全 文 ：热带亚热带植物学报 2004，12(5)：393-398
Journa／of Tropical and Subtropical Botany
基于叶绿体DNA trnL内含子序列数据的檀香目
科间系统发育关系的研究 ·
韩荣兰， 郝 刚， 张奠湘‘
(中国科学院华南植物园，广东广州510650)
摘要：应用叶绿体DNAtrnL内含子序列分析檀香目科间的系统发育关系。取样研究的檀香目个体的trnL内含子序列
长度在科间呈现较大差异 (从291 bp到587 bp)。最大简约性分析产生的严格一致树与以前已发表的基于其它基因
的檀香目的分子系统学研究结果大体一致。香芙木属(铁青树科)是最早分支出的类群：桑寄生科、槲寄生科分别表现
为单系类群，檀香科为并系；桑寄生科和槲寄生科并不具密切亲缘关系，槲寄生科从檀香科内衍生出来。本研究表明，
具相对高的核苷酸替换率的叶绿体DNA trnL内含子序列可为高等级类群系统发育关系的研究提供更多的信息位点。
关键词：檀香目；桑寄生科：铁青树科；山柑科；檀香科；槲寄生科；trnL；系统发育关系 ’
中图分类号：949．741．01 文献标识码：A 文章编号：1005-3395(2004)05-0393-06
Interfamilial Relationships of Santalales as Revealed by
Chloroplast trnL Intron Sequences
HAN Rong一1 an， HA0 Gang， ZHANG D i an—x i ang,
(South China Botanical Garden,the Chinese Academy of Sciences，Ouangzhou 510650，China)
Abstract： Interfamilial relationships of Santalales were investigated using chloroplast intron sequences． The
lengths oftmL intron regions present considerable variation among the sampled Santalalean families，varying from
29 1 to 587 bp．The topology of strict consensus tree generated from parsimony analysis is largely congruent witll
trees previously published based on DNA sequences of other genes，which revealed the basal positon of&hoepfia
(Olacaceae)，the knonophyly of Loranthaceae and Viscaceae，and the paraphyly of Santalaceae．Loranthaceae are
distinct from Viscaceae，the latter is derived from within Santalaceae．Our study also demonstrates the utility of the
rapidly evolved chloroplast trnL intron for addressing relationships among the component taxa of Santalales． 。
Key words：Santalales；Loranthaceae；Olacaceae；Opiliaceae；Santalaceae；．Viseacea~tmL；Phylogenetic relationship
As traditionally defined, the order Santalales
consist of seven families：Eremolepidaceae，Loranth·
aceae， Misodendraceae， Olacaceae， Opiliaceae，
Santalaceae and Viscaceae t 1， and form a wel1．
supported clade in several broad phylogenetic analyses
of angiosperms[2-41． Santalales represent the most
diverse assemblage of life forms among angiosperms
as it includes members ranging from nonparasites to
hemiparasites， and from root parasites to aerial
parasites(mistletoes)．
Whereas the monophyly of Santalales is strongly
supported, the interfamilial relationships oftlle order
remain unclear． In the past decades phylogenetic
relationships of Santalales have been addressed by
diferent authors[~1(see review in Nickrent et a1． ．
Until recentl5 molecular phylogenetic investiga-
Recelved：2003-1O-16 Accepted：2004-02-16
Foundation item：Supported by the Field Frontiers Project(Director Foundation ofSouth China Institute ofBotany)and Knowledge Innovation
Project(kscxz-sw-101 A)ofTheChiueseAcademyofSciences． j
Co~esponding author
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394 热带亚热带植物学报 第 12卷
tions have been attempted to clarify phylogenetic
relationships among genera and families of Santalales
using nuclear 1 8S and plastid rbcL sequences~Ⅷ．The
most recent study【 】obtained 1 8S and rbcL sequences
from 66 of the 155 genera in the order； the strict
consensus parsimony tree was generally congruent
with trees previously reported~Ⅷ．which indicated the
monophyly ofOpiliaceae，Loranthaceae，and Viscaceae，
and the paraphyly of Santalaceae(including Eremole—
pidaceae)and Olacaceae．Nevertheless，support for
interfamilial relationships was moderate or low， thus
greater sampling of taxa and genes is needed to reveal
more robust phylogenetic relationships in the order．In
the present study we sequenced a more rapidly
evolving region，trnL intron of chloroplast DNA，and
sampled more taxa mainly from China， which were
poorly represented in the previous studies， to provide
additional phylogenetic signal， as a complement to
studies undertaken by Nickrent an d his collaborators．
1 Materials and methods
Plant sampling Of the seven traditionally
recognized families[~，five were sam pled in the present
study，e．g．，Loranthaceae(6 genera)，Opiliaceae(1
genus)，Olacaceae(1 genus)，Santalaceae(4 genera)，
and Viscaceae(2 genera)．Overal，27 accessions of
trnL intron sequences were acquired， while other 6
accessions were retrieved from GenBank． In previous
global analyses of angiosperms using multiple genes[4]，
Santalales appears(unresolved)atthebase ofthe core
eudicots． It is un clear at present which taxon is the
