Content uploaded by Maryam Khoshsokhan
Author content
All content in this area was uploaded by Maryam Khoshsokhan on Nov 12, 2016
Content may be subject to copyright.
ORIGINAL ARTICLE
Tribe Eritrichieae (Boraginaceae s.str.) in West Asia:
a molecular phylogenetic perspective
Maryam Khoshsokhan Mozaffar •Shahrokh Kazempour Osaloo •
Roghayeh Oskoueiyan •Kosar Naderi Saffar •Atefe Amirahmadi
Received: 13 March 2012 / Accepted: 4 October 2012 / Published online: 13 November 2012
ÓSpringer-Verlag Wien 2012
Abstract A selection of Boraginaceae genera was used to
obtain a framework for the phylogenetic position of some
tribes belong to subfamily Boraginoideae and genera
within tribe Eritrichieae (Heterocaryum,Rochelia,Eri-
trichium,Lappula,Lepechiniella, and Asperugo) and rela-
ted species. Our results were produced on the basis of
nrDNA ITS and cpDNAtrnL-F sequences. The combined
nrDNA ITS trnL-F data confirm four main clades of Bor-
aginoideae comprising Echiochileae, Boragineae, Litho-
spermeae, and Cynoglosseae s. l. (including Eritrichieae,
Cynoglosseae s. str., and Myosotideae). The tribe Eri-
trichieae itself at the current status is paraphyletic; some
members, for example Asperugo procumbens,Lepechini-
ella inconspicua,Myosotidium hortensia, and Cryptantha
flavoculata are placed out of the core tribe Eritrichieae. The
genus Heterocaryum is monophyletic and allied with a
subclade of genera Lappula,Lepechiniella,Eritrichium,
and Rochelia.Rochelia is monophyletic, but Eritrichium
and Lappula are non-monophyletic. Lepechiniella is nested
among a group of Lappula species.
Keywords Boraginoideae Eritrichieae Heterocaryum
Lappula Lepechiniella nrDNA ITS cpDNAtrnL-F
Molecular phylogeny
Introduction
Members of the Boraginaceae s. str. (=subfamily Boragi-
noideae) have a worldwide distribution with ca 130 genera
and 2,300 species (Mabberley 1997). This family is very
easily recognized by their vegetative and floral characters
(Popov 1953; Riedl 1967; Nasir 1989; Khatamsaz 2002).
However, they form a very heterogeneous group with a
wide range of variation, particularly in their floral and fruit
characters. For this reason, the family has been variously
divided into groups whose number and limits are not very
clearly defined. Boraginaceae has variously been divided
into 4–20 tribes (e.g. Popov 1953, 12 tribes) even though
some authors have accepted only four to seven tribes
(Bentham and Hooker 1873; Gurke 1897). The main tribes
of this family are Boragineae DC., Lithospermeae (DC.)
Gurke, Cynoglosseae DC., Eritrichieae Benth. and Hook.
Myosotideae Reichenb., Trigonotideae (M. Pop.) H. Riedl,
and Trichodesmeae Zak. (Riedl 1997). On the basis of atpB
gene sequences, Langstrom and Chase (2002), established
a new tribe Echiochileae (Riedl) Langstrom and Chase
composed of Echiochilon Desf., Antiphytom DC. ex
Meisn., Ogastemma Brummitt., and Sericostoma Stocks ex
Wight. Weigend et al. (2010), on the basis of trnL-F data,
M. Khoshsokhan Mozaffar (&)
Department of Biology, Qom Branch, Islamic Azad University,
Qom, Islamic Republic of Iran
e-mail: m.khoshm@gmail.com
S. Kazempour Osaloo (&)K. Naderi Saffar A. Amirahmadi
Department of Plant Biology, Faculty of Biological Sciences,
Tarbiat Modares University, 14115-154 Tehran, Iran
e-mail: skosaloo@modares.ac.ir
K. Naderi Saffar
e-mail: k_naderi_12@yahoo.com
A. Amirahmadi
e-mail: atefeamirahmadi@yahoo.com
R. Oskoueiyan
Department of Biological Sciences, Ayatollah Amoli Branch,
Islamic Azad University, Amol, Iran
e-mail: ro.osko@gmail.com
A. Amirahmadi
Biology Department, School of Biology,
Damghan University, Damghan, Iran
123
Plant Syst Evol (2013) 299:197–208
DOI 10.1007/s00606-012-0715-4
revealed the polyphyly of the tribe Trigonotideae and
accepted four tribes Boragineae s. l., Lithospermeae s. lat.,
Cynoglosseae s. l., and Echiochileae. In the most classifi-
cations, Eritrichieae has been recognized as a distinct tribe
(Bentham and Hooker 1873; Baillon 1888; Gurke 1897;
Al-Shehbaz 1991; Riedl 1997; Takhtajan 1997).
Several studies based on micromorphology, for example
the columnar and/or pyramidal gynobase and appendaged
nutlets (Johnston 1924; Hilger 1985), palynology, for
example heterocolpate pollen (Ahn and Lee 1986; Diez and
Benito 1991; Khatamsaz 2001), and karyology, for exam-
ple x=12 and small chromosome size (Luque 1992;
Ghaffari 1996; Coppi et al. 2006) have provided evidence
of the close relationship of Eritrichieae with Cynoglosseae.
In recent molecular systematic studies (Langstrom and
Chase 2002; Khoshsokhan et al. 2008a,b; Weigend et al.
2010) Eritrichieae and Cynoglosseae are closely related
and intermixed taxa.
The tribe Eritrichieae is mainly distributed in the
mountains in Eurasia, from the Arctic to the Himalayas, and
in the west of North America (Popov 1953; Ovchinnikova
2009). This tribe is characterized by flowers frequently
brachymorphic, anthers subsessile, style short, hidden in
corolla tube, nutlets four (sometimes in fruit rudiments of
1–3 nutlets) with wings or spines or tubercles and columnar/
pyramidal gynobase. Tribe Eritrichieae in the worldwide
distribution has 19 genera and 449 species (Al-shehbaz
1991, cited in Langstrom and Chase 2002; Ovchinnikova
2009).
The tribe has been studied from various standpoints,
including gross morphology (Riedl 1967; Kazempour
Osaloo 1993; Khatamsaz 2002), nutlet micromorphology
and ontogeny (Hilger 1985; Ovchinnikova 2006a), paly-
nology (Diez and Benito 1991; Kazempour Osaloo 1993;
Khatamsaz 2001; Ovchinnikova 2006b), karyology (Luque
1992), and molecular phylogeny (Langstrom and Chase
2002; Weigend et al. 2010).
