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A molecular phylogenetic study of the Ephedra distachya / E. sinica complex in Eurasia


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This study provides new information on phylogenetic relationships in the Ephedra distachya / E. sinica complex (Ephedraceae, gymnosperms) based on sequence variation and morphology-based delimitation of individual species. Altogether we have included 50 samples from this complex and closely related species with 39 samples sequenced for the first time. Our sampling scheme provides a much broader sampling both in respect of the number of species as well as the number of samples per species than has been accomplished in previous studies. Sequences for E. dahurica and E. pseudodistachya are given here for the first time. We sequenced the nuclear ribosomal internal transcribed spacer regions (nr ITS1+ITS2) as well as the chloroplast intergenic spacer between rnL and trnF (cp trnL-F), but only ITS1 was sufficiently informative for phylogeny reconstruction. Our data show, for the first time, (1) a well supported E. strobilacea / E. sarcocarpa / E. transitoria clade as sister to the “L” or “Asia 2” clade, furthermore (2) a distinct E. distachya clade, (3) a composite E. dahurica clade that also includes E. sinica and a fraction of E. intermedia, (4) a well supported E. regeliana clade and (5) an unresolved agglomeration of E. intermedia, E. przewalskii, E. lomatolepis and E. pseudodistachya. Most noteworthy is the support derived from the molecular data for (1) the separation of the hitherto doubtful E. pseudodistachya from E. distachya and E. dahurica and (2) the high degree of sequence similarity of E. sinica and E. dahurica. Based on morphological, molecular, ecological and biogeographical evidence, E. sinica is reduced to a subspecies of E. dahurica.
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Annals of the Botanic Garden and Botanical Museum Berlin-Dahlem
41 · 2
Phylogeny and plant molecular systematics
Molecular phylogenetic study of the Ephedra distachya / E. sinica complex in Eurasia
Taxonomy of plants of the Americas
Theophrastaceae cubanae novae
Discovery, naming and typifi cation of Euphorbia pulcherrima (Euphorbiaceae)
E-taxonomy and biodiversity informatics
FloraGREIF – virtual guide and plant database as a practical approach to the fl ora of Mongolia
December 2011
203Willdenowia 41 – 2011
A molecular phylogenetic study of the Ephedra distachya / E. sinica complex in Eurasia
Kakiuchi N., Mikage M., Ickert-Bond S., Maier-Stolte M. & Freitag H.: A molecular phylogenetic study of the
Ephedra distachya / E. sinica complex in Eurasia. Willdenowia 41: 203 215. December 2011. – Online ISSN
1868-6397; © 2011 BGBM Berlin-Dahlem.
Stable URL:
This study provides new information on phylogenetic relationships in the Ephedra distachya / E. sinica complex
(Ephedraceae, gymnosperms) based on sequence variation and morphology-based delimitation of individual species.
Altogether we have included 50 samples from this complex and closely related species with 39 samples sequenced
for the first time. Our sampling scheme provides a much broader sampling both in respect of the number of species as
well as the number of samples per species than has been accomplished in previous studies. Sequences for E. dahurica
and E. pseudodistachya are given here for the first time. We sequenced the nuclear ribosomal internal transcribed
spacer regions (nr ITS1+ITS2) as well as the chloroplast intergenic spacer between rnL and trnF (cp trnL-F), but
only ITS1 was sufficiently informative for phylogeny reconstruction. Our data show, for the first time, (1) a well
supported E. strobilacea / E. sarcocarpa / E. transitoria clade as sister to the “L” or “Asia 2” clade, furthermore (2)
a distinct E. distachya clade, (3) a composite E. dahurica clade that also includes E. sinica and a fraction of E. inter-
media, (4) a well supported E. regeliana clade and (5) an unresolved agglomeration of E. intermedia, E. przewalskii,
E. lomatolepis and E. pseudodistachya. Most noteworthy is the support derived from the molecular data for (1) the
separation of the hitherto doubtful E. pseudodistachya from E. distachya and E. dahurica and (2) the high degree of
sequence similarity of E. sinica and E. dahurica. Based on morphological, molecular, ecological and biogeographical
evidence, E. sinica is reduced to a subspecies of E. dahurica.
Additional key words: gymnosperms, Ephedra dahurica, Ephedra intermedia, Ephedra pseudodistachya, Ephedra
strobilacea, Ma-huang, nr ITS, morphology, phylogeny, cp trnL-F.
The Ephedra distachya L. / E. sinica Stapf complex is de-
fined here somewhat arbitrarily as a group of morpholog-
ically similar and closely related species that additionally
also include E. dahurica Turcz., E. lomatolepis Schrenk
and E. pseudodistachya Pachom. Taxa in this complex
share the same habit, range from 10 40 cm in size, bear
2-seeded female cones which become fleshy at maturity,
generally have a rather short micropylar tube as well as
paired leaves (Fig. 1). Species delimitation in this com-
plex is difficult, particularly in the vegetative stage which
dominates in the field and in the majority of herbarium
specimens. The centre of distribution is the steppe and
semidesert area of Eurasia, further extending from the
Atlantic and Mediterranean to the eastern margin of the
continent. Recent molecular work (Long & al. 2004;
1 Faculty of Pharmaceutical Sciences, Kyusyu University of Health and Welfare, 1714-1 Yoshinotyo, Nobeoka, 882-8508 Japan;
*e-mail: (first author for correspondence).
2 Graduate School of Natural Science and Technology, Kanazawa University, Kana-zawa, 920-1192 Japan.
3 Department of Biology and Wildlife & University Museum Herbarium (ALA), University of Alaska, 907 Yukon Drive, Fair-
banks, AK 99775-6960, USA.
4 Institute of Biology, Section Systematics and Morphology of Plants, University of Kassel, 34132 Kassel, Germany; **e-mail: (second author for correspondence).
204 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
Ickert-Bond & Wojciechowski 2004; Huang & al. 2005;
Ickert-Bond & al. 2009; Rydin & Korall 2009; Rydin &
al. 2010; Ickert-Bond & Rydin 2011) has shown that spe-
cies in the E. distachya / E. sinica complex belong to the
“L” or “Asia 2” clade, one of three Asian clades in Ephe-
dra. However, E. pseudodistachya and E. dahurica are
here included in a phylogenetic study for the first time.
These two taxa and their taxonomic rank are particularly
difficult (see below) as is evident from the differing treat-
ments in checklists and floristic accounts of China (as,
e.g. Fu & al. 1999, Yang 1993, 2002), Russia (as, e.g.
Czerepanov 1995, Peschkova 2005, Galanin 2008) and
Mongolia (Grubov 1982; Freitag & Maier-Stolte 2009).
One member of the group, Ephedra sinica, is of out-
standing pharmaceutical importance. Its aerial parts have
been used as a staple crude drug since ancient times. In
the Shen-nong-ben-cao-jing, one of the oldest herbals,
written in Chinese Han Dynasty (1st century AD), is de-
scribed under its today’s name “Ma-huang” as a remedy
for perspiration, to cure a feverous state, for cough sup-
pression and relief from chills and fevers (Tang 2002).
Thanks to its analgesic, antipyretic and antitussive ef-
fects, Ephedra is still a constitutive part in traditional me-
dicinal prescriptions in China and Japan, and their active
ingredients, ephedrine alkaloids, are contained in modern
pharmaceuticals against colds worldwide. Besides E. si-
nica, E. equisetina Bunge and E. intermedia Schrenk &
C. A. Mey. are considered the most useful for crude drugs
and are prescribed in both Chinese and Japanese phar-
macopoeias (Chinese Pharmacopoeia Commission 2005;
Japanese Ministry of Health 2006). Among them, E. si-
nica dominates the crude drug market in both countries
due to its relatively rich natural resources.
The species of the Ephedra distachya / E. sinica com-
plex and related taxa
In the following we introduce the species under consid-
eration in the Ephedra distachya / E. sinica complex and
its relatives.
Ephedra distachya This species occupies a very wide
area ranging from Spain and western France, scattered
through the Alps and along the northern Mediterranean
coast to eastern Europe and the Near East, from where
it continues to western Central Asia and southwestern
Siberia (Freitag & Maier-Stolte 1994: map 3; Peschkova
2005: fig. 4). Plants from the dry Rhône valley in the
Swiss Alps, differing from typical E. distachya by a long
and twisted micropylar tube, were described as E. hel-
vetica C. A. Mey. Apart from the characteristics of the
microphylar tube, they are otherwise identical with typi-
cal E. distachya and are now generally classified as E.
distachya subsp. helvetica (C. A. Mey.) Asch. & Graebn.
(e.g. Freitag & Maier-Stolte 1993, Govaerts 2010a, b).
