Phylogeny of Ononis L. (Fabaceae) in Iran using nuclear ribosomal DNA and chloroplast sequence data

Abstract

The genus Ononis, embraces more than 85 species worldwide. In the present study, materials of two subspecies of O. spinosa from different localities of Iran alongside some other native species of the genus were included in phylogenetic analyses. In addition, over 50 accessions were obtained from GenBank. In order to clarify the exact number of subspecies of O. spinosa in Iran, datasets were obtained from sequencing of nuclear ribosomal ITS (nr DNA ITS) and trnL-F plastid (cp DNA) regions and analysed. Three taxa belonging to O. spinosa L. complex (O. spinosa subsp. leiosperma, O. spinosa subsp. antiquorum, and O. arvensis=O. spinosa subsp. arvensis) were previously reported from Iran. Based on Maximum Parsimony and Bayesian analyses of the molecular datasets, it is shown that, unlike the previous reports, Iranian O. spinosa complex belongs to only two subspecies (i.e. leiosperma and arvensis subspp.). In addition, we also found that, O. spinosa subsp. antiquorum is not present in Iran. It was also demonstrated that, seed sculpturing does not provide valuable characters in diagnosing different subspecies of O. spinosa.

Full text

Rostaniha 17(2): 95–107 (2016) ﺎﻬﯿﻨﺘﺳر17)2 :(107
-
95)1395(
Phylogeny of Ononis in Iran using nuclear ribosomal DNA and chloroplast sequence data
Received: 04.04.2016 / Accepted: 02.10.2016
Golaleh Mostafavi:Assistant Prof., Department of Biology, Yadegar-e-Imam Khomeini (RAH), Shahr-e Rey
Branch, Islamic Azad University, Tehran, Iran (golaleh.m@gmail.com, g.mostafavi@iausr.ac.ir)
Mostafa Assadi: Prof., Research Institute of Forests and Rangelands, Agricultural Research Education and Extension
Organization (AREEO), P.O. Box 13185-116, Tehran, Iran
Iraj Mehregan: Assistant Prof., Department of Biology, Science and Research Branch, Islamic Azad University,
Tehran, Iran
Abstract
The genus Ononis, embraces more than 85 species worldwide. In the present study, materials of two subspecies
of O. spinosa from different localities of Iran alongside some other native species of the genus were included in
phylogenetic analyses. In addition, over 50 accessions were obtained from GenBank. In order to clarify the exact
number of subspecies of O. spinosa in Iran, datasets were obtained from sequencing of nuclear ribosomal ITS
(nr DNA ITS) and trnL-F plastid (cp DNA) regions and analysed. Three taxa belonging to O. spinosa L. complex
(O. spinosa subsp. leiosperma,O. spinosa subsp. antiquorum, and O. arvensis=O. spinosa subsp. arvensis) were
previously reported from Iran. Based on Maximum Parsimony and Bayesian analyses of the molecular datasets, it is
shown that, unlike the previous reports, Iranian O. spinosa complex belongs to only two subspecies (i.e. leiosperma
and arvensis subspp.). In addition, we also found that, O. spinosa subsp. antiquorum is not present in Iran. It was
also demonstrated that, seed sculpturing does not provide valuable characters in diagnosing different subspecies
of O. spinosa.
Keywords: nrDNA ITS, molecular phylogeny, species diversity, systematics, trnL-F
ﺲﻨﺟ ﯽﯾازرﺎﺒﺗOnonisﯽﻟاﻮﺗ سﺎﺳاﺮﺑ يﺎﻫDNAﻪﺘﺴﻫ ﯽﻣوزﻮﺒﯾرناﺮﯾا رد ﯽﺘﺳﻼﭘوﺮﻠﮐ و يا
ﺖﻓﺎﯾرد :16/01/1395 /شﺮﯾﺬﭘ :11/07/1395
يﻮﻔﻄﺼﻣ ﻪﻟﻼﮔ:ﺖﺴﯾز رﺎﯾدﺎﺘﺳاﺖﺴﯾز هوﺮﮔ ،ﯽﻫﺎﯿﮔ ﮏﯿﺗﺎﻤﺘﺴﯿﺳ ،ﯽﺳﺎﻨﺷ ﯽﻨﯿﻤﺧ مﺎﻣا رﺎﮔدﺎﯾ ﺪﺣاو ،ﯽﺳﺎﻨﺷ)هر (،ير ﺮﻬﺷ هﺎﮕﺸﻧاد
دازآﯾا ،ناﺮﻬﺗ ،ﯽﻣﻼﺳا ناﺮ)golaleh.m@gmail.com, g.mostafavi@iausr.ac.ir(
يﺪﺳا ﯽﻔﻄﺼﻣ:ﻞﮕﻨﺟ تﺎﻘﯿﻘﺤﺗ ﻪﺴﺳﺆﻣ دﺎﺘﺳاﯽﺘﺴﭘ قوﺪﻨﺻ ،يزروﺎﺸﮐ ﺞﯾوﺮﺗ و شزﻮﻣآ ،تﺎﻘﯿﻘﺤﺗ نﺎﻣزﺎﺳ ،رﻮﺸﮐ ﻊﺗاﺮﻣ و ﺎﻫ
116
-
13185 ،ناﺮﯾا ،ناﺮﻬﺗ
نﺎﮔﺮﻬﻣ جﺮﯾا:ﺖﺴﯾز رﺎﯾدﺎﺘﺳاﺖﺴﯾز هوﺮﮔ ،ﯽﻫﺎﯿﮔ ﯽﻟﻮﮑﻟﻮﻣ ﯽﻧژﻮﻠﯿﻓ و ﯽﺳﺎﻨﺷ ،ﯽﻣﻼﺳا دازآ هﺎﮕﺸﻧاد ،تﺎﻘﯿﻘﺤﺗ و مﻮﻠﻋ ﺪﺣاو ،ﯽﺳﺎﻨﺷ
ناﺮﯾا ،ناﺮﻬﺗ
ﻪﺻﻼﺧ
ﺲﻨﺟOnonis)نﺎﯿﯾﻼﻗﺎﺑ( زا ﺶﯿﺑ ياراد ،85ﯽﻣ ﺎﯿﻧد ﺮﺳﺎﺗﺮﺳ رد ﻪﻧﻮﮔﺪﺷﺎﺑ .ﻧ زا ﻪﻧﻮﮔﺮﯾز ود داﻮﻣ ،ﺮﺿﺎﺣ ﻪﻌﻟﺎﻄﻣ رد توﺎﻔﺘﻣ طﺎﻘ
ﻪﻧﻮﮔ زا يداﺪﻌﺗ هاﺮﻤﻫ ﻪﺑ ناﺮﯾاﺪﻨﺘﻓﺮﮔ راﺮﻗ ﯽﮑﯿﺘﻧژﻮﻠﯿﻓ ﻞﯿﻠﺤﺗ درﻮﻣ ﺲﻨﺟ ﺮﮕﯾد ﯽﻣﻮﺑ يﺎﻫ. زا ﺶﯿﺑ ،هوﻼﻋ ﻪﺑ50ﯽﻟاﻮﺗ نژ ﮏﻧﺎﺑ زا ﺰﯿﻧ
ﺪﻣآ ﺖﺳد ﻪﺑ .ﻪﻧﻮﮔﺮﯾز ﻖﯿﻗد داﺪﻌﺗ ﻦﯿﯿﻌﺗ رﻮﻈﻨﻣ ﻪﺑ يﺎﻫO. spinosaهداد ﻪﻋﻮﻤﺠﻣ ،ناﺮﯾا ردﯽﻟاﻮﺗ زا ﻞﺻﺎﺣ يﺎﻫ ﯽﺑﺎﯾ ﯽﺣاﻮﻧيآ.ﯽﺗ.سا.
زايد.نا.يا .ﯽﻣوزﻮﺒﯾرﻪﺘﺴﻫ يﺪﯿﺘﺳﻼﭘ ﻪﯿﺣﺎﻧ و ياtrnL-F)يد.نا.يا.ﯽﺘﺳﻼﭘوﺮﻠﮐ (ﺪﺷ ﻞﯿﻠﺤﺗ .ﻪﻧﻮﮔ ﺲﮑﻠﭙﻤﮐ ﻪﺑ ﻖﻠﻌﺘﻣ ﻪﯾارآ ﻪﺳيا
O. spinosa)O. spinosa subsp. leiosperma،O.spinosa subsp. antiquorum وO. arvensis=O.spinosa subsp. arvensis( زا ﻼﺒﻗ
ﺷ شراﺰﮔ ناﺮﯾاﺪﻧدﻮﺑ هﺪ .ﻞﯿﻠﺤﺗ سﺎﺳاﺮﺑﻪﻓﺮﺻ ﻪﻨﯿﺸﯿﺑ يﺎﻫهداد ﻪﻋﻮﻤﺠﻣ ﻦﯾﺰﯿِﺑ طﺎﺒﻨﺘﺳا و ﯽﯾﻮﺟ ﻪﮐ ﺪﺷ هداد نﺎﺸﻧ ،ﯽﻟﻮﮑﻟﻮﻣ يﺎﻫ
شراﺰﮔ فﻼﺧﺮﺑﻪﻧﻮﮔ ﺲﮑﻠﭙﻤﮐ ،ﻦﯿﺸﯿﭘ يﺎﻫ ياO. spinosa ﺎﻬﻨﺗ ﻖﻠﻌﺘﻣ ﺖﺳا ﻪﻧﻮﮔﺮﯾز ود ﻪﺑ)ﻪﻧﻮﮔﺮﯾز يﺎﻫleiosperma وarvensis .(
هوﻼﻋﺪﺷ ﺺﺨﺸﻣ ،ﻦﯾا ﺮﺑ ﻪﻧﻮﮔﺮﯾز ﻪﮐO. spinosa subsp. antiquorumﯽﻤﻧ ناﺮﯾا ردﺪﯾور . ﺢﻄﺳ تﺎﻨﯿﯾﺰﺗ ﻪﮐ ﺪﺷ هداد نﺎﺸﻧ ،ﻦﯿﻨﭽﻤﻫ
ﯽﻤﻧ ﻪﻧادﻪﻧﻮﮔﺮﯾز ﺺﯿﺨﺸﺗ رد ار ﯽﺒﺳﺎﻨﻣ تﺎﻔﺻ ﺪﻧاﻮﺗ ﻒﻠﺘﺨﻣ يﺎﻫO. spinosaﺪﯾﺎﻤﻧ ﻦﯿﻣﺎﺗ.
هژاويﺎﻫيﺪﯿﻠﮐ: ،ﯽﻟﻮﮑﻟﻮﻣ ﯽﯾازرﺎﺒﺗعﻮﻨﺗﻪﻧﻮﮔ،يايآ.ﯽﺗ.سا.ﻪﺘﺴﻫ ﯽﻣوزﻮﺒﯾر،يا،ﮏﯿﺗﺎﻤﺘﺴﯿﺳtrnL-F
ROSTANIHA
ROSTANIHA
DOI: http://dx.doi.org/10.22092/botany.2017.109402
Archive of SID
www.SID.ir

