systematic position of cyathocline cass. (asteraceae): evidences from molecular, cytological and...
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ORIGINAL ARTICLE
Systematic position of Cyathocline Cass. (Asteraceae): evidencesfrom molecular, cytological and morphological data
Wei-Ping Li • Feng-Ming Qian • Xiu-Lin Yang •
San-Mao Chen
Received: 9 August 2012 / Accepted: 14 August 2013 / Published online: 30 August 2013
� Springer-Verlag Wien 2013
Abstract Cyathocline, a small genus, has been treated as
a member of subtribe Grangeinae of tribe Astereae
(Asteraceae), but has been neglected in molecular phylo-
genetic analyses of Astereae. Plastid trnL-F and nuclear
ribosomal DNA ITS sequences were used to carry out
phylogenetic analyses of Cyathocline (represented by
C. purpurea) through maximum parsimony and Bayesian
analyses. In addition, its karyotype, morphology and
micromorphology were also investigated. The results show
that in our three phylogenetic trees, C. purpurea is deeply
nested within the Blumea clade and/or the Inulinae clade,
and is closest to Blumea balsamifera (Inuleae, Inulinae). C.
purpurea is similar to Blumea in chromosome size bimo-
dality and to Inulinae in one single large oxalate crystal
within each cell of the cypsela epidermis, which, together
with molecular evidences, suggests strongly that Cyatho-
cline should be transferred from Astereae to Inuleae sub-
tribe Inulinae. Although C. purpurea has many anomalous
features, its most characters still are within a wide range of
morphological variations of Blumea. DNA data and the
karyotypic character support to merge C. purpurea into
Blumea. As a result, the new combination Blumea purpu-
rea (O. Kuntze) W.P. Li was made.
Keywords Astereae � Cyathocline � Morphology �Molecular evidence � Inuleae � Blumea �Blumea purpurea
Introduction
Tribe Astereae Cass. is the second largest tribe of family
Asteraceae and characterised by the combined traits such
as ecaudate (tailless) and obtuse anther bases and disc floret
style arms with lanceolate to deltoid apical appendages that
are glabrous adaxially and covered with sweeping hairs
abaxially (Ling et al. 1985; Nesom 1994a; Nesom and
Robinson 2007; Brouillet et al. 2009b). These diagnostic
characters cannot fully define this tribe, however. On one
hand, based on the morphological definition, some genera
of Astereae were misplaced in other tribes. DNA sequence
data supported strongly to transfer Printzia, Nannoglottis,
Welwitschiella and Sheareria from other tribes to tribe
Astereae (Bayer and Cross 2002; Liu et al. 2002; Brouillet
et al. 2009a; Gao et al. 2009; Li et al. 2012). On the other
hand, some genera were removed from Astereae.
Psednorrichia with ecaudate anthers was traditionally
placed in Astereae until Anderberg and Karis (1995)
transferred it to tribe Senecioneae. Similarly, Pseud-
oconyza viscosa (reviewed by Carr et al. 1999) and many
Conyza species (Chen and Anderberg 2011) were removed
from Astereae to Inuleae. In our phylogenetic study on
Chinese Astereae, we found that genus Cyathocline Cass.
should be excluded outside tribe Astereae.
Cyathocline Cass. is a small genus with two (Ling et al.
1985) or three (Bremer 1994; Nesom and Robinson 2007;
Chen et al. 2011) species and distributed in tropical Asia.
The genus was described in 1829 by Cassini and typified
by Cyathocline lyrata Cass. (reviewed by Ling et al. 1985;
Nesom and Robinson 2007), a synonym of C. purpurea
(Buch.-Ham. ex De Don) O. Kuntze. (Ling et al. 1985;
Chen et al. 2011). C. purpurea is native to India and
southern and south-western China and is a traditional
medicine. In south-western China, it, known as ‘‘Hong Hao
W.-P. Li (&) � F.-M. Qian � X.-L. Yang � S.-M. Chen
College of Life Sciences, Hunan Normal University,
Changsha 410081, China
e-mail: [email protected]
123
Plant Syst Evol (2014) 300:595–606
DOI 10.1007/s00606-013-0905-8
Zi’’ (red artemisia) in Chinese, is a folk drug used to treat
inflammations and pulmonary tuberculosis (Li et al. 2005),
and is used as an herbal remedy for human tuberculosis,
malaria, bleeding, rheumatism, swelling and inflammatory
diseases (Yu et al. 1993; Li et al. 2003), and various can-
cers (Ma et al. 2009), while in India, essential oil from its
stems, leaves and flowers acts as appetiser and anthelmintic
against tapeworms and hookworms (Shrivastava 1979;
Joshi et al. 2011; Tandon et al. 2011).
Ling et al. (1985) and Nesom and Robinson (2007)
placed Cyathocline within subtribe Grangeinae Benth. of
tribe Astereae. Cyathocline was considered to belong to
Astereae because it has truncate anther bases (Ling et al.
1985; Chen et al. 2011) and a chromosome base number of
x = 9 (Nesom and Robinson 2007). It was treated as a
member of subtribe Grangeinae based principally on dis-
ciform capitula with outer multiple-seriate filiform pistil-
late florets and inner tubular functionally male disc florets.