Hitherto, all molecular phylogenetic studies of the
whole of Boraginaceae s. str. at the tribal level utilized only
two cpDNA fragments, rbcL-atpB intergenic spacer
Table 1 List of Iranian genera (tribe Eritrichieae) studied in this article
List of genera Number of species
in the world/Iran
Distribution Taxonomic placement
(tribe/subtribe)
Asperugo 1/1 Eurasia, North America North Africa; Boreal,
Tethyan, and Madrean subkingdoms
1. Asperugeae/-
2. Eritricieae/Asperuginae
3. Eritrichieae/-
4. Asperugeae/-
Eritrichium 76/1 Cold parts of Asia, partly in Europe and the
western part of North America
1. Eritrichieae/Cynoglossinae
2. Eritrichieae/Eritrichiinae
3. Eritrichieae/-
4. Eritrichieae/Eritrichiinae
Heterocaryum 6/5 South–West Asia, Tethyan subkingdom 1. Heterocaryeae/-
2. Eritrichieae/Heterocaryinae
3. Eritrichieae/-
4. Heterocaryeae/-
Lappula 70/10 Eurasia, North America, Africa, Australia;
Boreal, Tethyan and Madrean subkingdoms
1. Eritrichieae/Cynoglossinae
2. Eritrichieae/Eritrichiinae
3. Eritrichieae/-
4. Eritrichieae/Echinosperminae
Lepechiniella 16/3 Eurasia, North America, Africa, Australia;
Boreal, Tethyan, and Madrean subkingdoms
1. Eritrichieae/Cynoglossinae
2. Eritrichieae/Eritrichiinae
3. Eritrichieae/-
4. Eritrichieae/Echinosperminae
Rochelia 15–20/7 Europe, South-West and Central Asia, Tethyan
subkingdom
1. Rochelieae/ -
2. Eritrichieae/ Rocheliinae
3. Eritrichieae/ -
4. Rochelieae/ -
Number of species, distribution and some various treatments following different authors 1. Popov (1953), 2. Riedl (1967), 3. Khatamsaz (2002),
4. Ovchinnikova (2009)
198 M. Khoshsokhan Mozaffar et al.
123
(Langstrom and Chase 2002) and trnL intron and trnL-trnF
intergenic spacer (hereafter as trnL-F) (Weigend et al.
2010), to elucidate relationships within it. However,
nrDNA ITS alone or in combination with trnL intron/trnL-
F also was used for phylogenetic analysis of some tribes,
for example Boragineae and Lithospermeae (Weigend
et al. 2010; Hilger et al. 2004).
In this research, we used sequence data from the nrDNA
ITS plus the trnL-F sequences:
1. To elucidate the phylogenetic relationships of some
Boraginoid tribes;
2. To reveal phylogenetic relationships within the tribe
Eritrichieae in west Asia; and
3. To clarify monophyly and relationships within the
multi-specific genera Lappula and Heterocaryum
(Table 1).
Materials and methods
Selection of taxa
Sixty-eight taxa (71 accessions) for nrDNA ITS analysis,
53 taxa (54 accessions) for cpDNAtrnL-F, and 51 taxa for
combined analyses were selected to represent six tribes
currently recognized within Boraginaceae s. str. Two out-
group species, Tournefortia rubicunda Salzm. ex DC. and
Heliotropium bacciferum Frossk. (Heliotropiaceae) were
chosen in accordance with previous works (Langstrom and
Chase 2002; Weigend et al. 2010). Both nrDNA ITS and
trnL-F were newly sequenced for 28 species in this study.
The remaining sequences were obtained from GenBank.
Information concerning voucher specimens or previously
published sequences is presented in the Appendix.
DNA extraction
The leaf materials were prepared either from silica-gel
dried leaves of specimens collected in the wild or herbar-
ium specimens (TARI, FUMH, TMUH). The modified
CTAB method of Doyle and Doyle (1987) was used for
extraction of DNA.
Amplification, sequencing, and alignment of target
regions
The nrDNA ITS region was amplified using as primers both
ITS4 and ITS5 (White et al. 1990) or ITS5m (Sang et al.
1995) or AB101F and AB101R (Douzery et al. 1999). The
cpDNAtrnL-F were amplified using primers ‘‘trnc’’ and
‘‘trnf’’ of Taberlet et al. (1991). PCR amplification of the
selected markers used 20-ll reactions containing 7.2 ll
deionized water, 10 ll29Taq DNA polymerase master mix
red (Amplicon, cat. no. 180301; 150 mM Tris-HCl pH 8.5,
40 mM (NH
4
)
2
SO
4
, 3.0 mM MgCl
2
, 0.4 mM dNTPs,
0.05 units ll
-1
Amplicon Taq DNA polymerase, inert red
dye and a stabilizer), 0.5 ll of each primer (5 pmol/ll), 1 ll
DMSO, and 0.8 ll template DNA (20 ng/ll). The PCR
profile consisted of an initial 5-min premelt at 94 °C and
33–35 cycles of 1:10 min denaturation at 94 °C, annealing at
a temperature depending on the region in 50 s (53 °C for
nrDNA ITS and 57–59 °C for cpDNAtrnL–F), and 1 min
extension at 72 °C, followed by a final extension of 5 min at
72 °C. PCR products were sequenced by use of big dye
terminator cycle sequencing ready-reaction kits, with the
same primers as used for PCR, in an ABI Prism 3,7309l
DNA analyzer (Applied Biosystems, USA).
Sequences were edited using BioEdit ver. 7.0.9.0 (Hall
1999) and aligned using MUSCLE (Edgar 2004) followed
by manual adjustment. The alignment of the datasets
required the introduction of numerous single and multiple-
base indels (insertions/deletions). Positions of indels were
treated as missing data for all datasets.
Phylogenetic analysis
Maximum parsimony (MP) analysis was conducted using
PAUP* (Swofford 2002). The heuristic search option was
used for each of the two single region datasets, using tree
bisection–reconnection (TBR) branch swapping, with
1,000 replicates of the random addition sequence. Unin-
formative characters were excluded from the analysis.