sister group to San talales； in the present study two
species ofA lnus(Betulaceae，Fagales)were designated
as outgroups【”． ·
Genomic DNA extraction,PCR amplification,
and sequencing Total DNA was extracted from
fresh or silica dried leaves， following the method of
Doyle and Doyle【 ”．Th e trnL intron was amplified by
the polymerase chain reaction (PCR) wim primers
“c’ an d “d’ of Taberlet et ai r习．Th e PCR products
were purifed using QIAquick Gel Extraction Kit
(QIAGEN)． Sequencing reactions were performed
using the dye-terminator cycle-sequencing ready-
reaction kit following the manufacturer’s protocol，an d
analyzed on an ABI 377 Automated DNA Sequencer
(Applied Biosystems)．
Phylogenetic analysis Sequences of the trnL
intronwere preliminarilyalign edwithClustalXⅡ an d
then manualy adjusted to accommodate indel events
otherwise not properly recognized． Mul邱le
alignments applying diferent parameters in Clustal X
have been explored to investigate their consequence
on phylogenetic inference． Th e lengths of trnL intron
regions present considerable variation among the
sampled fam ilies of San talales：samples of Loran th—
aceae un exceptionally have shorter sequences，ranging
from 291 to 356 bp，while the remaining samples of
Santalaleshavelongerones,rangingfrom443to 587bp．
Th e alignment was thus dificult due to the great
length diferences．When introducing indels，Clustal X
always placed a long deletion(up to 267 bp)in the end
part ofalignment matrix for the Loranthaceae samples，
which was apparently incorrect． Th e alignment thus
needed careful manual adjustment， and we used the
software of Se—AI Sequence Alignment Editor to
properly place gaps． In addition to minimizing the
number of informative characters(indels and su}sti—
tutions)， other criteria of alignment and mutational
interpretation outlined by Oxelman et a1．【 an d
Sim ons and Ochoterena[ were adopted．Potentially
inform ative and un am biguously assessed indels of
trnL region were scored as binary characters(1 for
insertion，0 for gap) regardless of their length and
added to the data matrix(61 such characters in tota1)．
M aximum parsimony an alysis was perform ed on
the data matrix using PAUP v4．0b8目q．Th e analysis
used heuristic searches with random addition and TBR
bran ch swapping． Clade robusmess was evaluated by
bootstrap analysis[ J7】using 1 000 replicates of heuristic
searches， with simple addition sequence an d TBR
bran ch swapping．
2 Results and discussion
2．1 Sequence characteristics of the trnL intron
All the newly acquired sequences have been
submited to GenBank (Table 1)．The lengths ofthe
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第 5期 韩荣兰等：基于叶绿体DNA trnL内含子序列数据的檀香目科间系统发育关系的研究 395
unaligned trnL intron fragments range from 29 1 to
587 bp； the aligned sequences had 766 positons．
These data resulted in uncorrected pairwise sequence
divergence ranged from 0．6％ (between Cansjera
rheedi 1 and C rheedi 2、 to 59．7％ (between
Konhalsel~ coraplanata and Schoepfia jasminodora)
among the ingroup (distance matrix not shown)．
W in major groups(families)，distances ranged from
1．0％ to 30．2％ in Loran thaceae． from 1．1％ to 38．4％
in Viscaceae．and from 0．7％ to 21．8％ in Santalaceae．
While the chloroplast trnL intron was、 dely
used to resolve phylogenetic relationships at the
species or among closely related genera[12,18-2q． in
some studies the intron was employed to reveal higher
level relationships in some large fam ilies or orders
(e．g．，am ong orders and families of asteridst2 ；am ong
tribes of Asteraceae[221； among palm tribes[231
，
．
among genera of Acan thaceae口棚： am ong genera of
Leguminosaet2 司)，although these studies always also
included sequences of廿1e adjacent and leSS conserved
Table 1 Samples used in the phylogenetic analysis of Santalales．GenBank accession numbers marked、 th ‘’’were relrieved liom
thedatabase，andthe othersWCle acquiredbythepresent study
“
一 ”notavailfible
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396 热带亚热带植物学报 第 l2卷
region of trnL·F intergenedc spacer． In the study of
Legurninosae using the trnL intron ． pairwise
distances among the three subfamilies vary from 7％
to 9％，while within major groups(subfamilies，tribes，
subtribes)distances range from 1．8％ to 5-3％．While
