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Phylogenetics of the Irano-Turanian taxa of Limonium (Plumbaginaceae) based on ITS nrDNA sequences and leaf anatomy provides evidence for species delimitation and relationships of lineages

March 2013
Botanical Journal of the Linnean Society 171(3):519-550

March 2013
171(3):519-550

DOI:10.1111/boj.12015

Authors:

Hossein Akhani

University of Tehran

Maryam Malekmohammadi

Freie Universität Berlin

Parastoo Mahdavi

Thünen Institute

Show all 5 authorsHide

In a taxonomic and molecular phylogenetic study using nuclear ribosomal internal transcribed spacer (ITS) DNA sequences and anatomical data, the taxonomic status and relationships of Irano‐Turanian Limonium spp. were investigated. The results of molecular phylogenetic analysis and anatomical synapomorphies showed that the Iranian Limonium spp. can be grouped into four major clades: (1) an unresolved clade including species of section Pteroclados as sister to all other Limonium spp.; (2) the L. axillare clade as sister to all Irano‐Turanian and Mediterranean species; (3) a poorly supported clade consisting of species of section Nephrophyllum, L. caspium, L. bellidifolium and L. iconium of section Limonium subsection Hyalolepidae and the isolated species L. sogdianum (section Siphonocalyx) and L. nudum (section Platyhymenium); and (4) a well‐supported clade including species of section Limonium subsection Limonium, part of section Sarcophyllum and L. lilacinum of section Sphaerostachys. The most diverse Mediterranean clade with many microspecies and apomictic taxa has no representatives in the Irano‐Turanian area. The ITS results agree with distribution and some morphological and anatomical characters, giving strong support for separating L. perfoliatum and L. reniforme that have been considered conspecific in all recent taxonomic treatments. An updated key to all known Iranian Limonium spp., a synopsis of all species, with distribution maps, and descriptions and illustrations of Iranian species of section Nephrophyllum are provided. © 2013 The Linnean Society of London

See caption on next page.

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Limonium otolepis. A, habit. B, spikelet from two views. C, outer bract. D, middle bract. E, inner bract. F, bracteole. G, calyx. H, petal and stamen. I, gynoecium. J, fruit. K, seed. (Chopanov 7.6.1967).

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Limonium perfoliatum. A, habit. B, spikelet. C, outer bract. D, middle bract. E, inner bract. F, bracteole. G, calyx. H, petal and stamen. I, gynoecium. J, fruit. K, seed. (Akhani et al. 18815).

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Epidermal cells of adaxial leaf sides of Limonium. Fig. 1. L. nudum (Akhani et al. 18804). Fig. 2. L. perfoliatum (greenhouse cultivated plant, no voucher). Fig. 3. L. caspium (Akhani et al. 18888). Fig. 4. L. sogdianum (Akhani & Memariani 19059). Fig. 5. L. iranicum (Malekmohammadi et al. 3826). Fig. 6. L. suffruticosum (Akhani & Joharchi 35188). Fig. 7. L. gmelinii (Akhani et al. 18946). Fig. 8. L. stocksii (Akhani 9750). Fig. 9. L. lobatum (Hekmatara 3978). S., stomata; s.g., salt gland; s.c. subsidiary cells. Scale bars, 100 mm.

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Leaf cross section of some representative Limonium species from Iran. Fig. 10. L. perfoliatum (Akhani et al. 18808). Fig. 11. L. gmelinii (Akhani et al. 18946). Fig. 12. L. sogdianum (Akhani & Memariani 19059). Fig. 13. L. suffruticosum (Akhani & Joharchi 35188). Fig. 14. L. axillare (Malekmohammadi et al. 3974). Fig. 15. L. stocksii (Akhani 9750). Sc., sclerenchyma; e.l., epidermal layer; v.b., vascular bundle; p.c., palisade cells; Sp.c., spongy cells; Ph., phloem; X, xylem. Scale bars, 100 m.

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Phylogenetics of the Irano-Turanian taxa of Limonium

(Plumbaginaceae) based on ITS nrDNA sequences and

leaf anatomy provides evidence for species delimitation

and relationships of lineages

HOSSEIN AKHANI1*, MARYAM MALEKMOHAMMADI1, PARASTOO MAHDAVI1,

AROOTIN GHARIBIYAN1and MARK W. CHASE2

1Department of Plant Sciences, School of Biology, Center of Excellence in Phylogeny of Living

Organisms, College of Sciences, University of Tehran, PO Box 14155-6455, Tehran, Iran

2Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

Received 4 May 2012; revised 22 October 2012; accepted for publication 4 December 2012

In a taxonomic and molecular phylogenetic study using nuclear ribosomal internal transcribed spacer (ITS) DNA

sequences and anatomical data, the taxonomic status and relationships of Irano-Turanian Limonium spp. were

investigated. The results of molecular phylogenetic analysis and anatomical synapomorphies showed that the

Iranian Limonium spp. can be grouped into four major clades: (1) an unresolved clade including species of section

Pteroclados as sister to all other Limonium spp.; (2) the L. axillare clade as sister to all Irano-Turanian and

Mediterranean species; (3) a poorly supported clade consisting of species of section Nephrophyllum,L. caspium,

L. bellidifolium and L. iconium of section Limonium subsection Hyalolepidae and the isolated species L. sogdianum

(section Siphonocalyx) and L. nudum (section Platyhymenium); and (4) a well-supported clade including species of

section Limonium subsection Limonium, part of section Sarcophyllum and L. lilacinum of section Sphaerostachys.

The most diverse Mediterranean clade with many microspecies and apomictic taxa has no representatives in the

Irano-Turanian area. The ITS results agree with distribution and some morphological and anatomical characters,

giving strong support for separating L. perfoliatum and L. reniforme that have been considered conspeciﬁc in all

recent taxonomic treatments. An updated key to all known Iranian Limonium spp., a synopsis of all species, with

distribution maps, and descriptions and illustrations of Iranian species of section Nephrophyllum are provided.

ADDITIONAL KEYWORDS: Anatolia – Caryophyllales – endemism – ﬂora of Iran – halophytes

–Mediterranean – south-west Asian ﬂora – taxonomy.

INTRODUCTION

The genus Limonium Mill. (Plumbaginaceae) is a

cosmopolitan halophytic genus, with c. 350 species;

it is most diverse in the Mediterranean area

(Kubitzki, 1993). In the Middle Eastern deserts and

salt marshes, this genus is represented in Anatolia

by c. 22 species (Bokhari & Edmondson, 1982; Tan &

Sorger, 1984, 1986; Yıldırımlı & Dog˘ru-Koca, 2006;

Dogan, Duman & Akaydin, 2008) and in the Flora

Iranica area (including Iran, Afghanistan, western

Pakistan and adjacent areas of Iraq and Turkmeni-

stan) by 14 species (Rechinger & Schiman-Czeika,

1974). Additional species have been reported to occur

in Iran in some post-Flora Iranica studies (Bokhari,

1982; Assadi, 1989; Akhani & Ghorbanli, 1993;

Assadi, 2005). It is counter-intuitive that, in spite of

a rich halophytic and xerophytic ﬂora in the Middle

East and Central Asia (Khan et al., 2006), Limonium

is comparatively much less diverse than in the Medi-

terranean area. There are also several genera related

to Limonium that have representatives in the Irano-

Turanian area, but these have been subject to differ-

ent generic interpretations in taxonomic treatments;

*Corresponding author. E-mail: akhani@khayam.ut.ac.ir

bs_bs_banner

Botanical Journal of the Linnean Society, 2013, 171, 519–550. With 46 ﬁgures

these include: Cephalorhizum Popov & Korovin, Dic-

tyolimon Rech.f., Aeoniopsis Rech.f., Chaetolimon

Lincz., Eremolimon Lincz., Goniolimon Boiss., Ikon-

nikovia Lincz. Popoviolimon Lincz., Neogontscharovia

Lincz., Bamiania Lincz., Bukiniczia Lincz. and Vas-

silczenkova Lincz. (Linczevski, 1971, 1979, 1985;

Rechinger & Schiman-Czeika, 1974).

Some Limonium spp. in Flora Iranica were

described by Rechinger as section Nephrophyllum

Rech.f., which comprises two species L. otolepis

(Schrenk) Kuntze and L. reniforme (Girard) Lincz.

This section is characterized by round–reniform

amplexicaul cauline leaves, reduced and ephemeral

rosulate leaves and an obconical calyx with narrow

limbs. Limonium otolepis has a Central Asian distri-

bution ranging from Turkmenistan, through Kaza-

khstan, Kyrgyzstan and Tajikistan, to northern China

in Gansu and northern Xinjiang (Linczevski, 1952;

Pen & Kamelin, 1996). This species has been intro-

duced into North America (Hickman, 1993; Smith,

2005). Limonium reniforme in its former circumscrip-

tion has a similar range but extends into south-

central Iran. Earlier morphological and geographical

studies by the ﬁrst author (H.A.) showed that these

last populations of L. reniforme (sensu Rechinger &

Schiman-Czeika, 1974) differ markedly from the

northern populations in Iran by their sterile branches.

The presence of such sterile branches caused Assadi

(2005) to classify these plants as L. otolepis. In order

to clarify this problem, we conducted more studies of

Limonium in Iran, including detailed morphological,

anatomical and molecular studies. The aims of this

paper are to: (1) test the application of internal trans-

cribed spacer (ITS) sequence data in reconstruction of

phylogenetic relationships and as taxonomic markers

for the Irano-Turanian lineages of Limonium; (2)

test the congruence of morphological and molecular

data with the traditional classiﬁcation of the genus;

(3) clarify the phylogeography of Iranian Limonium

spp. and their putative origin; and (4) present an

updated synopsis of the genus in Iran, an identiﬁca-

tion key and distribution maps.

MATERIAL AND METHODS

MORPHOLOGICAL AND TAXONOMICAL STUDIES

Natural populations of these species have been inves-

tigated during several excursions throughout Iran

since 1988 and to Turkmenistan during 1994, mostly

by the ﬁrst author (H.A.), but joined in 2007 by the

second author (M.M.). Herbarium specimens from

several herbaria in Iran and Europe (including B,

IRAN, K, TARI, TUH, P and W; abbreviations follow-

ing Index Herbariorum, (Thiers, 2009) and the private

herbarium of the ﬁrst author (Hb. Akhani, currently

housed in the Halophytes and C4Plants Research

Laboratory, School of Biology, University of Tehran),

have been studied. An updated key to identiﬁcation

and a synopsis of all accepted species growing in Iran,

distribution maps and descriptions of three species

with new taxonomic status and their illustrations are

provided, according to data obtained by the authors.

Many type specimens have been examined by the ﬁrst

author in short herbarium visits since 1990 in B, G,

K, P and W. However, we are unable to lectotypify

most names, as these short visits did not have this as

a primary aim.

ANATOMICAL STUDIES

Anatomical studies were made on leaf epidermis,

lamina and petiole cross sections. Forty-two samples

belonging to 14 species were studied. Fresh leaves

were ﬁxed during ﬁeld studies in FAA (one part

formalin, one part glacial acetic acid, 18 parts

70% ethanol). The samples were transferred to 70%

ethanol after 1 week. Using a razor blade, cross

sections were cut from the middle of the leaf lamina

and from the lower half of the petiole. Sections were

stained in aqueous methylene blue/carmine and dehy-

drated through an ethanol series and xylene. The

dehydrated samples were permanently mounted in

Euparal. Epidermal characters were studied on her-

barium samples following the method described by

Bokhari (1972): leaves were soaked overnight in 10%

potassium hydroxide (KOH) solution with a few drops

of hydrogen peroxide (H2O2), washed with distilled

water and allowed to stand in water with a few drops

of H2O2for 2 h. They were then thoroughly washed

with water and placed in ‘Eau de Javelle’ for bleach-

ing. After washing with distilled water, the upper

epidermis became loosened and was easily peeled off.

The epidermal peels were stained with toluidine blue.

After thorough dehydration, the peels were mounted

in Euparal. Sclereids were studied in both cross and

longitudinal sections to get a three-dimensional view.

Observations were made with a Nikon Optiphot-2

microscope, and photographs were taken with a Motic

image plus 2.0 camera; measurements were made

using a calibrated Motic image plus 2.0 software. Line

drawings of the sclereids are included here.

DNA SEQUENCING

Most samples in this study were collected during

various excursions in Iran, Turkey, Uzbekistan,

France and Italy by the ﬁrst author. In most cases,

leaves were collected in silica gel during ﬁeld studies

(Chase & Hills, 1991). Additional samples were taken

from herbarium specimens. DNA was extracted using

AccuPrep GMO DNA Extraction Kit according to

the manufacturer’s protocol. Ampliﬁcation of the ITS

region (ITS1 and ITS2 spacers plus the 5.8S gene)

520 H. AKHANI ET AL.

used the ITS5 and ITS4 primers of White et al.,

(1990) and followed the protocol of Van den Berg

et al. (2000). Ampliﬁed products were puriﬁed using

NucleoSpin PCR puriﬁcation columns in accordance

with the manufacturer’s protocols. Cycle sequencing

reactions were performed using the BigDye Termina-

tor Kit ver. 3.1 (Applied Biosystems Inc., ABI, War-

rington, UK) following the ABI protocols. Cycle

sequencing products were cleaned using Magnesil

(Promega, Southampton, UK) on a Beckman Coulter

robot (Biomek NX S8, Buckinghamshire, UK) follow-

ing the manufacturer’s protocols. Cleaned products

were then sequenced on an ABI 3730 Genetic Ana-

lyser following the manufacturer’s protocols.