Branch support values were calculated using a full heuristic
search with 1,000 bootstrap replicates (Felsenstein 1985)
each with a simple addition sequence. Combinability of
these two datasets was assessed by use of the partition
homogeneity test (the incongruence length difference test
(ILD) of Farris et al. (1995)) as implemented in PAUP*
(Swofford 2002). The test was conducted with invariant
characters excluded (Cunningham 1997) using the heuristic
search option involving 100 replicates of the random
addition sequence and TBR branch swapping with 1,000
homogeneity replicates. The maximum number of trees
was set to 1,000.
The model of sequence evolution for each dataset was
selected by use of the software MrModeltest v. 2.3
(Nylander 2004) as implemented in MrMTgui (Nuin 2005)
based on the Akaike information criterion (AIC) (Posada
and Backley 2004). All datasets were analyzed as a single
partition with the GTR ?I?G model by Bayesian
inference (BI) using the software MrBayes version 3.12
(Ronquist and Huelsenbeck 2003). Posteriors on the model
parameters were estimated from the data, using the default
priors. The analysis was performed with 4 million gener-
ations, using Markov chain Monte Carlo search. MrBayes
Tribe eritrichieae 199
123
performed two simultaneous analyses starting from dif-
ferent random trees (Nruns =2) each with four Markov
Chains trees sampled every 100 generations. The first 25 %
of trees were discarded as the burn-in. The remaining trees
were then used to build a 50 % majority rule consensus tree
accompanied by posterior probability (PP) values. Tree
visualization was performed by use of Tree View version
1.6.6 (Page 2001).
Results
Size and structure of molecular data sets
The aligned, nrDNAITS data set was 698 nucleotide sites
long, of which 348 nucleotide sites were parsimony
informative. The length of the nrDNA ITS region varies
from 577 bp in Heliotropium bacciferum to 627 bp in
Lappula semiglabra (Ledeb.) Gurke. Most sequences (63
sequences) for the taxa included in this study were between
606 and 620 bp long. Almost 48 ambiguous sites were
found in nrDNA ITS sequences overall. These sites were
eliminated before the phylogenetic analysis. The aligned
trnL-F data set was 1,102 nucleotide sites long, of which
225 were parsimoniously informative. The length of the
trnL-F sequences varied from 799 bases in Myosotis
lithospermifolia Hornem. to 923 bases in Heliotropium
bacciferum. These data sets differed in their taxon sam-
pling, with 71 accessions for nrDNA ITS and 54 for trnL-F.
The aligned combined nrDNA ITS-trnL-F data set for 51
taxa was 1,800 bp long, of which 532 were parsimoniously
informative.
Analysis of nrDNA ITS dataset
MP analysis of the dataset resulted in 120 shortest trees of
length (L) =1,980 steps, CI =0.373, and RI =0.688
(tree not shown). The Bayesian tree is topologically similar
to the MP tree (Fig. 1). All tribes (Echiochileae, Boragi-
neae, Lithospermeae, and Myosotideae) with the exception
of Cynoglosseae and Eritrichieae are well supported in the
monophyletic groups.
Asperugo procumbens L., Myositidium hortensia (Dec-
ne) Baill., Cryptantha flavoculata Payson, and Lepechini-
ella incospicua (Brand.) Riedl are positioned far from the
core Eritrichieae. This assemblage is, in turn, composed of
two subclades. One subclade comprises solely Heterocar-
yum A. DC. species; the second contains Lappula Gilib.,
Eritrichium Schrad., Rochelia Reichenb., and Lepechini-
ella M.pop. Rochelia is monophyletic but Lappula and
Eritrichium are non-monophyletic. Three Lepechiniella
species sampled here nested among a group of Lappula
species (L. barbata (M. B.) Gurke and L. microcarpa
(Ledeb.) Gurke), and another species, Lepechiniella
inconspicua, is nested in a clade of Paracaryum (DC.)
Boiss., Cynoglossum L. and Lindelofia Lehm.
Analysis of chloroplast trnL-F dataset
The parsimony search of the trnL-F dataset retained 100
trees with length (L) =386 steps, CI =0.775, and
RI =0.880 (tree not shown). Again, the Bayesian tree is
topologically similar to the MP tree (Fig. 2). With the
exception of the tribes Boragineae, Cynoglosseae, and
Eritrichieae, the three remaining ones (Echiochileae,
Lithospermeae, and Myosotideae) are well supported
monophyletic groups. Asperugo procumbens and Lepechi-
niella inconspicua arise in different branches far from the
core Eritrichieae. Heterocryum and Rochelia are mono-
phyletic whereas Lappula is not monophyletic. The two
Lepechiniella species analyzed are nested among a group of
Lappula species.
Analysis of the combined ITS-trnL-F dataset
The ILD test suggested that the trnL-F and nrDNA ITS
datasets were not incongruent (p\0.1). MP analysis of the
aligned combined ITS-trnL-F sequences (1,800 nucleotide
positions) for 27 taxa of Eritrichieae and related tribes
resulted in 10 most parsimonious trees, each of 1,890 steps
(CI =0.499, RI =0.708, tree not shown). Once again,
similar to the nrDNAITS tree, progressing upward from the
base, tribe Echiochileae is sister to all remaining tribes. In
the next clade, Boragineae and Lithospermeae are sister
taxa. Tribe Myosotideae, is monophyletic and sister group to
some members of Cynoglosseae/Eritrichieae, but Cyno-
glosseae and Eritrichieae are not monophyletic (Fig. 3). The
resulting tree from the combined dataset is better resolved
and supported than both nrDNA ITS and trnL-F trees.
Discussion
The phylogenetic status of Boraginoid tribes
This study, in agreement with previous work (Langstrom
and Chase 2002; Weigend et al. 2010), indicates that the
tribes Eritrichieae and Cynoglosseae, at the current status,
are not monophyletic. Four members of the former tribe are
allied with the two subclades of Cynoglosseae out of the
core Eritrichieae in a large clade. Myosotis (Myosotideae)
plus Trigonotis Stev. (Trigonotideae) are also nested within
this clade. Hence, all of these tribes are united in a single
tribe, called Cynoglosseae s. l. (sensu Weigend et al. 2010).
A subclade comprising Myosotis species and Trogonotis in
nrDNAITS tree, have the closest relationship of these two
200 M. Khoshsokhan Mozaffar et al.
123
genera (Weigend et al. 2010). Both are characterized by
nutlets without glochids or wings (Zhu et al. 1995).