96
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Introduction
Ononis L. (Fabaceae) is a genus with more than
85 annual and perennial herbaceous species distributed in
North Africa, West Asia, the Mediterranean region and
Europe.
Linnaeus first described 12 species of Ononis in
the Species Plantarum (Linnaeus 1753). Širjaev (1932)
divided the genus into two major sections (i.e. Ononis
and Natrix) and 22 subsections, while Turini et al. (2010)
recognized five major lineages within the genus based on
molecular phylogenetic approaches. In his later study,
most of the subsections were well reflected by the
molecular data (Turini et al. 2010). Furthermore, Turini
et al. (l.c.) confirmed the results of Steele &
Wojciechowski (2003) who had distinguished Ononis as
a monophyletic group.
Rechinger (1984) reported 11 species of Ononis
from the area of “Flora Iranica”. According to him (l.c.),
seven species i.e. O. reclinata L., O. viscosa L., O. sicula
Guss., O. pusilla L., O. arvensis L., O. spinosa L., and
O. serrata Forssk., occur in north, west and south of Iran.
Ononis spinosa is the only perennial spinose species
within the genus reported from Iran. This widespread
species, which is known also from Europe, has lateral
uniflorus flowers with sessile or sub-sessile glands on its
calyx-lobes (Huber-Morath 1970). Overall, eight
subspecies are reported for O. spinosa in the world
(Kalvij 2012).
Greuter et al. (1989) considered four taxa under
Ononis spinosa, i.e. O. spinosa subsp. arvensis (L.)
Greuter & Burdet, O. spinosa subsp. australis (Širj.)
Greuter & Burdet, O. spinosa subsp. masquillierii
(Bertol.) Greuter & Burdet and finally O. spinosa subsp.
spinosiformis (Simk.) Greuter & Burdet, but later, two of
them i.e. subsp. arvensis and subsp. spinosiformis were
considered as synonyms (Kalvij 2012).
Boissier (1872) introduced two further distinct
perennial species, i.e. O. Antiquorum, and O. leiosperma
for Iran which were later reduced to subspecies under
O. spinosa, i.e. O. spinosa subsp. antiquorum (L.) Briq.,
and O. spinosa subsp. leiosperma (Boiss.) Širj.,
respectively. The two subspecies are distinguished from
each other by the presence of tuberculate seeds in subsp.
antiquorum and the smooth ones in subsp. leiosperma as
the only morphological key character. Rechinger (1984)
reported O. spinosa subsp. leiosperma, originally
described from south Turkey, as the only subspecies that
grows wildly in Iran.
Akhani (1996) reported O. spinosa subsp.
antiquorum from Golestan National Park (northeast of
Iran) and Yusef Naanaie (2009) confirmed the presence
of this subspecies in central and western part of Iran.
Akhani (l.c) also stated that, O. spinosa group in Asia,
Afghanistan and NE Iran presents a complicated
taxonomic problem. Ononis spinosa subsp. antiquorum,
has a Mediterranean distribution and it is retained as a
subspecies of O. spinosa in many European publications
(Širjaev 1932, Greuter et al. 1989), while most of the
Russian botanists such as Muravjeva (1945) and Nikitin
&Gel’dihanov (1988) preferred using the specific level
for this taxon.
In this study, we have performed the phylogenetic
analyses of datasets containing nr DNA ITS (Internal
Transcribed Spacer) and trnL-F spacer sequences of
Ononis taxa including all its Iranian members to clarify
their taxonomic position. Subsequently, we have found
the exact number of Ononis species and subspecies in
Iran. Due to some uncertainty and ambiguity about the
taxonomic status of O. spinosa in Iran, we especially
focused on the material representing different subspecies
of this species in our analyses, in order to find out
whether those reports of different subspecies of
O. spinosa are correct or not and also how many
subspecies are distributed in Iran.
Materials and Methods
Leaf samples were taken from herbarium
specimens deposited at TARI and IAUH (abbreviations
according to Thiers, 2015+). Ten samples from six
Ononis spp. were sequenced especially for this study:
Ononis serrata, and O. spinosa from different localities
of Iran (with either smooth or tuberculate seeds),
O. arvensis (syn.: O. spinosa subsp. arvensis), O. pusilla,
O. reclinata and finally O. sicula. Table 1 represents a
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