Sequencing output ﬁles were edited and contigs

assembled either by using Sequencher 4.0 (Gene-

codes, Ann Arbor, MI, USA) or PhyDe. Sequences

were aligned using Muscle and manually improved

following the guidelines of Kelchner (2000). Trees

were produced using the maximum likelihood method

(ML) with 1000 replicates in MEGA5 (Tamura et al.,

2011). The Tamura Nei model (Tamura, 1992) with

gamma rate (TN93+G) was found to ﬁt best with the

ITS data nucleotide matrix. The tree with the highest

likelihood is generated. Initial tree(s) for the heuristic

search were obtained automatically as follows. When

the number of common sites was <100, or less than

25% of the total number of sites, the maximum par-

simony method was used; otherwise the BIONJ

method with Markov linkage clustering (MCL) dis-

tance matrix was used. Bootstrapping was calculated

according to Felsenstein’s (1985) method. Maximum

parsimony (MP) produced a similar topology with

small differences in bootstrap support, which are not

discussed in this paper.

RESULTS

ANATOMICAL DESCRIPTIONS

Epidermal cells and trichomes

The periclinal cell walls of the abaxial and adaxial

leaf surfaces of Iranian Limonium spp. fall into two

categories: (1) species with wavy or sinuous epidermal

cells including L. caspium (Willd.) Gams, L. nudum

(Boiss. & Buhse) Kuntze, L. otolepis,L. perfoliatum

(C.A.Mey ex Boiss.) Kuntze, L. reniforme and L. sog-

dianum (Pop.) Ikonn.-Gal. (Table 1, Figs 1–4); and (2)

species with entire epidermal cell walls including

L. carnosum (Boiss.) Kuntze, L. iranicum (Bornm.)

Lincz., L. suffruticosum (L.) Kuntze, L. gmelinii

(Willd.) Kuntze, L. meyeri (Boiss.) Kuntze, L. axillare

(Forssk.) Kuntze, L. stocksii (Boiss.) Kuntze and L. lo-

batum (L.f.) Kuntze (Table 1, Figs 5–9).

Stomata and salt glands: Stomata in all species

are anisocytic with three subsidiary cells; rarely in

Table 1. The epidermal characters of basal leaves in Iranian Limonium. Data on subsidiary cells and cells around salt glands refer to their size in comparison

with other epidermal cells

Species

Epidermal cells wall Stomatal density (500 ¥500 mm) Subsidiary

cells/epidermal

cells

Cells around

salt glands/

epidermal cells

Stomata

level

Salt gland

levelAbaxial Adaxial Abaxial Adaxial

L. axillare Entire Entire (39) 40.66 ± 1.52 (42) (36) 39.6 ± 3.04 (43) Similar Similar Sunken Sunken

L. carnosum Entire Entire (20) 21.5 ± 1.29 (23) (24) 25.75 ± 2.21 (29) Similar Similar Sunken Sunken

L. caspium Wavy Wavy (21) 24.62 ± 2.87 (29) (18) 19.8 ± 1.78 (22) Similar Longer At surface At surface

L. iranicum Entire Entire (22) 24.66 ± 2.59 (29) (21) 26.28 ± 3.4 (31) Similar Similar Sunken Deeply sunken

L. lobatum Entire Entire (23) 26.85 ± 2.47 (31) (20) 21 ± 1.01 (23) Smaller Longer At surface At surface

L. gmelinii s.l. Entire Entire (22) 26.16 ± 5.78 (41) (15) 24.5 ± 7.27 (36) Smaller Longer At surface At surface

L. nudum Wavy Wavy (26) 31.89 ± 2.65 (35) (26) 27.33 ± 1.15 (28) Similar Longer At surface At surface

L. otolepis Wavy Sinuous (16) 22.75 ± 3.77 (27) (13) 17 ± 4.61 (21) Similar Longer At surface At surface

L. perfoliatum Sinuous Sinuous (18) 20.2 ± 2.4 (24) (20) 21.77 ± 1.56 (25) Similar Longer At surface At surface

L. reniforme Sinuous Sinuous (19) 22.25 ± 2.81 (24) (27) 27.5 ± 0.7 (28) Similar Longer At surface At surface

L. sogdianum Wavy Wavy (27) 29 ± 1.41 (31) (38) 42.4 ± 3.04 (46) Similar Longer At surface At surface

L. stocksii Entire Entire (21) 21.6 ± 0.89 (23) (15) 16 ± 0.81 (17) Similar Similar Sunken Sunken

L. suffruticosum Entire Entire (16) 25.3 ± 8.73 (36) (13) 20 ± 6.48 (32) Similar Similar Sunken Deeply sunken

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 521

L.axillare,L.carnosum,L.iranicum,L.perfoliatum

(C.A.Mey.ex Boiss.) Kuntze, L.reniforme,L.sogdi-

anum and L.suffruticosum four and ﬁve subsidiary

cells have been observed (Figs 2, 4, 5). Subsidiary cells

in most species are the same size as other epidermal

cells, with the exception of L. gmelinii and L. lobatum,

in which they are smaller (Table 1, Figs 7, 9).

Stomata on the adaxial and abaxial lamina surface

are almost the same size. Their density ranges from

an average of 16 per 500 ¥500 mm on the adaxial

surface of L. stocksii to 40.6 on the abaxial side of

L. axillare (Table 1). In the majority of species, sto-

matal density on the abaxial surface is higher than

the adaxial surface (Table 1). Stomata and salt glands

occur mostly on the same level as other epidermal

cells, except in L. axillare,L. carnosum,L. iranicum,

L. stocksii and L. suffruticosum where they are

sunken (Table 1). Cells adjacent to the salt glands are

Figures 1–9. Epidermal cells of adaxial leaf sides of Limonium. Fig. 1. L. nudum (Akhani et al.18804). Fig. 2. L. per-

foliatum (greenhouse cultivated plant, no voucher). Fig. 3. L. caspium (Akhani et al.18888). Fig. 4. L. sogdianum (Akhani

& Memariani 19059). Fig. 5. L. iranicum (Malekmohammadi et al.3826). Fig. 6. L. suffruticosum (Akhani & Joharchi

35188). Fig. 7. L. gmelinii (Akhani et al.18946). Fig. 8. L. stocksii (Akhani 9750). Fig. 9. L. lobatum (Hekmatara 3978).

S., stomata; s.g., salt gland; s.c. subsidiary cells. Scale bars, 100 mm.

522 H. AKHANI ET AL.

either the same size as other epidermal cells or

remarkably larger; for example, in species with

sinuous epidermal cells and L. lobatum,L. gmelinii

and L. sogdianum (Table 1, Figs 1–4, 7, 9).

All species are glabrous except L.lobatum, which

has unicellular, non-glandular and conical trichomes

on midrib, secondary ribs and margins of leaves.

Leaf anatomy

All species are characterized by having a one-layered

epidermis followed by the palisade tissue. Based on

number and position of palisade cells, the studied

species can be grouped into three categories: (1) a

bifacial group with one or two palisade layers occur-

ring only in the adaxial leaf lamina, e.g. L.caspium,

L.nudum,L.otolepis,L.perfoliatum and L.reniforme

(Table 2, Fig. 10); (2) a sub-bifacial group with two or

three palisade layers on the adaxial leaf lamina and

a single-layered palisade tissue on the abaxial side

(the palisade cells of the abaxial sides are shorter

than those of adaxial side and therefore have an

intermediate form between spongy cells and palisade

cells such as L. gmelinii) (Fig. 11); and (3) an isobi-

lateral group in which a more or less uniform palisade

tissue consisting of two to four layers is present

on both sides of the lamina, as in L.axillare,L.car-

nosum,L.iranicum,L.sogdianum,L.stocksii and

L.suffruticosum (Table 2, Figs 12–15).

Arrangement of vascular bundles (VB) in leaf

lamina and petiole cross sections is more or less

similar (see below). An incomplete arc-like scleren-

chymatous bundle sheath tissue covers the phloem in

L. carnosum,L. gmelinii,L. perfoliatum,L. stocksii

and L. suffruticosum, and a complete or partially com-

plete sclerenchymatous tissue encircles the vascular

bundles in L.axillare,L.iranicum,L.otolepis and

L.reniforme (not illustrated).

Petiole anatomy

The outline of petiole transections of studied species

are narrowly elliptic (L. nudum, Fig. 16) or arc-like to

crescent-shaped (L. capsicum, L. otolepis,L. perfolia-

tum and L. reniforme, Figs 17, 18), semicircular–

ovate (L. sogdianum, Fig. 19), circular to semicircular

with intruded margin (L.carnosum,L.iranicum and

L.suffruticosum, Figs 20, 21), more or less fusiform

(L.gmelinii, Fig. 22, partially shown) or elliptic

(L.axillare and L. stocksii, Figs 23, 24).

With regard to presence and position of palisade

tissue in the petiole, Iranian Limonium can be

grouped into four categories: (1) a bifacial group with

one to three layers of palisade cells on the adaxial

side of the petiole in L. lobatum (not shown), which

might be small palisade-like cells as in L. gmelinii

(Fig. 22); (2) a sub-bifacial category with two or three

palisade layers on the adaxial side and a discon-

tinuous layer of palisade-like tissue on the edges of

abaxial side, as in L.sogdianum and L.nudum,

(Figs 16, 19); (3) an isobilateral group with a well-

developed and more or less uniform two to four layers

of palisade tissue on both sides, as in L.axillare,

L.carnosum,L.iranicum,L.stocksii and L.suffruti-

Table 2. Leaf anatomical characteristics of studied species of Iranian Limonium. Abbreviations and symbols used in

position of sclerenchyma tissue: P, palisade; PS, between palisade and spongy tissues; S, spongy tissue; +, present; ±,

scarcely present; -, absent

Species

Palisade tissue layers

Position of sclerenchyma

cells Sclereid typeAbaxial Adaxial

L. axillare 43 P(+), SP (±), S (-) Fusiform

L. carnosum 2 (midvein) 3 (blade) 2 (midvein), 3 (blade) P (±), PS (+) Fusiform

L. caspium Absent 1 Absent Absent

L. iranicum 2 (midvein), 3 (blade) 2 (midvein), 3 (blade) PS (+), penetrating

sometimes to palisade

Fusiform

L. lobatum Absent 2 Absent Absent

L. gmelinii Absent 1–2 (midvein*), 3–4 (blade) P (+), midvein (very rare) Ramiform

L. nudum Absent 1 (midvein), 2 (blade) Absent Absent

L. otolepis Absent 0 (midvein), 1 (blade) Absent Absent

L. perfoliatum Absent 1–2 Absent Absent

L. reniforme Absent 1–2 Absent Absent

L. sogdianum 0 (midvein), 3 (blade) 3 (midvein), 4 (blade) P (±), PS (+) Fusiform

L. stocksii 3–4 layers 3 layers PS (+) Fusiform

L. suffruticosum 2 (midvein), 3 (blade) 2 layers P (±), PS (+) Fusiform

*Short palisade-like cells in midvein.

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 523

cosum (Figs 20–21, 23–24); and (4) species lacking

palisade tissue in petiole sections as in L.caspium,

L.otolepis,L.perfoliatum and L.reniforme (Figs 17–

18).

VASCULAR BUNDLES IN LEAF AND PETIOLE

The general pattern of vascular bundle distribution

in lamina and petiole is more or less similar. In

L. perfoliatum,L. caspium,L. otolepis, L. nudum and

L. reniforme there are three to ﬁve (rarely more) large

vascular bundles arranged in a line with smaller

accessory bundles between them (Table 3, Figs 10,

16–18). Limonium sogdianum is characterized by

having one main vascular bundle in the middle with

smaller accessory bundles nearby (Fig. 19). In L. car-

nosum,L. iranicum and L. suffruticosum there is a

large vascular bundle in the centre associated with

one to three smaller bundles on each side (Figs 13, 20,

21). In L. gmelinii there are several vascular bundles

in the central part that are located irregularly and

vary in size and orientation (Fig. 22). The related

species L. axillare and L. stocksii are characterized by

having three main vascular bundles in the middle

and additional peripheral accessory bundles in the

spongy tissue (Figs 14, 15, 23, 24).

SCLEREIDS

Diffuse foliar sclereids occur only in L. axillare,

L.carnosum,L.iranicum,L.sogdianum,L.stocksii,

L.suffruticosum and L.gmelinii, which could be clas-

siﬁed into two main groups using terminology sug-

gested by Rao & Das (1981) (Figs 25–30).

Fusiform: This kind of sclereid is spindle-shaped,

narrow at the end, without branches, straight or

variously bent into C, V or S shapes, arranged verti-

cally or horizontally. These are located in spongy

and/or palisade tissue, as in L.axillare,L.carnosum,

L.iranicum,L.sogdianum,L.stocksii and L.suffru-

ticosum (Figs 25–28). There are differences including

length, width and cell-wall thickness in different

species: (1) in L.axillare they are mostly needle-like,

straight, with thin and ± smooth cell walls, 300–

600 mm in length, and with an average outer diameter

of 20 mm in the middle. They occur singly in palisade

tissue (Figs 23, 25); (2) in L. stocksii the cell walls of

Figures 10–15. Leaf cross section of some representative Limonium species from Iran. Fig. 10. L. perfoliatum (Akhani

et al. 18808). Fig. 11. L. gmelinii (Akhani et al. 18946). Fig. 12. L. sogdianum (Akhani & Memariani 19059). Fig. 13.

L. suffruticosum (Akhani & Joharchi 35188). Fig. 14. L. axillare (Malekmohammadi et al. 3974). Fig. 15. L. stocksii

(Akhani 9750). Sc., sclerenchyma; e.l., epidermal layer; v.b., vascular bundle; p.c., palisade cells; Sp.c., spongy cells; Ph.,

phloem; X, xylem. Scale bars, 100 ⬀m.