More recently, Nouviant (1993, 1997) described popula-
tions from the Italian and the French Alps as E. negrii
Nouviant. They differ from the Swiss E. helvetica by
shorter micropylar tubes, thus bridging the gap to typical
E. distachya (Fig. 1C).
Small plants with delicate twigs and often single-
seeded cones from Siberia were described as Ephedra
mo no stachya L., but monographers (Meyer 1846; Stapf
1889) have shown that Linné’s E. monostachya was an
admixture of dwarfed E. distachya and of E. monosperma
Gmel. ex C. A. Mey. Nevertheless, while excluding E.
monosperma, Riedl (1967) and Jarvis (2007) maintained
E. monostachya at the subspecies level and Nouviant
(1998) at the species level claiming that the taxon replac-
es the northern Mediterranean E. distachya from south-
eastern Central Europe to Siberia.
Doubtful species closely allied or belonging to Ephe-
dra distachya are E. aurantiaca Takht. & Pachom. from
the Caucasus, which differs by having orange to yellow
coloured female cones (Takhtajan & Pachomova 1967),
and E. vvedenskyi Pachom. (1968) from the Kopet Dagh
region southeast of the Caspian Sea, which approaches
E. intermedia with its coarser stature and a slightly elon-
gated micropylar tube. However, E. aurantiaca and E.
vvedenskyi are maintained as distinct species in the ac-
count of Govaerts (2010a, b).
Ephedra pseudodistachya The species grows in east-
ern Kazakhstan, southern Siberia and northern Mon-
golia (Peschkova 2005: fig. 4). It can be distinguished
from E. distachya by the reddish-brownish base of leaf
sheaths, and a more delicate habit, with thinner twigs and
being usually only 5 20 cm high. Until its description
by Pachomova (1968) the respective populations from
the former USSR were included in E. distachya, while
those from Mongolia were classified as E. sinica or E.
sinica var. pumila Florin (Florin 1933; Pachomova 1971;
Grubov 1982; Gubanov 1996). In Chinese floras (e.g. Liu
1985, Cheng 1978, Fu & al. 1999) the plants were named
E. distachya, although in Fu & al. (1999) the conspecifi-
city with true E. distachya was questioned, but E. sinica
var. pumila was suggested to occur in the country. Even
in Russia E. pseudodistachya was not unambiguously ac-
cepted, e.g. Czerepanov (1995) placed it in synonymy of
E. dahurica. At the beginning of our study we also were
not convinced that the separation of E. pseudodistachya
and E. distachya is justified.
Ephedra lomatolepis The species is restricted to an
area from western Kazakhstan and Uzbekistan to north-
western China (Xinjiang province) and southwestern
Mongolia (Freitag & Maier-Stolte 1994: map 7). E. lo-
matolepis differs from all other species of the complex
by the wide membranous margins of the bracts in the fe-
male cones, an olive-green colour of the stems and leaves
partly arranged in whorls of three. However, in areas of
overlap, such as along the lower reaches of the Syrdar’ya
river in western Kazakhstan where both species have
been seen growing side by side, it was difficult to sepa-
rate them in the vegetative stage.
205Willdenowia 41 – 2011
Ephedra sinica The species is distributed from cen-
tral China (Gansu province) and Mongolia eastward up
to the Gulf of Bohai (Hebei province) in northeastern
China (Cheng 1978; Fu & al. 1999), with an extension
to southeastern Siberia (Peschkova 2005: fig. 3). Pecu-
liar characters of E. sinica are comparatively long leaves
(up to 7 mm), whitish leaf sheaths, flattened basal in-
ternodes and completely glabrous twigs. According to
the literature, E. sinica appeared to be an easily recog-
nisable species, until we started to identify collections
from Mongolia and southeastern Siberia, which forced
us (Freitag & Maier-Stolte 2009) informally to reduce E.
sinica to a subspecies of E. dahurica. It is noteworthy,
that Stapf (1927) made the original description of E. si-
nica from raw material imported to the United States of
America from China. In 1999, the Chinese Government
proclaimed that Ephedra resources in the territory need
protection against desertification and over-collecting.
According to its significance as a medicinal plant, E. si-
nica and related species are of special interest.
Ephedra dahurica The species has been described by
Turczaninow (1854: 421) from the Transbaikal region
of Siberia. It was mistaken by Stapf (1884) and Florin
(1933) as E. monosperma J. G. Gmel. ex C. A. Mey. and
neglected until its revival by Siplivinskiy (1973). E. da-
hurica is widely sympatric with E. sinica in southeast-
ern Siberia and northeastern Mongolia (Peschkova 2005:
fig. 2; Galanin 2008). According to the description given
by Peschkova (2005), E. dahurica differs from E. sinica
by slightly shorter leaves united basally into a brown-
ish sheath and by the twig surface, which is more or less
rough due to the presence of short but hard papillae. E.
dahurica was included by Galanin (2008) as a subspecies
of E. sinica. Subspecies rank for both taxa was also pro-
posed by Freitag & Maier-Stolte (2009) under the older
name E. dahurica.
Related species Additional species of the “L” (or
Asia 2”) clade (Rydin & al. 2010) that have not been
dealt with in detail in this study because of their clear
morphological separation are:
Ephedra intermedia Schrenk & C. A. Mey.: coarse
subshrub, usually 30 80 cm high, female cones with
very long, screw-like micropylar tube; semideserts from
Iran to China.
Ephedra przewalskii Stapf: delicate to robust sub-
shrub, usually 30 80 cm high, brown, with adherent sand
grains, leaves usually in 3s, female cones at maturity with
completely membranous bracts; sand deserts from Uz-
bekistan to Mongolia and China.
Ephedra regeliana Florin: prostrate subshrub, usu-
ally 2 5 cm high; alpine steppes of Central Asian high
Here also belong the three stout Irano-Turanian spe-
cies Ephedra strobilacea Bunge, E. sarcocarpa Aitch. &
Hemsl. and E. transitoria Riedl.
Our study aims to determine whether the mostly weak
morphological differentiation in the species complex, in
particular regarding the species pairs Ephedra sinica / E.
dahurica and E. distachya / E. pseudodistachya can be
supported by molecular data. In order to get most mean-
ingful results, we sampled throughout the distributional
areas of the individual species as complete as possible.
Besides, we expected to get insights into the genetic vari-
ability of widely distributed Ephedra species along geo-
graphical gradients and to resolve phylogenetic relation-
ships in the species group.
From previous phylogenetic studies in Ephedra (e.g.
Ickert-Bond 2004, Rydin & Korall 2009, Rydin & al
2010) we were aware of the comparatively low amount of
sequence divergence in Ephedra and particularly among
more closely related groups. Nevertheless, from the to-
pology of the hitherto published phylogenies (the most
refined in Rydin & al. 2010) we expected that by means
of extended sampling and inclusion of species which
were not sequenced yet we could get additional support
for taxonomic decisions made based on morphology.
The morphologically well-separated species of the
“L” clade (Rydin & al. 2010) are also included in our
study in order to elucidate their relationships to the spe-
cies under particular consideration. However, these spe-
cies are represented by exemplary samples only. Further-
more, to show the position of the Ephedra distachya / E.
sinica group among the other Eurasian Ephedra clades,
selected species from all clades are likewise included in
our analysis.
Material and methods
Plant material Field surveys of Ephedra dahurica in
southeastern Siberia and E. sinica in Mongolia were con-
ducted in July and August 2006, respectively, by the first
two authors (Kakiuchi & Mikage 2007; Mikage 2009).
Helmut Freitag studied and collected E. pseudodistachya
and E. dahurica in southern and southeastern Siberia in
August and September 2003, as well as E. lomatolepis
and E. distachya in various earlier field campaigns in
other parts of Eurasia. The specimens were deposited in
the herbaria of Kanazawa University (KANP) and Kassel
University (KAS). Additionally, a few specimens from
other herbaria and selected GenBank sequences with
reliable identification and traceable provenance infor-
mation were included. Herbarium abbreviation follow
Thiers (2008+). Taxonomic identifications were made
and/or verified by D. Chimitov (Ulan-Ude), H. Freitag,
M. Maier-Stolte and M. Mikage except for the GenBank
material. In Appendix 1 we provide a complete list of
the 59 samples of Ephedra included in this study, with
geographic origin, voucher information and GenBank ac-
cession numbers for all sequences. Several samples from
adjoining locations had identical sequences and only one
sequence was submitted for Genbank registration. Thus
the 59 sequences are in fact based on a total of 80 vouch-
206 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
ers. In Fig. 2 only the samples of the E. distachya / E.
sinica group sequenced here for the first time are given.