Its most unique autapomorphy is cup-shaped receptacles.
However, in taxonomy, the genus Cyathocline has been
paid less attention to. Hitherto, no any DNA sequence data
of this genus have been reported and no molecular phylo-
genetic analyses of Astereae (Noyes and Rieseberg 1999;
Brouillet et al. 2009b; Li et al. 2012) have included it.
Furthermore, its some morphological features such as
anther bases and ovaries of disc florets have not been
observed and recorded exactly (Ling et al. 1985; Zhuang
2004; Nesom and Robinson 2007; Chen et al. 2011). Dif-
ferent chromosome base numbers of x = 9 (Koul and
Wakhlu 1975; Koul et al. 1976, 1986; Marhold et al. 2011)
and x = 11 (reviewed by Koul and Wakhlu 1975; Grau
1977) were reported for Cyathocline.
Based on plastid trnL-F and nuclear ribosomal DNA
internal transcribed spacer (ITS) sequence data, the present
study carried out phylogenetic analyses of Cyathocline to
determine its systematic position. In addition, its karyo-
type, morphology and micromorphology were also inves-
tigated to provide evidences for its systematic position.
Materials and methods
Taxon sampling
Cyathocline purpurea is the only representative of this
genus in China and can represent the genus in phylogenetic
analyses, although the other species are unavailable. Syn-
apomorphy such as the cup-shaped receptacles supports
strongly monophyly of the genus. Furthermore, C. purpu-
rea is the generic-type species because the genus was
established in 1829 and typified by C. lyrata that is a
synonym of C. purpurea (Ling et al. 1985; Bien 2007;
Chen et al. 2011).
Two populations of C. purpurea were sampled for
morphological, cytological and molecular investigation.
One occurs in fallow field (Fig. 1a) in Xishuangbanna,
southern Yunnan Province, China and at 600 m alt.,
22�000N and 100�440E (voucher lwp0804070, HUUN). The
other population is located in field (Fig. 1b) in Luodian
County, southern Guizhou Province, China and at 430 m
alt., 25�200N and 106�530E (voucher lwp1205010, HUUN).
For morphological comparison, Grangea maderaspa-
tana (L.) Poir. and Blumea balsamifera (L.) DC. were
observed. G. maderaspatana, a representative of subtribe
Grangeinae of Astereae, was sampled in Zhaoqing City,
Guangdong Province, China (voucher lwp0708234,
HUUN), while B. balsamifera, a putative closest relative to
C. purpurea, was sampled in the same site (voucher
lwp1205005, HUUN) as the Luodian population of
C. purpurea.
Three designed datasets were plastid DNA trnL-F
region (trnLUAA-trnLUAA-trnFGAA), ITS (nrDNA internal
transcribed spacer) and combined trnL-F/ITS dataset. Taxa
and GenBank accession numbers for all 86 trnL-F and ITS
sequences as well as voucher information for newly gen-
erated sequences are listed in Appendix. All the plant
materials include 40 in-group accessions from 40 species
and 3 out-group species. Except a few tribes were
unavailable for trnL-F and ITS sequence data, all the other
12 tribes of Asteroideae were sampled. Since C. purpurea
belongs traditionally to Astereae subtribe Grangeinae, two
species of subtribe Grangeinae (G. maderaspatana and
Dichrocephala auriculata Druce) were sampled. Other two
Astereae species sampled were Printzia polifolia (L.)
Hutch. (a representative of basal lineages of Astereae) and
Aster amellus L. The Basic Local Alignment Search Tool
(BLAST) was used to find which taxa are similar to
C. purpurea in trnL-F and ITS sequences, and the results
showed that B. balsamifera and other Blumea species
(Inuleae subtribe Inulinae) are closest to C. purpurea.
Therefore, 24 taxa were selected and represented the dif-
ferent Inuleae clades of the phylogenies of Pornpongrun-
grueng et al. (2007) and Englund et al. (2009).
In all three phylogenetic analyses, Lactuca canadensis
L. (tribe Cichorieae), Vernonia gigantea (Walt.) Trel. (tribe
Vernonieae) and Erato polymnioides DC. (tribe Liabeae) of
Cichoroideae were selected as outgroups for the rooting of
the phylogenetic trees because in molecular phylogenetic
analyses Cichoroideae is a sister to Asteroideae (Funk et al.
2009).
DNA extraction, polymerase chain reaction (PCR)
and sequencing
Newly generated sequences include only trnL-F and ITS
sequences of two accessions from the two C. purpurea
596 W.-P. Li et al.
123
populations. Total genomic DNA was isolated from silica
gel-dried leaves using a modified cetyltrimethylammonium
bromide procedure (Doyle and Doyle 1987). Amplification
and sequencing were performed using the primers ITS1 and
ITS4 (White et al. 1990) for the ITS region, and c and f
(Taberlet et al. 1991) for the plastid DNA trnL-F region.