524 H. AKHANI ET AL.

sclereid cells are thickened and pitted and the length

varies from 300 to 600 mm, with an average of 50 mm

diameter in the middle; they occur both singly or in

groups in the lamina, but are mostly seen in groups in

the petiole (Figs 24, 26); (3) L.sogdianum is charac-

terized by polymorphic sclereids with thickened cell

walls, and length varies from 200 to 550 mm and an

average of 70 mm outer diameter in the middle

(Figs 19, 27); and (4) L.carnosum,L.iranicum and

L.suffruticosum have fusiform sclereids mostly C, V

or S- shape, rarely erect, with pitted and pointed

appearance in the cell wall, 100–250 mm in length

and with an average of 20 mm outer diameter in the

middle (Figs 13, 20, 21, 28).

Figures 16–24. Petiole cross section of representative Limonium species from Iran. Fig. 16. L. nudum (Akhani et al.

18804). Fig. 17. L. perfoliatum (Akhani et al. 18808). Fig. 18. L. caspium (Akhani et al. 18888). Fig. 19. L. sogdianum

(Akhani & Memariani 19059). Fig. 20. L. iranicum (Malekmohammadi et al. 3809). Fig. 21. L. suffruticosum (Akhani &

Joharchi 35188). Fig. 22. L. gmelinii (Malekmohammadi et al. 3827). Fig. 23. L. axillare (Malekmohammadi et al. 3974).

Fig. 24. L. stocksii (Akhani 9750). Sc., sclerenchyma; e.l., epidermal layer; v.b., vascular bundle; p.c., palisade cells; Sp.c.,

spongy cells; Ph., phloem; X, xylem. Scale bars, 100 mm.

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 525

Table 3. Petiole anatomical characteristics of studied species of Iranian Limonium. Abbreviations and symbols used in position of sclerenchyma tissue: P,

palisade; PL, palisade-like; PS, between palisade and spongy tissues; S, spongy tissue; +, present; ±, scarcely present; -, absent

Species

Palisade tissue layers

Arrangement of vascular bundles (VB)

Position of

sclerenchyma cellsAbaxial Adaxial

L. axillare 4 3–4 3 large VB in centre, some smaller VB in spongy tissue P (+), PS (±), S (–)

L. carnosum 2 (midvein),

3 (blade)

2 (midvein), 3 (blade) 1 large VB, smaller VB in line with the main and

dispersed in spongy tissue

P (±), PS (+)

L. caspium 0 0 3 large VB, small intercalary VB occur between them on

the same line

Absent

L. gmelinii 0 0 (midvein), 2 (blade),

3 (edges)

Irregularly distributed in central part, secondary veins are

horizontally derived from central bundles

P(+), PS (rare), S (+)

L. iranicum 2 (midvein),

3 (blade)

2 (midvein), 3 (blade) 1 large VB, smaller VB in line with the main and

dispersed in spongy tissue

PS (+), penetrating

sometimes to palisade

L. lobatum 0 2 PL 1 large, small VB dispersed in spongy tissue Absent

L. nudum 0 (midvein),

2 (blade edges)

2 3 large VB, small intercalary VB occur between them on

the same line

Mostly absent, rarely

occur in S

L. otolepis 0 0 3 large VB, small intercalary VB occur between them on

the same line

Mostly absent, rarely occur in S

L. perfoliatum 0 0 3 large VB, small intercalary VB occur between them on

the same line

Mostly absent, rarely occur in S

L. reniforme 0 0 3 large VB, small intercalary VB occur between them on

the same line

Mostly absent, rarely occur in S

L. sogdianum 0 2–3 1 large VB, smaller VB occur on the same line and are

distributed on adaxial side of spongy tissue

P (–), PS (±), S (+) (only on

adaxial side)

L. stocksii 3 2 (midvein), 3 (blade) 3 large VB in centre, some smaller VB in spongy tissue P (–), PS (+), S (±)

L. suffruticosum 3 2 1 large VB, smaller VB in line with the main and

dispersed in spongy tissue

P (–), PS (+), S (–)

526 H. AKHANI ET AL.

Figures 25–30. See caption on next page.

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 527

Rhizosclereids and ramiform sclereids: root-like

sclereids, short, with two or three branches at one end

only and thick and pitted cell walls are characteristic

of palisade tissue in L. gmelinii s.l. (Fig. 29). Rami-

form sclereids are similar to short rhizosclereids, with

thick cell walls that are pitted but with forking in

both ends. This type also occurs only in L. gmelinii s.l.

(Fig. 30).

PHYLOGENETICS

New ITS sequences were provided in this study for 74

accessions belonging to 19 Limonium spp., one Cepha-

lorhizum sp. and two Psylliostachys spp. Sequences of

eight species were included from GenBank (accession

numbers are provided in the Appendix). After trim-

ming the ends of the matrix, the analysis involved

847 characters including 397 variable sites, among

which 300 sites were potentially parsimony informa-

tive. The ML tree showing highest likelihood

(ln =-4771.1757) is presented in Figure 31. Some

important anatomical characters are shown or plotted

on the tree with informal clade names. We apply the

following descriptions for bootstrap percentage (BP):

50–74 poorly supported, 75–84 moderately supported

and 85–100 well supported.

Apart from species of section Pteroclados, the

remaining Limonium spp. (here called core Limo-

nium) form a strongly supported clade (BP 99). The

genera Cephalorhizum and Psylliostachys (outgroups)

form a well-supported clade (BP 95). Internal rela-

tionships are largely resolved with ﬁve clades: (1) the

L. axillare clade, sister to all other clades; (2) a mod-

erately supported clade (BS 81) comprising species of

section Limonium subsections Dissitiﬂorae Boiss. and

Steirocladae Boiss. and L. echioides (subgenus Limo-

nium section Schizhymenium) marked as ‘Mediterra-

nean clade’; (3) a poorly supported clade (BP 62)

consisting species of section Nephrophyllum and

L. caspium,L. bellidifolium (Gouan) Dumort and

L. iconium (Boiss. & Heldr.) Kuntze of section Limo-

nium subsection Hyalolepidae,L. sogdianum (section

Siphonocalyx) and L. nudum (section Platyhy-

menium), all of which are collectively termed here the

‘L. reniforme clade’; (4) a well-supported clade (BP 91)

including species of section Limonium subsection

Limonium, part of section Sarcophyllum (sensu

Rechinger & Schiman-Czeika, 1974) and L. lilacinum

(Boiss. & Bal.) Wagenitz of section Sphaerostachys

(sensu Bokhari & Edmondson, 1982), termed here the

‘Limonium-type clade’. This clade is subdivided into a

L. vulgare subclade (BP 69) and L. suffruticosum sub-

clade (BP 90).

DISCUSSION

PHYLOGENY

Our ITS tree includes almost all Limonium spp.

in Iran, except L. stocksii (Rechinger & Schiman-

Czeika, 1974); in addition, some endemic species from

Turkey, some Mediterranean species, two species of

Psylliostachys and one species of Cephalorhizum are

included. The sampling includes representatives

of main infrageneric taxa of Limonium from south-

western Asia according to Boissier (1848). ITS nuclear

ribosomal DNA (nrDNA) provides enough variation

for inferring phylogenetic relationships among

major clades, but it also has good potential for being

a useful molecular marker even among the most

closely related species (see also Palacios, Rosselló &

González-Candelas, 2000).

Our results conﬁrm previous molecular studies

based on plastid rbcL,trnL intron and the trnL–trnF

intergenic spacer by Lledó et al. (2005); Limonium,

as circumscribed in Flora Iranica and Flora of Turkey

(Rechinger & Schiman-Czeika, 1974; Bokhari &

Edmondson, 1982), is monophyletic. The situation in

section Pteroclados requires more study (but see

Lledó et al., 2011). Our results do not support split-

ting the genus into smaller genera, such as Eremoli-

mon, as proposed by Linczevski (1979). Additionally,

the independent status of Cephalorhizum and Psyl-

liostachys as distinct from Limonium is strongly sup-

ported. However, additional sequences are required to

clarify the relationships of some monotypic and oligo-

typic genera from Central Asia and Afghanistan, such

as Dictyolimon,Aeoniopsis,Chaetolimon, Ikonniko-

via,Popoviolimon,Neogontscharovia,Bamiania,

Bukiniczia and Vassilczenkova (Linczevski, 1971,

1985), some of which are well supported as distinct in

plastid trees (cf. Lledó et al., 2005).

The phylogenetic status of section Pteroclados

remains unresolved. The sister position of this section

to Limonium was only supported by 60 BP in the

plastid analysis of Lledó et al. (2005), although the

internal relationship of the two resulting groups

(subsections Odontolepideae and Pteroclados) was

strongly supported (100 BP). This section with dis-

tinctive winged stems comprises c. 15 species of

Figures 25–30. Sclereid illustrations. Fig. 25. Fusiform sclereids in L. axillare (Malekmohammadi et al., 3974). Fig. 26.

Fusiform sclereids in L.stocksii (Akhani 9750). Fig. 27. Fusiform sclereids in L.sogdianum (Akhani & Memariani 19059).

Fig. 28. Fusiform sclereids in L.iranicum (Akhani & Malekmohammadi 18842). Fig. 29. Rhizoscelereids in L.gmelinii

(Akhani & Malekmohammadi 18852). Fig. 30. Ramiform sclereids in L.meyeri (Akhani & Malekmohammadi 18852).

Scale bar, 60 mm.

528 H. AKHANI ET AL.

L iranicum 3986

L iranicum 3826

L iranicum 15760

L iranicum 18851

L. iranicum

type

L iranicum 3972

L iranicum 19133

L iranicum 20723

L iranicum 3823

L iranicum 21058

L iranicum 3976

L iranicum 3977

L iranicum 5563

L iranicum 18805

L iranicum 58

L iranicum 16499

L iranicum 4813

L iranicum 3975

L iranicum 20820

L carnosum 18997

L carnosum 18891

L carnosum 15625

L carnosum 18894

L carnosum 18904

L anatolicum 17999

L suffruticosum 8108

L suffruticosum 40226

L suffruticosum 35188

L suffruticosum 10160T

L suffruticosum 12277

L suffruticosum 20364

L suffruticosum 20382

L suffruticosum 28088

L suffruticosum 17282

L suffruticosum 41

L. suffruticosum

subclade

L lilacinum 17930

L gmelnii3 1792

L gmelimi 10637

L gmelinii 12229

L gmelinii 18948

L narbonense AJ222838

L vulgare AJ222839

L latifolium 20504

L gmelinii 19013

L gmelinii 18996

L gmelinii 18887

L gmelinii1 5636

L gmelinii 18852

L gmelinii 15740

L gmelinii 3827

L gmelinii 3825

L. vulgare subclade

Limonium Type

clade

L sogdianum 19055

L nudum 18804

L caspium 18888

L bellidifolium 20511

L iconium 17961

L iconium 17921

L iconium 18011

L reniforme 3970

L reniforme 30.3.2004

L reniforme 66965

L otolepis 20367

L otolepis 3897

L otolepis 10185

L perfoliatum 10057

L perfoliatum 10130

L perfoliatum 24567

L perfoliatum 18815

L perfoliatum 18808

L perfoliatum 10169T

L. reniforme clade

L echioides AJ222861

L delicatulum AJ222851

L furfuraceum AJ222856

L cf.cumanum 16815

L minutum AJ132332

Mediterranean clade

L axillare 3974

L axillare 3974 L. axillare clade

Core Limonium

L arborescens EU410356

L lobatum AJ132333

L lobatum 3978 Pteroclados clade

Cephalorhizum turcomanicum 28978

Psylliostachys spicata 14878

Psylliostachys leptostachys 57

Outgroup

100

0,001

Figure 31. See caption on next page.

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 529

annual herbaceous to shrubby species distributed

from the Macaronesian to the Mediterranean and the

Saharo-Sindian ﬂoristic regions (Karis, 2004). Ana-

tomically, species of section Pteroclados subsection

Odontolepidae contain no sclereids (see also Bokhari,

1970; Rao & Das, 1981) and have isobilateral leaf and

bifacial petiole anatomy. This section is represented

in southern Iran only by L. lobatum, the widespread

annual species ranging from the Canaries through

North Africa and the Mediterranean to southern

Iran. The antiquity of extant species of some isolated

groups of Limonium, such as species of sections

Pteroclados and Limoniodendron in the Canaries

and north-western Africa, was demonstrated based on

morphological data by Karis (2004) and plastid phy-

logenetics by Lledó et al. (2005, 2011).

Limonium axillare is sister to the remaining

species of the core-Limonium clade, which agrees

with its position in analyses of plastid DNA sequences

(Lledó et al., 2005). This species belongs to a small

group of Saharo-Sindian species that were classiﬁed

by Rechinger & Schiman-Czeika (1974) in section

Sarcophyllum (Boiss.) Lincz. (including species of the

L. suffruticosum subclade). Limonium axillare and its

close relative L. stocksii are short shrubby species of

hypersaline soils characterized by ± cushion habit

and short ﬂowering branches. They are restricted in

their range in southern Iran along the shores of the

Persian Gulf and Baluchestan. The most obvious ana-

tomical feature separating L. axillare and L. stocksii

from other species already classiﬁed in section Sar-

cophyllum is the presence of three main vascular

bundles in petioles that are more or less the same

size and separated by spongy tissue (Fig. 24). Fur-

thermore, the leaf lamina has three main veins and

petioles are ﬂattened. In L. iranicum,L. suffrutico-

sum and L. carnosum, the petiole cross section

is characterized by three closely spaced bundles, of

which the central one is larger than the laterals

(Figs 20–21). The leaf lamina has a central midrib

and the petioles are almost terete.