Molecular sequencing and initial phylogenetic analyses
were carried out at Kanazawa University, while addition-
al Bayesian inference and Maximum Likelihood analyses
were completed at the University of Alaska Fairbanks.
Morphological comparisons were done in Kassel. Eco-
logical data are based on field observations made by H.
Freitag, unless explicitly citing other sources.
DNA preparation and PCR amplification Dry twigs
were cut into 2 mm long pieces, frozen in liquid nitrogen
and ground into powder. Using a DNeasy Plant Mini Kit
(Qiagen), DNA was extracted according to the manufac-
turer’s protocol. Total DNA was used as a template for
amplifying the nuclear ribosomal internal transcribed
spacer regions (nr ITS1 and ITS2) and the chloroplast
intergenic spacer region between trnL and trnF (cp trnL-
F) by PCR. The primers were designed based on 18S
Fig. 1. A C: Ephedra distachya subsp. helvetica – A: with mature female cones; Suisse, Valais, Tourbillon hill near Sion, on S
exposed rocky outcrops (with Sedum album); phot. H. Freitag, August 1990; B: shoot with male cones; Italy, S Tyrol, Vinschgau,
S-exposed slopes above Schlanders; phot. H. Freitag, July 1990; C: shoot with immature female cones, the comparatively short
micropylar tubes correspond to E. “negrii”; France, Alpes de Hautes Provence, Larange-Montéglin; phot. E. Zippel, May 2005. –
D: E. dahurica with mature female cones; the distorted twigs are artefacts due to unknown injury that also occur in other species;
Russia, Buryatia, c. 100 km S Ulan-Ude, in Stipa krylovii steppe near Sulfat’noe Ozero; phot. H. Freitag, September 2003.
207Willdenowia 41 – 2011
and 26S nuclear ribosomal DNA from Genbank (Eph-
1F:D38242, Eph-1R:U90708) and previous works (5.8S-
R, 5.8S-F: Long & al 2004, Aco-1F, Aco-2R: Taberlet &
al. 1991). The primer sets of Eph-1F (GAC GTC GCG
used for the amplification of ITS1 and ITS2, respectively.
and Aco-2R (ATT TGA ACT GGT GAC ACG AG), were
used to amplify the trnL-F region. Standard PCR was per-
formed in 25 µl reaction mixture containing 2.5 µl of 10 ×
PCR buffer for KOD-Plus, 0.2 mM each of dNTP, 1 mM
MgSO4, 0.4 M of each primer, approximately 100 ng
of the DNA sample and 0.5 units of KOD-Plus DNA
polymerase (Toyobo). PCR was carried out as follows:
hot start at 94 °C for 2 min, 30 cycles of denaturation at
94 °C for 15 sec, annealing at 55 °C for 30 sec and elon-
gation at 68 °C for 45 sec, and a final elongation at 68 °C
for 5 min. Three microliters of the PCR product was used
for agarose gel electrophoresis and the remaining product
was purified using the QIA quick PCR Purification Kit
SequencingThe purified PCR products were se-
quenced using a BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems) on an ABI PRISM 310 Genetic
Analyzer (Applied Biosystems). The primers, Eph-1F,
5.8S-R, Eph-1R and 5.8S-F, were used for the sequenc-
ing reaction of the ITS 1 and 2 regions, and Aco-1F and
Aco-2R were used for sequencing the trnL-F region.
Phylogenetic analysis The nuclear ribosomal (nr)
ITS2 sequences were invariable across all the taxa sam-
pled here and we thus only included the nr ITS1 sequenc-
es in our analysis. In the cp trnL-F sequences we also
detected only very few nucleotide changes among the
species under consideration. They were likewise omit-
ted. In several DNA samples multiple banding patterns
indicating a putative hybrid origin of the sample or its
ancestors were found. Subsequent cloning results were
inconclusive and these samples were later excluded. Se-
quence alignment was done manually in MacClade 4.08
(Maddison & Maddison 2005). We scored gap charac-
ters using modified complex-indel-coding (Simmons &
Ocho terena 2000; Müller 2006; Simmons & al. 2007). A
total of 17 parsimony informative complex-indel-coding
gap characters were scored from unambiguously aligned
regions. Maximum likelihood tree searches (ML) and
ML bootstrapping were performed using RAxML 7.2.7
(Stamatakis & al. 2008; Pfeiffer & Stamatakis 2010).
RAxML searches were performed using the general
time-reversible (GTR) model with among-site rate het-
erogeneity modelled by a gamma distribution with 25
rate categories, which sufficiently account for rate het-
erogeneity (Stamatakis & al. 2008). Non-parametric
bootstrapping was used as implemented in the rapid
bootstrap option of RAxML on the CIPRES portal using
500 replicates. The trees were rooted with Ephedra foe-
minea Forssk. and E. fragilis Desf., members of the most
basal clade of the genus in recent phylogenetic analyses
(Ickert-Bond & al. 2009; Rydin & Korall 2009). We also
included several accessions of the two other Asian Ephe-
Fig. 2. Distribution map of new sequences (nr ITS1+ITS2 and cp trnL-F) generated for accessions of the Ephedra distachya / E.
sinica complex in the molecular analysis.
208 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
dra clades (Long & al. 2004; Ickert-Bond & al. 2009;
Rydin & Korall 2009).
Bayesian inference using the GTR + G model plus a
proportion of invariable sites was performed using Mr-
Bayes 3.1.2 (Ronquist and Huelsenbeck 2003). Markov
chain Monte Carlo (MCMC) runs started from independ-
ent random trees were repeated twice and extended for 1
million generations, with trees sampled every 1000 gen-
eration. We used the default priors in MrBayes, namely a
flat Dirichlet prior for the relative nucleotide frequencies
and rate parameters, a discrete uniform prior for topolo-
gies, and an exponential distribution (mean 1.0) for the
gamma-shape parameter and branch lengths. Stationarity
of the Bayesian MCMC runs was assessed by checking
that (1) the standard deviations of split frequencies were
less than 0.01; (2) the log probabilities of the data given
the parameter values fluctuated within narrow limits; (3)
that the convergence diagnostic (the potential scale re-
duction factor [PSRF] given by MrBayes) approached 1
and (4) by examining the plot provided by MrBayes of
the generation number versus the log probability of the
data. Trees sampled prior to convergence were discarded
as burn-in (2500 trees) and a majority rule consensus
tree was constructed from the remaining trees with clade
credibility values (posterior probabilities; PP).
The DNA sequence matrix covering 57 accessions from
16 taxa yielded a reasonably well-resolved phylogram
(Fig. 3). The outgroup taxa Ephedra fragilis and E. foe-
minea (Outgroup, Fig. 3) are sister to an unambiguously
supported clade of the three accessions of E. likiangensis
Florin (ML BS 100 % / PP 1.00). This clade is in turn
Fig. 3. Maximum likelihood tree for Ephedra obtained from nr ITS1 sequences. Likelihood bootstrap values are indicated above
branches, Bayesian posterior probability values below branches, and the geographic origin of each accession follows species
names. Our own sequences from species of the E. distachya / E. sinica group are marked by a geographical abbreviation, those from
related species by an asterisk, while data from GenBank is shown in gray font (E. dahurica Brt 3 7 represents five accessions with
identical ITS1 sequence from Buryatia; E. sinica Chn 1 5 represent five accessions from China and E. sinica Mng 3 7 represents
five accessions from Mongolia with identical ITS1 sequences; GenBank E. intermedia AY394070 represents four accessions with
identical ITS1 sequences). Branches with hash marks have been shortened to allow for enlargement of ingroup relationships.
209Willdenowia 41 – 2011
marginally well supported (ML BS 59 % / PP 0.65) to
be the sister of the remaining taxa that form two unam-
biguously supported clades (ML BS 100 % / PP 1.00).
The first of these two highly supported clades (E. mono-
sperma clade, Fig. 3) is comprised of E. monosperma,
E. saxatilis (Stapf) Royle ex Florin and E. gerardiana
Wall. ex Florin. The second, larger clade comprises three
subclades: The first, unambiguously supported clade (E.
strobilacea clade, Fig. 3) consists of E. transitoria, E.
strobilacea and E. sarcocarpa (BS 100 % / PP 1.00). The
second, marginally supported clade (E. distachya clade,
Fig. 3) comprises all 20 accessions of E. distachya ex-
cept for the easternmost accession of E. distachya from
Uzbekistan (ML BS 52 % / PP 0.53). The individual E.
distachya samples differ from each other by up to three
nucleotide changes. The third subclade shows a basally
branching E. distachya (GU065261) from Uzbekistan as
sister to the remaining species. Within the remaining spe-
cies a polytomy shows a well supported clade made up of
7 accessions of E. dahurica representing 8 samples and
3 accessions of E. sinica (representing 11 samples) that
have almost identical sequences. These 10 sequences of
E. dahurica and E. sinica are placed in a well supported
clade (ML BS 92 % / PP 0.99) together with a sequence
of E. intermedia (AY394070, representing 4 accessions).
