2003-5-30-a, Guangdong, China

Eurya laotica Gagnep

AY943256 Tsou et al. 1620, Sapa, Vietnam

Eurya macartneyi Charmp

AY943257 Tsou et al. 1579, Guangdong, China

Eurya nitida Korthals

AY943258 Tsou et al. 1826, Hong Kong

Eurya quinquelocularis Kobuski

AY943259 Ye, Hua Gu 2002-6-29-b, Guangdong, China

Eurya rubiginosa Chang

AY943260 Tsou et al. 1580, Guangdong, China

Eurya strigillosa Hayata

AY943263 Tsou et al. 1335, Nantou, Taiwan

Eurya subintegra Kobuski

AY943264 Ye, Hua Gu 2002-6-29-c, Guangdong, China

Eurya weissiae Chun

AY943262 Ye, Hua Gu s.n., Guangdong, China

Euryodendron exclesum Chang

AY943269 Tsou et al. 1590, Guangdong, China

Ternstroemia gymnanthera (Wight et Arn.) Sprague AY943270 Tsou et al. 1098, Taipei, Taiwan

Ternstroemia kwangtungensis Merr.

AY943271 Tsou et al. 1585, Guangdong, China

Ternstroemia luteoflora Ling

AY943272 Tsou et al. 1587, Guangdong, China

TrnL-trnF

Eurya emarginata Makino

AY943273 Tsou et al. 1716, Turtle Is., Taiwan

Eurya glaberrima Hayata

AY943274 Tsou et al. 1603, Yilan, Taiwan

Eurya strigillosa Hayata

AY943275 Tsou et al. 1650, Pingdung, Taiwan

Eurya acuminata var. wallichiana Dyer

AY943276 Maxwell 2004-233, N Thailand

116

Botanical Studies, Vol. 48, 2007