全 文 ：　 　第 38 卷　第 1 期
1999 年　1月
中山大学学报 (自然科学版)
ACTA　SCIENTIARUM　NATURALIUM
UNIVERSITATIS　SUNYATSENI
　
Vol.38　No.1
Jan.　1999
　
文章编号:0529-6579 (1999)01-0034-38
The Phylogenetic Relationships Between Mytilarioideae and
the Other Subfamilies of Hamamelidaceae Inferred from
the ITS Sequences of Nuclear Ribosomal DNA

SHI Suhua , HUANG Yelin , ZHANG Qun ,
QU Lianghu , ZHANG Hongda (CHANG Hungta)
(School of Life Sciences , Zhongshan University , Guangzhou 510275)
Abstract:The phylogenetic relationships between Mytilarioideae and the other subfamilies of Hamameli-
daceae based on internal transcribed spacers (ITS)and 5.8S coding region of nuclear ribosomal DNA were
shown in this study.The monophyly of Mytilarioideae including Mytilaria and Chunia was strongly support-
ed , which suggests that Mytilarioideae is an independent subfamily.The ITS data indicated that there was a
sister relationship between Exbucklandioideae and Rhodoleioideae(with 95% bootstrap value supporting),
and Hamamelidioideae and Disanthoideae(with 58% bootstrap value supporting) respectively.All taxa of
Hamamelidaceae sampled in this study formed a monophletic group and Liquidambar formosana was cladisti-
cally basal within the family , which supported the result of our last study in 1997.
Keywords:phylogenetic relationship , Mytilarioideae , ITS sequence
Classification number:Q 949.751.4 , Q 751
The comprehensive classification of the family Hamamelidaceae was proposed by Harms[ 1] .He
recognized the five subfamilies , i.e., Disanthoideae , Hamamelioideae , Rhodoleioideae , Bucklan-
dioideae (=Exbucklandioideae)and Liquidambaroideae (=Altingioideae).This classification was
adopted by several authors(with minor changes), such as Vink[ 2] and Bogle[ 3] .However , Chang[ 4]
separated the subfamily Exbucklandioideae into two subfamilies:Mytilarioideae for Mytilaria and Chu-
nia , the latter was the new genus described by Chang[ 5] in 1948 , and Exbucklandioideae for only
Exbucklandia.Endress[ 6] provided an emended classification of the family with four subfamilies ,
which combined Exbucklandioideae , Mytilarioideae , and Disanthoideae into the subfamily Exbucklan-
dioideae.The objectives of this study were to reconstruct the phylogenetic relationships between Myti-
larioideae and the other subfamilies of Hamamelidaceae based on the divergences data of ITS se-
quences , and to discuss if the subfamily Mytilarioideae is an independent subfamily.
1　Materials and methods
1.1　DNA isolation and PCR amplification
Eight taxa representing four subfamilies and an outgroup were sampled in this study (Tab.1).
The sequences of ITS regions of Hamamelis mollis and Liquidambar formosana [ 7] , which representing
基金项目:国家自然科学基金 (39570052 , 39970057);广东省自然科学基金 (970190)
收稿日期:1998-10-15　　第一作者简介:施苏华 , 女 , 42 岁 , 教授
the subfamilies Hamamelioideae and Altingioideae respectively , were used in this study.Total DNAs
were extracted with the CTAB method of Doyle and Doyle[ 8] followed by purification with DPS (DNA
purification system)kit(made by our laboratory).DNA amplification was performed in 2×60μL re-
actions following Wen and Zimmer[ 9] .The PCR products of ITS were purified with the centrifugal filter
units(UFC3LTKNB , Millipore).