Another small well supported clade (E. regeliana clade,
Fig. 3) consists of the two accessions of E. regeliana (ML
BS 90 % / PP 0.98). Relationships between the remaining
accessions of E. intermedia, E. lomatolepis, E. przewals-
kii and E. pseudistachya are not well supported and the
taxa cannot be reliably placed, except for two accessions
of E. intermedia, which are highly supported in a clade
(E. intermedia clade, Fig. 3, ML BS 85 % / PP 0.99), as
well as another well supported clade of two accessions
of E. lomatolepis (BS 89 % / PP 1.00). Three additional
accessions of E. lomatolepis group together with four ac-
cessions of E. pseudodistachya in a weakly to moderately
supported clade (Fig. 3, ML BS 54 % / 0.88 PP).
Relationship of the Ephedra distachya / E. sinica group
to other species of the “L” clade
The phylogenetic analysis corroborates the monophyly
of the “L” clade (Rydin & al. 2010, identical with the
second Asia clade in Rydin & Korall 2009) that was pre-
viously sampled by Ickert-Bond & al. (2009), Rydin &
Korall (2009), Rydin & al. (2010) and Ickert-Bond &
Rydin (2011). Likewise its position related to the “K”
clade (= first Asia clade, respectively), which widely cor-
responds to our Ephedra monosperma clade, and the “H”
clade (= China clade), here named E. likiangensis clade,
remains basically unchanged (Fig. 3). An interesting and
well supported difference (see Results) is the clear separa-
tion of the E. strobilacea clade. While in earlier analyses
E. strobilacea, E. sarcocarpa and E. transitoria formed
a subclade together with E. distachya in the “M” clade
(Rydin & al. 2010), in our phylogeny they appear as a
distinct clade at the base of the “L” clade. The distinct po-
sition of these taxa is further supported by sharing a very
stout habit and inhabiting extremely arid semideserts of
the Irano-Turanian region. It is interesting to note that
the two accessions of E. strobilacea are not resolved as
monophyletic, rather one of the accessions (AY599162)
appears basally to the remainder, possibly due to the fact
that it belongs to the morphologically very similar E. mi-
crobracteata Ghareman ( E. strobilacea subsp. micro-
bracteata (Ghareman) Freitag & Maier-St.).
Previously, the “N” clade has been shown to include
only Ephedra distachya, E. sinica and E. lomatolepis, but
now we show conclusively that E. pseudodistachya and
E. dahurica also belong here (Fig. 3). However, as in phy-
logenies published before, the morphologically defined
Ephedra distachya / E. sinica group is not monophyletic
because E. intermedia, E. regeliana and E. przewalskii
Stapf, which on morphological grounds are well separat-
ed, remain nested among E. sinica, E. dahurica, E. pseu-
dodistachya and E. lomatolepis. On the other hand, E.
fedtschenkoi Paulsen, which is shown as a member of the
“N” clade in Rydin & Korall (2009), is according to our
own sequences (not shown) from material collected near
the type locality, instead well supported as a member of
the “M” clade. The material analysed by Rydin & Korall
(2009) is from a cultivated plant of unknown provenance
and likely misidentified.
Ephedra intermedia — This is the most puzzling spe-
cies because the different accessions included in this study
are placed in three different clades with two of them un-
ambiguously supported and one (AY394070, representing
three different vouchers from Xinjiang province, Long
& al. 2005) having the same ITS signature as E. sinica
and E. dahurica. Our results indicate that E. intermedia
is polyphyletic. Rydin & al. (2010) already detected that
their four accessions of E. intermedia were placed in two
different clades. However, at this point no conclusions can
be drawn because the hitherto analysed accessions cover
only small sections of the vast distributional area (western
Iran to central China). It is noteworthy that seven segre-
gate species have been described by different authors, of-
ten from the same or adjoining areas for E. intermedia:
E. glauca Regel (1880), E. heterosperma Nikitin (1957),
E. microsperma Nikitin (1957), E. ferganensis Nikitin
(1957), E. rituensis Yang & al. (2003), E. sumlingensis
Sharma & Uniyal (2009), E. kardangensis Sharma & al.
(2010), E. khuri kensis Sharma & al. (2010). Except for
E. glauca, none of these species are generally accepted.
Further studies are required to disentangle the conflicting
morphological and molecular diversity of the E. interme-
dia species complex.
Ephedra regeliana — The two accessions of E. rege-
liana unambiguously group together (Fig. 3). They are
equally well separated from the related Central Asian
species by the ITS signature as by their morphology and
210 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
Ephedra przewalskii The two accessions se-
quenced here are included in a weakly supported clade
(ML BS 51 %, PP 0.57) together with E. intermedia, E.
lomatolepis and E. pseudodistachya, but their position
within this clade is not well supported, contrary to the
unmistakable morphological characters, particularly the
distinct membranous bracts.
Relationships among the taxa of the Ephedra dis ta-
chya / E. sinica group
Ephedra distachya We included 17 sequences repre-
senting 21 accessions, by far the broadest sampling ever
analysed not only of E. distachya but of any species of
the genus. 16 sequences (20 accessions) form a margin-
ally supported clade (ML BS 52 %, PP 0.53), while one
sample from western Uzbekistan appears well supported
as the basally diverging species in the E. dahurica clade,
separated from all other E. distachya accessions. Sequenc-
es included for E. distachya show the highest degree of
variation in the ITS1 sequences studied. It is noteworthy,
that the E. distachya sequences sampled come from a vast
distributional area, which extends over several floristic
regions with rather diverse climatic conditions. However,
a detailed look at the site conditions reveals fundamental
similarities. From eastern Europe to western Siberia, E.
distachya is a component of zonal dry steppes and semi-
desert communities, prefers coarse-textured soils and is
often associated with species such as Stipa capillata L.,
Artemisia campestris L., Kochia prostrata (L.) Schrad.,
Krascheninnikowia ceratoides (L.) Gueldenst. and Sal-
sola laricina Pall. The isolated occurrences in Central
Europe are typical azonal islands on skeletal soils with
impoverished steppe vegetation. In the northern Mediter-
ranean as well as along the Atlantic coast E. distachya
grows in sand dunes or on dry pebble layers. This distri-
butional pattern can best be explained by extensive plant
migrations followed by partial extinctions caused by cli-
matic fluctuations during the Pleistocene (Frenzel 1992;
Lang 1994).
Interestingly, no trend towards geographical clustering
of mutations is recognisable (see Fig. 3). Instead, some-
times samples from widely separated locations group to-
gether (e.g. GU065257 from Kazakhstan and GU065272
from southern France), while on the other hand samples
from adjoining places differ more conspicuously in their
sequences (e.g. the three Turkish samples). Even the three
samples of the morphologically distinct Ephedra dis-
tachya subsp. helvetica, which include the most doubtful
E. negrii (our sample Itl1), do not group together though
probably it represents the youngest taxon of the genus. As
the area of the Alps was completely covered by ice during
long intervals of the Pleistocene, most likely the endemic
subspecies has originated from re-migrating subspecies
distachya only after the retreat of the glaciers that hap-
pened c. 15 000 years ago. Detailed molecular studies,
which include a broad sampling of the populations in and
around the Alps are underway by E. Zippel (Berlin Bo-
tanic Garden and Botanical Museum).
The morphologically doubtful subdivision of Ephe-
dra distachya into a Mediterranean subsp. distachya and
an eastern European to Siberian subsp. monostachya
(L.) Riedl (see p. 204) is not supported by our data. An
eastern segregate of E. distachya could be presumed from
the phylogeny by Rydin & al. (2010), where a collection
made by F. K. Karo (no. 236) differs considerably from
the otherwise rather homogeneous cluster. The sample
is cited as having been collected in southeastern Russia,
but at that time Karo collected in southeastern Siberia,
mainly in Dahuriya (Chaudhri & al. 1972), far outside of
the area of E. distachya. Most likely that sample belongs
to E. dahurica, which otherwise is omitted from their
sampling. E. vvedenskyi and E. aurantiaca (see p. 204)
were not included in our sampling. However, a GenBank
accession (GU968571, Rydin & al. 2010) under E. dis-
tachya (Fig. 3) could well represent E. vvedenskyi as it
was taken from its core area, Turkmenistan. It agrees
with other samples of E. distachya from the Ukraine.