97
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
list of taxa already reported from Iran. No herbarium
specimen matching the description of O. viscosa was
found in two mentioned above herbaria and also those
which we had access to.
Moreover, 51 and 53 accessions from GenBank
were included in the analyses of trnL-F and ITS regions,
respectively (Appendix 1). Trigonella caerulea (L.) Ser.
was elected as outgroup (see Steele & Wojciechowski
2003, Wojciechowski et al. 2004, Turini et al. 2010).
The sources of plant material for DNA isolation and
GenBank accession numbers for ITS and trnL-F
sequences used in the analyses are given in Appendix 1.
Following manufacturer’s protocol, total genomic
DNA was extracted from dried leaf specimens
(approximately 0.5 g material per sample) using
NucleoSpin Plant-Kit (Macherery-Nagel, Düren,
Germany). The complete Internal Transcribed Spacer
(ITS) region was amplified using forward primer
AB101(5’-ACGAATTCAAGGTCCGGTGAAGTGTTCG-3’)
and reverse primer AB102 (5’-T A G A A T T C C C C G
G T T C G C T C G C C G T T A C-3’) (Douzery et al.
1999). The trnL-F spacer region was amplified using
forward (5’-CGAAATCGGTAGACGCTACG-3’) and
reverse (5’-ATTTGAACTGGTGACACGAG-3’) primers
(Taberlet et al. 1991). Following program used for
amplification of nuclear regions (ITS1 and ITS2) in a
PCR reaction: a pretreatment at 94° C for 2 min 30s; 40
cycles of denaturation at 94° C for 30s, annealing at
54.5° C for 30s and extension at 72° C for 1 min 15s; and
a final extension at 72° C for 10 min. To amplify the
plastid region trnL-F, following PCR parameters used:
40 cycles, each comprised of denaturation at 94° C for
30s, annealing at 55.3° C for 30s and extension at 72° C
for 1 min 30s, the other steps stayed unchanged.
Amplification products were purified using NucleoSpin®
Extract II-Kit (Macherey-Nagel, Düren, Germany)
following manufacturer’s protocol. Purified PCR
products were sequenced on an ABI 3730 automatic
sequncer machine (Applied Biosystems/Hitachi) and
achieved chromatograms were edited using Sequencher
v. 4.8 software (Gene Codes Corporation). Manual
sequence alignment was carried out using Macclade
v. 4.08a software (Maddison & Maddison 2005). After
editing and aligning the sequences, resulting data matrix
was analyzed using PAUP* v.4.0b10 (Swofford 2003)
and MrBayes v.3.1.2 program (Huelsenbeck & Ronquist
2001, Ronquist & Huelsenbeck 2003). Maximum
Parsimony (MP) analysis was carried out in PAUP*
using heuristic search strategies that following these
settings: simple sequence addition, TBR branch
swapping. All characters were unordered and equally
weighted and the relative level of support was assessed
by bootstrap analysis for individual clades in strict
consensus tree (Felsenstein 1985). The ModelTest
software v. 3.7 was used in order to select the best
substitution model (Posada & Crandall 1998, Posada &
Buckley 2004). Bayesian analysis of the ITS1+ITS2, trnL-F
and combined ITS1+ITS2+trnL-F datasets were performed
using following parameters: for ITS datasets: [Model
selected: TVM+I+G, frequency of base A=0.2293,
C=0.2174, G=0.2425 and T=0.3107; Substitution model:
(A-C=1.3972, A-G=2.9763, A-T=1.2096, C-G=0.6831,
C-T=2.9763, G-T=1.0000), Invariable sites proportion (I):
0.2736, Gamma distribution shape parameter: 0.5878]; For
trnL-F datasets: [Model selected: TVM+I+G, The base
frequencies (A=0.3616, C=0.1739, G=0.1649 and
T=0.2995); Substitution model: (A-C=0.7082, A-G=0.6724,
A-T=0.4147, C-G=0.8420, C-T=0.6724, G-T=1.0000),
Invariable sites proportion (I): 0.4553; Gamma distribution
shape parameter: 0.9248]; For combined datasets: [Model
selected: TIM+I+G; Base frequencies: A=0.2955,
C=0.1929, G=0.2091 and T=0.3025; Substitution model:
(A-C=1.0000, A-G=1.6847, A-T=0.7499, C-G=0.7499,
C-T=2.3530, G-T=1.0000), Invariable sites proportion (I):
0.4227, Gamma distribution shape parameter: 0.6795]. The
Markov Chain Monte Carlo process was set and four chains
were run for the total of 3,000,000 generations
simultaneously. Overall, 30,000 trees were saved in the
primary choices (one tree out of 100 generations). After
discarding25% of primary trees, posterior probability of the
phylogeny and its branches was determined from the
remaining 22500 trees. Internodes with ≥0.95 posterior
probabilities were considered as well supported. Bayesian
analysis led to a 50% majority rule consensus tree.
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