Tab.1　Accessions of Hamamelidaceae and an outgroup Platanus sampled for the ITS studies1)
Species Subfamily Voucher Geographical origin
Chunia bucklandioides Mytilarioideae Zhou/974301 Jianfengling ,
Hainan , China
Disanthus cercidifolius
var.longipes Disanthoideae Hao/974001 Cultivated in ZhongshanBotanical Garden , China
Exbucklandia populnea Exbucklandioideae Chen/9703056 Cultivated in South China
Botanical Garden , China
Exbucklandia tonkinensis Exbucklandioideae Chen/9703057 Cultivated in South ChinaBotanical Garden , China
Mytilaria laosensis Mytilarioideae Chen/9703054 Cultivated in South China
Botanical Garden , China
Mytilaria laosensis Mytilarioideae Shi/9704118 Heishiding ,Guangdong , China
Platanus acerifolia Platanaceae Shi/974338 Cultivated in Wuhan
Botanical Garden , China
Rhodoleia championii Rhodolioideae Xing /6742 Dawushan ,Hong Kong , China
　　　1)The classification system was based on Chang H T (1979)
1.2　Sequencing of the PCR products
The ITS regions were sequenced following the dideoxy chain termination method of Sanger
[ 10]
us-
ing the Sequenase Version 2.0 DNA sequencing kit(US70770 , Amersham)and alpha 35S-dATP as a
radioactive tracer.C5.8S , ITS4 , N5.8S andN18L18[ 9] were used as sequencing primers to obtain the
entire ITS and 5.8S regions from both directions.Products of the sequencing reactions were separated
on a 6%polyacrylamide gel at 50 W.Gels were transferred to 3mm Whatman paper , dried in an oven
at 45 ℃for 3 hours , and exposed to an X-ray film(Kodak XAR)for 3 ～ 4 days at room temperature.
About 320 nucleotides were obtained per sequencing run.
1.3　Sequence analysis and phylogenetic reconstruction
The DNA sequences were assembled and the boundaries between the coding and spacer regions
were determined by comparison with the sequences of carrot (Daucus carota[ 11]).The assembled se-
quences were aligned using CLUSTAL X[ 12] .
Phylogenetic analyses were performed using PAUP 3.1.1[ 13] .The amount of support for mono-
phyletic groups revealed in the most parsimonious tree(MPT)was examined with 100 bootstrap repli-
cates with the random addition and the heuristic search options.The phylogenetic tree was rooted using
Corylus chinensis (Betulaceae)and Platanus acerifolia (Platanaceae)as the complex outgroup.
2　Results and discussion
The total length of the ITS1 , 5.8S and ITS2 regions of Mytilaria , Chunia and Exbucklandia are
672 , 684 and 680 ～ 687 bp respectively.The Kimura two-parameter distances are presented in Tab.2.
The maximum parsimony analysis generated two MPTs with a total length of 1 173 steps , a consistency
index(CI)of 0.743 , a homoplasy index (HI)of 0.257 , a retention index (RI)of 0.542 and a
35第 1 期　　施苏华等:壳菜果亚科(金缕梅科)与相关亚科的核糖体 DNA ITS区序列分析及系统发育关系
rescaled consistency index (RC)of 0.403 , treating gaps as missing data.The strict consensus tree
from the two MPTs is shown in Fig.1.
Tab.2　Kimura two-parameter sequence divergence values of taxa in Mytilarioideae and the other subfamilies
of Hamamelidaceae.Below the diagnoal are the absolute distances;and above the diagonal are the
Kimura two-parameter distances.Gaps were eliminated from the comparison
　　Species 1 2 3 4 5 6 7 8 9 10 11
P.acerifolia213 - 0.425 0.396 0.399 0.380 0.380 0.372 0.377 0.379 0.396 0.423
Cor.chinensis 321 - 0.393 0.383 0.364 0.370 0.371 0.375 0.376 0.381 0.407
H.mollis58 299 297 - 0.240 0.250 0.262 0.257 0.249 0.250 0.262 0.327
D.cercidifolius153 301 289 181 - 0.208 0.228 0.229 0.238 0.238 0.244 0.322
E.populnea124 287 275 189 157 - 0.032 0.164 0.215 0.216 0.232 0.310
E.tonkinensis125 287 279 198 172 24 - 0.172 0.226 0.228 0.240 0.315
R.championii110 281 280 194 173 124 130 - 0.233 0.234 0.242 0.314
M.laosensis123 285 283 188 180 162 171 176 - 0.001 0.131 0.298
M.laosensis140 286 284 189 180 163 172 177 1 - 0.132 0.299
C.bucklandioides157 299 288 198 184 175 181 183 99 100 - 0.319
L.fomosana63 319 307 247 243 234 238 237 225 226 241 -
　P .acerifolia213
　Cor.chinensis
　L.formosana65
　H.mollis58
　D.cercidifolius153
　C.bucklandioides157
　M.laosensis123
　M.lasensis140
　R .championii110
　E .populnea124
　E .tonkinensis125
175
171
79
99
54
80
43
95
35
58
101
80
53
100
8
16
65
61
100
43
100
0
1
56
132
Fig.1　The strict consensus tree of the two most
parsimonious trees of Mytilarioideae and
the other subfamilies of Hamamelidaceae
based on the ITS sequences of nuclear ri-
bosomal DNA(1 173 steps ,CI=0.743 ,RI
=0.542 , RC = 0.403).The tree was
rooted using Corylus chinensis and Pla-
tanus acerifolia as the complex outgroup.