The general topology resulting from our datasets is
similar to that of studies by Langstrom and Chase (2002)
and Weigend et al. (2010), confirming the four main clades
of Boraginaceae s. str., corresponding to Echiochileae,
Boragineae, Lithospermeae, and Cynoglosseae s. l.
Echiochileae, with the two species analyzed herein, are
Fig. 1 Phylogeny of nrDNA ITS data from 68 taxa using Bayesian
inference. Posterior probability (PP) and bootstrap (BP) value are next
to the nodes (PP/BP), some indicated by arrows. Delimitation of four
confirmed clades is indicated in the filled boxes (Lith Lithospermeae,
Bor Boragineae, EC Echiochileae). Nodes E,C,M,L,B, and EC
correspond to tribes Eritrichieae, Cynoglosseae, Myosotideae, Litho-
spermeae, Boragineae, and Echiocileae, respectively. Species names
in bold face represent members of Eritrichieae. Accession numbers
for some species retrieved from GenBank are given in front of their
name
Tribe eritrichieae 201
123
202 M. Khoshsokhan Mozaffar et al.
123
retrieved at the base of the nrDNA ITS and combined trees
as revealed by Weigend et al. (2010). The members of this
tribe are shrubs and weedy plants whereas the remaining
tribes are herbaceous plants. Boragineae and Lithosper-
meae seem to be closest sister tribes (Figs. 1,3). They
commonly have the same base chromosome number
(x=8) (Luque and Valdes 1984). Morphologically, Bor-
agineae is characterized by usually well-developed faucal
appendages, a simple style with one or two stigmas, nutlets
with a basal attachment scar often with an annular rim
around it, and a flat gynobase. Lithospermeae has the style
usually divided at the apex with two to four stigmas,
combined with nutlets with a broad basal attachment scar
and a flat gynobase.
Fig. 2 Phylogenetic tree from Bayesian analysis of the cpDNAtrnL-
F sequences. Posterior probability (PP) and bootstrap (BP) values are
next to the nodes (PP/BP), some indicated by arrows. Species names
in Bold face represent members of Eritrichieae. Nodes E,C,M,L,B,
and EC correspond to tribes Eritrichieae, Cynoglosseae, Myosotideae,
Lithospermeae, Boragineae, and Echiocileae, respectively. Accession
numbers for some species retrieved from GenBank are given in front
of their name
Fig. 3 Bayesian inference tree resolved using nrDNA ITS-
cpDNAtrnL-F sequences data for 51 species. Posterior probability
(PP) and bootstrap (BP) values are next to the nodes (PP/BP), some
indicated by arrows. Delimitation of four confirmed clades indicated
in the filled boxes.(Lith Lithospermeae, Bor Boragineae, EC
Echiochileae)
b
Tribe eritrichieae 203
123
The relationships within Eritrichieae
Asperugo procumbens, Lepechiniella inconspicua, Cryp-
tantha flavoculata, and Myosotidium hortensia (the last two
species were analyzed for nrDNA ITS, only) are placed out
of the core Eritrichieae within Cynoglosseae s. l. clade. A.
procumbens positioned almost at the base of Cynoglosseae
s. l. This taxon with calyx divided into linear-lanceolate
lobes, accrescent and saucer-like in the fruiting stage, and
unusual nutlet arrangement differed from other taxa of
Eritrichieae (Riedl 1967; Hilger 1985). Popov (1953), and
after that Ovchinnikova (2009), classified Asperugo L. in
the monotypic tribe Asperugeae Zak. This is not, however,
consistent with our finding. Lepechiniella inconspicua,an
annual species, is nested in a clade with Paracaryum,
Cynoglossum, and Lindelofia. It was originally described as
Paracaryum inconspicuum Brand (cited in Riedl 1967).
Heterocaryum is positioned as a sister to the remainder
core Eritrichieae. Our analysis showed that the five mem-
bers of the genus formed a distinct clade. This genus with
six species ephemerals grows in deserts and semi-deserts,
especially in mountainous semi-deserts of Iran, southern
Russia, and Turkey to Central Asia and the Himalayas
(Popov 1953; Riedl 1967; Khatamsaz 2002; Ovchinnikova
2009) (Table 1). Morphologically its species are charac-
terized by four zygomorphous and unequal nutlets. The
nutlets are small, flat, dorsiventrally compressed, with
dentate wing. All species but H. laevigatum (Kar. and Kir.)
DC. are covered by thin bristles. Based on the last char-
acter, Riedl 1967 divided the genus into two sections
Heterocaryum and Laevigata (M. Pop.) H. Riedl. H. lae-
vigatum belongs to the monotypic section Laevigata and
forms a sister group relationship with the monophyletic
section Heterocaryum (including H. subsessile Vatke., H.
szovitsianum (Fitch. and Mey.) DC., H. macrocarpum Zak.,
and H. rigidum DC.) (Figs. 1,2,3).
Genus Eritrichium is a perennial plant distributed
mainly in the cold parts of Asia, some partially in Europe,
and some in the western part of North America. It is rep-
resented here by two species which seem to be non-
monophyletic and accompanied by Rochelia and Lappula
species (Table 1).
Rochelia is a small Eurasian genus with 15–20 species
(Mabberly 1997; Ovchinnikova 2009) (fruits with two
nutlets), of which seven species are represented herein. It is
monophyletic and well allied with the two Lappula species,
L. sessiflora (Boiss.) Gurke and L. drobovii M. Pop. ex Pavl.
Khoshsokhan et al. (2010), on the basis of nrDNA ITS and
the trnL-F sequences, revealed the monophyly of Rochelia.
Their analysis indicated that Rochelia sect. Rochelia,
because of inclusion of the monotypic R. sect. Cryptocarpa
(R. cardiosepala Bge.), is not monophyletic. Likewise, its
subsections, Rochelia and Pedunculares, are paraphyletic.
The genus Lappula is a difficult genus of 70 species
(Ovchinnikova 2009) with two sections: Lappula and
Sclerocaryum (DC.) Post and Kuntze (Riedl 1967). Section
Lappula comprises four subsections, Lappula,Macranthae
(M. Pop.) H. Riedl., Sinaicae (M. Pop.) H. Riedl. and
Anomalolappula M. Pop. They are distributed mainly in
temperate Europe and Asia, Australia, and America (Nasir
1989). These taxa are usually annual with small and thin
plants whose limits are not well defined, possibly because
of hybridization. The nutlet characteristics are of impor-
tance in the delimitation of the various species. Nutlets are
tuberculate–verrucate, bearing marginal glochidiate acule-
ate appendages. Nutlets are winged or wingless. The genus
at the current status is not a monophyletic group. Lappula
sect. Lappula is not monophyletic, whereas, L. sect. Scle-
rocaryum is monophyletic (see below). In our analysis,
nine Lappula species sampled formed three distinct bran-
ches. Lappula sinaica (M. Pop.) H. Riedl (of the sect.