98
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Table 1. List of Ononis species accompanied by their morphology and distribution in Iran
Taxon
Morphology
Distribution
in Iran
Reference
O. pusilla L.
Perennial, stems erect to ascending erect, glandular-hairy. Flowers axillary, overtopped by
floral leaves, forming dense terminal racemes. Peduncle inconspicuous. Calyx lobes ovate -
lanceolate to lanceolate. Corolla yellow. Legume ovate-rhomboid. Seeds orbicular, slightly
tuberculate.
N, W
Rechinger
1984,
Huber-
Morath
1970
O. reclinata L.
Annual, without spines, mainly branched from the base, glandular-villous. Flowers scattered
or forming a short leafy raceme. Calyx lobes sub equal, linear-lanceolate, peduncle
conspicuous (5-10mm), awnless. Corolla pink to purple. Legume linear-oblong, glandular-
villous. Seeds orbicular, rough-tubercled.
N, W, S
"
O. serrata Forssk.
Annual, viscid pubescent, branched from the base, with decumbent or prostrate stems,
without spines. Flowers sessile or sub sessile, forming dense oblong terminal racemes.
Peduncle conspicuous. Calyx lobes lanceolate-subulate, corolla pink, glandular-hairy.
Legume ovate, glandular-pubescent. Seeds elliptic to sub-orbicular, tuberculate.
W, S
"
O. sicula Guss.
Annual, commonly erect, branched from the base, with dense glandular hairs. Flowers
solitary, arranged in a leafy raceme. Peduncle conspicuous (10-30 mm). Corolla yellow, 6-9
mm long, legume linear-oblong. Seeds reniform-orbicular.
W, S
"
O. arvensis L.
[= O. spinosa
subsp. arvensis (L.)
Greuter & Burdet]
Perennial, spinose, with erect ascending stems. Flowers shortly pedicellate, mainly
congested into ovate terminal raceme. Calyx lobes linear-lanceolate, with numerous long
eglandular and shorter glandular hairs. Corolla pink with purple stripes. Seeds orbicular,
tuberculate.
C
"
O. spinosa L.
subsp. leiosperma
(Boiss.) Širj.
Perennial, spinose, with erect ascending stems. Flowers shortly pedicellate, solitary or
raceme. Calyx lobes with sessile or sub-sessile glands, sometimes glabrescent. Seeds
smooth.
W, S
"
O. viscosa L.
Annual, often branched from the base. Flowers forming long leafy racemes. Peduncle
conspicuous (10-25 mm), long awned, one flowered. Calyx lobes linear acuminate. Corolla
pale yellow, often striped with red. Legume linear to linear oblong, glandular- hairy. Seeds
kidney-shaped to orbicular, tuberculate.
W, S
"
Results
- ITS datasets
In Maximum Parsimony (MP) analysis of ITS
datasets, 10000 shortest trees with the length of 577
evolutionary steps, CI=0.633 and RI=0.867 were
combined to form a strict consensus tree with 588 steps,
CI=0.621 and RI=0.860. For trnL-F datasets, 10000
shortest trees with the length of 164 evolutionary steps,
CI=0.793 and RI=0.911 were combined to form a strict
consensus tree of 178 steps, CI=0.730 and RI=0.874. In
analysis of combined (ITS1+ITS2+trnL-F) datasets
10000 shortest trees with 670 steps length, CI=0.676 and
RI= 0.874 were obtained and combined into a strict
consensus tree with 680 evolutionary steps length,
CI=0.666 and RI=0.869.
The strict consensus tree obtained from Maximum
Parsimony (MP) analysis of the nrDNA ITS, is shown in
Fig. 1. Both bootstrap support (BS) (derived from MP
analysis) and posterior probability (PP) (resulted from
Bayesian analysis) are also shown in Fig. 1. In the strict
consensus tree, Ononis taxa including the Iranian ones
and those obtained from GenBank are grouped into two
major clades: 1) Clade A: This well-supported clade
(BS: 100%, PP: 1.00) comprise three species i.e.
O. rotundifolia L., O. fruticosa, and O. tridentata subsp.
tridentata. All members of this clade belong to the same
section, i.e. section Natrix. 2) Clade B: This clade is
divided into four subclades (i.e. B1, B2, B3 and B4).
Two sub-clades, B1 and B3, are strongly supported (BS:
100%, PP: 1.00). Clade B1 consists of O. reuteri Boiss.
& Reuter and O. speciosa Lagasca, both belonging to
subsection Chrysanthae Willkomm. Clade B3 contains
only one Iranian specimen of O. pusilla along with
O. pusilla L. subsp. saxicola (Boiss.) Malag.,
O. cephalotes Boiss., O. minutissima L. and finally,
O. striata Gouan, all obtained from GenBank. All these
taxa belong to the same subsection i.e. Bugranoides (see
Table 1). Clade B2 is weakly supported and includes
only one species i.e. O. ornithopodioides L. Clade B4
consists of two major lineages among which only B4a is
well supported (BS: 100%, PP: 1.00) and contains only
one Iranian specimen of O. sicula but also O. aurasiaca
Förther & Podlech, O. ramosissima Desf., O. tazaensis
Förther & Podlech, O. hesperia (Maire) Förther &
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