Bootstrap confidence values(100 repli-
cates) are given below the branches ,
whereas the branch lengths are indicated
above the branches.
2.1　The phylogenetic relationships between Mytilarioideae and the other subfamilies
In the strict consensus tree(Fig.1), the monophyletic group of Mytilarioideae that includes Myti-
laria and Chunia was shown(the bootstrap value is 100%).A sister group , i.e., Hamamelidioideae
and Disanthoideae , was suggested , but not very strong (58%)bootstrap analysis supported.However
they had a long branch length respectively (101 in Hamamelidioideae and 80 in Disanthoideae , see
Fig.1).This result agreed to the morphological results of Huang[ 14] and Pan et al[ 15] , which suggested
that Disanthoideae was more closely related to Hamamelidioideae than the other subfamilies based on
the characters of paracytic stomatal apparatuses and pentamerous flowers.
It should be indicated here because no any samples of Exbucklandia was compared in our former
publication① , the wrong Exbucklandia and Hamamelioideae formed a rather weak clade (50%boot-
strap value supported)in our former publication[ 7] .After the data of the two species of the Exbucklan-
36 中山大学学报 (自然科学版)　　　　　　　　　　　　　第 38 卷
① The species Exbucklandia tonkinensis in our last publication[ 7] was sampled by mistake.It should be the
species Mytilaria laosensis according to our present experimental tests.
dia were analyzed in this study , the strict consensus tree showed that a sister group relationship be-
tween the Exbucklandioideae and the Rhodoleioideae was strongly supported(95%)by the bootstrap
analysis(Fig.1).This result agreed with Pan et al[ 15] , who indicated that Exbucklandioideae and
Rhodoleioideae had the same cyclocytic stomatal apparatuses.The ITS data of this study also suggested
that all taxa of Hamamelidaceae sampled here formed a monophyletic group and the genus Liquidambar
was cladistically basal within the family(99%), which is the same as our former study[ 7] .There are
the sister relationships among the clades of Hamamelidioideae and Disanthuoideae , Mytilarioideae , and
Exbucklandioideae and Rhodoleoideae supported by the bootstrap value of 80%.
2.2　Mytilarioideae is an independent subfamily
Controversies concerning the classification of Mytilaria and its phylogenetic position have been
existing for a long time.Harms[ 1] recognized the five subfamilies.However , the position of the Myti-
laria was insufficiently known at Harms time.Chang[ 5] segregated Mytilaria and Chunia in the new
(and six)subfamily Mytilarioideae , leaving only Exbucklandia in Exbucklandioideae.Huang and
Lee[ 16] supported the segregation on the basis of wood anatomy.Takhtajan[ 17] , on the other hand , list-
ed subfamily Chunioideae (rather than Mytilarioideae)as one of the six subfamilies in Hamameli-
daceae , but did not indicated which of the three genera of Exbucklandioideae he would assign to it or
his reasons for removing them from the Exbucklandioideae.Pan et al[ 15] and Pan and Yang[ 18] support-
ed the treatment of Mytilaria as an independent subfamily on the features of stephanocytic stomatal ap-
paratuses and the basic chromosome number(χ=13).In this study the phylogenetic analysis based on
the ITS data supports strongly that Mytilarioideae is considered as an independent subfamily.