Lappula subsect. Sinaicae) is the first diverging branch of
the Lappula/Eritrichium/Rochelia clade. The species is
characterized by small nutlets ca 2 mm long, with an
indistinct margin, bearing few appendages, minutely verr-
ucate–tuberculate.
The other branch is, in turn, composed of two subclades
of seven Lappula and two/three Lepechiniella species. One
subclade comprises Lappula spinocarpos (Forssk.) Asch-
erson and Kuntze and L. ceratophora (M. Pop.) M. Pop.
(both of the sect. Sclerocaryum). These species have large
nutlets with uniform, thick, and stonelike tubercles, with
high grumose walls and disk nearly or completely covered
by its adjacent thickened margins, sometimes protruding
like a keel. Because of these morphological characteristics,
Sadat (1989) transferred the two species to a separate
genus, Sclerocaryopsis Brand.
The second subclade contains Lappula patula (Lehm.)
Ascherson ex Gurke (of sect. Lappula subsect. Macranthae)
and Lappula squarrosa (Retz.) Durmort (of sect. Lappula
subsect. Lappula), as sister taxa, allied with L. semiglabra,
L. microcarpa (both of sect. Lappula subsect. Macranthae),
and L. barbata (of sect. Lappula subsect. Lappula) and
Lepechiniella. It is noteworthy that three of four Lepechi-
niella species (Le. albiflora Riedl, Le. persica (Boiss.) Riedl
and Le. wendelboi Riedl) sampled here well nested among
these Lappula species. Lepechiniella is morphologically
similar to Lappula, both have homomorphic, equal-sized,
and detachable nutlets (Popov 1953; Riedl 1967; Kazempour
Osaloo 1993; Khatamsaz 2002). In Flora of USSR, Popov
(1953) established Lepechiniella with eight species of which
five species were transferred from other genera, for example
Paracaryum,Echinospermum, and Eritrichium. The genus
at the current status, has 16 species (Ovchinnikova 2009)
distributed in Iran, Afghanistan, and C. Asia. According to
nomenclatural priority, we suppose that the type species of
204 M. Khoshsokhan Mozaffar et al.
123
the genus might be Le. omphaloides (Schrenk) M. Pop (see
Popov 1953), which is not included in our analysis. The
genus is characterized by perennial, rarely annual, habit and
winged nutlets. Nevertheless, in terms of perennial habit, the
genus is similar to Eritrichium and Paracaryum, but, having
a pyramidal gynobase, is much related to Lappula. With the
exception of Le. albiflora, the two Lepechiniella (Le. persica
and Le. wendelboi) do not have nucleotide substitutions with
Lappula microcara and L. barbarta. These species should be
synonymized with Lappula species L. barbata. Other spe-
cies (Le. inconspicua) as noted above, are placed with the
Paracaryum, Cynoglossum, and Lindelofia clade.
Lappula barbata and L. microcarpa have been mor-
phologically distinguished by discrepancy in fruit length,
number and size of anchorlike spinules along margin of
disk, and different apparent color of plants. Ripe fruit
samples are needed for identification. Also, investigation of
the phenol chemistry of populations in the two species in
Turkey revealed the same phenolic bands (Apaydin and
Bilgener 2000).
The third branch comprises L. sessiliflora (subsect.
Anomalolappula M. Pop.), and L. drobovii. They are sister
to the Rochelia clade. This subclade is placed out of the
Lappula species core and settled in the Rochelia species
clade. In all analyses, these two species are placed sister to
Rochelia with strong bootstrap and Bayesian posterior
probability support. These two species have some diag-
nostic characters: in L. sessiliflora pedicels are very short
and nutlets sessile, as if adhering to the thick axis of
raceme, and in L. drobovii fruits usually have 1 or 2 broad-
winged nutlets. Popov (1953) reported that L. sessiliflora is
rather more related to Rochelia, because the flowers, at
least, are similar to those of Rochelia; he also noted that in
L.sessiliflora (similar to the Rochelia), rarely, two nutlets
develop in the fruit and the disk of nutlets was completely
covered by adjacent margins. Khatamsaz (2002) introduced
L. drobovii from northeastern Iran. This species is also
distributed in C. Asia and the type species has been
reported from Tashkent, Uzbekistan (Popov 1953). These
two species have low nucleotide differences in both nrDNA
and trnL-F sequences.
Conclusion
On the basis of nrDNA ITS, cpDNAtrnL-F, and combi-
nation of the two sequences, four tribes in previous studies
(Echiochileae, Boragineae, Lithospermeae, and Cynoglos-
seae s. l.) are confirmed. Eritrichieae is placed within
Cynoglosseae and none of them is monophyletic. Bulk
Eritrichieae formed a monophyletic group. Two genera,
Heterocaryum and Rochelia were supported well in a
distinct subclade; Lappula is not monophyletic. It seems
that Eritrichium in the current status is not monophyletic.
Three species of Lepechiniella settled within Lappula
species, and one species (Lepechiniella inconspicua)isa
part of the Paracaryum and its allies. To summarize, two
single sequences (nrDNA ITS and trnL-F regions) are
congruent, however, nrDNA ITS segment resolved the
relationships and better supported the branches than the
trnL-F.
Acknowledgments We thank the herbariums TARI and FUMH for
providing leaf materials.
Appendix
Genbank accession numbers for nrDNA ITS and
cpDNAtrnL-F sequence data, and voucher information with
herbarium where specimen is deposited. The accession
numbers for nrDNA ITS (reference or Location)/ and trnL-
F (reference or location) are given in front of the species
names. The dash symbol (–) shows that the sequence is not
accessible. Species with a star symbol (*) have been
sequenced in this article. TMUH: Tarbiat Modares University
Herbarium, TARI: Herbarium of Research Institute of Forests
and Rangelands, FUMH: Ferdowsi University of Mashhad
Herbarium.
Anchusa officinalis L., AF531080 (Papini et al. 2002)/
AY045703 (Bigazzi et al. 2002); Anchusa thessala Boiss. and
Spruner, AF531084 (Papini et al. 2002)/AF530599 (selvi
et al. 2002); Arnebia guttata Bge., EF199862 (Hu et al.