Amplification of the ITS region (ITS1, 5.8S rDNA and
ITS2) was performed as described by Li et al. (2012). PCR
products were purified using a UNIQ-10 Spin Column PCR
Product Purification Kit (Sangon Biotech Co., Ltd,
Shanghai, China) following the manufacturer’s instruc-
tions. Sequencing reactions were performed in both
directions by Sangon Biotech Co., Ltd.
Sequence alignment and phylogenetic analyses
According to Pornpongrungrueng et al. (2007) and Engl-
und et al. (2009), boundaries of the trnL-F and ITS regions
were determined. All DNA sequences were aligned ini-
tially using Clustal X1.83 (Jeanmougin et al. 1998) and
then adjusted manually using BioEdit version 7.0.1 (Hall,
1999). The trnL-F regions, ITS regions and the combined
trnL-F/ITS sequences were analysed, respectively. The
incongruence length difference test (Farris et al. 1994) was
carried out to test the homogeneity between data sets using
PAUP* version 4.0b10 with 1,000 replicates. Maximum
parsimony (MP) and Bayesian inference (BI) methods were
performed for the data sets using PAUP* version 4.0b10
(Swofford 2001) and MrBayes version 3.1.2 (Ronquist and
Huelsenbeck 2003), respectively. In the MP analysis, gaps
were treated as missing data, all characters were equally
weighted and treated as unordered, and a heuristic search
was implemented with 1,000 random additional sequence
replicates and sub-tree pruning–regrafting branch swap-
ping. Bootstrap analyses based on 1,000 replicates with ten
random additions per replicate were used to estimate the
confidence of the clades. The MaxTrees setting in PAUP*
was set to 5,000 for the searches and bootstrap tests.
Cytological observation
For chromosome observations, actively growing root tips
were cut from the seedlings from the achenes of the two
C. purpurea populations. The root tips were pretreated with
0.1 % colchicine at 8–12 �C for 4 h and then fixed in
Carnoy I (3:1 95 % ethanol:glacial acetic acid) at
20 ± 5 �C for 12 h. They were then macerated in 1 M
hydrochloric acid at 60 �C for 8 min, stained and squashed
in Carbol fuchsin, and photographed under a digital
imaging microscope (Nikon E200). The chromosome
number of each plant was determined from at least 20 cells
at mitotic metaphase.
Observation of morphology and micromorphology
Fresh materials of C. purpurea, B. balsamifera and
G. maderaspatana for morphological observations were
collected in the field. Under a digital imaging microscope
(Nikon E200), cypsela wall epidermis of marginal florets
and hairs of corolla, anthers and styles of disc florets were
observed and photographed, and under a SZX10 stereo
microscope with DP72, longitudinal section of a head,
receptacle, phyllaries and florets were observed and
photographed.
Results
Characterization of nucleotide data
The trnL-F and ITS sequences are completely identical
between the two accessions from the two populations of C.
Fig. 1 Habitat of Cyathocline purpurea. a Xishuangbanna population; b Luodian population
Systematic position of Cyathocline Cass. (Asteraceae) 597
123
purpurea, so only one accession was included in the
matrices (Appendix). Sequence lengths ranged from 747 to
843 bp in trnL-F matrix, 581 to 646 bp in ITS matrix and
1,387 to 1,499 bp in the combined trnL-F/ITS matrix. Of
985 total characters in the trnL-F matrix, 763 characters are
constant, 130 variable characters are parsimony-uninfor-
mative and 92 characters are parsimony-informative. Of
703 total characters in the ITS matrix, 237 characters are
constant, 77 variable characters are parsimony-uninfor-
mative and 389 characters are parsimony-informative.
Base-pair difference in ITS is 14.06 % between C. pur-
purea and B. balsamifera and 16.64 between C. purpurea
and B. virens DC. The incongruence length difference test
indicated that the data sets were not significantly hetero-
geneous (P = 0.301). Therefore, a combined analysis of
the two regions was performed using PAUP* and MrBayes.
Of 1,688 total characters in the combined matrix, 1,000
characters are constant, 207 are variable and parsimony-
uninformative and 481 characters are parsimony-informa-
tive. Therefore, in the combined analysis, the number of
phylogenetically informative sites of ITS is more than four
times as high as in trnL-F region.
Phylogenetic analyses
Phylogenetic analyses using trnL-F, ITS and combined
data sets yielded generally consistent phylogenetic trees
(Bayesian trees; see Figs. 2, 3, 4). In all the phylogenetic
trees (Figs. 2, 3, 4), C. purpurea is nested within the
Blumea clade rather than the Astereae clade, while in the
ITS and the combined trees, it is closest to B. balsamif-
era, the type species of Blumea. The trnL-F and com-
bined trees have the Blumea clade with strong Bayesian
posterior probability (PP) and weak bootstrap support
(BS) (PP = 0.94 and BS = 49 in Fig. 2; PP = 0.97 and
BS = 50 in Fig. 4), whereas the Blumea clade has no
significant support (PP = 0.72; BS = 38) in the ITS tree
(Fig. 3). The ITS and combined trees have the Inulinae
clade with the highest support (PP = 1.00 and BS = 100
in Figs. 3, 4), although there is no clade in the trnL-F
tree (Fig. 2). Similarly, the Inuleae clade occurs in the
ITS and combined trees with strong to weak support
(PP = 0.98 and BS = 72 in Fig. 3; PP = 0.97
and BS = 79 in Fig. 4), whereas the trnL-F tree has no
Inuleae clade. Therefore, the combined data analysis was
more resolved than a single sequence data analysis,
while ITS data yielded trees with higher resolution and
stronger support than those based on trnL-F data except
for the Blumea clade (Figs. 2, 3, 4). The present results
about molecular phylogenetic analyses of Inuleae
are largely in agreement with the previous work by
Pornpongrungrueng et al. (2007) and Englund et al.