After all, at least one definite conclusion can be drawn
from our results: Ephedra pseudodistachya and E. dahu-
rica, which are morphologically more similar to E. dis-
tachya, are clearly confirmed as distinct from E. distachya
based on their differing molecular signatures. From the
localities cited by the pre-Linnean botanists Amman
(1739) and Gmelin (1747) from Siberia it is evident that
they have lumped together all three species that later were
named E. monostachya, but by chance unfortunately only
one specimen of E. distachya was sent to Linné.
Ephedra sinica and E. dahurica The 19 accessions
of E. sinica and E. dahurica used in this study showed
extremely low sequence divergence and only 7 distinct
sequences are included (several samples had identical
sequences, Fig. 3), but form a well supported clade (E.
dahurica clade, Fig. 3). Unexpectedly they are joined by
one sequence of E. intermedia that represents 4 acces-
sions with identical nucleotide sequences from Xinjiang
province in western China beyond the known area of E.
sinica and E. dahurica (see p. 205). The three taxa have
almost identical ITS1 sequences except for one Chita ac-
cession, which differs in one base position (Fig. 3). This
low sequence divergence contrasts with the intraspecific
sequence variation found in E. distachya. It is even more
surprising when considering that the 19 specimens of
the E. dahurica / E. sinica group span a rather large area
from near Lake Baikal in Buryatia to eastern China.
These molecular results are somewhat disappoint-
ing because they do not give any support to the morpho-
logically based discrimination of the three species. The
deviating Ephedra intermedia accession AY394070 can
be clearly distinguished from E. sinica and E. dahurica,
but not from other populations of E. intermedia, by the
corkscrew-like bent micropylar tube, a mutation that
parallels E. distachya subsp. helvetica, and furthermore
211Willdenowia 41 – 2011
by the stout habit and the distinct geographical distri-
bution. The taxonomy of E. sinica and E. dahurica are
further complicated. Though the differential characters
are well expressed in the authentic material, the plants
in the field and in herbarium collections from Mongolia
and southeastern Siberia show all transitions in length
of leaves, colour of leaf sheaths and structure of twig
surface, so that many plants could be assigned to the
one or the other species only statistically. At first, when
comparing the type material, we expected that their dif-
ferent habit could serve as a good additional character
for identification. While E. dahurica (one specimen in
LE) shows a typical chamaephytic habit, with annual
twigs arising from much stronger and woody perennial
branches, the very many original specimens (lectoypifi-
cation in prep. by Freitag & Maier-Stolte) of E. sinica (F,
GH, K, LE, US) are almost hemicryptophytic, with per-
ennial stems more or less missing and the annual twigs
arising in dense bunches at the level of the soil surface.
However, in the field (Buryatia) both types occur in ad-
joining habitats: the subshrubby plants on rocky slopes
with Caragana pygmaea (L.) DC. and Artemisia gmeli-
nii Web. ex Stechm. and the hemicryptophytic plants on
gravelly lower slopes and in plains covered by steppes
with dominating Stipa krylovii Roshev. Obviously the
rocky slopes offer a better water supply and the steep
topography protects the plants better against browsing
by goat and sheep. Intermediates between both growth
forms are common.
For similar reasons Galanin (2008) already reduced
both taxa to subspecies. However, his new combination
Ephedra sinica subsp. dahurica (Turcz.) Galanin is for-
mally incorrect because E. dahurica is the earlier validly
published name. Therefore, if the two taxa are considered
as subspecies, they must be named E. dahurica subsp.
dahurica and E. dahurica subsp. sinica (Stapf) Freitag
& Maier-St., respectively. Anyhow, E. dahurica seems
to be more or less restricted to southeastern Siberia and
northern Mongolia, whereas E. sinica is common in the
steppe areas of eastern and northeastern China. More de-
tailed morphological studies in the area of subsp. sinica
are warranted.
It has been shown that also the ephedrine content var-
ies considerably in Ephedra sinica s.l., from 1.2 1.3 %
for Mongolia, 0.76 0.83 % for Inner Mongolia and
0.45 % for Buryatia (Wang & al. 2010).
Our results somewhat contrast with those of Rydin
& al. (2010). Out of their 4 samples of Ephedra sinica,
No. 151 from Inner Mongolia takes a separate position,
which could reflect either intraspecific variation not ob-
served in our larger sampling, or incorrect identification.
Ephedra lomatolepis Our phylogenetic analysis un-
fortunately does not resolve the relationships in the
composite clade consisting of E. lomatolepis, E. pseudo-
distachya, E. przewalskii and one accession of E. inter-
media. Only one small subclade made up of two samples
of E. lomatolepis is well supported and its placement
among several accessions of E. przewalskii confirms the
close morphological affinity to that species. However,
three other accessions of E. lomatolepis cluster together
in a weakly supported subclade with the morphologically
differing E. pseudodistachya, with which it is widely
sympatric, and one accession even with E. intermedia.
We still hesitate to conclude that E. lomatolepis is a
polyphyletic taxon because the danger of misidentifica-
tions is apparent, particularly in the information taken
from GenBank. In contrast to our results, the five samples
of E. lomatolepis included by Rydin & al. (2010) cluster
together and appear as sister to E. przewalskii. However,
these authors did not sample E. pseudodistachya.
Ephedra pseudodistachya The four samples of E.
pseudodistachya appear indistinguishable from E. lo-
matolepis in the respective heterogeneous clade. Nev-
ertheless, a most interesting result of our study is the
distinct position of E. pseudodistachya as compared
to E. distachya and E. dahurica. By that, the ITS data
support its status as a distinct species, which so far is
not generally accepted by taxonomists. The three spe-
cies are morphologically and ecologically very close to
each other. E. pseudodistachya was collected from stony
slopes in steppe areas with dwarf shrubland communities
of Spiraea hypericifolia M. Bieb., S. media F. Schmidt,
Berberis sibirica Hort. ex Schult. f. and Stipa capillata L.
in Tuva and from extremely dry rocky slopes in southern
It is noteworthy that the low amount of nr ITS1 sequence
divergence and the almost complete lack of variation in
ITS2 and cp trnL-F sequences corroborates findings and
conclusions in other studies that species of the Ephedra
distachya / E. sinica complex are comparatively young.
Probably they have evolved only from the late Miocene
and early Pliocene onwards (e.g. Ickert-Bond & al. 2009).
This is also reflected by the comparatively small amount
of morphological variation, but contrasts with the bioge-
ographical and ecological specialisation in the Eurasian
Ephedra species.
Because of the generally low or missing sequence
divergence in traditionally used molecular markers, the
search for more highly variable regions should be inten-
sified. However, particular attention also should be given
to unambiguously identified plant material in order to
prevent an increase of confusion in GenBank data. For
the time being, the taxonomy of the respective groups
can be improved by further morphological studies only.
With regard to the “N” clade this is warranted especially
in case of Ephedra intermedia, but also in the eastern pop-
ulations of E. distachya (W Kazakhstan and Uzbekistan)
as well as in E. pseudodistachya and E. lomatolepis in
Central Asia.
212 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
This study was supported by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan. We gratefully acknowledge also
the funding of field work by the Deutsche Forschungsge-
meinschaft (DFG): Kazakhstan 1992, grant Fr 438; Cas-
pian Lowland 1998, grant Fr 222/14-1; Uzbekistan 2000,
grant We 1830/2-1; S Siberia 2003, grant We 1839/2-3,
and by the National Science Foundation (USA), grant
NSF-0629657. We like to thank Dr Dava Chimitov for
support during the field work in Buryatia, Ms Miyuki
Sakai, Mr Yuhki Abiru and Mr Jordan Metzgar for tech-
nical assistance and Dr Elke Zippel for providing the im-
age reproduced in Fig. 1C. Finally, we want to thank two
reviewers for their helpful comments.
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Y. (ed.), Flora xinjiangensis 1.Wulumuqi: Xinjiang
Science, Technology and Hygiene Publishing.
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(Ephedraceae) from China. – Novon 13: 153 155.
214 Kakiuchi & al.: Molecular phylogenetic study of the Ephedra distachya / E. sinica complex
Appendix 1. List of taxa sampled with voucher and
GenBank accession numbers
Full voucher data include collector and collecting number,
collecting date, collection locality and, in brackets, her-
barium code (following Thiers 2008+) and herbarium ac-
cession number, if any. The last part of an entry are the
GenBank accession numbers, referring to the ITS1 and
trnL-F sequences (for Brt 3 7, Chn 1 5 and Mng 3 7
only one accession was submitted to GenBank since the
data were identical for each of the respective accessions).