The Mediterranean species form a moderately

supported clade (BP 81). Considering the unresolved

relation with other clades, more studies are required,

including a wider sampling of central and eastern

Mediterranean taxa to clarify the origin of this rapidly

diversifying lineage in the Mediterranean basin. Fre-

quent apomixis and hybridization in this clade are

further complications for resolving their phylogenetic

relationships (Palacios et al., 2000; Lledó et al., 2005).

The ‘L. reniforme clade’ is poorly supported (BP 62)

and consists of endemic species of the Irano-Turanian

area belonging to sections Nephrophyllum Rech.f.

and Platyhymenium (Boiss.) Lincz. (according to Flora

Iranica, Rechinger & Schiman-Czeika, 1974) or section

Limonium subsection Platyhymenium (according

to Boissier, 1848), in addition to species of the L. bel-

lidifolium complex (L. caspium and L. iconium) and

L. sogdianum. Except for L. nudum and L. sogdianum,

all these species were classiﬁed in section Limonium

subsection Hyalolepidae by Boissier (1848). Limonium

nudum and L. sogdianum are successively sister to

a strongly supported group (BP 97). Both species are

isolated strictly Irano-Turanian species restricted to

gypsum hills.

The synapomorphies of the wavy to sinuous epider-

mal cell walls link all species of L. reniforme-clade.

However, the epidermal cells of L. sogdianum are

slightly wavy (Fig. 4) compared with those of L. per-

foliatum and L. reniforme, which are clearly sinuous

(Fig. 2). Other characters, such as bifacial leaf

anatomy, absence of palisade cells in petiole cross

section and absence of diffuse sclereids, are additional

synapomorphies linking members of this clade, except

for L. sogdianum. This last species differs anatomi-

cally from other species of this clade by having diffuse

sclereids that are uniquely straight, thickened and

unbranched and arranged only on the adaxial side

(Tables 3 and 4; Figs 12, 27). Linczevski (1979) trans-

ferred L. sogdianum and other species of section

Siphonocalyx Lincz. to his new genus Eremolimon

because of the tubulate calyx and erect or slightly

dilated calyx limb with its peculiar habit. Neither

morphology nor our molecular analysis supports its

segregation.

Limonium nudum is an isolated and geographically

restricted species (Fig. 36). This is a typically unique

plant among Iranian Limonium in having rosulate

leaves and glomerate spikelets and large ﬂowers

(with petals up to 10 mm long).

The internal relationships of the L. reniforme

clade show that three closely related western Irano-

Turanian/Mediterranean species (L. caspium,L. bel-

lidifolium and L. iconium) form a strongly supported

group (BP 99), sister to three other closely related

central Irano-Turanian species. The morphological

and sequence differences between three species from

Iran (L. caspium), Turkey (L. iconium) and France

(L. bellidifolium) are minimal (see notes in taxonomic

treatment).

Figure 31. Maximum likelihood tree of Irano-Turanian lineages of Limonium obtained from analysis of the nuclear

ribosomal internal transcribed spacers (nrITS) using TN93+G model (ln =-4771.1757). Numbers on branches are

bootstrap percentages. An asterisk indicates occurrence of foliar sclereids and an empty square indicates presence of

sunken stomata and isobilateral leaf anatomy.

530 H. AKHANI ET AL.

Table 4. Comparison of morphological and anatomical characters of the three closely related species, L. perfoliatum,L. otolepis and L. reniforme. SD, stomatal

density; SG, salt gland

Species/characters L. otolepis L. reniforme L. perfoliatum

Height 40–50 (–90) cm 35–70 (–90) cm 50–95 (–150) cm

Sterile branches Sterile branches numerous Some lower branches sterile Without sterile branches

Cauline leaves position Only occurring on the lower nodes of

main stem, rarely 1–2 small leaves

present on nodes of lateral branches

Present on all nodes of main and

lateral branches

Present on all nodes of main

and lateral branches

Cauline leaf size 0.5–1.5 ¥0.8–1.5 cm 0.5–2.5(–3.5) ¥0.5–2.0(–3.0) cm 1.8–3.4 ¥1.2–2.2 cm

Cauline leaf margin Entire or slightly undulate Entire Entire and undulate

Length of inﬂorescence 17–30 cm 10–25 cm 24–70(–130) cm

No. of spikes per cyme 4–9 (1–)4–6 (5–)7–18

No. of ﬂowers per spike 1(–2) (1–)2 (2–)3

Outer bract size 0.5–1.0 ¥1.0–1.5 mm 0.5–1.0 ¥1.0–1.5(–1.7) mm 1.0–1.5(–1.7) ¥1.0–1.5(–1.7) mm

Length of herbaceous part of

outer bract

(0–)0.5 ¥0–1 mm (0–) 0.5–0.7 mm ¥(0–) 0.5–0.7

(–1.2)mm

1.0–1.5 mm ¥(0.2) 0.5–0.7 (1.0)

Middle bract length 0.5–1.0 mm ¥0.5–1.0 (0.7-)1.0–1.5 ¥1.0–1.2 mm 1.0–1.5 ¥1.0–1.5 mm

Middle bract colour Membranous, without nerve Membranous with one or two

brown nerves

Membranous usually with 1–2

narrow brown nerves

Inner bract length 1.5–2.0 ¥2.0–2.5 mm (1.5-)2.0–2.5 ¥2.0–2.5 mm 2.5–3.0(-3.5) ¥2.0–2.5 mm

Inner bract length of herbaceous part 1 mm (1.0–) 1.5-2 mm (1.7–)2.0–2.5(–2.7) mm

Bracteole shape Obovate Elliptic or orbicular rarely rhombic Elliptic or broadly elliptic,

rarely obovate

No. of bracteoles per spike (0–)1 2–5(–7) (1–)3–5(–8)

Calyx shape Obconical Obconical Funnel form

Calyx length 2.0 mm 2.5–3.0 mm (2.5–)3.5 mm

Calyx indumentum Mostly with short or sometimes sparse

long hairs on the basal 0.7 mm, hairs

often conﬁned to one side

Dense and short or more or less

long hairs between nerves and

on two nerves, other nerves

glabrous or with short and lax

hair, rarely calyx glabrous

Dense or sometimes lax long

hairs on two or three of

nerves, other nerves glabrous

Calyx diameter at apex 1.2–1.7 mm (1.2–)1.5(–1.7) mm 2.0–3.0 mm

Calyx nerve 1.5 mm 2.0–2.5 mm (2.0–)2.2–2.5(–3.0) mm

Length of calyx lobes 0.5 mm 0.5–0.7 mm (0.5–)0.7–1.0 mm

Achene shape Obovate Fusiform Obovate

Epidermal cells of leaf abaxial side Wavy Sinuous Sinuous

SD* (abaxial) 30.28 ± 5.28 16.33 ± 2.88 25.77 ± 1.39

SD* (adaxial) 22.80 ± 5.77 20.33 ± 1.15 29.50 ± 2.25

SG* (abaxial) 3.16 ± 0.4 3.66 ± 0.57 4.55 ± 0.52

SG* (adaxial) 3.00 ± 0.87 3.33 ± 0.57 6.16 ± 1.47

* 500 ¥500 mm.

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 531

30N

35N

40N

45E 50E 55E 60E 65E

32. L. lobatum

30N

35N

40N

45E 50E 55E 60E 65E

33. L. axillare

30N

35N

40N

45E 50E 55E 60E 65E

34. L. stocksii

30N

35N

40N

45E 50E 55E 60E 65E

35. L. sogdianum

30N

35N

40N

45E 50E 55E 60E 65E

37. L. caspium

30N

35N

40N

45E 50E 55E 60E 65E

38. L. otolepis

30N

35N

40N

45E 50E 55E 60E 65E

39. L. perfoliatum (dot) and L. reniforme (triangle)

30N

35N

40N

45E 50E 55E 60E 65E

40. L. carnosum

30N

35N

40N

45E 50E 55E 60E 65E

41. L. iranicum

30N

35N

40N

45E 50E 55E 60E 65E

42. L. suffruticosum

30N

35N

40N

45E 50E 55E 60E 65E

43. L. gmelinii s.l.

30N

35N

40N

45E 50E 55E 60E

36. L. nudum

Figures 32–43. Distributions of Limonium species in Iran.

532 H. AKHANI ET AL.

In one cluster, all six samples of L. perfoliatum are

grouped into two smaller sets each having three rep-

resentatives. In one set, samples from the Golestan

province and adjacent Turkmenistan are grouped,

corresponding to the type locality of this species. The

second set includes three samples from the north-

easternmost parts of Iran. The sequences of the

Golestan and Khorassan groups have two base pair

differences. Morphologically, the populations from

Khorassan are taller and their leaves are strongly

undulate compared with those from Golestan. ITS

sequence and morphological differences are compat-

ible with subspeciﬁc rank for these geographically

separated populations. However, because of the wide

distribution of L. perfoliatum in Central Asia and

Afghanistan, these questions should be addressed in a

study including samples from across the entire range

of the species.

In the second strongly supported cluster (BP 97),

three accessions of L. reniforme (Girard) Lincz.

(according to the circumscription accepted in this

paper) are grouped apart from other species. The third

cluster includes L. otolepis with a low support (BP 75).

This result solves a complicated taxonomic problem,

because the distinction between L. reniforme and

L. perfoliatum has long been confused in the literature.

Limonium reniforme (as Statice reniformis Girard) has

been described from the saline depressions in Fars

province (de Girard, 1844), and L. perfoliatum (S. per-

foliata) was known from a saline depression in Hassan

Kuli in the south-eastern lowlands of the Caspian Sea,

a locality near the Iranian border in Turkmenistan

(Karelin, 1839). This name was later validated by

Boissier (1848), who also accepted Girard’s species as

var. reniformis (Girard) Boiss., although he later syno-

nymized both names (Boissier, 1879). Ledebour (1849)

also supported recognition of S. reniformis.Inthe

standard ﬂoras of this area, these names have vari-

ously been used. Boissier (1879) accepted the name

Statice perfoliata C.A.Mey. and considered S. reni-

formis L. a synonym. In the two main ﬂoras of the area,

Flora Iranica (Rechinger & Schiman-Czeika, 1974)

and Flora of URSS (Linczevski, 1952), the two species

are considered conspeciﬁc and, based on priority,

L. reniforme was accepted. More recently, Assadi

(2005) erroneously applied L. otolepis to the popula-

tions of southern central Iran in Fars Province without

providing any argument as to how populations of the

locus classicus of L. reniforme can be attributed to

L. otolepis described from Central Asia. As it is evident

from molecular analysis (Fig. 31), morphological

studies and distribution (Table 4, Figs 38–39, 44–46,

and the taxonomic treatment below), these three taxa

as treated here are distinct species.

The Limonium-type clade (BP 91) includes two main

morphologically distinct complexes. The L. vulgare

subclade (BP 69) includes broad-leaved hemicrypto-

phyte and hygrohalophytic species, with a distribu-

tion spanning the Euro-Siberian, Mediterranean

and Irano-Turanian areas. Limonium lilacinum,an

endemic species from central Anatolia, is sister to

other species. This is morphologically the most distinct

species of this complex. Limonium lilacinum is char-

acterized by more or less ﬂeshy leaves with indistinct

lateral veins and petioles with hyaline margin and a

loose inﬂorescence (Bokhari & Edmondson, 1982). The

anatomical studies of Bokhari (1970) showed that

L. lilacinum has numerous clusters of sclereids dis-

persed in the lacunose spongy tissue of the petiole,

which is different from L. gmelinii and related species.

Accordingly, following Boissier (1848), Bokhari (1973)

classiﬁed this species with another closely related

Anatolian-Syrian species, L. globuliferum (Boiss. &

Heldr.) Kuntze, in section Sphaerostachys (Boiss.)

Bokhari.

In Flora Iranica,L. meyeri and L. gmelinii are dis-

tinguished by minor differences: the former with con-

gested spikelets and the latter by loose inﬂorescences

and cymes. However, in herbarium specimens it is

always difficult to separate both species conﬁdently.

The molecular analysis gives a complicated topology in

which most branches have only low support. The

Iranian species are distributed into two clades: one

clade includes samples from the northern sector of Iran

(BP 50) and the other samples from central, southern

and north-western parts of Iran (BP 61). The two

Mediterranean and European representatives of the

complex (L. vulgare Mill. and L. narbonense Mill.) are

closely related and form a clade (BP 76), which is

congruent with plastid analyses by Lledó et al. (2005,

2011). Despite several attempts, we were unable mor-

phologically to separate the Iranian members of this

complex and therefore prefer to treat them all under

L. gmelinii s.l. The congested vs. loose inﬂorescence

seems to be related to the age of collection and appears

in both groups in the phylogenetic tree. This group

needs additional investigation, including the microspe-

cies described from Central Asian, Anatolia and the

Mediterranean; for example, L. vanense Kit Tan &

Sorger from eastern Anatolia (Tan & Sorger, 1984).

The L. suffruticosum clade is well supported

(BP 90), but its internal relationships are not well

resolved. In a large polytomy, three groups are dis-

tinguished: one includes all samples of L. suffru-

ticosum, the second includes all specimens of L.

carnosum with L. anatolicum Hedge as their sister,

and the third includes four samples of L. iranicum

from central Iran (Arak, Hamadan, Delijan and

Esfahan); Arak is the locus classicus of L. iranicum.