(2009).
Cytological feature
In the two populations, root tip cells of C. purpurea have
18 chromosomes with 4 (2 pairs) long chromosomes and 14
(7 pairs) short ones (Fig. 6), meaning that its chromosome
base number is 9 (x = 9; 2n = 2x = 18) and its karyotype
is bimodal.
Morphological and micromorphological data
Morphological and micromorphological traits (Fig. 5a–k)
of C. purpurea were observed, many of which have not
been recorded up to now. Firstly, a pistillate marginal floret
has three corolla lobes (Fig. 5e) and a corolla base covered
Fig. 2 The 50 % majority rule consensus tree from the Bayesian
analysis of plastid genome DNA trnL-F sequences. Bayesian
posterior probabilities (C0.89) and bootstrap values (C49 %) are
indicated above the branches; ‘–’ indicates that Bayesian posterior
probabilities are \0.89 or bootstrap percentages are \49 %. Accord-
ing to Pornpongrungrueng et al. (2007) and Englund et al. (2009),
genus Blumea, subtribe Inlulinae and Plucheinae and tribe Inuleae are
defined. Filled triangle indicates the place of Cyathocline purpurea
598 W.-P. Li et al.
123
with glandular hairs (Fig. 5e). Secondly, one large, stick-
like oxalate crystal occurs in each cell of cypsela wall
epidermis (Fig. 5f). Thirdly, hairs on the lobes of disc
florets are a single line of cells and have an obtuse tip,
waveform surface and obvious nodes (Figs. 5d, g).
Fourthly, disc florets have a fleshy secretory disc around
the base of a style (Fig. 5i) and no ovary (Fig. 5a, g, i).
Fifthly, in disc florets, sweeping hairs on the styles are
obtuse and reach below the furcation when the stigma is
divided into two short branches (Fig. 5k) though most
styles are undivided (Fig. 5j). Sixthly, the anther base of
disc florets is not truncate but slightly tailed (Fig. 5h), and
the tails is in length between those of G. maderaspatana
(Fig. 7f) and B. balsamifera (Fig. 7e). Finally, fruits are
(0.3–) 0.5–0.7 mm long.
B. balsamifera has a flat receptacle (Fig. 7a, b) with
scale-like paleae, i.e. degenerate bracts (Fig. 7b) and many
marginal female florets and many disc florets with acute
sweeping hairs on style branches (Fig. 7a, c). There is one
large, stick-like oxalate crystal (Fig. 7d) in each cell of
Fig. 3 The 50 % majority rule consensus tree from the Bayesian
analysis of nuclear ribosomal DNA internal transcribed spacer
sequences. Bayesian posterior probabilities (C0.89) and bootstrap
values (C49 %) are indicated above the branches; ‘–’ indicates that
Bayesian posterior probabilities are \0.89 or bootstrap percentages
are \49 %. According to Pornpongrungrueng et al. (2007) and
Englund et al. (2009), genus Blumea, subtribe Inlulinae and Pluchei-
nae and tribe Inuleae are defined. Filled triangle indicates the place of
Cyathocline purpurea
Systematic position of Cyathocline Cass. (Asteraceae) 599
123
cypsela wall epidermis, which is the most important syn-
apomorphy of Inuleae subtribe Inulinae. Anther base is
obviously tailed in B. balsamifera (Fig. 7e) and truncate in
G. maderaspatana (Fig. 7f).
Discussion
Tribal and subtribal position of the genus Cyathocline
Cyathocline has a well-defined generic circumscription and
has been treated as a member of Astereae (Grau 1977; Fayed
1979; Ling et al. 1985; Zhang and Bremer 1993; Bremer
1994; Nesom and Robinson 2007; Chen et al. 2011). Diag-
nostic features of Astereae are principally truncate or
slightly auricular anther bases and style branches with two
adaxial stigmatic lines and lanceolate to deltoid, hairy and
apical appendages, while Cyathocline was described as
having disc florets with truncate anther bases like Grangea
maderaspatana (Fig. 7f) and linear-lanceolate style branch
(Ling et al. 1985; Chen et al. 2011). C. purpurea lacks the
typical tribal characteristics however, because our obser-
vation shows that, in its functional male disc florets, anther
bases are not truncate but slightly tailed (Fig. 5h). Like
Grangea and Dichrocephala (subtribe Grangeinae: tribe
Astereae), Cyathocline has a few series of marginal filiform
female florets (Fig. 5a, e), which may be one of the most
important reasons why the genus has been placed within
tribe Astereae and within subtribe Grangeinae. Neverthe-
less, multiseriate pistillate marginal florets with filiform
corolla also occur in Inuleae (Fig. 7a, c; Pornpongrungrueng
et al. 2007). Chromosome base number x = 9 is one of the
features of Eurasian Astereae and subtribe Grangeinae,
while x = 9 is also one of the chromosome base numbers of
Inuleae (Anderberg and Eldenas 2007). Therefore, it is
questionable to place Cyathocline within Astereae.
