全 文 ：Circumscriptions and Phylogenetic Relationships of Primula Sects.Auganthus
and Ranunculoides :Evidence from nrDNA ITS Sequences
HAO Gang
1 , HU Chi_Ming1 ＊ , LEE Nam_Sook2
(1.Center for Systematic and Evolutionary Botany , South China Institute of Botany , The Chinese Academy of Sciences , Guangzhou 510650, China;
2.Department of Biology , Ewha Womans University , Seoul 120_750, Korea)
Abstract:　The circumscriptions of sections Auganthus Pax ex Balf.f.and Ranunculoides Chen et C.M.
Hu in the genus Primula L.have been controversial , due to the different treatments of P .filchnerae Knuth.
We conducted a phylogenetic analysis using sequences from the ITS region of nuclear ribosomal DNA.All five
species of these two sections and some representatives of other related sections were sampled.The ITS trees
show that P .filchnerae could be convincingly placed in sect.Auganthus , together with P.sinensis Sabine ex
Lindl.and P .rupestris Balf.f.et Farrer , whereas sect.Ranunculoides contains two species:P.cicutari-
ifolia Pax and P.merrilliana Schltr.These two sections are distantly related and their association with other
sections needs further studies.This study also demonstrated that ITS sequences would be a suitable marker for
the reconstruction of Primula phylogeny.
Key words:　Primula;sect.Auganthus;sect.Ranunculoides;Phylogeny;ITS;Primulaceae
　　Primula L., with ca.500 species , is the largest
genus of Primulaceae
[ 1] .It is mainly distributed in the
temperate and arctic areas of the northern Hemisphere ,
with only a few outliers on the mountainous regions of Af-
rica (Ethiopia), tropical Asia (Java and Sumatra), and
South America.About 300 species are native to China ,
with the centre of diversity in western Sichuan , eastern
Xizang , and northwestern Yunnan.Many species of
Primula are cultivated for their attractive flowers as pot
plants , in rock gardens , or in garden borders.
The first comprehensive taxonomic study of Primula
was completed by Smith and Fletcher
[ 2] , who presented a
31_section system , which was a revision of an earlier 32_
section system of Smith and Forrest
[ 3] .In an attempt to
clearly interpret the phylogenetic relationships , Wende-
lbo
[ 4]
proposed an infrageneric classification by assigning
Smith and Fletcher s 31 sections into seven subgenera.
Six of the seven subgenera consist of a few morphologically
cohesive sections , respectively;the remainder of all those
uncertain sections , however , were included in a large ,
heterogeneous subgenus Aleuritia Wendelbo.Richards[ 5]
modified Wendelbo s system and recognized the six sub-
genera.In the revision of Chinese Primulaceae , Hu[ 1] de-
limited 24 sections , but the subgenus concept was not
adopted.
Traditionally the morphological characters employed
in section delimitations of Primula were those of the inflo-
rescence , farinose , leaf hairs , bracts , etc.The evolu-
tionary trends of these characters , however , are somewhat
less clear in this genus.Different viewpoints may result in
conflict taxonomical treatment.This situation was well re-
flected in sections Auganthus Pax ex Balf.f.and Ra-
nunculoides Chen et C.M.Hu.
In Smith and Fletcher′s[ 6] monograph , sect.Augan-
thus includes two species , P .sinensis Sabine ex Lindl.
and P.rupestris Balf.f.et Farrer , and is characterized
by plants pubescent , leaves palmately lobed , lobes not
reaching midvein and calyx inflated at base;sect.Pin-
natae Knuth includes four species:P.cicutariifolia Pax ,
P.filchnerae Knuth , P .merrilliana Schltr.and P.
erodioides Schltr.(subsequently reduced to P.cicutari-
ifolia by Hu
[ 1]), with the diagnostic characters of plants
glabrous or pubescent , leaves pinnately lobed to midvein ,
and calyx not inflated at base.The character state of calyx
for P.filchnerae , however , was incorrectly defined ,
largely because of the meager materials in the period of
Smith and Fletcher
[ 6] .Hu and Kelso[ 7] rediscovered this
species in Sichuan.Based on morphological characteris-
tics(plant pubescent , calyx inflated at base , etc.)and
observation under cultivation , he consequently transferred
it into sect.Auganthus.As P.filchnerae is the type of
the section Pinnatae , a new sectional name , Ranuncu-
loides Chen et C.M.Hu , was proposed instead of Pin-
natae.This treatment was followed by Halda[ 8] .Howev-
er , Richards[ 5] still agreed with Smith and Fletcher[ 6] that
P.filchnerae is a rather clear member of section Pin-
natae.