Endress[ 7] recognized that the genera of Mytilaria , Chunia , Exbucklandia and Disanthus
seemed to form a good natural group by many morphological charactersistics.So he combined those four
genera (three subfamilies)into the subfamily Exbucklanioideae.Bogle[ 3] indicated that the fact of
Chunia shared multilacunar nodal anatomy with Mytilaria provided evidence to support the close rela-
tionship between the two genera implied by Chang.However , on the basis of other morphological evi-
dence , he would prefer to see Chunia and Mytilaria maintained within the subfamily Exbucklan-
dioideae as a tribe Mytilareae rather than segregated as a subfamily Mytilarioideae.The phylogenetic
analyses based on ITS sequence data in this study shows that there are the high level divergences a-
mong the Disanthus , Exbucklandia , and Mytilaria and Chunia(see the branch lengths in Fig.1 and
the Kimura two-parameter distances in Tab.2)and they are in the different clades respectively with
strong bootstrap values supporting (Fig.1).Therefore the data of ITS sequences in this study do not
support the treatment of combining those four genera into the subfamily Exbucklandioideae.
Acknowledgement　This work was supported by the National Natural Foundation of China(39570052 , 39970057),
the Natural Science Foundation of Guangdong Province(970190).We are grateful to Liu Nian and Xing Fuwu at South
China Botanical Garden , and the South China Institute of Botany , and Zhou Tiefeng at Jianfengling Natural Reserve ,
Hainan Province for providing leaf tissues.Special thanks to Dr.Kim Youngdong for helping to align the sequence data ,
and Professor Zhong Yang for revising the manuscrit.
References:
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mann:Leipzig , 1930.303～ 343
[ 2] 　VINK W.Hamamelidaceae.In:VAN STEENIS C G G J ed.FloraMalesiana Ⅰ .Groningen:Nijhoff , 1957.363～
379
[ 3] 　BOGLE A L.A second multilacunar nodal type in Hamamelidaceae.Harvard Paper , 1991(3):65～ 70
37第 1 期　　施苏华等:壳菜果亚科(金缕梅科)与相关亚科的核糖体 DNA ITS区序列分析及系统发育关系
[ 4] 　CHANG H T.A revision of the hamamelidaceous flora of China.Acta Sci Nat Univ Sunyatseni(in Chinese), 1973
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[ 12] 　THOMPSON J D, GIBSON T J , PLEWNIAK F.The CLVSTAL_X windows interface:flexible strategies for multiple
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壳菜果亚科(金缕梅科)与相关亚科的核糖体
DNA ITS区序列分析及系统发育关系
施苏华 ,黄椰林 ,章　群 , 屈良鹄 , 张宏达
摘　要:测定和分析了金缕梅科(Hamamelidaceae)壳菜果亚科等各亚科代表属的核糖体 DNA
ITS区和 5.8S 编码区序列.应用最大简约法构建的分子系统树表明:壳菜果亚科 (Mytilari-
oideae)植物形成 1个单系类群[含壳菜果属(Mytilaria)和山桐材属(Chunia)] ,支持壳菜果亚
科作为1个独立的亚科.本研究所得到的 ITS 数据表明马蹄荷亚科(Exbucklandioideae)与红
花荷亚科(Rhodoleioideae),金缕梅亚科(Hamamelidioideae)与双花木亚科(Disanthoideae)分别
为姐妹群关系(其自展数据支持率各为 95%和 58%).分子系统树还表明 ,测得的所有金缕
梅科植物形成一单系类群(其自展数据支持率为 99%),枫香亚科(Liquidambaroideae)为该科
最基础的 1个分支.这结果与 1997年所得到的结论相一致.
关键词:系统进化关系 ,壳菜果亚科 , ITS区
分类号:Q 949.751.4 ,Q 71　　文献标识码:A
38 中山大学学报 (自然科学版)　　　　　　　　　　　　　第 38 卷
中山大学生命科学学院 , 广州 510275