99
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Podlech, O. mogadorensis Förther & Podlech,
O. paralias Förther & Podlech, O. angustissima Lam.
subsp. longifolia (Willd.) Förther & Podlech,
O. pubescens L., O. antennata Pomel subsp. antennata,
O. pseudoserotina Batt. & Pit., O. serotina Pomel,
O. zygantha Maire & Wilczek, O. polyphylla Ball and
finally O. polysperma Barratte & Murb. In subclade B4b,
O. adenotricha Boiss., is weakly supported as sister to
other species nested in this clade. The secondary lineage
(B4b) is also divided into two clades, one of which is
strongly supported (BS: 90%). Ononis christi Bolle, and
O. laxiflora Desf. are supported as sisters (Fig. 1; BS:
100%, PP: 1.00). In this clade, the two species
O. leucotricha Coss., and O. pinnata Brot., belonging to
the subsection Pinnatae, are sisters (MP BS= 100%, PP:
1.00). Group I, placed in clade B4b (BS: 100%, PP:
1.00), is divided into two subgroups, i.e. Ia and Ib.
Ononis alba Poir. subsp. monophylla (Desf.) Murb. is the
only species placed in subgroup Ia, while other Ononis
species containing O. nuristanica Podlech (group A),
O. spinosa subsp. spinosa (group B), O. spinosa subsp.
masquilierii,O. spinosa subsp. maritime, and O. spinosa
subsp. antiquorum (group C), four specimens of
O. spinosa species (group D), with either smooth or
tuberculate seeds, collected from different localities of
Iran and finally three specimens of O. spinosa subsp.
arvensis (two of which are Iranian samples shown in
bold in bold in Fig. 1; groups F, G) are placed in clade
Ib, with high bootstrap support (BS: 100%). Four
specimens of O. spinosa strongly nested in a separate
group (group D; BS: 80%, PP: 0.95) and three O. spinosa
subspp. i.e. masquilierii,antiquorum, and maritima
(group C) (obtained from GenBank) are clustered in a
separate clade with moderate bootstrap support (BS:
80%) and full maximum posterior probability (PP: 1.00),
among which two first subspecies were sisters (BS: 70%,
PP: 0.95). Moreover, the clade encompassing all
O. spinosa subspecies accompanied by O. alba subsp.
monophylla, and O. nuristanica are well supported by
full Bayesian posterior probabilities (PP: 1.00) (Fig. 1).
- trnL-F datasets
The trnL-F matrix included 51 Ononis taxa and
only one species as outgroup. The species were grouped
into six major lineages (A–F) (Fig. 2). Most of the results
of trnL-F datasets were in line with those obtained from
ITS.
Clade C, a well-supported clade, (BS: 100%, PP:
1.00) shows polytomy. Three taxa i.e. O. biflora Desf.,
O. polysperma and O. zygantha with strong bootstrap
support and posterior probability are placed in this clade
(BS: 70%, PP: 0.56) one of which i.e. O. biflora was
absent in ITS topology. All Ononis spinosa subspecies
containing Iranian ones (subspp. leiosperma,arvensis,
maritime, and spinosa), together with O. nuristanica,
have constructed a polytomy (BS: 80%, PP: 0.82).
- Combined (ITS+trnL-F) datasets
The results of combined datasets are shown in
Fig. 3. Species are divided into two major clades A and
B. Clade B is divided into four subclades B1, B2, B3 and
B4. The Iranian O. spinosa subspp., i.e. O. spinosa
subsp. arvensis,O. spinosa and O. spinosa subsp.
leiosperma, together with O. spinosa subsp. maritima,
O. spinosa subsp. masquilierii,O. spinosa subsp.
spinosa,O. nuristanica, and O. alba subsp. monophylla
gathered from GenBank, are placed in the same clade and
are well supported (B4bII) (BS: 100%, PP: 1.00). In
Figure 3, it is quite obvious that, four specimens of
Ononis spinosa collected from different localities of Iran
(two of which were previously distinguished as
O. spinosa subsp. antiquorum by morphological
characters) are grouped in a well-supported monophyletic
clade (BS: 100%, PP: 1.00). Ononis spinosa subsp.
masquilierii, and O. spinosa subsp. maritima are also
placed in the same subgroup (BS: 90%, PP: 1.00).
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

100
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Fig. 1. Strict consensus tree resulted from the Maximum Parsimony analysis of the nrDNA ITS dataset (The newly
sequenced accessions are in Bold-Italic). All other sequences are taken from the GenBank. Numbers above branches
represent Bootstrap values (BS) and numbers below branches show posterior probabilities (PP) (only values greater
than 50 % for BS and greater than 0.50 for PP are shown). Trigonella caerulea is outgroup.
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

101
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Fig. 2. Strict consensus tree resulted from the Maximum Parsimony analysis of the plastid trnL_F spacer dataset (The
newly sequenced accessions are in Bold-Italic). All other sequences are taken from the GenBank. Numbers above
branches represent Bootstrap values (BS) and numbers below branches show posterior probabilities (PP) (only values
greater than 50 % for BS and greater than 0.50 for PP are shown). Trigonella caerulea is outgroup.
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

102
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Fig. 3. Strict consensus tree resulted from the Maximum Parsimony analysis of the combined nrDNA
ITS+plastid trnL_F spacer datasets (The newly sequenced accessions are in Bold-Italic). All other sequences are taken
from the GenBank. Numbers above branches represent Bootstrap values (BS) and numbers below branches show
posterior probabilities (PP) (only values greater than 50 % for BS and greater than 0.50 for PP are shown). Trigonella
caerulea is outgroup.
Discussion
The results of this study corroborate those of
Turini et al. (2010) in following points: placing the
Iranian Ononis serrata (subsect. Diffusae) close to
O. mitissima (subsect. Mitissimae) and O.tournefortii
(subsect. Diffusae); placing the Iranian O. reclinata close
to O. macrosperma and O. verae, and finally, placing
O. pusilla close to O. pusilla subsp. saxicola,
O. cephalotes, and O. minutissima in the combined
datasets. Four specimens of O. spinosa from different
parts of Iran were investigated. These specimens were
previously distinguished as two separate subspecies (i.e.
O. subsp. leiosperma with “smooth” seed coat and subsp.
antiquorum with “tuberculated” seed coat) (Akhani
1996). The phylogenetic results showed that, all the
mentioned specimens formed a monophyletic clade with
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