It is apparent that additional studies are needed with
new data taken into account for the sectional integrity in
Primula.In the present study we use sequence compari-
son of the rapidly evolving ribosomal internal transcribed
spacer(ITS)region to assess the monophylies of sects.
Auganthus and Ranunculoides.Species of these two sec-
tions were chosen as a core of taxa , as well as some other
Received:2000-12-11　Accepted:2001-04-17
Supported by the National Natural Science Foundation of China(39970053), the Chinese Academy of S ciences(KSCX2_1_06B), and Korea Research Foundation
(KRF_1999_015_DI 0085).
＊Author for correspondence.E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(1):72-75
related species , to evaluate their putative systematic posi-
tions.This study can provide references for the recon-
struction of the infrageneric phylogeny of genus Primula.
1　Materials and Methods
All five species of sects.Auganthus and Ranuncu-
loides were sampled in this study (Table 1).Their affini-
ties with other sections are unknown , insofar no phyloge-
netic framework of Primula is available.Putative close
relatives include sects.Monocarpicae Franch.ex Pax ,
Obconicolisteri Balf.f., Malvacea Balf.f., Proliferae
Pax , Primula , Aleuritia Duby and Cortusoides Balf.f.
Five more species representing five sections were also in-
cluded in this study.They are P.malacoides Franch.(sect.Monocarpicae), P.obconica Hance (sect.Ob-
conicolisteri), P.veris L.(sect.Primula), P.pulver-
ulenta Duthie (sect.Proliferae), and P .farinosa L.(sect.Aleuritia).Sequence of P .farinosa was acces-
sioned from Genbank.For P.sinensis , two accessions
were sampled to assess infraspecific variation.Androsace
paxiana Knuth was selected as the outgroup.Recent mo-
lecular studies
[ 9 , 10]
have confirmed the close relationships
between Primula and Androsace .In this study ITS region
of A.paxina was readily alignable with those from Prim-
ula species.In addition , a midpoint rooting approach
with exclusion of A.paxiana did not affect the resulting
ingroup tree topology.　　Total DNA was extracted from fresh or silica_gel
dried leaves , following the method of Doyle and
Doyle
[ 11] . Double_strand polymerase chain reaction(PCR)was applied to amplify the entire ITS region , us-
ing universal primers “ ITS 5” and “ ITS 4”[ 12] .PCR
product was purified using the QIAquick Gel Extraction
Kit (QIAGEN).Automated sequencing was performed
using fluorescent dye_labeled nucleotides on an ABI 377
DNA Sequencer , using at least two of the four primers
N18L18
[ 13] , ITS2 , ITS3 and ITS4[ 12] .
The combined sequences of ITS1 , 5.8S and ITS2
were aligned using Clustal W
[ 14] , adjusted manually
where necessary.Maximum parsimony analyses were per-
formed by PAUP version 3.1.1[ 15] using a BRANCH AND
BOUND search with MULPARS “on” and furthest addit-
ion sequence options.Bootstrap analysis was conducted
with 500 replicates to assess relative support for the differ-
ent clades within each tree.
2　Results
The length of the entire ITS region in Primula spe-
cies surveyed ranges from 618 to 655 bp;the 5.8S region
is consistently 163 bp long , whereas the ITS1 and ITS2
regions range between 233-261 and 220-231 bp , re-
spectively.P .merrilliana has a notably long deletion of
18 bases in ITS1 region.The two accessions of P.sinen-
sis are identical in sequences.The aligned ITS sequence
matrix is 666 bp in length , of which 267 sites are vari-
able.There are 104 phylogenetically informative sites in
ITS1 , 2 in 5.8S , and 65 in ITS2.