2006)/EF199872 (Hu et al. 2006); Arnebia euchroma (Royle)
Johnst., EF199848 (Hu et al. 2006)/EF199874 (Hu et al.
2006); Arnebia szechenyi Kanitz, EF199863 (Hu et al. 2006)/
EF199871 (Hu et al. 2006); Asperugo procumbens L.,
AB758290 *(Iran: Kazempour, 2007-3TMUH)/AB758320
*(Iran: Kazempour, 2007-3TMUH); Borago officinalis L.,
AF091151 (Ferguson 1998)/–; Brunnera orientalis (Schenk)
Johnst., AY383289 (Hilger et al. 2004)/AF530601 (Selvi
et al. 2002); Buglossoides purpurocaerulea (L.) Johnst.,
AJ555897 (Langstrom and Chase 2002)/–; Cerinthe minor
L., AB758291 *(Iran: Kazempour, 2008-6 TMUH)/L43203
(Bohle et al. 1996); Cryptantha flavoculata Payson.,
AF091154 (Ferguson 1998)/–; Cynoglossum creticum Miller,
FR715303 (Selvi et al. 2011)/–; Cynoglossum officinale L.,
AB758292 *(Iran: Assadi, 73526TARI)/ AB758321 *(Iran:
Assadi, 73526TARI); Cynoglottis barrelieri (All.) Vural and
Kit, AF531081 (Papini et al. 2002)/AY045708 (Bigazzi et al.
2002); Echiochilon fruticosum Desf., EU044843 (Thomas
et al. 2007)/EU044881 (Thomas et al. 2007); Echiochilon
persicum (Burm.f.) Johnst., AB758293 *(Iran: Mozaffarian,
49917TARI)/AB758322 *(Iran: Mozaffarian, 49917TARI);
Echium vulgare L., AJ555896 (Langstrom and Chase 2002)/–;
Tribe eritrichieae 205
123
Eritrichium canum (Benth.) Kitam., AB758294 *(Ger-
many: cultivated in Munich Botanical Garden)/AB758323
*(Germany: cultivated in Munich Botanical Garden); Eri-
trichim nanum (Vill.) Schrad., AY092901 (Winkworth
et al. 2002)/–; Gastrocotyle macedonica (All.) Vural and
Kit, AF531086 (Papini et al. 2002)/AY045706 (Bigazzi
et al. 2002); Heliotropium bacciferum Frossk., AB758295
*(Iran: Sonboli, 01TMUH)/AB758324 *(Iran: Sonboli,
01TMUH); Heterocaryum laevigatum (Kar. et Kir.) DC.,
AB758296 *(Iran: Faghihna and Zangooei, 25349FUMH)/
AB758325 *(Iran: Faghihna and Zangooei, 25349FUMH);
Heterocaryum subsessile Vatke., AB758297 *(Iran: Fag-
hihna and Zangooei, 28193TMUH)/AB758326 *(Iran:
Faghihna and Zangooei, 28193TMUH); Heterocaryum
szovitsianum (Fisch. et Mey.) DC., AB758298 *(Iran:
Kazempour Osaloo, 2007-5TMUH)/AB758327 *(Iran:
Kazempour Osaloo, 2007-5TMUH); Heterocaryum rigi-
dum DC., AB758299 *(Iran: Shafaghi and Borhan,
371TARI)/AB758328 *(Iran: Shafaghi and Borhan,
371TARI); Heterocaryum macrocarpum Zak., AB758300
*(Iran: Joharchi and Zangooei, 19639TMUH)/AB758329
*(Iran: Joharchi and Zangooei, 19639TMUH); Lappula
barbata (M. B.) Gurke, AB564703 (Khoshsokhan et al.
2010)/AB564713 (Khoshsokhan et al. 2010); Lappula
ceratophora (M. Pop.) M. Pop., AB758301 *(Iran: Moz-
affarian, 58407TARI)/AB758330 *(Iran: Mozaffarian,
58407TARI); Lappula drobovi M. Pop. ex Pavl.,
AB758302 *(Iran: Faghihnia and Zangooei, 26636FUMH)/
AB758331 *(Iran: Faghihnia and Zangooei, 26636FUMH);
Lappula microcarpa (Ledeb.) Gurke, AB758303 *(Iran:
Kazempour Osaloo, 2007-6TMUH)/AB758332 *(Iran:
Kazempour Osaloo, 2007-6TMUH); Lappula microcarpa
(Ledeb.) Gurke, AB758304 *(Iran: Assadi, 75090TARI)/–;
Lappula patula (Lehm.) Ascherson ex Gurke, AB758305
*(Iran: Assadi, 73860TARI)/AB758333 *(Iran: Assadi,
73860TARI); Lappula semiglabra (Ledeb.) Gurke,
AB758306 *(Iran: Mozaffarian, 42998TARI)/AB758334
*(Iran: Mozaffarian, 42998TARI); Lappula squarrosa
(Retz.) Durmort., AB758307 *(Germany: cultivated in
Munich Botanical Garden)/AB758335 *(Germany: culti-
vated in Munich Botanical Garden); Lappula sessiliflora
(Boiss.) Gurke, AB564704 (Khoshsokhan et al. 2010)/
AB564714 (Khoshsokhan et al. 2010); Lappula sinaica (DC.)
Ascherson ex Schweinf., AB758308 *(Iran: Kazempour
Osaloo, 2007-7TMUH)/AB758336 *(Iran: Kazempour Osa-
loo, 2007-7TMUH); Lappula spinocarpos (Forssk.) Ascher-
son et Kuntze, AB758309 *(Iran: Kazempour Osaloo,
2007-4TMUH)/AB758337 *(Iran: Kazempour Osaloo,
2007-4TMUH); Lepechiniella albiflora Riedl., AB758310
*(Afghanistan: Rechinger, 31424TARI)/-; Lepechiniella
inconspicua (Brand) Riedl, AB758311 *(Iran: Joharchi and
Ayatollahi, 10711FUMH)/AB758338 *(Iran: Joharchi and
Ayatollahi, 10711FUMH); Lepechiniella persica (Boiss.)