All other specimens of L. iranicum are unresolved

and not related to either of the ﬁrst two clades

(above). Limonium iranicum,L. suffruticosum and

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 533

L. carnosum are widespread in Iran. They occur in

almost all saline depressions and salt marshes of the

Persian Gulf. Morphologically, these species are sepa-

rable based on the treatment of Flora Iranica (see key

below). Two other previously described species belong-

ing to this complex are L. gabrielii (Bornm.) Rech.f.,

from Iranian Baluchestan between Nehbandan and

Zahedan (Rechinger & Schiman-Czeika, 1974), and

L. failachicum Erben & Mucina, which was recently

described from Failacheh Island in Kuwait (Erben &

Mucina, 2006). Two of the most isolated samples of

L. suffruticosum are from the southernmost part of

the range of this species, southern Khorassan (35188)

and northern Baluchestan (8108), and in morphology

and geography match L. gabrielii. This taxon is

similar to L. suffruticosum and differs only in its

tomentose calyx and pilose bracts and bracteoles. In

L. suffruticosum the calyx is pilose, bracts are usually

glabrous and bracteoles are always glabrous. Taking

into account the molecular, geographical and morpho-

logical features, L. gabrielii could merit subspeciﬁc

rank. Limonium failachicum is close and morphologi-

cally similar to L. iranicum. Study of the type of this

species shows no reliable differences from the Iranian

populations of the species along the Persian Gulf. Two

sequenced samples collected from Ghabre Nakhoda

(20723) and Deyreh Islands (20820) in the Persian

Gulf belong to the large polytomy, in line with all

other samples. They do not have unique sequence

differences from other samples from southern

Iran. However, further studies and plastid sequences

are required to give a better picture of species

delimitation and phylogenetic relationships in this

complex.

Presence and shape of diffused sclereids have been

considered to be important taxonomic characters

in Limonium (Bokhari, 1970; Rao & Das, 1981). The

phylogenetic tree shows that these are homoplastic

characters occurring in all species of the Limonium-

type clade, L. sogdianum belonging to the L. reni-

forme clade and the L. axillare clade (Fig. 31). These

might be useful taxonomic characters to separate

closely related species (e.g. L. axillare and L. stocksii,

Figs 25, 26), but they should be used with caution.

The presence of rhizosclerids and ramiform sclereids

in L. gmelinii s.l. and the large variation in the form

and size of sclereids in L. sogdianum are two exam-

ples (Figs 27, 29, 30).

Isobilateral leaf anatomy is another homoplastic

character occurring in three clades of Iranian Limo-

nium (Fig. 31). The presence of this character is

congruent with sunken stomata in these species.

Both features occur in xerohalophytic species of these

clades, indicating putative adaptive value in coping

with water stress and high temperature (Fahn &

Cutler, 1992).

BIOGEOGRAPHICAL REMARKS

The sampling of Limonium from the Middle East in

this paper is not sufficient to give a detailed phylo-

geographical interpretation, but it seems clear from

these results that the Iranian species of Limonium

have four phytogeographic origins.

1. Almost all species of the L. reniforme clade are

Irano-Turanian species. Limonium bellidifolium

occurs in the Mediterranean area and is closely

related, and in a broad interpretation it is conspe-

ciﬁc with L. caspium. In the results of Lledó et al.

(2005), L. bellidifolium,L. tetragonum (Thunb.)

Bullock (East Asia), L. sinense (Girard) Kuntze

(China) and L. tenellum (Turcz.) Kuntze (China,

Mongolia) form a clade. Except for L. bellidifolium,

all these other species have eastern and central

Asian distributions. Therefore, two possibilities

with regard to their origin and diversiﬁcation can

be considered: either the Mediterranean distribu-

tion of this group might be secondary; or the

migration of the Mediterranean ancestor into the

Irano-Turanian area resulted in further divergence

and speciation.

2. Limonium lobatum is the only Iranian representa-

tive of section Pteroclados subsection Odontolepi-

dae (Karis, 2004). This is also the only annual

species of Limonium in Iran. This section has a

north-western African and Canarian centre of

diversity, in which only L. sinuatum (L.) Mill. and

L. lobatum have a widespread Mediterranean dis-

tribution. An extension of Mediterranean elements

into western, south-western and northern Iran has

already been reported in several groups: Cercis

siliquastrum L., Myrtus communis L., Jasminum

fruticans L., Cupressus sempervirens L., Pteris

cretica L., Hibiscus cannabinus L., Cistus salviifo-

lius L., Caucalis platycarpos L., Convolvulus can-

tabrica L., Cynoglossum creticum Mill., Asteriscus

spinosus (L.) Sch.-Bip., Petrorhagia prolifera (L.)

P.W.Ball & Heywood, Vicia amphicarpa Dorthes,

V. lutea L., Stipa bromoides (L.) Dörﬂ., Carex

hallerana Asso, Daucus guttatus Sm., Laser tri-

lobum (L.) Borkh., Orlaya daucoides (L.) Greuter,

Fumana arabica (L.) Spach, Ononis pusilla L.,

Sternbergia lutea (L.) Ker Gawl. ex Spreng., some

species of Orchis L and halophytic communities of

south-western Iran (Zohary, 1973; Akhani &

Ziegler, 2002; Akhani & Deil, 2012).

3. Limonium axillare and the closely related L. stock-

sii are clearly isolated with an Afro-Arabian origin.

They belong to L. subsection Sarcophyllae p.p.

Restriction of these species to the frost-free zone of

southern Iran with a tropical desert bioclimate

close to sea shores indicates that this group is

cold-sensitive. Field observations and greenhouse

534 H. AKHANI ET AL.

cultivation indicate that these species have a con-

tinuous life cycle over the year. The presence of

thick cuticle and sunken chalk glands are morpho-

logical traits that occur in L. axillare and L. stock-

sii in addition to species of the L. suffruticosum

complex.

4. The biogeographic origin of the species belonging

to core-Limonium (L. iranicum,L. suffruticosum

and L. gmelinii s.l.) is more complicated because

(1) these clades do not have strong support and

(2) our sampling covers only part of this clade

that is widely distributed over Eurasia. All these

species are common in both inland and coastal

saline soils.

In conclusion, ITS sequences provide useful

taxonomic–phylogenetic data for studying sexual

Limonium spp. and the results indicate that previous

classiﬁcations of the genus in the literature require

some alterations. It will be highly valuable to include

plastid markers and extend this study into the south-

western and central Asian taxa of Limonium.

TAXONOMIC TREATMENT

The taxonomic treatment here includes an updated

key to all Limonium spp. accepted by us in Iran, a

synopsis of all species with maps and descriptions

and line drawings of the two species that are recir-

cumscribed in this paper.

KEY TO SPECIES OF LIMONIUM IN IRAN

1. Annual herbs; stem and ﬂowering branches with wing...........................................................1. L. lobatum

1. Perennial herbs or shrublets; stem and branches without wing................................................................2

2. Perennial herbs; leaves in a rosette, with or without cauline leaves..........................................................3

2. Shrublets; leaves mostly cauline .........................................................................................................9

3. Plants with many well-developed cauline, ± ﬂeshy, rotundate–reniform and amplexicaul leaves.....................4

3. Plants without cauline leaves, often with scale-like reduced leaves on nodes ..............................................6

4. Stem without sterile branches; cauline leaves undulate at margins, basal cauline leaves auriculate.................

................................................................................................................................8. L. perfoliatum

4. Stem with sterile branches; cauline leaves entire, not auriculate..............................................................5

5. Sterile branches numerous; cauline leaves only on lower part of main stem, rarely on ﬁrst to second nodes of

lateral branches; spikes ± compact, 1–3 cm long, with four to nine spikelets (only cultivated or escaped from

cultivation in Iran) ........................................................................................................... 7. L. otolepis

5. Sterile branches scarce and laxly disposed, only on lower parts of lateral branches; cauline leaves on all nodes both

main stem and lateral branches; spikes ± lax, 1–3 cm long, with two to six spikelets.................9. L. reniforme

6. Sterile branches numerous, slender, occurring along the lower 2/3 of the plant height; inﬂorescence corymbose-

paniculate; spikelets up to 5.5 mm long...............................................................................6. L. caspium

6. Plants without sterile branches; combination of characters not as above....................................................7

7. Basal leaves small, spathulate, up to 7 cm long; inﬂorescence subcapitate or spicate................................... 8

7. Basal leaves large, obovate or spathulate–obovate, 8–38 cm long; inﬂorescence much branched, paniculate.......

...............................................................................................................................13 L. gmelinii s.l.

8. Inﬂorescence subcapitate, at the end of dichotomous stem branches; calyx funnel-shaped, scarious parts of calyx

pale violet; outer and middle bracts densely pilose..................................................................5. L. nudum

8. Inﬂorescence laxly spiciform, spikelets at intervals along spike; calyx tubular, scarious parts of calyx whitish;

bracts glabrous............................................................................................................4. L. sogdianum

9. Flowering branches elongate, often longer than half of the plant height; leaves with single midrib...............10

9. Flowering branches short, often shorter than one third of plant height; leaves with three main veins...........12

10. Leaves with a scarious semicircular auricle at the base; inﬂorescence spicate; spikelets forming dense glomerules

on elongated inﬂorescence branches, spikelets occurring only on lateral cymes...................12. L. suffruticosum

10. Leaves without auricles; inﬂorescence paniculate; spikelets occurring on lateral and terminal cymes ............ 11

11. Inﬂorescence shortly paniculate, cymes mostly condensed at the terminal branches of inﬂorescence; spikelets

mostly 6.0–6.5 mm; calyx 5.5–6.0 mm long.........................................................................10 L. carnosum

11. Inﬂorescence widely paniculate, cymes ± distanced along inﬂorescence branches; spikelets up to 5.5 mm long; calyx

3.0–4.0 mm long............................................................................................................11. L. iranicum

12. Leaves oblanceolate or spathulate–oblanceolate, acute or subacute at the apex; calyx funnel-shaped, with short

hairs (0.5 mm) occurring only on the lower 1.5–2.0 mm of calyx tube; calyx lobes semicircular, rounded at apex,

tube nerves ending below the base of calyx lobes...................................................................2. L. axillare

12. Leaves spathulate, rounded or obtuse at the apex; calyx obconical, with longer hairs (0.7 mm) occurring all along

calyx nerves; calyx lobes triangular, acute at apex, tube nerves reaching the tip of calyx lobes........3 L. stocksii

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 535

SYNOPSIS OF IRANIAN SPECIES OF LIMONIUM

Limonium lobatum (L.f.) Kuntze

Rev. Gen. Pl. 2: 395. 1891. Statice lobata L.f., Suppl.

Pl.: 187. 1781 [basionym]. Limonium thouinii (Viv.)

Kuntze, Rev. Gen. Pl. 2: 395. 1891.

Distribution: Southern Spain, southern Greece,

northern Africa, Syria, Palestine, Saudi Arabia, Iraq,

southern Iran (Bushehr, Fars, Hormozgan, Khuz-

estan provinces, Fig. 32).

Note: L. lobatum is the only annual Iranian

Limonium and a typical southern Mediterranean

element occurring in southern and south-western

Iran (Fig. 32). The species is easily distinguished by

its annual life form, the winged stems and pedicel,

horned middle bracts, pubescent leaves and coloured

calyces bigger than corolla.

Limonium axillare (Forssk.) Kuntze

Rev. Gen. Pl.: 395. 1891. Statice axillaris Forssk., Fl.

Aegypt.-Arab. 58.: 1775 [basionym]. S. bovei Jaub. &

Spach, Ill. Pl. Or. 1: 157, t. 86. 1884. L. wendelboi

Bokhari in Notes Roy. Bot. Gard. Edinburgh 40: 93.

1982.

Distribution: Egypt, Arabian Peninsula, Qatar and

Iran (salty plains and Persian Gulf salt marshes in

Bushehr and Hormozgan Provinces; Fig. 33).

Note: L. axillare is a rare plant restricted to Hormoz-

gan and Bushehr Provinces (Fig. 33). Based on avail-

able data and ﬁeld observations, its status according

to IUCN (2001) has been evaluated as endangered in

Iran. The species was not rediscovered in some of the

previously known localities cited in the published

literature (Rechinger & Schiman-Czeika, 1974).

Limonium axillare is closely related to L. stocksii.

The main differences include the shape of calyx lobes,

which are triangular in L. axillare and rounded in

L. stocksii. However, the petiole anatomy of the

former is characterized by individually dispersed

needle-like sclereids, in contrast to the latter with

fusiform sclereids that are mostly clustered (Figs 14,

15, 23, 24, 25–26).

Bokhari (1982) described the new species L. wen-

delboi Bokhari from southern Iran and compared

it with L. stocksii based on the shape of sclereids.

Earlier studies of L. wendelboi and L. axillare showed

evidence for their synonymy (Akhani & Ghorbanli,

1993), and this is conﬁrmed by detailed studies in this

paper and comparison of material from North Africa

and Saudi Arabia.

Limonium stocksii (Boiss.) Kuntze

Rev. Gen. 365. 1891. Statice stocksii Boiss. in DC.

Prodr. 12: 664. 1848 [basionym]. TYPE: In Salsugino-

sis Prov. Scinde Indiae Borealis, Stocks (K!, G, W!).

Distribution: Afghanistan, western Pakistan and

south-eastern Iran (Sistan-o Baluchestan Province,

Fig. 34).

See notes under L. axillare.

Limonium sogdianum (Pop.) Ikonn.-Gal.

Trudy Bot. Inst. Akad. Nauk S.S.S.R. Ser. 1, Fl. Sist.

Vyssh. Rast. 2: 268. 1936. Statice sogdiana Pop.

in Bot. Geogr. Issled. V Turkest. 51: 1915 [basionym].

L. piptopodum Nevski in Tr. Bot. Inst. AN SSSR, Ser.

I, 4. 313. 1937. Eremolimon sogdianum (Ikonn.-Gal.)