The equally weighted parsimony analysis resulted in
two most parsimonious trees of 495 steps , with a CI of
0.77(0.70 excluding uninformative characters)and an
RI of 0.73.The strict consensus tree (Fig.1)is a di-
chotomy:one clade , with moderate bootstrap support(71%), furthermore includes two subdivisions.In the
first subdivision (supported at 65%), the two accessions
of P.sinensis , P .rupestris and P.filchnerae comprise
a well supported (100%)clade , which in turn becomes
Table 1　Taxa included in the molecular study of Primula
Taxon　　　　 Source voucher GenBank Accession No.
sect.Auganthus Pax ex Balf.
　P.rupestris Balf.f.et Farrer Yichang , Hubei;Hao Gang 210 AF323697
　P.sinensis Sabine ex Lindl.(1) Guanxian , sichuan;Hu Chi_Ming s.n. AF323695
　P.sinensis Sabine ex Lindl.(2) Chongqing;Hao Gang 216 AF323696
　P.filchnerae Knuth Chongqing;Hao Gang 214 AF323702
sect.Ranunculoides Chen et C.M.Hu
　P.cicutariifolia Pax Shitai , Anhui;Guo Xin_Hu 200011 AF323703
　P.merrilliana Schlt r. Ningguo , Anhui;Guo Xin_Hu 200026 AF323704
sect.Monocarpicae Franch.ex Pax
　P.malacoides Franch Chongqing;Hao Gang 215 AF323700
sect.Obconicolisteri Balf.f.
　P.obconica Hance Yichang , Hubei;Hao Gang 211 AF323698
sect.Primula
　P.veris L. Chongqing;Hao Gang 217 AF323701
sect.Proliferae Pax
　P.pulverulenta Duthie Guanxian , Sichuan;Hao Gang 230 AF323699
sect.Aleuritia Duby
　P.farinosa L. 　　　　　- AF164014
Androsace paxiana Knuth Omei , Sichuan;Hao Gang 222 AF323705
Supraspecif ic classif ication follows Hu[ 1] .Voucher specimens deposited in IBSC herbarium.Sequence of P.farinosa is from GenBank.
HAO Gang et al:Circumscriptions and Phylogenetic Relationships of Two Primula Sections:Evidence from nrDNA ITS Sequences 73　
Fig.1.　Strict consensus tree of the two most parsimonious trees
from entire ITS sequences.
Each tree has a length of 495 steps , a consistency index (CI)of
0.77 , and a retention index(RI)of 0.73.Numbers above lines
represent bootstrap values in 500 replicates.Supraspecific classifica-
tion follows Hu[ 1] .
the sister to the clade containing P.malacoides (sect.
Monocarpicae)and P.obconica (sect.Obconicolisteri),
which has strong support (95%).In the other subdivi-
sion (supported at 98%), P.cicutariifolia and P.
merrilliana constitute a monophyletic group with 100%
support , with P.pulverulenta (sect.Proliferae)as its
sister group , supported at 98%;the other clade consists
of P.veris (sect.Primula)and P .farinosa (sect.
Aleuritia), with 99% support.
3　Discussion
The ITS trees are consistent with Hu s[ 1] interpreta-
tion of sectional limits of sects.Auganthus and Ranuncu-
loides.Primula filchnerae could convincingly be treated
as a member of sect.Auganthus.It shares all morpholog-
ical features with P.rupestris and P.sinensis , except
the pinnately rather than palmately lobed leaves.The
character of pinnately lobed leaves may have undergone
parallel evolution in sect.Auganthus and Ranunculoides.