Riedl, AB758312 *(Iran: Assadi and Maassumi,
51278TARI)/AB758339 *(Iran: Assadi and Maassumi,
51278TARI); Lepechiniella wendelboi Riedl, AB758313
*(Iran: Assadi and Maassumi, 21064TARI)/AB758340
*(Iran: Assadi and Maassumi, 21064TARI); Lepechiniella
wendelboi Riedl, AB758314 *(Iran: Kazempour Osaloo,
2008-7TMUH)/–; Lindelofia longiflora Baill., AJ555895
(Langstrom and Chase 2002)/–; Myosotidium hortensia
(Decne) Baill, AY092902 (Winkworth et al. 2002)/–;
Myosotis abyssinica Boiss. and Reuter, AY092904
(Winkworth et al. 2002)/–; Myosotis arvensis (L.) Hill.,
AY092908 (Winkworth et al. 2002)/–; Myosotis asiatica
Schischk, AB758315 *(Iran: Serkart and Olfat, 167TARI)/
AB758341 *(Iran: Serkart and Olfat, 167TARI); Myosotis
australis R. Br., AY092910 (Winkworth et al. 2002)/–;
Myosotis lithospermifolia (Willd.) Hornem., AY092923
(Winkworth et al. 2002)/AB758342 *(Iran: Babakhanloo
and Amin, 18179TARI); Myosotis olympica Boiss., –/
AB758343 *(Iran: Assadi and Shahsavari, 65961TARI);
Myosotis sylvstica Her. ex Hoffm., AY092935 (Winkworth
et al. 2002)/–; Omphalodes japonica Maxim., DQ320747
(Serrano et al. 2005)/–; Omphalodes kuzinskyanae Willk.,
AY837595 (Serrano et al. 2004)/–; Omphalodes linifolia
L., AB758316 *(Germany: cultivated in Munich Botanical
Garden)/AB758344 *(Germany: cultivated in Munich
Botanical Garden); Omphalodes linifolia L., AY837597
(Serrano et al. 2004)/–; Omphalodes nitida Hoffmanns and
Link, AY837615 (Serrano et al. 2004)/–; Onosma hookeri
Nyman., EF199847(Hu et al. 2006)/–; Onosma panicula-
tum Bueau and French., EF199859 (Hu et al. 2006)/
EF199851 (Hu et al. 2006); Onosma waltoni Duthic,
EF199846 (Hu et al. 2006)/–; Paracaryum persicum
(Boiss.) Boiss., AB758317 *(Iran: Kazempour Osaloo,
2007-8TMUH)/AB758345 *(Iran: Kazempour Osaloo,
2007-8TMUH); Rochelia bungei Trautv., AB564695
(Khoshsokhan et al. 2010)/AB564705 (Khoshsokhan et al.
2010); Rochelia cardiosepala Bge., AB564601 (Khoshsok-
han et al. 2010)/AB564711 (Khoshsokhan et al. 2010); Roc-
helia disperma (L. F.) Koch., AB564698 (Khoshsokhan et al.
2010)/AB564708 (Khoshsokhan et al. 2010); Rochelia mac-
rocalyx Bge., AB564600 (Khoshsokhan et al. 2010)/
AB564710 (Khoshsokhan et al. 2010); Rochelia mirheydari
Reidl et Esfandiari, AB564696 (Khoshsokhan et al. 2010)/
AB564706 (Khoshsokhan et al. 2010); Rochelia persica Bge.
ex. Boiss., AB564697 (Khoshsokhan et al. 2010)/AB564707
(Khoshsokhan et al. 2010); Rochelia peduncularis
Boiss., AB564699 (Khoshsokhan et al. 2010)/AB564709
(Khoshsokhan et al. 2010); Solenanthus circinatus Ledeb.,
AB758318 *(Iran: Khoshsokhan, 2007-9TMUH)/AB758346
*(Iran: Khoshsokhan, 2007-9TMUH); Symphytum 9uplan-
dicum, AY092903 (Winkworth et al. 2002)/–; Trichodesma
206 M. Khoshsokhan Mozaffar et al.
123
aucheri DC., AB758319 *(Iran: Mozaffarian, 57195TARI)/
AB758347 *(Iran: Mozaffarian, 57195TARI); Tournefortia
rubicunda Salzm.ex DC., EF688852 (Luebert et al. 2008)/
EF688799 (Luebert et al. 2008); Trigonotis peduncularis
(Trevir.) Benth., DQ320740 (Serrano et al. 2005)/–.
References
Ahn YM, Lee S (1986) A polynotaxonomic study of the Korean
Boraginaceae. Kor J Plant Tax 16(3):199–215
Al-Shehbaz I (1991) The genera of Boraginaceae in the southeastern
United States. J Arnold Arbor Suppl Ser 1:1–169
Apaydin Z, Bilgener M (2000) The phenolic chemistry of Lappula
squarrosa (Retz.) Dumort., L. barbata (Bieb.) Gurke and L.