Fig. 4 The 50 % majority rule
consensus tree from the
Bayesian analysis of the
combined data set (trnL-F/ITS).
Bayesian posterior probabilities
(C0.89) and bootstrap values
(C49 %) are indicated above
the branches; ‘–’ indicates that
Bayesian posterior probabilities
are \0.89 or bootstrap
percentages are \49 %.
According to
Pornpongrungrueng et al.
(2007) and Englund et al.
(2009), genus Blumea, subtribe
Inlulinae and Plucheinae and
tribe Inuleae are defined. Filled
triangle indicates the place of
Cyathocline purpurea
600 W.-P. Li et al.
123
It is worth noting that the karyotype in C. purpurea is
quite different from that in Astereae but similar to that in
Blumea of Inuleae subtribe Inulinae. C. purpurea has a
bimodal karyotype with 2 distinctly different size classes, 4
(2 pairs) long chromosomes and 14 (7 pairs) short ones
(Fig. 6). In Astereae, the chromosomes showed a steady
gradation in length from the longest to the shortest (Wa-
tanabe et al. 1999; Li 2002, 2005, 2006; Li and Liu 2005a,
b; Li et al. 2011) or even medium size or a similarly small
size (S-type) (Nesom 1994b; Li 2006), while chromosome
size bimodality occurs only in polyploid hybrids between
Aster ageratoides Turcz. and A. incisus Fisch. (=Kalimeris
incisa DC.) (Tara 1996) and between Aster ageratoides and
A. indicus L. (=Kalimeris indica Bch.-Bip.) (Li 2006). By
contraries, Blumea (Inuleae: Inulinae) has a distinct
karyotype characterised by having one to several pairs of
long chromosomes among the predominantly short ones in
its complement (Peng and Hsu 1978). C. purpurea is in
accordance with Blumea in chromosome size bimodality,
which supports that Cyathocline should be transferred from
Astereae to Inuleae and close to Blumea of Inulinae.
Our morphological and micromorphological investiga-
tions also suggest to transfer Cyathocline to Inuleae sub-
tribe Inulinae. The most important discovery is that in each
cell of cypsela wall epidermis of C. purpurea, there is one
large, stick-like oxalate crystal (Fig. 5f), which is absent in
that of Astereae (Li et al. 2008). The presence of a single
large oxalate crystal in cypsela epidermal cells (Fig. 7d;
Anderberg 1991, 2009; Eldenas et al. 1999; Anderberg
et al. 2005; Anderberg and Eldenas 2007; Englund et al.
2009; Chen and Anderberg 2011) is a diagnostic synapo-
morphy for the Inuleae-Inulinae (Pornpongrungrueng et al.
2007) and may be the most useful for delimiting subtribe
Inulinae (except Caesulia axillaris) (Anderberg et al.
2005). Therefore, Cyathocline should be placed within
subtribe Inulinae of Inuleae. The slightly tailed anther
bases (Fig. 5h) implied that it is not improper to place
Cyathocline within Inuleae, because in tribe Inuleae anther
Fig. 5 Some morphological and micromorphological features of
Cyathocline purpurea, of which a–c, e, g and i were photographed
under a stereo microscope and the others under a digital imaging
microscope. a Longitudinal section of a head (bar 500 lm). b A head
from which fruits had dispersed (arrow indicates a cup-shaped and
naked receptacle; bar 500 lm). c A part of receptacle (bar 100 lm).
d Hairs on the outside of corolla of disc florets (bar 50 lm). e A
marginal female floret (arrow indicates a magnified glandular hair;
bar 200 lm), f Epidermis cells of cypsela wall with one large, stick-
like oxalate crystal (bar 20 lm), g A disc floret with no ovary (bar
200 lm). h A stamen (arrows indicate a slightly tailed anther base;
bar 150 lm). i A disc floret style with linear-lanceolate apical
appendages and a base embedded by a fleshy disc (arrow indicates a
disc; bar 100 lm). j The upper part of a style with sweeping hairs
(arrow indicates a few further magnified obtuse sweeping hairs; bar
150 lm). k The upper of a style with sweeping hairs from the top of
short style branches to below the style bifurcation (arrow indicates a
further magnified obtuse sweeping hair; bar 150 lm)
Systematic position of Cyathocline Cass. (Asteraceae) 601
123
tails are variable in length (Anderberg and Eldenas 2007;
Pornpongrungrueng et al. 2007; Chen and Anderberg
2011). For example, Laggera aurita (L.f.) Benth. ex
C.B.Clarke (subtribe Inulinae) has a short acute anther base
(Wild 1969), which is similar to C. purpurea.