103
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
robust support (Figs 1 & 3). It is shown that, Iranian
samples of O. spinosa with different seed coat
ornamentation could not be separated as different taxa.
However, O. spinosa subsp. antiquorum, from GenBank,
was placed in a different clade in the ITS analysis (Fig. 1).
This indicates that, despite a few morphological
differences among the Iranian taxa, there is no
considerable phylogenetic difference between them (Figs
1 & 3). Having granular or smooth seed surface is not
regarded as a diagnostic character to differentiate these
closely related Iranian taxa into two separate subspecies
(subspp. leiosperma and antiquorum). Therefore, we
concluded that, identification of these subspecies is not
correct and both must be identified under a single
subspecies. Considering the geographical distribution of
O. spinosa subsp. leiosperma, which is described based
on the type material collected from southwest Asia
(Boissier 1872), it is more reliable to accept the presence
of this subspecies instead of subsp. antiquorum. Type
material of O. spinosa subsp. antiquorum is collected
from south Europe (Mediterranean region; Huber-Morath
1970). Therefore, Ononis spinosa subsp. leiosperma
could be considered as the only subspecies occurs in Iran.
Further investigations based on the materials from south
Europe, Iran, Afghanistan and central Asia are needed to
clarify taxonomic position of subspecific taxa of
O. spinosa complex. One of the Ononis species that is
endemic to Afghanistan and Pakistan, i.e. O. afghanica
(Syn.: O. spinosa subsp. afghanica (Širj. & Rech.f.)
Kitamura) reported in Flora Iranica is very complicated
and could not be easily separated from those reported
from the Mediterranean region under the name
O. spinosa subsp. antiquorum. Ononis spinosa subsp.
arvensis (Syn.: O. arvensis) that was collected from
northwest Iran is placed in a group encompassing all
Ononis spinosa subspp. This issue confirms the
morphological results of Greuter et al. (1989) who have
introduced a new combination, i.e. O. spinosa subsp.
arvensis (L.) Greuter & Burdet. With respect to the
results obtained in this study, two subspecies have been
recognized for O. spinosa (i.e. subsp. arvensis, and
subsp. leiosperma), and O. spinosa subsp. antiquorum is
not present in Iran. In conclusion, our molecular
phylogenetic data suggest the occurrence of three species
of Ononis in Iran among which, O. spinosa itself is
further represented by three subspecies.
Acknowledgments
This work is supported by Islamic Azad
University, Yadegar-e-Imam Khomeini (RAH), Shahr-e
Rey Branch (Tehran, Iran). Authors wish to thank the
Herbarium curator of Research Institute of Forests and
Rangelands Herbarium (TARI), Tehran, Iran and also the
Islamic Azad University, Science and Research Branch
(Tehran, Iran), for providing the laboratory equipment
for this investigation. Authors also appreciate the
assistance of Dr. Y. Shahali, Miss M. Keivani and Mrs.
D. Yeganeh for their laboratory helps in this research.
References
Akhani, H. 1996. Studies on the flora and vegetation of
the Golestan National Park, NE Iran I: A new
species and some new plant records. Annalen des
Naturhistorischen Museums in Wien 98: 97–105.
Boissier, E. 1872. Ononis L. Pp. 526–553. In: Flora
Orientalis (Boissier, E., ed.). Basilease &
Lugundi, Genevae. Vol. 2.
Douzery, E.J.P., Pridgeon, A.M.A., Kores, P., Linder,
H.P., Kurzweil, H. & Chase, M.W. 1999.
Molecular phylogenetics of Diseae
(Orchidaceae): a contribution from nuclear
ribosomal ITS sequences. American Journal of
Botany 86: 887–899.
Felsenstein, J. 1985. Confidence limit on phylogenies:
An approach using the bootstrap. Evolution 38:
383–391.
Greuter, W., Bürdet, H.M. & Long, G. 1989. Med-
Checklist 4: Dicotylédones (Lauraceae-
Rhamnaceae). Conservatoire et Jardin botaniques,
Genève.
Huber-Morath, A. 1970. Ononis L. (Leguminosae-
Ononideae). Pp. 373–384. In: Flora of Turkey and
the East Aegean Islands (Davis, P.H.,
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

104
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Chamberlain, D.F. & Matthews, V.A., eds),
Edinburgh University Press, Edinburgh.
Huelsenbeck, J.P. & Ronquist, F. 2001. MRBAYES:
Bayesian inference of phylogeny. Bioinformatics
17: 754–755.
Kalvij, J.M. 2012. Review of ‘The plant list, a working
list of all plant species’. Journal of Vegetation
Science 23(5): 998–1002. Online at:
http://www.theplantlist.org/.
Linnaeus, C. 1753. Species plantarum. Laurentius
Salvius, Stockholm. 2 Vols.
Maddison, W.P. & Maddison, D.R. 2005. MacClade 4:
analysis of phylogeny, and character evolution.
Sunderland, Massachusetts: Sinauer Associates.
Muravjeva, O.A. 1945. Ononis L. Pp. 94–102. In: Flora
of the USSR (Schischkin, B.K., ed.). National
Science Foundation, Washington D.C.
Nikitin, V.V. & Gel’dihanov, A.M. 1988. Opredelitel’
rastenii Turkmenistana. Nauka, Leningrad.
Posada, D. & Crandall, K.A. 1998. Modeltest: Testing
the model of DNA substitution. Bioinformatics
14: 817–818.
Posada, D. & Buckley, T.R. 2004. Model selection and
model averaging in phylogenetics: Advantages of
Akaike Information Criterion and Bayesian
approaches over Likelihood Ratio Tests.
Systematic Biology 53(5): 793–808.
Rechinger, K.H. 1984. Ononis (Papilionaceae II). Pp.
189–199. In: Flora Iranica (Rechinger, K.H., ed.).
Akademische Druck, Graz, Austria. Vol. 157.
Ronquist, F. & Huelsenbeck, J.P. 2003. MRBAYES 3:
Bayesian phylogenetic inference under mixed
models. Bioinformatics 19: 1572–1574.
Širjaev, G. 1932. Generis Ononis L., revisio critica.
Beihefte zum Botanischen Centralblatt 49:
381–665.
Steele, K.P. & Wojciechowski, M.F. 2003. Phylogenetic
analysis of tribes Trifolieae and Vicieae, based on
sequences of the plastid gene matK
(Papilionoideae:Leguminosae). Pp. 355–370. In:
Advances in Legume Systematics, Higher Level
Systematics (Kligaard, B.B. & Bruneau, A., eds)
Royal Botanic Gardens, Kew, Vol. 10.
Swofford, D.L. 2003. PAUP*: Phylogenetic analysis
using Parsimony (*and other methods), version
4.0b10 for 32-bit Microsoft Windows. Sinauer,
Massachusetts, Sunderland.
Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991.
Universal primers from amplification of three
non-coding regions of chloroplast DNA. Plant
Molecular Biology 17: 1105–1109.
Thiers, B. 2015 [+continuously updated]. Index
herbariorum: a global directory of public herbaria
and associated staff. New York Botanical
Garden’s virtual herbarium. Published at
http://sweetgum.nybg.org/ih/ [accessed 1 Apr.
2015].
Turini, F.G., Bräuchler, C. & Heubl, G. 2010.
Phylogenetic relationships and evolution of
morphological characters in Ononis L.
(Fabaceae). Taxon: 59(4): 1077–1090.
Wojciechowski, M.F., Lavin, M. & Sanderson, M.J.
2004. A phylogeny of legumes (Leguminosae)
based on analysis of the plastid matK gene
resolves many well-supported subclades within
the family. American Journal of Botany 91:
1846–1862.
Yusef Naanaei, S. 2009. A correct record for Flora of
Iran: Ononis spinosa subsp. antiquorum
(Fabaceae). Iranian Journal of Botany 15(2):
181–182.
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