microcarpa (Ledeb.) Gurke species. Turk J Bot 24:169–17600
Baillon H (1888) Boraginacees. In: Baillon H (ed) Hist P1 (Baillon
H). Librairie Hachette & Cie, Paris, pp 343–402
Bentham G, Hooke JD (1873) Genera Plantarum (Bentham &
Hooker). Lovell Reeve & Co, Williams & Norgate, London
Coppi A, Selvi F, Bigazzi M (2006) Chromosome studies in
Mediterranean species of Boraginaceae. Fl Medit 16:253–274
Cunningham CW (1997) Can three incongruence tests predict when
data should be combined? Mol Biol Evol 14:733–740
Diez MJ, Benito V (1991) Pollen morphology of the tribes Eritrichieae
and Cynoglosseae (Boraginaceae) in the Iberian Peninsula and its
taxonomic significance. Bot J Linn Soc 107:49–66
Douzery JP, Pridgeon M, Kores P, Linder HP, Kurzweil H, Chase
MW (1999) Molecular phylogenetics of diesae (orchidaceae): A
contribution from nuclear ribosomal ITS sequences. Am J Bot
86(6):887–899
Doyle JJ, Doyle JS (1987) A rapid DNA isolation procedure for small
quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Edgar RC (2004) Muscle: multiple sequence alignment with high
accuracy and highthroughput. Nucleic Acids Res 32(5):1792–1797
Farris JS, Kallersjo M, Kluge AG, Bult C (1995) Testing significance
of incongruence. Cladistics 10:315–319
Felsenstein J (1985) Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39:783–791
Ghaffari SM (1996) Chromosomes studies in some species of
Boraginaceae from Iran. Iran J Bot 7(1):81–93
Gurke M (1897) Boraginaceae. In: Engler A, Prantl K (eds) Die
natu
¨rlichen pflanzenfamilien IV, vol 4., pp 71–131
Hall TA (1999) BioEdit: a user-friendly biological sequence align-
ment editor and analysis program for Windows 95/98/NT. Nucl
Acids Symp Ser 41:95–98
Hilger HH (1985) Ontogenie, Morphologie und systematische
Bedeutung geflu
¨gelter und Glochidien tragender Cynoglosseae
und Eritrichieae Fru
¨chte (Boraginaceae). Bot Jahrb Syst
105:323–378
Hilger HH, Selvi F, Papini A, Bigazzi M (2004) Molecular
systematics of Boraginaceae tribe Boragineae based on ITS1
and trnL sequences, with special reference to Anchusa S. l. Ann
Bot 94:201–212
Johnston IM (1924) Studies in the Boraginaceae. III.1. The old World
genera of the Boraginoideae. Contr Gray Herb 73:42–78
Kazempour Osaloo S (1993) Revision of the tribe Eritrichieae
(Boraginaceae) in Iran. M.S. thesis (unpubl.), Tarbiat Modares
University, Tehran
Khatamsaz M (2001) Pollen morphology of Iranian Boraginaceae
family and its taxonomic significance. Iran J Bot 9:27–40
Khatamsaz M (2002) Boraginaceae in M. Assadi et al. Flora of Iran
no. 39. Research Institute of Forests and Rangelands, Tehran
Khoshsokhan M, Kazempour Osaloo S (2008a) Phylogenetic analysis
of tribe Eritrichieae (Boraginaceae) based on cpDNA (trnLin-
tron/trnLtrn Fintergenic spacer) sequences. 15th nationaland 3rd
international conference of biology, 37(abstract)
Khoshsokhan M, Kazempour Osaloo S, Attar F, Saadatmand S,
Nejadsattari T (2008b) Phylogeny of tribe Eritrichieae (Bora-
ginaceae) based on nrDNA ITS. 10th congress Iranian Genetics
Society, 273(abstract)
Khoshsokhan M, Kazempour Osaloo S, Saadatmand S, Attar F (2010)
Molecular phylogeny of Rochelia (Boraginaceae) based on
nrDNA ITS and cpDNAtrnL-F sequences. Iran J Bot 16(1):22–29
Langstrom E, Chase MW (2002) Tribes of Boraginoideae (Boragin-
aceae) and placement of Echiochilon, Ogastemma and Sericos-
toma: a phylogenetic analysis based on atpB plasted DNA
sequence data. Plant Syst Evol 234:137–153
Luque T (1992) Karyological studies on Spanish Boraginaceae. VI.
Contribution to the tribe Eritrichieae. Bot J Linn Soc 110:77–94
Luque T, Valdes B (1984) Karyological studies on Spanish Boragin-
aceae: lithospermum L. sensu lato. Bot J Linn Soc 88(4):
335–350
Nasir YJ (1989) Boraginaceae in S. I. Ali, Y.J. Nasir (eds.) Flora of
Pakistan. No.191 printed at pangraphics (PVT) Ltd., Islamabad
Nuin P (2005) MrMTgui 1.0 (version 1.6). Program distributed by the
author at http://www.genedrift.org/mtgui.php
Nylander JAA (2004) MrModeltest v2. Program distributed by the
author. Evolutionary Biology Centre, Uppsala University
Ovchinnikova SV (2006a) Ultrasculptural features of fruit surface in
subtribe Echinosperminae (tribe Eritrichieae, Boraginaceae). Bot
Zhurn 91:105–116
Ovchinnikova SV (2006b) Palynomorphology of the representatives
of the tribe Eritrichieae (Boraginaceae). In: Kamelin RV (ed)
Problems of botany of south Siberia and Mongolia, Barnaul.,
pp 173–180
Ovchinnikova SV (2009) On the position of the tribe Eritrichieae in
the Boraginaceae system. Bot Serbica 33:141–146
Page DM (2001) Treeview (Win32) version 1.6.6. Available at:
http://taxonomy.zoology.gla.ac.uk/rod/treeview.html
Popov MG (1953) Boraginaceae in B. K. Shischkin Flora of USSR.
Leningrad, Moskva, pp 97–691
Posada D, Backley TR (2004) Model selection and model averaging
in phylogenetics: advantages of akaike information criterion and
Bayesian approaches over likelihood ratio tests. Sys Biol
53:793–808
Riedl H (1967) Boraginaceae. In: Rechinger KH (ed) Flora Iranica,
No. 48. Akademische Druck, Graz
Riedl H (1997) Boraginaceae. In: Kalkman C, Noteboom HP, de
Wilde WJ, Kirkup DW, Stevens PF (eds) Flora Malesiana. Ser.
I. Spermatophyta Publications Department, Rijksherbarium,
Leiden, pp 43–168
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phyloge-
netic inference under mixed models. Bioinformatics 19:1572–
1574
Sadat F (1989) Revision ausgewahlterkritischergattungen der Borag-
inaceenaus der flora Afganestans. Mitt Bot staatssmml Munchen
28:1–210
Sang T, Crawford DJ, Stuessy T (1995) Documentation of reticulate
evolution in peonies (Paeonia) using internal transcribed spacer
sequences of nuclear ribosomal DNA: implications for bioge-
ography and concerted evolution. Proc Natl Acad Sci
92:6813–6817
Swofford DL (2002) PAUP*: phylogenetic analysis using parsi-
mony (* and other methods), Version 4. Sinauer Associates,
Sunderland
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for
amplification of three non-coding regions of chloroplast DNA.
Plant Mol Biol 17:1105–1109
Tribe eritrichieae 207
123
Takhtajan AL (1997) Diversity and classification of flowering plants.
Columbia University Press, New York
Weigend W, Gottschling M, Selvi F, Hilger HH (2010) Fossil and
extant western hemisphere Boragieae, and the polyphyly of
Trigonotideae Riedl (Boraginaceae: Boraginoideae). Sys Bot
35(2):409–419
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
In: Innis DH (ed) PCR protocols: a guide to methods and
applications. Academic Press, San Diego, pp 315–322
Zhu G, Riedl H, Kamelin RV (1995) Boraginaceae. Flora of China.
Science Press, Beijing
208 M. Khoshsokhan Mozaffar et al.
123