It is the first time that Cyathocline was included within
molecular phylogenetic analyses. Like oxalate crystal,
molecular data support strongly that Cyathocline belongs
to tribe Inuleae rather than to tribe Astereae or tribe Ant-
hemideae (represented by Chrysanthemum indicum L.).
C. purpurea was at first published as a member of tribe
Anthemideae and named Tanacetum purpureum Bucha-
nan-Hamilton ex D. Don (Ling et al. 1985; Chen and
Anderberg 2011), but in all the trees, it is far away from C.
indicum of Anthemideae. Similarly, the ITS and combined
phylogenetic trees (Figs. 3, 4) show that C. purpurea is not
close to the other Astereae species but deeply nested inside
the Inuleae clade (PP = 0.98 and BS = 72 in Fig. 3;
PP = 0.97 and BS = 79 in Fig. 4). In the trnL-F tree
(Fig. 2), four Astereae species include the type genus
(Aster), a basal taxon (Printzia) of Astereae and two rep-
resentatives of subtribe Grangeinae and is sister to a Ant-
hemideae species with a high Bayesian posterior
probability (PP = 0.99) and low bootstrap support
(BS = 62), whereas C. purpurea is nested within the
Blumea clade (PP = 0.94 and BS = 49). The Blumea
clade is combined first with two Inlulinae species and then
belongs to a polytomy with no Astereae clade (Fig. 2).
Biogeography also supports the new systematic position
of Cyathocline for it is distributed in tropical Asia that is
one of the major diversity centers of subtribe Inulinae
(Anderberg 2009; Englund et al. 2009).
Taxonomic relationship of Cyathocline with Blumea
Should Cyathocline be maintained as a genus or treated as
species of genus Blumea? C. purpurea is closest to B.
balsamifera and these two species form a subclade
(PP = 1.00 and BS = 50 in Fig. 4) of the Blumea clade
(PP = 0.99 and BS 49 in Fig. 2; PP = 0.97 and BS = 50
in Fig. 4) that is equal to Blumea s.l. (Pornpongrungrueng
et al. 2007; Englund et al. 2009). Nevertheless, no mor-
phological and micromorphological synapomorphies have
been found to belong to Cyathocline and B. balsamifera,
and Cyathocline is so much different from B. balsamifera
Fig. 6 A micrograph of somatic metaphase chromosomes of Cya-
thocline purpurea from Luodian population (voucher lwp1205010;
HUUN; bar 5 lm). Arrows indicate four long chromosomes
Fig. 7 Some morphological and micromorphological features of
Blumea balsamifera (a–e) and Grangea maderaspatana (f), of which
a–c were photographed under a stereo microscope and the others
under a digital imaging microscope. a Longitudinal section of a head
(bar 1 mm). b A part of receptacle and phyllaries (bar 200 lm). c A
marginal female floret (left) and a disc floret (right) (arrow indicates a
further magnified acute sweeping hair of the disc floret; bar 500 lm).
d Epidermis cells of cypsela wall with one large, stick-like oxalate
crystal (bar 20 lm). e A stamen (bar 500 lm). f A stamen (bar
150 lm)
602 W.-P. Li et al.
123
and other Blumea species. Firstly, phyllaries are 2 or 3
seriate in Cyathocline (Fig. 5a, b), but 4 or 5 seriate in
Blumea (Chen and Anderberg 2011). Secondly, Cyatho-
cline has peculiarly and exclusively cup-shaped and com-
paratively smooth receptacles (Fig. 5a–c), whereas Blumea
possesses flat receptacles with scale-like ridges (Fig. 7a, b;
Anderberg and Eldenas 2007; Chen and Anderberg 2011).
Thirdly, disc florets of C. purpurea are functional male
with slightly tailed anther bases (Fig. 5h) and have special
hairs (Fig. 5d, g) on corolla lobes and, in particular, no
ovary (Fig. 5a, g, i) as opposed to the condition in
B. balsamifera and other Blumea species, where disc florets
are perfect (Fig. 7a, c) with obviously tailed anther bases
(Fig. 7e) and with no such hairs as those on corolla lobes of
C. purpurea. Fourthly, in C. purpurea, sweeping hairs on
styles are obtuse and reach below the furcation (Fig. 5i–k),
whereas in Inulinae sweeping hairs are usually acute and
do not reach the furcation (Pornpongrungrueng et al. 2007).
Fifthly, there is no pappus in Cyathocline (Fig. 5a, e, g),
and in contrast, pappi are barbellate, capillary bristles in
Blumea (Fig. 7a, c; Anderberg and Eldenas 2007; Chen and
Anderberg 2011). Finally, there is very high ITS sequence
divergence between C. purpurea and Blumea. For example,
the ITS sequence divergence between C. purpurea and B.
balsamifera, which are closest to each other, is up to
14.06 %, and the divergence between C. purpurea and B.
virens is high up to 16.64. Moreover, the Blumea clade has
no significant support in the ITS tree (PP = 0.72; BS = 38
in Fig. 3) and very low bootstrap support in the trnL-F tree
(BS = 49; Fig. 2) and the combined tree (BS = 50;
Fig. 4), showing that monophyly of the Blumea clade needs
further study. If it is reasonable to maintain the generic
status of Cyathocline, currently delimited Blumea (Porn-
pongrungrueng et al. 2007; Chen and Anderberg 2011) is
not monophyletic but polyphyletic (Figs. 2, 3, 4) and
should be split into three genera.