105
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Appendix 1. List of the 67 Ononis species and one outgroup, accompanied by other information of each specimen
[including GenBank accession number, locality (for Iranian species), collector name, collection number,
section/subsection]. The two last column on the right show the presence of each species in either ITS or trnL-F
sequences analyses (by the signs of + and -)
No.
Taxon
GenBank No.
(ITS/trnL-F)
Collector &
collection No.
Locality
Section/subsection
ITS
seq.
trnL-
Fseq.
1
O. spinosa L.subsp. leiosperma
(Boiss.) Širj.
KM113804-
KM204129
Nowroozi &
Etemadi, 1280
(TARI)
Isfahan: above
Boin Cheshmeh
Rashid, 2475 m
Ononis / Ononis
+
+
2
O. spinosa L.subsp. leiosperma
(Boiss.) Širj.
KM113806-
KM204130
Buttler &
Buthmer, 22817
(TARI)
Tehran: Firuzkuh-
Pol-e sefid, C. 4
km NE of
Firuzkuh. 1950 m
Ononis / Ononis
+
+
3
O. spinosa L.subsp. leiosperma
(Boiss.) Širj.
KM113805-
KM204127
Sanandaji, 1700
(IAUH)
Kurdestan:
Sanandaj, Saral-
Chattan, 2041 m.
Ononis / Ononis
+
+
4
O. spinosa L.subsp. leiosperma
(Boiss.) Širj.
KM113803-
KM204128
Rahmani, 7680
(IAUH)
Qazvin: Bashgol,
1900 m
Ononis / Ononis
+
+
5
O. spinosa L.subsp. arvensis (L.)
Greuter & Burdet
GQ488523-
GQ488595
Heubl, n/a
Spain
Ononis / Ononis
+
+
6
O. spinosa L.subsp. antiquorum
(L.) Arcang.
GQ488522
Berger, 4284
Spain
Ononis / Ononis
+
-
7
O. spinosa L.subsp. maritima
(Dumort.) P. Fourn.
GQ488524-
GQ488596
Schuhwerk,
05/272
Spain
Ononis / Ononis
+
+
8
O. spinosa L. subsp. masquillieri
(Bertol.) Greuter & Burdet
GQ488525-
GQ488597
Ferrari, s.n.
Spain
Ononis / Ononis
+
+
9
O. spinosa L. subsp. spinosa
GQ488521-
GQ488594
Heubl, n/a
Spain
Ononis / Ononis
+
+
10
O. spinosa L.subsp. arvensis (L.)
Greuter & Burdet
KM113807-
KM204122
Bairami, 5937
(TARI)
Azarbayjan:
Kaleybar, Khoda
Afarin Arasbaran
protected area,
Kalaleh village,
1200 m
Ononis / Ononis
+
+
11
O. spinosa L.subsp. arvensis (L.)
Greuter & Burdet
KM113802-
KM204121
Mozaffarian,
43600 (TARI)
Azarbayjan: 90 km
from Varzeghan to
Jolfa, 1000 m
Ononis / Ononis
+
+
12
O. serrata Forssk.
KM113799-
KM204125
Mozaffarian,
62663 (TARI)
Khuzestan:
Susangerd, Bostan,
Allahoakbar, 60 m
Ononis / Diffusae
Širj.
+
+
13
O. pusilla L.
KM113800-
KM204123
Hamze & Asri,
81601 (TARI)
Azarbayjan:
Arasbaran
protected area,
Vaighan to
Kalaleh, Research
forests, 1150 m
Ononis /
Bugranoides
Willk.
+
+
14
O. pusilla L.subsp. saxicola (Boiss.
& Reut.) Malag.
GQ488510-
GQ488584
Reverchon, 1333
Spain
Ononis /
Bugranoides
Willk.
+
+
15
O. reclinata L.
KM113798-
KM204124
Mozaffarian,
63202 (TARI)
Khuzestan: Elize
Parke Jangali, 850
m
Natrix (Moench)
Griseb /
Reclinatae Širj.
+
+
16
O. sicula Guss.
KM113801-
KM204126
Mozaffarian,
59161 (TARI)
Bandar-Abbas:
Genu Mts., 1200-
2200 m
Natrix /Reclinatae
Širj.
+
+
17
O. sicula Guss.
GQ488518-
GQ488591
Lobin, 7
Morocco
Natrix /Reclinatae
Širj.
+
+
18
O. angustissima Lam.
subsp. longifolia (Willd.) Förther &
Podlech
GQ488550
Ginovés et al.,
16.222
Tenerife
Natrix /Natrix
-
+
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