Data for own sequences from taxa of the Ephedra dis-
tachya / E. sinica group are preceded by a geographical
abbreviation. Data for sequences (ITS1) taken from Gen-
Bank are preceded by an asterisk.
Ephedra dahurica Turcz.: Brt 1, H. Freitag 33.121
(KAS), 3.9.2003, Russia, Buryatia, Gusinoozersk,
GU065255, GU111509; Brt 2, H. Freitag 33.119 (KAS),
3.9.2003, Russia, Buryatia, c. 95 km SW Ulan-Ude,
GU065267, GU111521; Brt 3, M. Mikage & al. (KANP
060804337), 4.8.2006, Russia, Buryatia, suburb of
Ulan-Ude, GU186919, GU186921; Brt 4, 5, M. Mikage
& al. (KANP 060809358, KANP 060809361), 9.8.2006,
Russia, Buryatia, Tugnuiskii Steppe, GU186919,
GU186921; Brt 6, 7, M. Mikage & al. (KANP 060810366,
KANP 060809370), 10.8.2006, Russia, Buryatia,
Khoshun-Uzur near Ulan-Ude; Cht, H. Freitag 33.130
(KAS), 11.9.2003, Russia, Chita dist., Daurya Biosphere
reserve, GU065253, GU111507. E. distachya L.
subsp. distachya: Spn, H. Freitag s.n. (KAS), 30.5.1963,
E Spain, Dehesa de Valencia, GU065271, GU111524;
Frc, P. König s.n. (KAS), 26.8.1981, S France, Roussil-
lon, St. Cyprien Plage, GU065272, GU111525; Trk 1,
H. Freitag 28.772 (KAS), 5.10.1997, Turkey, C Anato-
lia, Konya prov., GU065256, GU111510; Trk 2, M. Ny-
degger 45.473 (KAS), 19.5.1990, Turkey, E Anatolia,
near Aralik, GU065258, GU111512; Trk 3, H. Freitag
28.834 (KAS), 8.10.1997, Turkey, Ankara prov., E side
of Tuz Gölü Lake, GU065260, GU111514; Ukr 1, H.
Freitag 33.259 (KAS), 12.10.2003, Ukraine, S Crimea,
Ayudag Mt., GU065252, GU111506; Ukr 2, H. Freitag
33.231 (KAS), 9.10.2003, Ukraine, NE Crimea, Ara-
batskaya Strel’ka, GU065254, GU111508; * Ukraine,
Pidopliczka, s.n. (UPS), 1925, FJ958013; Syr, H. Freitag
30.144 (KAS), 17.10.2001, Syria, 40 km NE Damascus,
GU065273, GU111526; Uzb, W. Wucherer 646 (KAS),
1.10.1994, Uzbekistan, Aral Lake, cliff on W side,
GU065261, GU111515; Kzh 1, H. Freitag 26.506 (KAS),
26.9.1992, Kazakhstan, Kzyl-Orda dist., 54 km E No-
vokazalinsk, GU065257, GU111511; * W. Wucherer 453
(KAS), S Kazakhstan, coast of Aral Lake, AY599135; *
Kolakovsky 3651 (S), 1951, Turkmenistan, GU968571;
(Rydin) 03-684 (S), AY755769. — E. distachya subsp.
helvetica (C. A. Mey.) Asch. & Graebn.: Itl 1 (“E. negrii
Nou viant”), H. Freitag 26.678 (KAS), 22.5.1993, N Ita-
ly, S Tyrol, Vinsch gau, GU065259, GU111513; Sws 1-5,
N. Kakiuchi & al. 1-5 (KANP 08080801-3, 08080805-6),
8.8.2008, Switzerland, Valais, Sion Castle, GU065276,
GU111529; * H. Freitag 20.332 (KAS), 4.8.1990, Swit-
zerland, Wallis, Mont Orges, AY599133. — E. gerardi-
ana Wall. ex Florin: G. & S. Miehe 3.329 (herb. Miehe),
9.7.1990, Pakistan, Yasin, HQ876913; H. Freitag 34.019
(KAS), 3.9.2004, India, Himachal Pradesh, HQ 876914.
E. intermedia Schrenk & C. A. Mey.: * Bartolo mew
8.211 (MO), 2001, China, Xinjiang, GU968563; * C.
Long & al. (KANP 03071510), 17.7.2003, China, Xin-
jiang, Guhe Zhen, AY730604; * C. Long & al. (KANP
02309, 02341, 02363, 02364), 23.7.2003, China, Xin-
jiang, Aerjin Mountain, AY394070; * [Rydin] 03-925 (S),
AY755741. E. likiangensis Florin: M. Mikage & al.
707063 (KANP), 22.7.2007, China, Sichuan, FJ868729;
M. Mikage & al. 707026 (KANP), 16.6.2007, China,
Yunnan, FJ868730; G. & S. Miehe 04-165-17 (herb.
Miehe), 20.9.2004, China, SE Xizang, HQ876918. — E.
lomatolepis Schrenk: Kzh 2, H. Freitag & S. Rilke 26.507
(KAS), 26.9.1992, Kazakhstan, Kzyl-Orda dist., 54 km E
Novokazalinsk, GU065262, GU111516; Kzh 4, H. Frei-
tag & S. Rilke 26.343a (KAS), 18.9.1992, Kazakhstan,
Dzhambul dist., 74 km ESE Ulanbel’, GU065263,
GU111517; Kzh 5, H. Freitag & S. Rilke 26.355 (KAS),
24.8.1992, Ka zakhstan, Dzhambul dist., 72 km SE
Ulanbel’, GU065269, GU111522; * I. O. Baitulin & al.
(UPS), 1997, Ka zakhstan, FJ958006; * I. M. Krasnoborov
192 (MO), 1995, Siberia, Novosibirsk, GU968564; * A.
Bosshard & al. 803.24 (Z), 1989, Pakistan, GU968562.
E. mono sperma J. G. Gmel. ex C. A. Mey.: H. Frei-
tag 33.031 (KAS), 22.8.2003, Buryatia, 30 km S Kyzyl
beside track towards Duz Khol, HQ876927; * H. Hurka
& B. Neuffer 12.142 (KAS, OSBU), Mongolia, Gobi
Altai, AY599139. E. przewals kii Stapf: * C. Long & al.
(KANP 02321, 02326, 02340), 9.7.2003, China, Xin-
jiang, AY394072; * C. Long & al. (KANP 03071131),
13. 22.7.2003, China, Xinjiang, AY730601. E.
pseudodis tachya Pachom.: Kzh 3, H. Freitag 27.000
(KAS), 1.5.1994, Kazakhstan, Chimkent dist., Alimtau,
GU065264, GU111518; Tuv, H. Freitag 33.002 (KAS),
20.8.2003, Russia, C Tuva, near Kyzyl, GU065266,
GU111520; Mng 1, W. Hilbig & Z. Schams ran s.n. (HAL),
14.8.1976, Mongolia, Altai dist., E border of Schargain
Gobi, GU065275, GU111528; * H. Hurka & B. Neuffer
10.242 (OSBU), Mongolia, Hovd Aimak, AY599169. —
E. regeliana Florin: * U. Wündisch 956 (KAS), 6.8.1998,
China, SW Xinjiang, AY599160; G. Miehe 5.318 (herb.
Miehe), 23.7.1991, W China, Xinjiang, upper Gez Gorge,
HQ876924. — E. sarcocarpa Aitch. & Hemsl: * H.
Freitag 13.988 (KAS), 2.8.1976, Iran, Kavir desert near
Mobarakiyeh, AY599137; * K. H. Rechinger & al. 2.786
(S), 14.4.1948, Iran, prov. Semnan, inter Veramin et Sem-
nan, FJ958017. E. saxatilis (Stapf) Royle ex Florin:
U. Wündisch 1.090 (KAS), 9.8.1995, Nepal, Langtang
valley, HQ876916; G. Miehe 95-18-11 (herb. Miehe),
4.9.1995, China, SE Xizang, AY599140; R. Singh & P.
Radha (KAS), 6.7.2004, India, Uttanranchal, Deoban,
HQ876915. — E. sinica Stapf: Mng 2, M. Mikage & al.
215Willdenowia 41 – 2011
(KANP 20531001), 29.7.2005, Mongolia, Tov dist., Lun,
GU186918, GU186920; Mng 3, M. Mikage & al. (KANP
20531062), 6.8.2005, Mongolia, Tov dist., Baruun haraa;
Mng 4, M. Mi kage & al. (KANP 20531081), 8.8.2005,
Mongolia, Tov dist., Lun; Mng 5, 6, M. Mikage & al.