Lincz. in Novosti Sist. Vyssh. Rast. 22: 206. 1985.

Distribution: Central Asia (Uzbekistan, Turkmeni-

stan), north-eastern Iran in Khorassan province

(Fig. 35).

Note: The habitat of L. sogdianum is restricted to

the gypsum hills in the eastern parts of Khorassan

Province. Associated species include Salsola montana

Litw., Asparagus breslerianus Schult. & Schult.f.,

Cleome quinquenervia DC., Kaviria tomentosa (Moq.)

Akhani, Krascheninnikovia ceratoides (L.) Gueldenst.

and Artemisia sp.

Limonium nudum (Boiss. & Buhse) Kuntze

Revis. Gen. 2: 395. 1891. Statice nuda Boiss. & Buhse

in Nouv. Mem. Soc. Nat. Mosc. 12: 184. 1860 [basio-

nym]. SYNTYPES: Iran: Albursgebirge bei Radkann,

Juli 1848; bei der Mineralquelle Gendäb auf steini-

gem Kalkboden in grosser Menge, 28 Juli 1848. Mon-

tibus Alborz, prope Pagum Radkann, Gendab, Buhse

1039 (W!, K!, G-photograph!).

Distribution: Southern parts of Alborz in Semnan

Province and Republic of Azerbaijan near Jolfa

(Fig. 36).

Note: The locality of one of the syntypes near Gendab

is located c. 70 km north-east of Semnan. There is

another village named Kendab near Radkann, which

is located in Golestan Province. Radkan area has

been visited by the ﬁrst author, without any evidence

on the occurrence of this species. All localities where

this species was found recently are on gypsum

hills c. 15–20 km north of Damghan, c. 40 km from

Gendab. The second locality ‘An bergigen Orten bei

Kaschan, Mai 1849. no. 1039.’ cited in the original

protologue and Rechinger & Schiman-Czeika (1974)

is suspicious and probably incorrect. Kashan is a

city located in Esfahan Province, c. 250 km far from

Gendab. Limonium nudum has never been found in

the Kashan area. The picture of the type specimen

kindly provided by the authorities of Conservatoire

and Jardin botaniques de la Ville de Genève (G) bears

the locality of Gendab, 28.8.1948, Buhse 1039. It is

536 H. AKHANI ET AL.

unlikely that two specimens bear the same number.

Based on personal communication by Fernand Jac-

quemoud (G), there is no specimen of L. nudum in

Boissier’s herbarium collected by Buhse from Kashan.

The occurrence of L. nudum in Republic of Azerbai-

jan, near Jolfa is documented by a specimen collected

by T. Heideman & H. Alive on 27.5.1932 (HAM).

Limonium caspium (Willd.) Gams

In Hegi, Ill. Fl. Mittleleur. 5, 3: 1880. 1927. Statice

caspia Willd., Enum. Pl. Hort. Berol. 1: 336. 1809.

Type: Regio Caspica. (LECTOTYPE: B-Willdenow!)

[basionym]. St.caspia var. urumiensis Bornm. in

Verh. Zool. Bot. Ges. Wein 60: 165. 1910. L. bellidifo-

lium auct. non (Guan) Dumort.

Distribution: Central Asia, Dsungaro-Kashgar, Mon-

golia, Turkey and Iran (restricted to the surrounding

area of the Uromieh Lake in eastern Azerbaijan,

Fig. 37).

Note: L. bellidifolium and L. caspium have been vari-

ously interpreted in many taxonomic treatments of

the Irano-Turanian and Mediterranean areas. Several

authors considered L. caspium as a synonym of L. bel-

lidifolium described from southern France (Bokhari

& Edmondson, 1982; Assadi, 2005). Earlier in Flora

Iranica (Rechinger & Schiman-Czeika, 1974), and

later in the supplement of Flora of Turkey (Davis

et al., 1988), the Irano-Turanian range of this complex

was separated as L. caspium, which is said to differ

from L. bellidifolium in its less developed sterile

branches, outer bracts with herbaceous part in the

middle, ﬁrst inner bract (middle bract) obovate–

oblanceolate and calyx tubular, as opposed to L. bel-

lidifolium with dense sterile branches, membranous

outer bracts and tubular–funnel-shaped calyx.

Comparison of Iranian plants with two specimens

collected from southern France near the type local-

ity of L. bellidifolium (France: Provence-Alpes-Cote

d’Azur, near Port-Saint-Louis, 43°21′5″N, 04°52′28″E,

sea level, 12.12.2008; Akhani 20511; ibid 1.10.1983,

Ponel s.n.), provides additional differences, including

slightly longer calyx (3.0–4.3 vs. 2.7–3.0 mm) and

shorter and sparser calyx hairs in L. caspium. The

ITS sequences of two species differ by eight nucleotide

changes, which is compatible with the concept that

they are distinct species.

Limonium otolepis (Schrenk) Kuntze

Rev. Gen. Pl. 2: 398. 1891. Statice otolepis Schrenk

in Bull. Acad. Petersb. 1: 362. 1843 [basionym].

Figure 44.

Distribution: Turkmenistan, through Kazakhstan,

Kyrgyzstan and Tajikistan, northern China in Gansu

and northern Xinjiang. In Iran, known only as a

cultivated plant for cut ﬂowers.

Examined specimens: IRAN: Tehran: Flower shop

in Tehran, 16.5.2008, Malekmohammadi 3897

(Hb. Akh.); ibid. 25.6.2008, 3898 (Hb. Akh.); ibid.

20.7.2009, Malekmohammadi 3899 (Hb. Akh.).

Additional specimens examined from outside Iran:

KAZAKHSTAN: Golodnaja Steppe, 9.1915, Dimo and

Popov (TARI). TURKMENISTAN: Charshanga Dis-

trict, Kelif, 7.6.1967, Chopanov s.n. (ASH, Hb. Akh.);

Ashghabad Province (=Ashkhabadskia oblasti):

2 km north of Ashghabad, along Kara-Kum Canal,

16.9.1994. Akhani 10185-T (Hb. Akh.).

Note: This species is known in Iran only from culti-

vated plants offered for sale in ﬂower shops. The

report of this species from Fars province according to

Assadi (2005) is attributable to misidentiﬁcation of

L. reniforme (see notes under L. perfoliatum).

Limonium perfoliatum (C.A.Mey. ex Boiss.) Kuntze

Rev. Gen. 2: 396 (1891). Statice perfoliata C.A. Mey. ex

Boiss. in DC., Prodr. 12: 663. 1848 [basionym]. Statice

perfoliata C.A.Mey. nom. nud. in Bull. Mos.: 167.

1839. Figure 45.

Description: Perennial herbs, 50–95(–150) cm tall;

caudex 0.7–2.5 cm in diameter; stem sometimes

branched from the base into two or three stems;

current year stems (0.5–)1.5–2.5(–3.0) mm in diam-

eter, green, glabrous, with salt glands, gland cavities

indistinct, sterile branches absent. Basal leaves

in a rosette, spathulate or spathulate–oblanceolate,

mostly dying off during ﬂowering time, trinerved,

green, glabrous with salt glands, without gland cavi-

ties, sheathing at the base, (1.5–)2.5–5.5(–8.5) ¥(0.5–)

1.0–2.0 cm, lamina (0.7–)1.0–2.0 cm long, petiole

length excluding sheath (0.2–)1.0–3.0 cm and includ-

ing sheath (0.7–) 1.5–3.5 cm, entire at margin,

rounded or acutish–rounded at apex, attenuate at

base. Cauline leaves more or less ﬂeshy, arranged on

nodes of main stem and lateral branches, decreasing

in size towards the upper parts, green, glabrous,

± palmately veined, veins with dichotomous ends,

with salt glands; lower half cauline leaves of main

stem obovate or spathulate, rarely reniform, 1.5–

3.5 ¥1.2–2.2 cm, ± undulate at margin, rounded or

rarely acute at apex, semiperfoliate or auriculate at

base; upper half cauline leaves of main stem orbicular

or reniform, 1.0–1.2(–2.2) ¥0.2–1.0(–1.2) cm, strongly

undulate at margin, perfoliate at base; cauline leaves

of lateral branches orbicular or reniform, 0.1–

0.7 ¥0.1–1.2 cm, entire, sometimes slightly undulate.

Inﬂorescence dense paniculate, 24–70 (–130) cm tall,

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 537

Figure 44. Limonium otolepis. A, habit. B, spikelet from two views. C, outer bract. D, middle bract. E, inner bract. F,

bracteole. G, calyx. H, petal and stamen. I, gynoecium. J, fruit. K, seed. (Chopanov 7.6.1967).

538 H. AKHANI ET AL.

Figure 45. Limonium perfoliatum. A, habit. B, spikelet. C, outer bract. D, middle bract. E, inner bract. F, bracteole. G,

calyx. H, petal and stamen. I, gynoecium. J, fruit. K, seed. (Akhani et al. 18815).

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 539

upper lateral branches 1.0–1.5(–2.0) cm tall; spikes

unilateral, (ﬁve–) seven to 18 spikelets per spike,

(two–) three-ﬂowered. Outer bracts triangular or

broad triangular, 1.0–1.5(–1.7) mm long, 1.0–1.5(–1.7)

mm broad, herbaceous part brown with an apical

and marginal 0.2–0.5 mm membranous band, gla-

brous, entire at margin, acute, rarely acuminate

at apex, semiamplexicaul or amplexicaul at base;

herbaceous part 1.0–1.5 mm long, (0.2–)0.5–0.7 mm

wide, apex acute. Middle bracts elliptic or orbicular,

rarely triangular–semicircular, 1.0–1.5 mm long, 1.0–

1.5 mm wide, membranous, with one or two narrow

brown nerves, glabrous, entire, rounded or abruptly

acute, rarely retuse at apex, semiamplexicaul,

rarely amplexicaul at base. Inner bracts elliptic or

orbicular–elliptic, rarely obovate, 2.5–3.0(–3.5) mm

long, 2.0–2.5 mm broad, herbaceous part brown with

a 0.5–1.0 mm apical and 0.5–0.7 mm marginal mem-

branous band, glabrous, entire, acutish–rounded or

retuse, rarely truncate at apex, semiamplexicaul at

base; herbaceous part (1.7–)2.0–2.5(–2.7) mm long,

(0.7–)1.0(–1.5) mm wide, rounded, or abruptly acute,

rarely truncate at apex. Bracteoles (two–)three to

ﬁve(–eight) per spikelet, elliptic or broadly elliptic,

rarely obovate, (1.5–)2.0–2.5 mm long, 1.0–1.5 mm

wide, membranous with a narrow brown nerve, gla-

brous, entire at margin, rounded, or obtuse–rounded,

rarely abruptly acute at apex, semiamplexicaul at

base. Calyx funnel-shaped, (2.5–)3.0–3.5 mm long,

limb 2.0–3.0 mm, base 0.5 mm and neck 1.0–1.5 mm

in diameter, 5-lobed; nerves brown, 2–3 mm long,

lobes and between nerves whitish, with dense or

sometimes lax long hairs on two or three nerves,

other nerves glabrous; calyx lobes (0.5–)0.7–1.0 mm,

triangular. Corolla: petals oblanceolate, (3.0–)3.5–

4.5 mm long, 1.0–1.2(–1.5) mm wide, glabrous, dark

purplish or purplish, entire at margin, retuse or

emarginate, rarely rounded at apex, slightly connate

at the base. Filaments adnate to the base of petals,

(2.0–)2.7–3.5(–4.0) mm long, in the middle 0.2–

0.5 mm wide. Anthers 0.7–1.0 mm long, reddish or

violet. Ovary elliptic to obovate, 0.7–1.0 mm long,

upper half or one third granulate. Style 1.5–3.2 mm

long, ﬁliform, free from base. Stigma 0.5–0.7(–1.0)

mm, ﬁliform. Fruit elliptic or fusiform, upper half

granulate, one-seeded, 2.0–2.5 mm long. Achene

obovate, 1.2–1.5 mm long. Funicle 1.5–2.0 mm long.

General distribution: Afghanistan, Iran (Golestan

and Khorassan Provinces, Fig. 39), Turkmenistan and

Uzbekistan.

Examined specimens: IRAN: Golestan: Golestan, near

the border of Turkmenistan, north-west of Alagol

wetland, salty soils in Tamarix thickets, 37°23′27″N,

54°34′42″E, 10 m, 7.7.2007, Akhani, Malekmoham-

madi & Toofani 18808 (Hb. Akh.); near Incheboroon,

just close to Iran-Turkmenistan border, North Ajigol

wetland, 37°26′18″N, 54°38′25″E, 12 m, 7.7.2007,

Akhani, Malekmohammadi & Toofani 18815 (Hb.

Akh.); Gorgan: 47 km north of Pahlavi-Dezh,

13.5.1966, Pobot 3377 (IRAN); Tangoly, 52 km

north of Pahlavi-Dezh, sea level, 13.10.1977, Hewer

4046 (TARI); 52 km from Gomishan to Inche-Boroon,

-20 m, 1.11.1983, Assadi & Abouhamzeh 43285

(TARI); Tangoly, near Ajigol salt lake, 0–10 m,

30.6.1988, Akhani 4297 (Hb. Akh.); Maraveh-Tappeh,

base of Atrak, 24.6.1956, Scharif 33078 (IRAN). Kho-

rassan: between Mashhad and Sarakhs, 18 km after

Mazdavand to Sarakhs, 650 m, 16.8.1994, Akhani &

Zangui 10057 (Hb. Akh); between Sarakhs and Kalat,

between Chahchaheh and main Mashhad–Kalat road,

750 m, 14.8.1993, Mozaffarian 72280 (TARI); 9 km

after Chahchaheh towards Kalat-e Naderi, in river

bed, 600 m, 17.8.1994, Akhani & Zangui 10130 (Hb.