However, C. purpurea should be a member of Blumea.
Blumea is heterogeneous in many important morphological
characters, so that some anomalous features of C. purpurea
still are within a wide range of morphological variations.
Like C. purpurea, B. flava (the former monotypic genus
Blumeopsis) has obtuse sweeping hairs extending from the
top of style branches to below the style bifurcation (An-
derberg and Eldenas 2007; Pornpongrungrueng et al.
2007). In addition, functionally male disc florets also occur
in B. bengutensis (the former monotypic genus Merrittia)
(Anderberg and Eldenas 2007), and cyathiform receptacles
and a lack of pappi occur in B. sericeus, too. A recent
taxonomic treatment of B. sericeus (Thomson) Anderb. and
A.K. Pandey (Anderberg and Pandey 2008) sets an exam-
ple for taxonomy of C. purpurea. B. sericeus, originally
described as a monotypic genus of the tribe Mutisieae, was
recently transferred into Blumea (Anderberg and Pandey
2008) based on DNA sequence data from the plastid gene
ndhF and ITS, although it is quite different from other
Blumea species by a number of autapomorphic character
states including its distinctly bilabiate corolla, sweeping
hairs reaching far below bifurcation, anthers with short tails
and epappose fruits, and no diagnostic synapomorphies
shared by it and other Blumea species have been reported.
Similarly, though C. purpurea has a number of characters
quite different from other Blumea species, C. purpurea is
nested within the Blumea clade (Figs. 2, 3, 4) and similar
to some Blumea species in chromosome size bimodality
(Fig. 6; Peng and Hsu 1978), and as a result C. purpurea
should be treated as a member of Blumea. It needs further
investigations to determine if the other species of Cya-
thocline should be moved to Blumea.
As C. purpurea is merged into the genus Blumea,
character evolution of Blumea should be re-evaluated. C.
purpurea brings to Blumea some new features, e.g. 2–3
seriate phyllaries, relatively smooth receptacles and disc
florets with no ovary. Obtuse sweeping hairs are possessed
by both C. purpurea and B. flava, and cyathiform recep-
tacles and a lack of pappi by C. purpurea and B. sericeus,
which imply that in Blumea the three traits have indepen-
dently evolved twice, respectively. And sweeping hairs
reaching below the furcation are shared by C. purpurea, B.
flava and B. sericeus have independently evolved thrice.
The lack of ovary in functional male disc florets of C.
purpurea is unique not only in the Inuleae but also in
Asteraceae. In C. purpurea, its disc florets must, at first,
have undergone loss of female function through disap-
pearance of two stigmatic lines and an ovule as in many
other plants of Asteraceae, and then further lost the empty
ovary. In functional male disc florets of Asteraceae, the
empty ovary could function as a pedicel, but in C. purpu-
rea, the marginal florets are so small that sweeping hairs of
the disc florets, without the help of ovaries, are higher than
the marginal florets (Fig. 5a) and adapt well to pollination.
The disappearance of pappus in C. purpurea makes
against long-distance dispersal of its fruits, whereas it is
widely distributed from southern Asia to southeastern Asia.
Its fruits are (0.3–) 0.5–0.7 mm long and it usually occurs
in open field, so its small size and open habitat perhaps
help it spread by wind.
Blumea purpurea (Buchanan-Hamilton ex D. Don)
W. P. Li, comb. Nov.
Tanacetum purpureum Buchanan-Hamilton ex D. Don,
Prodr. Fl. Nepal. 181, 1825; Cyathocline lyrata Cassini in
Ann. Sci. Nat. I. 17: 34, 1829; C. purpurea Kuntze, Revis.
Gen. Pl. 1: 333. 1891; Dichrocephala minutifolia Vaniot.
In Bull. Acad. Internat. Geogr. Bot. 12: 242, 1903.
Herbs: annual or biennial, low, strongly aromatic.
Stems: erect, branched from base, reddish purple or red
tinged, villous, short-stipitate glandular, more densely so
Systematic position of Cyathocline Cass. (Asteraceae) 603
123
upward. Leaves: alternate, sessile with basal auricles, bi-
pinnatifid; abaxially sparsely villous particularly along
midvein, stipitate glandular; adaxially sparsely villous or
glabrate, sparsely glandular; mid cauline ovate or obovate,
2.5–12 cm long; basal and lower cauline often deciduous
by flowering. Capitula: small, numerous or few in often
dense terminal corymbiform or corymbiform–paniculiform
synflorescences; peduncles: densely white villous, ±den-
sely stipitate glandular. Involucre: hemispheric, ca. 2 mm
in diam.; phyllaries 2- or 3-seriate, subequal, membranous,
abaxially sparsely white villous or glabrous, sometimes
sparsely glandular (mostly distally), margin hyaline, scar-
ious, ±fimbriate, ciliate, apex acuminate, purple. Recep-
tacles: cup-shaped, non-alveolate, naked, ±smooth.