(KANP 20531083, KANP 20531085), 8.8.2005, Mongo-
lia, Tov dist., Hustayn; Chn 1, M. Mikage & al. (KANP
2109), 5.6.2002, China, Inner Mongolia, Eerdousi,
AY394071, AY423430; Chn 2, M. Mikage & al. (KANP
2133), 8.6.2002, China, Shangxi prov., Datone; Chn 3,
M. Mikage & al. (KANP 2139), 10.6.2002, China, Hebei
prov. Chengde; Chn 4, M. Mikage & al (KANP 2143),
1.6.2002, China, Inner Mongolia, Chifeng; Chn 5, M.
Mikage & al. (KANP 2145), 11.6.2002, China, Inner
Mongolia, Tongliao; * J. Eriksson 05-9020 (S), 1926,
China, Inner Mongolia, GU968550. E. strobilacea
Bunge: * K. H. Rechinger & al. 2.703 (S), 21.4.1948,
Iran, Semnan and Yesd prov., between Nain and Aghda,
FJ 958018; * K. H. Rechinger 27.161 (US), 26.3.1965,
Iran, Kerman, near Fahraj, AY599162. E. transitoria
Riedl: * H. Freitag 30.123 (KAS), 12.10.2001, Jordan,
Shaumari Wildlife Res., AY599172.
Outgroup: Ephedra foeminea Forssk.: H. Freitag 19.807
(KAS), 20.9.1989, Greece, Kephallonia Is., GU065270,
GU111523. — E. fragilis Desf.: * H. Freitag 27.237
(KAS), 24.9.1995, SE Spain, AY 599130.
Contents of Willdenowia 41 · 2
Phylogeny and plant molecular systematics
n Kakiuchi N., Mikage M., Ickert-Bond S., Maier-Stolte M. & Freitag H.: A molecular phylogenetic study of the Ephedra
distachya / E. sinica complex in Eurasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
n Lomonosova M. & Freitag H.: Typification of plant names in Suaedoideae (Chenopodiaceae) published by P. Pallas,
C. A. Meyer & A. Bunge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
n Freitag H.: Typification of Salicornia perennans Willd. (Chenopodiaceae/Amaranthaceae) and the significance of
names by Pallas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Taxonomy of plants of Europe and the Mediterranean
n Dunkel F. G.: Typification of Ranunculus binatus Kit. ex Rchb. (Ranunculaceae) . . . . . . . . . . . . . . . . . . . . . 239
n Hand R.: The Euro+Med treatment of Apiaceae [Notulae ad floram euro-mediterraneam pertinentes 28] . . . . . . . . 245
Taxonomy of plants of Africa
n Otte V., Fleischer B., Stoll A. & Bräutigam S.: Type material of names based on plants from Namibia collected by
F. Schäfer and kept at the Senckenberg Museum of Natural History, Görlitz (GLM) . . . . . . . . . . . . . . . . . . . . 251
Taxonomy of plants of Asia
n Mehregan I.: Cousinia saloukensis (Asteraceae, Cardueae), a new species from NE Iran . . . . . . . . . . . . . . . . . 261
n Pahlevani A. H., Maaroofi H. & Joharchi M. R.: Notes on six endemic or rare species of Euphorbia subg. Esula
(Euphorbiaceae) in Iran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Taxonomy of plants of the Americas
n Lepper L. & Gutiérrez Amaro J. E.: Theophrastaceae cubanae novae IV [Novitiae florae cubensis 36] . . . . . . . . . . 277
n Bécquer E. R.: Calycogonium pseudofloribundum, a new species of Melastomataceae, Miconieae, from eastern
Cuba [Novitiae florae cubensis 37] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
n Szlachetko D. L., Mytnik-Ejsmont J., Dudek M. & Górniak M.: Three new species of the genus Takulumena
(Orchidaceae, Epidendrinae) from Ecuador and Peru . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
n Lack H. W.: The discovery, naming and typification of Euphorbia pulcherrima (Euphorbiaceae) . . . . . . . . . . . . . 301
Inventorying plant diversity of the Mediterranean
n Greuter, W. & Raus, Th. (ed.): Med-Checklist Notulae, 30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
n Barina Z., Pifkó D. & Mesterházy A.: Contributions to the flora of Albania, 3 . . . . . . . . . . . . . . . . . . . . . . . 329
n Hand R. (ed.): Supplementary notes to the flora of Cyprus VII. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Inventorying plant diversity of the Americas
n Cáceres González D. A., Schulte K., Schmidt M. & Zizka G.: A synopsis of the Bromeliaceae of Panama, including new
records for the country . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
E-taxonomy and biodiversity informatics
n Rilke S. & Najmi U.: FloraGREIF – virtual guide and plant database as a practical approach to the flora of Mongolia . . 371
n Hiepko P.: Wolfram Schultze-Motel (1934–2011) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
n Buchbesprechungen / Book reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
n Index to new names and combinations appearing in Willdenowia 41(2) . . . . . . . . . . . . . . . . . . . . . . . . . 389
n Index to typifications of names in Willdenowia 41(2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
n Reviewers of manuscripts submitted for publication during 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
n Contents volume 41 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
... Rbcl Huang et al., 2005Wang et al., 2005 12. rps4 Ickert-Bond and Wojciechowski 2004;Rydin et al., 2004 13. trn K Zhu et al., 2012 14. Trn L Kakiuchi et al., 2011;Long et al., 2004 15. ...
... Thus such characters had proven to be useful for monitoring genetic diversity in gene pools and to elucidate the factors controlling changes in life history of natural populations such as breeding system, inbreeding depression, effective population size, population structure and gene flows (Lynch and Milligan, 1994). In genus Ephedra, molecular taxonomy had helped in solving the taxonomic complexity and misidentification problems and reduction to synonyms (Kakiuchi et al., 2011;Long et al., 2004;Faried et al., 2018). In some cases, the very closely species had been reduced to a subspecies due to high degree of sequence similarity such as E. dahurica to E. sinica subsp. ...
... In some cases, the very closely species had been reduced to a subspecies due to high degree of sequence similarity such as E. dahurica to E. sinica subsp. dahurica (Kakiuchi et al., 2011) and recently E. dahurica had been reduced to E. sinica (Kakiuchi, 2017). In other cases, different species had been separated from species complex such as E. ...
The descriptions of dubious new species that lack robust taxonomic rigor create more confusion rather than bridging the Linnean shortfall in biodiversity. In an era of biodiversity crisis, it becomes urgent to undertake integrative taxonomic revisions to resolve taxonomic confusions in several plant taxa, even if that leads to drastic decline in the species number. Here I resolve the taxonomic conundrum of Ephedra in India by adopting an integrative taxonomic approach and using comprehensive set of characters from multiple lines of evidence (morphology, anatomy, palynology, seed micromorphology and molecular data). I reduce the number of Ephedra species in India from the currently known 16 to only 4 well-defined species: E. foliata, E. gerardiana, E. intermedia and E. regeliana. I provide proper species delimitations, detailed descriptions, taxonomic keys, photoplates of diagnostic characters, regional distributions and phylogenetic relationships of Ephedra in India, validated by robust empirical evidence. Our studies reveal that the previously reported three species: E. nebrodensis, E. pachyclada and E. przewalskii do not occur in India. The recently described four species (E. sumlingensis, E. pangiensis, E. khurikensis and E. yangthangensis) are synonymized with E. intermedia, and another E. kardangensis synonymized with E. gerardiana. Five recent designations viz. E. sheyensis, E. yurtungensis, E. yurtungensis var. lutea, E. lamayuruensis and E. khardongensis are recognized as nomen nodums due to lack of descriptions, diagnosis and type specimens. Our study provides a robust and reliable set of 16 morphological characters, validated by significant statistical support, which can prove useful for species delimitation in Ephedra. I also recorded few novel characters of evolutionary significance in Ephedra, which merit further investigation in future. Looking ahead, I believe that the methodological and data analytical learnings from this study can guide the future research direction in designing integrative taxonomic studies on such complex plant taxa elsewhere in the world.
... The cultivation of this plant was reported in few studies, but many fields contain other species of Ephedra. Genetic studies with ITS (internal transcribing sequence of nuclear ribosomal DNA) on Chinese Ephedra fields have shown that crops grown in six fields have been identified as Ephedra sinica and E. intermedia (Kakiuchi et al., 2011). In North African regions (native origin of this species), Louhaichi et al. (2011) mentioned the establishment of two field genebanks containing E. alata in arid zone of Libya. ...