Akh); 10 km from Chahchaheh to Sanganeh (north-

east of Mashhad), 600 m, 17.8.1994, Akhani & Zangui

24567 (Hb. Akh. and FUMH); In declivibus argillosis

a Mozduran boreo-orientem vs. c. 36°15′N, 60°34′E,

inter Mashhad et Sarakhs, 900–1000 m, 25.5.1977,

Rechinger 55671 (B).

Additional specimens examined outside Iran: TURK-

MENISTAN: Ashghabad Province (=Ashkhabadskia

oblasti): 2 km north of Ashghabad, along the Kara

Kum Canal, 16.9.1994, Akhani 10169-T (Hb. Akh.);

soviet of the village Surkhi, Chodzhambaz District,

Komsomol, nearby Amudarja River, 4.10.1970, Sejfu-

lin s.n. (ASH, Hb. Akh.).

Note: L. reniforme,L. perfoliatum and L. otolepis are

related Irano-Turanian species characterized by per-

foliate leaves. See Table 4 for distinguishing charac-

ters of these species and discussion above on the

history and reasons for recognizing L. reniforme and

L. perfoliatum as separate species.

Limonium reniforme (Girard) Lincz

Fl. USSR. 18: 456. 1952. Statice reniformis Girard in

Ann. Sci. Nat. Ser. 3 Bot. 2: 325. 1844 [basionym].

Figure 46. TYPE: Persia Australis, Shiraz, Aucher-

Eloy 5246 (LECTOTYPE: P!, ISOLECTOTYPE G!,

K!, W).

Description: Perennial herbs, 35–70(–90) cm tall;

caudex 1–2(–3) cm in diameter; stem pale green or

green, glabrous, with salt glands, glands small and

without cavities, branched from base into two or three

(to ten) main stems, current year stems 1.0–1.5(–

3.0) mm in diameter, sterile branches lax and just on

the lower branches of the stem. Basal leaves in a

rosette, spathulate, rarely oblanceolate, 3.0–4.0 (–7.0)

540 H. AKHANI ET AL.

Figure 46. Limonium reniforme. A, habit. B, spikelet. C, outer bract. D, middle bract. E, inner bract. F, bracteole. G,

calyx. H, petal and stamen. I, gynoecium. J, fruit. K, seed. (Akhani 7914).

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 541

cm ¥0.5–1.5 cm, lamina (1.0–)2.0–2.5(–4.0) cm,

petiole excluding sheath 1.0–2.5 cm and including

sheath 1.5–3.0 cm, almost completely dying off

about ﬂowering time, trinerved, green, glabrous, with

salt glands, without a cavity, entire, rounded, rarely

abruptly acute at apex, attenuate at base. Cauline

leaves ± ﬂeshy, on the nodes of main stem and lateral

branches, size decreasing upwards, ± palmately

veined, veins at margin dichotomous, green or pale

green, glabrous, with salt gland, without a cavity,

cauline leaves of main stem reniform, rarely obovate,

0.5–2.5(–3.5) ¥0.5–2.0(–3.0) cm, entire at margin,

rounded at apex, semiamplexicaul at base; semiam-

plexicaul to ± perfoliate at base; cauline leaves of

stem branches slightly ﬂeshy, orbicular or transverse

elliptic, 0.1–2.4 ¥0.1–1.3 cm. Inﬂorescence panicle,

10–25 cm tall, upper fertile lateral branches of

inﬂorescence 0.5–1.5 cm tall; spikes unilateral, ± lax,

four to six spikelets per spike, spikelets (one–) two-

ﬂowered. Outer bracts triangular, or rarely semicir-

cular, 0.5–1.0 mm long, 1.0–1.5(–1.7) mm broad, with

an apical 0.2 mm and marginal 0.2–0.5 mm membra-

nous band, sometimes without a ﬂeshy part and com-

pletely membranous, brown, glabrous, entire, acute,

rarely truncate at apex, semiamplexicaul, or amplexi-

caul at base; herbaceous part 0.5–0.7 mm long, 0.5–

0.7(–1.2) mm broad, acute, rarely caudate at apex.

Middle bracts triangular, elliptic, or orbicular, (0.7–

)1.0–1.5 mm long, 1.0–1.2 mm broad, membranous

with one or two narrow brown nerves, glabrous,

entire, rounded or deeply retuse at apex, semiam-

plexicaul at base. Inner bracts orbicular, rarely

transverse broadly elliptic, (1.5–)2.0–2.5 mm long,

2.0–2.5 mm broad, with an apical and marginal

0.5 mm membranous band, brown, glabrous, entire,

rounded, or truncate, rarely emarginate at apex,

amplexicaul at base; herbaceous part (1.0–)1.5–

2.0 mm long, 1.0–1.5 mm wide, rounded, rarely trun-

cate or praemorse at apex. Bracteoles elliptic, or

orbicular, rarely rhombic, 1.2–1.5(–2.0) mm long, 1.0–

1.5(–1.7) mm wide, membranous with a narrow brown

nerve, glabrous, entire, rounded or acute, rarely

obtuse at apex, semiamplexicaul at base, spikelets

with two to ﬁve (to seven) bracteoles. Calyx obconical,

sometimes obconical–funnel-shaped, ﬁve-lobed, 2.5–

3.0 mm long, calyx limb (1.2–)1.5(–1.7) mm, base

0.5 mm and neck 1.0–1.5 mm in diameter; nerves

2.0–2.5 mm long, brown or reddish–brown; lobes and

between nerves whitish, with dense and short to

± long hairs between and along two of nerves, other

nerves glabrous or with short, lax hairs, calyx rarely

glabrous; calyx lobes 0.5–0.7 mm long, triangular, or

semicircular–triangular, rarely semicircular. Corolla;

petals oblanceolate, rarely obovate, (2.5–)3.0–4.0

(–4.5) ¥1.0–1.2(–1.5) mm, purplish, glabrous, entire,

rounded, or slightly retuse at apex, slightly connate

at the base. Filaments adnate to the base of petals,

(2.0–)2.5–3.5(–4.0) mm long, up to 0.5 mm broad in

the middle. Anthers (0.7–)1.0 mm long, pale yellow.

Ovary obovate, 0.7–1.0 mm long, upper one third or

one quarter granulate. Styles ﬁliform, 1.5–3.0 mm

long, free from base. Stigma ﬁliform, 0.5 mm long.

Fruit obovate, 2.2 mm long, upper half granulate.

Achene fusiform, 1.5 mm long. Funicle 1.5 mm long.

Distribution: Endemic to southern–central saline

depressions of Fars Province in Iran (Fig. 39).

Examined specimens: IRAN: Fars: Margin of Tashk

Lake, 5 km south of Gomban, 1560 m, 17.11.1991,

Akhani 7914 (Hb. Akh.); Arsanjan, western shores of

Tashk lake, between the villages Gomban and Katak,

1700 m, 27.11.1987, Assadi & Akhani 61818 (TARI);

Kalagh Djiro, south-west of Tashk lake, 1380 m,

2.7.1989, Zehzad 66965 (TARI); between Arsanjan

and Tashk, Chaharghanat, c. 1700 m, 7.6.1992,

Mozaffarian 71316 (TARI); 24 km after Arsanjan

towards Gomban, before Charghanat Village,

Sarcheshmeh Gomban, 29°48′17″N, 53°28′17.8″E,

1582 m, 4.12.2007, Malekmohammadi, Khoshravesh

& Mahdavi 3970 (Hb. Akh.); Maharlu Lake, 1600 m,

2.6.1973, Bokhari 918 (SHIRAZ); Maharlu Lake,

c. 20 km east of Shiraz, 1450 m, 7.10.1974, Bokhari,

Hedge & Wendelbo 14843 (TARI); in desert of Fengn-

nek near Schiraz, 29.6.1885, Stapf 2172 (B); Bakht-

egan Lake, near Kharameh, 1570 m, 4.10.1974,

Foroughi & Assadi 15036 (TARI); 10–18 km east of

Kharameh, 26.10.1975, 1545 m, Bokhari & Wendelbo

19006 (TARI); Ad lacum Niriz, c. 1400 m, VIII.1949, F.

Starmuehlner 21 (B); Neyriz, 10.7.1949, Behboudi,

33079 (IRAN); 103 km south-east of Darab towards

Hajiabad, along salty river, 28°14′34″N, 55°14′41″E,

835 m, 25.11.2005, Akhani, Dehghani & Doulatyari

18308 (Hb. Akh.); Darab, 13.4 km after fork to Gha-

latooyeh, 55°14′N, 28°14′E, 750 m, 30.3.2004, Khos-

ravi & Tahari s.n. (SHIRAZ); Senjunek near Shiraz

29.6.1885, Stapf 2265 (K); south Iran, ad lacum

salsum Nemek–Derja prope urbis Schiraz, 1.6.1842,

Kotschy 464 (K); Maharlu Lake near Shiraz, 5000 ft,

3.8.1939, Davis 748 (B, K).

Note: See notes under L. perfoliatum and discussion.

Limonium reniforme is an endemic restricted to

margins of saline depressions in Fars Province

around Tashk, Bakhtegan and Maharloo Lakes. This

area is of great interest because of the presence of

other halophytic local endemics, the most peculiar

of which is Hypericopsis persica Boiss., an isolated

species of south-western Asian Frankeniaceae

(Chrtek, 1972). Recent anatomical studies and

molecular evidence show that this species belong in

542 H. AKHANI ET AL.

Frankenia L. (Olson, Gaskin & Ghahremani-nejad,

2003). Salicornia persica Akhani and S. perspolitana

Akhani are other species described from the same

area (Akhani, 2008).

Limonium carnosum (Boiss.) Kuntze

Rev. Gen. Pl. 2: 395 (1891). Statice carnosa Boiss. in

DC. Prod. 12: 663. 1848 [basionym]. TYPE: Aderbid-

jan [Azerabaijan], Persiae bor. Prope Khoi, Aucher-

Eloy 2505 (LECTOTYPE G!). S. suffruticosa var.

typica Trautv. in Bot. Sada. 7, 2: 504. 1881, non

Trautv. 1867. S. suffruticosa var. carnosa (Boiss.)

Kusn. in Mat., Fl. Kavk. 4, 1: 222. 1903.

Note: Limonium carnosum is a vicariant of the

widely distributed L. iranicum in north-western Iran

(Fig. 40). This species can easily be distinguished

from the latter by longer (4.5–5.0 mm) and funnel-

form calyx (in L. iranicum the calyx is 3.0–3.5 mm

long and its shape is usually tubulate). Furthermore,

the spikelets of L. carnosum are more condensed and

are borne at the end of inﬂorescence branches. The

character of glabrous vs. pilose calyx used to distin-

guish L. carnosum from L. iranicum by Assadi (2005)

cannot be conﬁrmed here. Several specimens, includ-

ing plants near the type locality (Akhani et al.18891,

18904,18894,19046,Assadi 60977,Zehzad 83/1089)

have a pilose or shortly pilose calyx. Considering the

calyx characters, two specimens collected from Gha-

havand in Hamedan and Semnan Provinces (Assadi

61097,Assadi & Hamdi 85415) were misidentiﬁed as

L.carnosum by Assadi (2005). Specimens with a gla-

brous calyx in L. iranicum occur in Semnan province

(Mirjalili 20729,Akhani & Salimian 15335,Akhani

16499,Akhani & Ghobadnezhad 15777).

Limonium iranicum (Bornm.) Lincz.

Fl. URSS. 18: 461. 1952. Statice leptophylla Schrenk

var. iranica Bornm. in Beih. Bot. Centrbl. 22, 2: 140

1907 [basionym]. TYPE: Sultanabad, Strauss ad

Teramis, 4.8.1809. Th. Strauss s.n. (LECTOTYPE B!,

ISOLECTOTYPE B!, W!, G, JE).

Distribution: Iran (Bushehr, Esfahan, Fars,

Hamedan, Hormozgan, Ilam, Kerman, Khorasan,

Ostan-e Markazi, Qom, Semnan, Tehran, Yazd. Prov-

inces, Fig. 41), Iraq.

Note: Limonium iranicum is the most frequent

Limonium sp. in central and southern Iranian saline

deserts and salt marshes. It is closely related to

L. leptophyllum (Schrenk) Kuntze and L. suffrutico-

sum. In most parts of Iran, there is no difficulty

in distinguishing L. suffruticosum and L. iranicum

using leaf and inﬂorescence characters (see key

above). However, in some parts of Iran (e.g. Kho-

rassan Province) where they occur together it is

difficult to separate them, probably because of

hybridization.

The southern Iranian populations of L. iranicum

differ from central Iranian plants in their clearly

woody base, smaller ﬂower segments, denser inﬂores-

cence and longer life cycle. Because of the arid tropi-

cal climate of southern Iran and absence of low winter

temperatures, many plant species including L. irani-

cum persist nearly throughout the year. Therefore,

they appear as a subshrub with more developed

woody parts. Erben & Mucina (2006) described

L. failachicum as an endemic species from Kuwait

(Failacheh Island). In a study of type specimen

(Kuwait, Failaka, east part of the island, 5 km from

the port, 2 m, very salty area, 4.X.1981, Rawi 10838

(MSB 86094!) and plants collected from Dara Island

(Akhani & Pahlevani 20820), c. 100 km north-east of

Failakeh, we found no reliable differences between

these populations. In our molecular results they fall

in an unresolved polytomy (Fig. 31). One alternative

option would be to combine the southern Iranian

plants and L. failachicum as a subspecies of L. irani-

cum, but we prefer to conduct more studies before

making this decision.