the patterns of substitution rate variation across
organellar gene loci are complex and rate acceleration
is not。a general evolutionary feature of Santalales[2]，
the present study demonstrates mat in the trnL intron
of plastid genome， rate acceleration has occurred in
the hemiparasitic San talales relative to autotrophic
plants．
2．2 Phylogenetic relationships
Heuristic search ofparsimony analysis ofthe trnL
intron data generates 3 equally most parsimonious
trees， m a length of 888 steps，CI=0．72(excluding
uninformative characters)．and RI=0．87．In the strict
consensus tree (Figure 1)，Schoepfia jasminodora
(Olacaceae)is placed at the basal position，sister to the
clade containing the remaining taxa of Santalales．The
later clade furtherm ore consists of two subclades．one
for Loranthaceae，the other for SantalaceaeNiscaceae／
Opiliaceae．
The strict consensus (Figure 1) of trnL intron
Viscum multinerve
Viscum articulatum
Viscum liquidambatfcolum
Vsicumsp．
Viscum ovalifolium
Viscum album
Viscum cruciatum
Korthalsela complanata
Korthalselajaponica
Korthalselalindsayi
Korthalsela papuana
Dendrotrophe polyneura
Santalum papuanum
Santalum album
OsyHs wightiana
Pyrulafia sinensis
Cansjera rheedi 1
Cansjera rheedi2
Scurrula parasitica 2
Scurrula parasilica 1
Taxilus sutchuensis
Scurrula sootepensis
Scurrula sp．
Scurrula notothixoides
Scurrula chincji
Helixanthera pierrei
Taxi#us chinenMs
Helixanthera parasitica
Macrosolen cochinchinensis
Dendrophthoe pentandra
Loranthus delavayi2
Loranthus delavayi 1
Schoepfia iasrninodora
Alnus glutinosa
Alnus firma
Viscaceae
Santalaceae
Opiliaceae
I Olacaceae
l Outgroups
Fig·l Strict consensus tree of the three most parsimonious trees from tmL intron sequences of Santalales species
．
Tree length 888 steps，CI 0．72，ILl 0．87．Numbers above lines represent bootstrap values in 1000 replicates
．
Familial classifcation system folows Kuijt ．Scum~ parasitica l，Scurrula parasiticavat．parasitica；
Scum~ parasitica2，Scurrula p~askicavar
．gr~iliflora．
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第5期 韩荣兰等：基于叶绿体DNA trnL内含子序列数据的檀香目科间系统发育关系的研究 397
sequences is topologically congruent with trees
previously published【。工旧． Monophyly of Santalales
is strongly supported (1 00％)． Santalales minus
Schoepfia (Olacaceae) are monophyletic with hi gh
bootstrap support (89％)．In previous studies【】一，
howeve~Schoepfia is not closely related to Olacacea~
it is sister to the mistletoe genus Misodendrum
(Misodendraceae)， and this clade is in turn sister to
Loranthaceae， although these relationships received
only moderate support． The limited sampling of
Olacaceae in this study prohibited the test of
paraphyly ofthis family,but revealedthatSchoepfiais
the first-branching member of Santalales sampled in
this study．
Loranthaceae,in the present study composed of
five genera， appears as a monophyletic group，
although the support is low(BS<50％)．Loranthaceae
minus Loranthus delarayi (two accessions) are
monophyletic with high BS support(95％)．The genera
Helixanthera an d Taxillus r、 th two species sam pled
each)are scatered in diferent clades，their monophyly
awaits further studies．Contrastingwith the hypothesis
ofBhandari and Vohra【9】’ and consistent with that of
KuijtM and Wiens and Barlow[sj，Loranthaceae are
distinct from Viscaceae．
Analyses of 18S rDNA and rbcL sequences[。2。01
show that San talaceae are not monophyletic but a
grade that culminates in Viscaceae． The present study
obtained a similar evolutionary patern (Figure 1)．
Carujera rheedi appearedasthe sisterto Santalaceae／
Viscaceae，although this relationship had low support
(BS<50％)． Sam ples of Viscaceae (two genera)
constitute aclade thfulbootstrap support．andwere
derived from within Santalaceae， with Dendrotrophe
polyneura (Santalaceae)as its sister，suggesting the
paraphyly of San talaceae．
As with previous studies， the present study fails
to resolve a monophyletic Santalaceae．This study also
demonstrates the utility of the rapidly evolved
chloroplast trnL intron for addressing relationships
am ong the component taxa of Santalales． Further
studies， particularly using greater sampling of taxa，
wil be required before the phylogeny and evolution of
this group ofparasitic plan ts is resolved．
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