Desert plants are a potentially sustainable source of bioactive molecules. This paper investigates phenolic profile, total polyphenols, flavonoids and condensed tannins in a collection of desert shrubs of Tunisian flora (Pituranthos tortuosus; Retama raetam; Ephedra alata; Ziziphus lotus; Capparis spinoza; Calligonum comosum). The collection was build according to people traditional tendency uses. The chemical composition was estimated in ethanol extracts of aerial parts together with a comparison of antioxidant activities. The results were significantly dependent on plant material source. Phenolic contents ranged from 15.52 to 37.5 mg of gallic acid equivalents (GAE)/g of Dry Weight (DW) of plant, and flavonoids ranged from 2.92 to 9.14 mg of rutin equivalents (RE)/g DW. The phenolic profile was composed of 23 compounds identified through LC-ESI-MS analysis. Five phenolic acids (quinic acid, gallic acid, syringic acid, p-coumaric acid, and trans-Ferulic acid) and five flavonoids (catechin, epicatechin, rutin, quercetrin and naringenin) were predominant and common between plants. Based on all data visualized within the heatmap, plants were clustered into two groups. The tow species Z. lotus and E. alata were selected for their highest antioxidant potential. These results allowed us to select the combination of three phenolic acids (rutin, qurectrin, quinic acid) that seems determinant in the strong antioxidant capacity. In addition, the possible domestication and cultivation of the two selected plants are discussed.
... It is partially because gross morphology does not reveal considerable interspecies variation, particularly in Ephedra as well in as some Gnetum lineages, e.g., Chinese lianoid Gnetum. In addition, intraspecific variation is also present in some species that possess a broad geographic distribution, e.g., E. distachya (Kakiuchi et al. 2011) and G. parvifolium (Huang et al. 2010), which further complicates taxonomic choices and assessments of species delimitation. In addition, vegetative parts of the Gnetales may exhibit plasticity as a response to the environment. ...
Full-text available
... A revision of the New World species is currently in preparation by the first author, and the Asian species present the greatest taxonomic challenge. New species of Ephedra continue to be described, mostly from India and China, although they appear morphologically close to E. intermedia and E. saxatilis (Yang et al., 2003;Yang, 2005;Sharma & Uniyal, 2009;Sharma et al., 2010;Sharma & Singh, 2015 (Freitag & Maier-Stolte, 1993, 1994Kakiuchi et al., 2011). Rydin et al., 2004;Huang et al., 2005;Rydin & Korall, 2009;Loera et al., 2015). ...
Full-text available
Ephedra, Gnetum, and Welwitschia constitute the gymnosperm order Gnetales of still unclear phylogenetic relationships within seed plants. Here we review progress over the past 10 years in our understanding of their species diversity, morphology, reproductive biology, chromosome numbers, and genome sizes, highlighting the unevenness in the sampling of species even for traits that can be studied in preserved material, such as pollen morphology. We include distribution maps and original illustrations of key features, and specify which species groups or geographic areas are under sampled.
... Previous phylogenetic studies on Ephedra have identifi ed low levels of molecular sequence divergence, suggesting a recent origin (Ickert-Bond and Wojciechowski 2004, Rydin et al. 2004, Huang et al. 2005, Wang et al. 2005, Rydin and Korall 2009, Kakiuchi et al. 2011, Qin et al. 2013. In this study, we sequenced additional molecular markers to estimate the divergence time of NW Ephedra species, generating an alignment of 7703 bp, improving resolution and providing a more suitable framework for the objectives proposed in this study. ...
Full-text available
In this study we selected the New World species of Ephedra to understand the ecological consequences of different dispersal syndromes. The twenty-three species of Ephedra in the New World have a disjunct distribution in North and South American arid and semi-arid habitats, exhibiting three dispersal syndromes related to dispersal by birds, wind and rodents. Using DNA sequence data we inferred phylogenetic relationships and lineage divergence times, and used these estimates to test different ecological assumptions. Using comparative methods we tested for correlations between dispersal syndromes and a set of ecological variables (niche breadth, niche evolution, distributional ranges and niche position). We found that speciation events in the New World coincided with the expansion of arid habitats in this region. We suggest that the bird dispersal syndrome is related with higher rates of climatic niche evolution for all variables used, including aridity index, mean annual temperature and mean annual precipitation. Distribution ranges were correlated with niche breadth, they were however not significantly different between dispersal syndromes. Species inhabiting the extremely arid regions on niche axes had narrower niche breadths. We conclude that species whose seeds are dispersed by birds have colonized a broader set of habitats and that those with wind and rodent dispersal syndromes might have promoted the colonization of more arid environments.
Full-text available
Anatomically modern humans are now believed to have arrived in Eurasia as much as 80,000- 120,000 years ago (e.g., see Callaway 2015), and in bands of hunters and gatherers these early people moved east and west progressively across this massive landmass. In the process of spreading out over this vast continental region, they developed very ancient and long-lasting paleoethnobotanical relationships with numerous species; the more significant of these prehistoric relationships in Eurasia included medicinally and psychoactively important genera such as Cannabis, Papaver, and Ephedra (for Cannabis, see chapter 1 in this volume; for Papaver somniferum L., the opium poppy, see chapter 2). In the case of Ephedra, species in this genus appear to have been used very early on in some regions of both the Old and New World beginning many thousands of years ago.
Although Kampo medicine is now fully integrated into the modern Japanese healthcare system, most Kampo formulations depend on imported crude drugs from limited foreign areas. To prepare for possible shortages of crude drugs in the future, a wider scope for the supply of medicinal plants is necessary. We conducted field research and collaborated with international laboratories for phylogenic analysis and evaluation of medicinal plant resources. Our research on ephedra plants from a wide region of Eurasia has, for example, confirmed their phylogenic structure: based on DNA sequencing analysis of nuclear ribosomal internal transcribed spacer region 1 (ITS1) as well as the chloroplast intergenic spacer between trnL and trnF (trnL-F), the 8 major Chinese species and related plants grown on the continent could be divided into 3 groups. Additionally, Ephredra sinica was found to be synonymous with Ephredra dahurica and was reduced to a subspecies of Ephredra distachya. Furthermore, Ephredra likiangensis and Ephredra gerardiana, which are grouped in separate phylogenic trees, would be good candidates for medicinal material. Aconites from Hokkaido, as an example of domestic plants reviewed, were collected for phylogenic and aconitine alkaloid content analysis. The phylogenic analysis of nr ITSs revealed that the majority of specimens were genetically similar. However, the aconitine alkaloid content of the tuberous roots demonstrated that specimens from different habitats had varying alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profiles of these genetically similar specimens to diversify.
Full-text available
Ephedrae Herba (ma-huang in Chinese), the herbal stems of Ephedra sinica Stapf, which belongs to the Ephedraceae family, is an important crude drug in traditional Chinese medicine. Some researchers think that this species is identical to E. distachya L., which mainly grows in Europe. Thus, in this study, we carried out anatomical, chemical, and molecular genetic studies on E. distachya plants collected in Switzerland, France, and Turkey to consider whether E. distachya could be used to produce Ephedrae Herba. As a result, we found that the morphology of the subepidermal fiber bundles in cross sections of the herbal stems of E. distachya and the frequencies of ephedrine alkaloids in E. distachya were quite different from those observed in E. sinica and that the DNA sequence of the ITS1 region of E. distachya contains 11 base changes compared with that of E. sinica. Based on these facts, we concluded that E. distachya and E. sinica should be treated as different taxa and that, according to the prescriptions of the current Japanese and Chinese pharmacopoeias, E. distachya is not suitable for producing Ephedrae Herba.
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Classification of Ephedra has long been a matter of debate, as Ephedra plants have few morphological characteristics to aid classification. In particular, the distinction between E. equisetina and E. major subsp. procera is uncertain. Thus, in this study, an attempt was made to clarify their relationship by molecular analysis and content of ephedrine alkaloids. Molecular analysis revealed that E. equisetina and E. major subsp. procera have a close relationship with differing matrilineage, and chemical analysis showed that they have the same characteristic of varied alkaloid composition ratio by collection site. We suggest that on the basis of molecular phylogeny that E. major subsp. procera should be treated as a synonym or subspecies of E. equisetina instead of a subspecies of E. major.
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A review of Ephedra species of the Western Himalayan region led to the recognition of two new species, Ephedra kardangensis Sharma & Uniyal sp. nov. and E. khurikensis Sharma & Uniyal sp. nov. The most distinctive characters observed are shape and size of male strobilus, number of bracts, length of synangiophore with synangia, shape and size of female strobilus, number of bracts, size and color of seeds, and several features of the micropyle for morphological and statistical analysis (Parametric MANOVA and Principal Components Analysis). These species are most similar to E. intermedia.
An analysis of the publication, in instalments, of Turczaninow's Flora in the Bulletin de la Société impériale des naturalistes de Moscou together with a statement of the way the type was used to produce the consolidated reprint in book‐form.