The specific status of P .rupestris and P.sinensis
has been controversial
[ 1 , 5 ,6] .They are nearly indistin-
guishable in vegetative and cytological aspects
[ 1 , 5 ,6] ;the
former has for long time been considered to be the wild
progenitor of the latter cultivated one.Any attempts of hy-
bridization between them , however , have never been suc-
cessful.Hu[ 1] and Halda[ 8] united P.rupestri with P.
sinensis.In the recent revision of Chinese Primula[ 7] they
were recognized as independent species.Our ITS data
demonstrated that P.rupestris and P.sinensis have dis-
tinctive sequences (differ in 15 bases), which seems in
favor of their species status , taking into account that the
two accessions of P .sinensis have identical sequences.
Further studies on infraspecific genetic variation are need-
ed for these two taxa.
Sect.Ranunculoides thus should contain only two
members , P .cicutariifolia and P.merrilliana , charac-
terized by highly divided pinnate leaves.Minor differenc-
es between them include (1 or)2-6 pairs of pinnae ,
with 2-4 teeth on each side;pink or pale lilac corolla
for P.cicutariifolia , whereas in P.merrilliana there
are 7-9 pairs of pinnae , pinnatified or pinnately dentate
and white or tinged with lilac corolla.
This two sections occur in the lowland area of central
and eastern China , deviating from the diversity centre of
Primula.They are actually distantly related with each
other , and can not readily be allied with any other sec-
tions in Primula .In the present study , the ITS trees sug-
gest that sect.Auganthus shares a more recent common
ancestor with sects.Monocarpicae and Obconicolisteri(although with moderate bootstrap support), than it does
with sect.Ranunculoides , while the latter becomes sister
to sect.Proliferae , with strong bootstrap support.This
study was focused on the testing of monophylies of sects.
Auganthus and Ranunculoides;patterns of phylogenetic
relationships among sections of Primula revealed in this
study may change when more of sets of data are available.
In genus Primula where vegetative features are of
limited value , molecular approaches may offer additional
data.To evaluate the major subdivisions in Primula large
scale of species should be screened.This preliminary
study demonstrated that ITS sequences would be a suitable
marker for the reconstruction of Primula phylogeny.
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[ 4] 　Wendelbo P.Stuides in Primulaceae.Ⅱ.An account of
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报春花属藏报春组 、毛茛叶报春组的界定和系统发育关系:
核糖体 DNA ITS序列证据
郝　刚1 　胡启明1＊　李南淑2
(1.中国科学院华南植物研究所系统演化植物学中心 , 广州 510650;
2.Department of Biology , Ewha Womans University , Seoul 120_750, Korea)
摘要:　报春花属(Primula L.)藏报春组(sect.Auganthus Pax ex Balf.f.)和毛茛叶报春组(sect.Ranunculoides Chen et
C.M.Hu)的界定一直是没有解决的问题。应用核糖体 DNA ITS 序列数据探讨其系统发育关系。取样包括藏报春
组和毛茛叶报春组的全部 5 个种以及其他一些相关组的代表种。 ITS 系统树表明 , 陕西羽叶报春(P.filchnerae
Knuth)应与藏报春(P.sinensis Sabine ex Lindl.)和野藏报春(P.rupestris Balf.f.et Farrer)一起置于藏报春组;毛茛叶
报春组只含两种:毛茛叶报春(P.cicutariifolia Pax)和安徽羽叶报春(P.merrilliana Schltr.)。这两组并不具密切亲
缘关系 ,它们在报春花属中与其他组的关系还需进一步研究。研究也表明 ITS 序列可以为报春花属的系统发育重
建提供大量可靠资料。
关键词:　报春花属;藏报春组;毛茛叶报春组;系统发育;ITS;报春花科
中图分类号:Q949　　　文献标识码:A　　　文章编号:0577-7496(2002)01-0072-04
收稿日期:2000-12-11　接收日期:2001-04-17
基金项目:国家自然科学基金(39970053)、中国科学院基金(KSCX2_1_06B)、韩国研究基金(KRF_1999_015_DI 0085)。
＊通讯作者。E_mai l:.
(责任编辑:梁　燕)
HAO Gang et al:Circumscriptions and Phylogenetic Relationships of Two Primula Sections:Evidence from nrDNA ITS Sequences 75