Marginal female florets: fertile, multiseriate on outer and
inner surfaces of cup, purple, tube filiform and short with
glandular hairs on base, 2–3 lobed; disc florets: 7–11 with
no ovary, purple or lobes purple, tube cylindric,
0.6–0.8 mm, limb funnelform, ca. 1.6 mm; lobes 5 and
triangular covering hairs with an obtuse tip and ±wave-
form surface; anther base: slightly tailed; style: undivided
or bifid, style branches with obtuse sweeping hairs abax-
ially reaching below the furcation; style base with promi-
nent disc. Pappus: absent. Achenes: oblong to fusiform,
glabrous, (0.3–) 0.5–0.7 mm long. Flowering and fruiting
nearly year-round. 2n = 2x = 18.
Blumea purpurea is widely distributed from southern
China (Guangdong and Guangxi) and south-western China
(Guizhou, Sichuan, Yunnan), to northwestern and north-
eastern India, and to Pakistan, Bhutan, Cambodia, Lao
PDR, Myanmar, Nepal, Thailand, Vietnam and Bangla-
desh, growing in forests, grasslands on slopes, ravines,
watersides, roadsides, fields.
Acknowledgments We thank the reviewers for their valuable
comments and suggestions; Prof. Fu-Sheng Yang for helping con-
struct the phylogenetic trees; Dr. Gen-Shen Yin and Mrs. Ping Zhang
for field assistance. This study was financed by the National Natural
Science Foundation of China (Grant Nos. 30470131 and 39899400),
the Hunan Provincial Construct Program of the Key Discipline in
Ecology, and the Scientific Research Fund of Hunan Provincial
Education Department (Grant No. 08A046).
Appendix
Taxa sampled and their GenBank accession numbers for
the trnL-F and ITS sequences. Only new sequences
obtained in this study include voucher information.
Antennaria microphylla Rydberg, HM364534,
HM244731; Anvillea garcinii subsp. radiata (Coss. and
Durieu) Anderb., EF211058, EF210963; Arnica cordifolia
Hook., EF104923, EF104922; Aster amellus L., JN543744,
JN543742; Blumea balsamifera DC., EF211053,
EF210958; Blumea densiflora DC., EF211029, EF210934;
1Blumea purpurea (Buchanan-Hamilton ex D. Don) W.
P. Li, LWP1205010 (HUUN); Blumea virens DC.,
EF211052, EF210957; Buphthalmum salicifolium L.,
EF211059, EF210964; Caesulia axillaris Roxb.,
EF211044, EF210949; Calendula officinalis L.,
JN315917, JN315941; Carpesium macrocephalum
Franch. et Savat., FM997839, FM995368; Chaenactis
douglasii (Hook.) Hook. and Arn., GU817985, GU818511;
Chrysanthemum indicum L., JN315916, JN315940;
Chrysophthalmum gueneri Aytac and Anderb.,
FM997842, FM995370; Dichrocephala auriculata
(Thunb.) Druce, JN315895, JN315919; Duhaldea eupato-
rioides (DC.) Steetz, EF211056, EF210961; Duhaldea
nervosa (Wallich ex Candolle) Anderberg, EF211027,
EF210932; Duhaldea rubricaulis (Wall. ex DC.) Anderb.,
FM997844, FM995372; Erato polymnioides DC.,
GU817999, AF539949; Eupatorium serotinum Michx.,
GU818001, DQ236176; Flaveria pubescens Rydb.,
DQ122575, DQ122498; Grangea maderaspatana (L.)
Poir., JN315896, JN315920; Helenium autumnale L.,
GU818007, GU818553; Helianthus tuberosus L.,
GU818008, AF047953; Inula confertiflora A.Rich.,
FM997847, FM995375; Inula helenium L., FM997850,
FM995377; Iphiona aucheri (Boiss.) Anderb., FM997855,
FM995382; Iphiona senecionoides (Baker) Anderb.,
FM997858, FM995385; Lactuca canadensis L.,
GU818025, GU818575; Laggera alata (D. Don) Sch.-Bip.
ex Oliv., EF211025, FJ980335; Lifago dielsii Schweinf.
and Muschl., FM997860, FM995386; Limbarda crithmo-
ides (L.) Dumort., FM997861, FM995387; Merrittia
benguetensis (Elmer) Merr., EF211050, EF210955; Pen-
tanema indicum (Linn.) Ling, EF211048, EF210953;
Pluchea carolinensis (Jacq.) G.Don, EU385104,
AF437851; Polymnia canadensis L., GU818051,
AF465876; Printzia polifolia (L.) Hutch., AF098816,
FJ457927; Pseudoconyza viscosa (Mill.) D’ Arcy,
EF211021, EF210926; Pulicaria dysenterica (L.) Bernh.,
FM997870, FM995395; Senecio squalidus L., JN790007,
JN789909; Stenachaenium campestre Baker, EF211026,
EF210931; Vernonia gigantea (Walter) Trel., GU818102,
AY142949.
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