106
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Appendix 1 (contd.)
19
O. angustissima Lam.
subsp. angustissima
GQ488549
Frahm et al., 9
Algeria
Natrix /Natrix
-
+
20
O. nuristanica Podlech
GQ488500-
GQ488575
Podlech, 727
Afghanistan
Ononis / Ononis
+
+
21
Ononis alba Poir.
subsp. monophylla (Desf.) Murb.
GQ488469-
GQ488547
Podlech, 39084
Algeria
Ononis /
Villosissimae Širj.
+
+
22
O. antennata Pomel
subsp. antennata
GQ488473-
GQ488551
Deil, 1023
Morocco
Natrix / Viscosae
Širj.
+
+
23
O. aurasiaca Förther & Podlech
GQ488475-
GQ488553
Podlech, 38634
Algeria
Natrix /Natrix
+
+
24
O. alopecuroides L.
subsp. alopecuroides
GQ488548
Erben, s.n.
Sicily
Ononis /
Salzmannianae
Širj.
-
+
25
O. rotundifolia L.
GQ488515-
GQ488588
Weimer, s.n.
Switzerland
Natrix / Antiquae
Širj.
+
+
26
O. atlantica Ball
GQ488552
Podlech, 47466
Morocco
Natrix /Natrix
+
27
O. dentata Sol. ex Lowe
GQ488484-
GQ488559
Gaisberg, 162
Canary Islands
Natrix /Reclinatae
Širj.
+
+
28
O. filicaulis Salzm. ex Boiss.
GQ488486-
GQ488561
Lobin, 321a
Spain
Ononis /
Villosissimae Širj.
+
+
29
O. hesperia (Maire) Förther &
Podlech
GQ488489-
GQ488564
Podlech, 48769
Morocco
Natrix /Natrix
+
+
30
O. laxiflora Desf.
GQ488492-
GQ488567
Breitfeld, s.n.
Gran Canaria
Natrix /Reclinatae
Širj.
+
+
31
O. biflora Desf.
GQ488555
Liston, s.n.
Israel
Natrix / Biflorae
Širj.
-
+
32
O. macrosperma Hub.-Mor.
GQ488477-
GQ488554
Hertel, 34.138
Turkey
Natrix /Reclinatae
Širj.
+
+
33
O. maweana Ball
GQ488494-
GQ488569
Lambinon &
Lewalle, 84/M/90
Morocco
Natrix / P ilosae
Širj.
+
+
34
O. megalostachys Munby
GQ488495-
GQ488570
Faure, s.n.
Algeria
Ononis /Verae
Širj.
+
+
35
O. minutissima L.
GQ488496-
GQ488571
Grau, et al., s.n.
Spain
Ononis /
Bugranoides
Willk.
+
+
36
O. mogadorensis Förther & Podlech
GQ488498-
GQ488573
Podlech, 46441
Morocco
Natrix /Natrix
+
+
37
O. ornithopodioides L.
GQ488502-
GQ488576
Zohary & Liston,
7-82-53
Israel
Natrix /Torulosae
Širj.
+
+
38
O. paralias Förther & Podlech
GQ488503-
GQ488577
Podlech, 48353
Morocco
Natrix /Natrix
+
+
39
O. pendula Desf. subsp. pendula
GQ488504-
GQ488578
Förther, 1297
Sicily
Natrix /Reclinatae
Širj.
+
+
40
O. pinnata Brot.
GQ488506-
GQ488579
Segura
Zubizarreta,
27464
Spain
Ononis / Pinnatae
Širj.
+
+
41
O. polyphylla Ball
GQ488519-
GQ488592
Podlech, 41542
Morocco
Natrix /Reclinatae
Širj.
+
+
42
O. polysperma Barratte & Murb.
GQ488507-
GQ488580
Podlech, 43002
Morocco
Natrix / Biflorae
Širj.
+
+
43
O. pseudoserotina Batt. & Pit.
GQ488508-
GQ488581
Krach & Koepff,
3784
Morocco
Natrix /Natrix
+
+
44
O. pubescens L.
GQ488509-
GQ488582
Kramer &
Kürschner, 4258
Turkey
Natrix /Viscosae
Širj.
+
+
45
O. ramosissima Desf.
GQ488511-
GQ488585
Erben, s.n.
Sardinia
Natrix /Natrix
+
+
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir

107
Mostafavi et al. / Phylogeny of Ononis in Iran …/ Rostaniha 17(2), 2016
Appendix 1 (contd.)
46
O. reuteri Boiss. & Reut.
GQ488513-
GQ488587
Huter, 34484
Spain
Ononis /
Chrysanthae
(Willk.) Širj.
+
+
47
O. serotina Pomel
GQ488516-
GQ488589
Faure, s.n.
Algeria
Natrix /Natrix
+
+
48
O. speciosa Lag.
GQ488520-
GQ488593
Nydegger, 33772
Spain
Ononis /
Chrysanthae
(Willk.) Širj.
+
+
49
O. cephalotes Boiss.
GQ488479-
GQ488556;
Lippert, W. & B.
9914
Spain
Ononis /
Bugranoides
Willk.
+
+
50
O. tazaensis Förther & Podlech
GQ488527-
GQ488600
Podlech, 46441
Morocco
Natrix /Natrix
+
+
51
O. thomsonii Oliv.
GQ488528-
GQ488601
Podlech, 47638
Morocco
Natrix /
Mauritanicae Širj.
+
+
52
O. tournefortii Coss.
GQ488529-
GQ488602
Podlech, 53259a
Morocco
Ononis / Diffusae
Širj.
+
+
53
O. tridentata L. subsp. tridentata
GQ488530-
GQ488603
Nydegger, 35832
Spain
Natrix /
Rhodanthae Willk.
+
+
54
O. fruticosa L.
GQ488487-
GQ488562
Lambinon,
00/F/250
France
Natrix /
Rhodanthae Willk.
+
+
55
O. baetica Clemente
GQ488476
Merxmüller &
Lippert, 23326
Spain
Ononis / Diffusae
Širj.
+
-
56
O. diffusa Ten.
GQ488485-
GQ488560
Baumann, 0/306
Rhodes
Ononis / Diffusae
Širj.
+
+
57
O. mitissima L.
GQ488497-
GQ488572
Baumann, s.n.
Tenerife
Ononis /
Mitissimae Širj.
+
+
58
O. cintrana Brot.
GQ488557
Nydegger, 33874
Spain
Ononis /
Intermediae Širj.
-
+
59
O. vilosissima Desf.
GQ488535-
GQ488608
Podlech, 45394
Morocco
Ononis /
Villosissimae Širj.
+
+
62
O. verae Širj.
GQ488534-
GQ488607
Širjaev, 12192
Crete
Natrix /Reclinatae
Širj.
+
+
63
O. christii Bolle
GQ488480
Bramwell, 10362
Fuerteventura
Natrix /
Canariensis Širj.
+
-
64
O. cossoniana Boiss. & Reut.
GQ488482
Conert et al., 682
Spain
Ononis / Diffusae
Širj
+
-
65
O. leucotricha Coss.
GQ488493
Segura
Zubizarreta,
42580
Spain
Ononis / Pinnatae
Širj.
+
-
66
O. striata Gouan
GQ488526
Dubuis, 7547
France
Ononis /
Bugranoides
Willk.
+
-
67
O. adenotricha Boiss.
GQ488466
Nydegger, 42160
Turkey
Natrix / Natrix
+
-
68
Trigonella caerulea Ser.
GQ488542-
GQ488615
Podlech, 37947
Austria
-
+
+
ROSTANIHA
ROSTANIHA
Archive of SID
www.SID.ir