Limonium suffruticosum (L.) Kuntze

Rev. Gen. Pl. 2: 396.1891. Statice suffruticosa L., Sp.

Pl. 276. 1753 [basionym]. L. gabrielii (Bornm.) Rech.f.

in Flora Iranica 108: 10. 1974. St. gabrielii Bornm. in

Repert. Spec. Nov. Regni Veg. 36: 170. 1934.

Distribution: Afghanistan, Iran (Golestan, Khorasan,

Sistan-o Balochestan, Yazd, Fig. 42), Mongolia, south-

eastern Russia, Siberia, Turkmenistan.

Note: Limonium suffruticosum extends from Central

Asia through eastern parts of Iran (Fig. 42). The

southernmost populations of L. suffruticosum col-

lected from southern Khorassan (Gabriel 10;Mobayen

8108;Akhani & Joharchi 17313;Foroughi 10562;

FOS 8109) differ from other plants studied in having

broader leaves, a tomentose calyx, hairs on bracts and

bracteoles that are tomentose on the outer bracts and

a slightly shorter corolla, 4.5 mm long, vs. narrower

leaves, shortly pilose calyx tube, and slightly longer

corolla, 5.5–6.0 mm long. The southern populations

match L. gabrielii (Gabriel 10, W!). The position of

this taxon as a subspecies or a distinct species is

under further investigation.

Limonium gmelinii (Willd.) Kuntze

Rev. Gen. Pl. 2: 395. 1891. Sensu lato. Statice gmelinii

Willd., Sp. Pl. 1: 1524. 1797 [basionym]. TYPE: USSR,

in Siberia locis salsis a Jaico ad Angaram Usque,

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 543

Gmelin 6174 (HOLOTYPE: B-Willd.!). Incl. L. meyeri

(Boiss.) Kuntze, Revis. Gen. Pl. 2: 395. 1891. St.

meyeri Boiss. in DC., Prodr. 12: 645. 1845. St. gmelinii

Willd. var. laxiﬂora Boiss., l. c. 646.; S. obovata

Ledeb., Fl. Ross. 3: 468. 1849.

Distribution: Central Asia, Balkans, Caucasia,

Crimea, Central Europe, Iran (Ardebil, Azarbaijan-e

Gharbi, Azarbaijan-e Sharghi, Bushehr, Esfahan,

Fars, Golestan, Khorasan, Khuzestan, Kohkiloye

va Boyer Ahmad, Lorestan, Mazandaran, Ostan-e

Markazi, Tehran Provinces, Fig. 43).

Note: Limonium gmelinii and L. meyeri belong to a

widely distributed species complex occurring in the

Mediterranean, Euro-Siberian and Irano-Turanian

areas (including L. vulgare Mill, L. narbonense Mill.

and L. latifolium (Sm.) Kuntze). In Iran, populations

of these two species occur in most parts of the country.

Rechinger & Schiman-Czeika (1974) separated them

using the densely arranged spikelets in L. gmelinii

and loose spikelets in L. meyeri,asintheFlora of

Turkey (Bokhari & Edmondson, 1982). We found that

dense and loose inﬂorescences can occur in the same

population (e.g. Akhani 12228,12229, collected from

Golestan National Park). Apparently plants growing

in areas with more water produce much longer and

looser inﬂorescence branches (the meyeri-form). Those

growing in drier soils usually develop smaller and

denser inﬂorescence (the gmelinii-form). Further-

more, younger plants have looser inﬂorescences than

older plants. Boissier (1848), who originally described

L. meyeri, later reduced it in Flora Orientalis (Bois-

sier, 1879) to a variety of L. gmelinii, which seems to

be more applicable based on present knowledge.

Analysis of ITS sequence data (Fig. 31) separated

several accessions into two major groups that are

distinct geographically: one group includes plants

from north-eastern Iran and northernmost parts of

Azerbaijan (close to the Aras River). They are char-

acterized by hairs on the calyx only on the basal-

most 1–3 mm and leaves that are spathulate or

spathulate–oblanceolate. The second group includes

populations from central Iran and near the Uromieh

Salt Lake in north-eastern Iran that are character-

ized by hairs on the calyx nerves and oblanceolate

leaves. In this latter group, two subgroups can be

distinguished based on our analysis. One includes

only the central and southern Iranian populations

and the second only plants occurring in saline soils

near Uromieh Salt Lake in eastern and western

Azerbaijan, but morphologically these two are not

easily distinguished. A revision of the whole group

across the Mediterranean, Euro-Siberian and Irano-

Turanian area using detailed morphological and

molecular analysis is required to solve the taxonomic

problems of this species complex.

ACKNOWLEDGEMENTS

This paper is the result of the research project

‘Geobotanical studies in different parts of Iran IV’,

supported partly by the Research Council of the Uni-

versity of Tehran. HA and PM received funds from the

Bentham-Moxon Trust (at the Royal Botanic Gardens,

Kew) to visit the Jodrell Laboratory at the Royal

Botanic Gardens, Kew, during 2008. During our visit,

several people helped us including Michael Frohlich,

James Clarkson, M. Dolores Lledó, Edith Kapinos

and Shahina Ghazanfar. Some DNA sequences were

generated during a sabbatical visit of the ﬁrst author

(H.A.) at the Botanical Garden and Botanical

Museum, Berlin-Dahlem invited by Prof. Thomas

Borsch, with support from the Alexander von Hum-

boldt Foundation. M.M. received a small student

grant to work on her M.Sc. from the College of

Science, University of Tehran. We thank Fernand

Jacquemoud (Geneva) for his help in clarifying the

ambiguity of the herbarium label of L. nudum,Dr

Susy Fuentes Bazan (Berlin) for her advice on

improving matrix aligments and Prof. Manuel B.

Crespo (Alicante) for the improvements he suggested

on the earlier version of this paper.

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Amsterdam: Gustav Fische.

APPENDIX

List of species used in phylogenetic analysis of Limonium in Iran with their classiﬁcation according to Boissier (1848),

and post-Boissier classiﬁcation, vouchers and GenBank accession numbers. Boissier’s special infrasectional rank is

considered, as for other authors, as subsection. The cases indicated by ‘L. SUBSECT.’ showed that the species was classiﬁed

under a subsection of section Limonium. Voucher details of two species described in taxonomic part are not repeated. They

are indicated by an asterisk after collector and herbarium number

Name

Boissier’s sections

and subsections

(sub Statice)

Sections or

subsections of

later authors Voucher and/or origin and [DNA accession no.]

Cephalorhizum

turcomanicum

Pop.

Cephalorhizum Khorasan: Kalat Naderi Archangan, km 10, 1100 m,

1.vi.1997, Faghihnia & Zanguei 28978-FUMH

[JX983658]

L. anatolicum

Hedge

–Sarcophyllum‡ Turkey-Aksaray: 10 km east of Eskil towards southern

saline shores of Tuz Gölü Lake, high salty soils,

38°25′5″N, 33°2952″E, 914 m, 28.viii.2004, Akhani et al.

17999 [JX983724]

L. axillare

(Forssk.) Kuntze

L. subsect.

Sarcophyllae

Sarcophyllum† Bushehr, Bidkhoon, Nayband National Park, 27°27′28″N,

52°40′31E, 7.xi.2007, Malekmohammadi et al. 3974

[JX983660, JX983661]

L. arborescens

(Brouss. ex

Webb &

Berthel.) Kuntze

Pteroclados

subsect.

Nobiles

(Brockington et al., 2008) EU410356

L. bellidifolium

(Gouan) Dumort

France: Provence-Alpes-Cote d’Azur, near Port-Saint-Louis,

43°21′5″N, 04°52′28″E, sea level, 12.xii.2008, Akhani

20511 [JX983722]

L. carnosum

(Boiss.) Kuntze

L. subsect.

Sarcophyllae

Sarcophyllum† Azerbaijan Sharghi: c. 20 km west of Marand, 2 km south

of Koshksaray towards Arland, 38°25′57″N, 45°33′39″E,

1260–1300 m, 15.ix.2007, Akhani et al. 18894

[JX983727]; 22 km south of Evoghli, in salty ﬂats along

the road towards Marand, 38°33′13″N, 45°19′22″E,

1034 m, 15.ix.2007, Akhani et al. 18904 [JX983728];

c. 30 km West Tabriz, 5 km West Qezel Dizej towards

Alishah, 38°5′35″N, 45°56′56″E, highly saline soils,

1310 m, 14.ix.2007, Akhani 18891 [JX983729];

Azerbaijan Gharbi: 10 km south of Khoy towards

Salmas, salty hills and saline ﬂats beside Khoy

industrial centre, 38°25′N, 44°54′E, 1197 m, 17.ix.2007,

Akhani et al. 18997 [JX983726]; East Azerbaijan: 10 km

south-west of Sarab on the road towards Asbforoushan,

8.ix.2001. Akhani 15625 [JX983725].

L. caspium (Willd.)

Gams

L. subsect.

Hyalolepidae

Limonium† Azerbaijan Sharghi: 22 km west of Tabriz, 11 km west of

Mayan-e Olia, saline ﬂats near Qezel Dizej, 38°04′29″N,

46°00′31″E, 1318 m, 14.ix.2007, Akhani et al. 18888

[JX983721].

546 H. AKHANI ET AL.

APPENDIX Continued

Name

Boissier’s sections

and subsections

(sub Statice)

Sections or

subsections of

later authors Voucher and/or origin and [DNA accession no.]

Limonium cf.

cumanum (Ten.)

Kuntze

Italy: Naples (Napoli), south-east of Naples, c.10km

south-west of Salerno, Amalﬁ, 11.v.2003, Akhani 16815

[JX983717].

L. delicatulum

(Girard.) Kuntze

L. subsect.

Dissitiﬂorae

L. subsect.

Dissitiﬂorae§

Palacios et al. 2000: Spain: Alicante (AJ222851)

L. echioides (L.)

Miller

Schizhymenium Schizhymenium‡ Palacios et al., 2000: Spain: Alicante (AJ222861)

L. furfuraceum

(Lag.) Kuntze

L. subsect.

Steirocladae

L. subsect.

Steirocladae§

Palacios et al., 2000: Spain: Alicante (AJ222856)

L. gmelinii (Willd.)

Kuntze s.l.

(Group A)

L. subsect.

Genuinae

Limonium†‡ Azerbaijan Gharbi: 40 km north-west of Poldasht, 10 km

west of Shotloo towards Aghghol, 39°30′43″N,

44°50′18″E, 788 m, 16.ix.2007, Akhani et al. 18948

[JX983715]; Khorassan: Jajarm to Chamanbid, Shurek,

20.ix.1998, Faghihnia & Zangui 31792-FUMH

[JX983713]; Khorassan: Golestan National Park: near

Cheshmekhan, 14.x.1995, Akhani 12229 [JX983714];

north-west of Bojnurd, Shahrabad, 800 m, 28.viii.1983,

Joharchi & Zangui 10637 [JX983716].

L. gmelinii (Willd.)

Kuntze s.l.

(Group B)

L. subsect.

Genuinae

Limonium‡ Arak: c. 18 km north-east of Arak, saline lands near the

southern margin of Arak salt lake, 34°12′10″N,

49°55′8″E, 1686 m, 27.viii.2007, Akhani &

Malekmohammadi 18852 [JX983691]; 25 km from

Delijan towards Esfahan, Roud-e Shour bridge,

33°47′33″N, 50°50′42″E, 1861 m, 3.x.2007,

Malekmohammadi et al. 3827 [JX983685]. Kohkiloye va

Boyerahmad: 35 km east of Lordegan, 21.ix.2001,

Akhani 15740 [JX983692]; Azerbaijan Sharghi: 23 km

north of Ajabshir, on the eastern salt marshes of

Orumieh lake, 37°42′34″N, 45°50′25″E, 1296 m,

18.ix.2007, Akhani et al. 19013 [JX983688]; 22 km west

of Tabriz, 11 km west of Mayan-e Olia, saline ﬂats near

Qezel Dizej, 38°4′42″N, 46°00′53″E, 1318 m, 14.ix.2007,

Akhani et al. 18887 [JX983690]; 8 km south-west of

Sarab, on the road towards Asbforoushan, 8.ix.2001,

Akhani 15636 [JX983686]; Azerbaijan Gharbi: 10 km

south of Khoy towards Salmas, salty hills and saline

ﬂats beside Khoy industrial centre, 38°25′N, 44°54′E,

1197 m, 17.ix.2007, Akhani et al. 18996 [JX983689];

Bushehr: 10 km to Dalaki from Borazjan, Cheshme

Googerdi, 29°20′35″N, 51°15′49″E, 2.x.2007,

Malekmohammadi 3825 [JX983687].

L. iconium (Boiss.

& Heldr.)

Kuntze

L. subsect.

Hyalolepidae

Limonium‡ Turkey: Aksaray: salt ﬂats south of Tuz Gölü Lake, 17 km

from Yenikent towards Sultanhani, 38°15′56″N,

33°38′51″E, 938 m, 27.viii.2004, Akhani et al. 17961

[JX983719]; Ankara: 28 km north of S¸ ereﬂikochisar

towards Ankara, north of Tzu Gölü Lake, saline soils

around Mogan Gol Lake, 39°8′50″N, 33°19′4″E, 895 m.

27.viii.2004, Akhani et al. 17921 [JX983720]; Konya:

west of Tuz Gölü Lake, 8 km north-west of Gölyazi,

38°37′3″N, 33°9′16″E, 920 m, 28.viii.2004, Akhani et al.

18011 [JX983718].

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 547

APPENDIX Continued

Name

Boissier’s sections

and subsections

(sub Statice)

Sections or

subsections of

later authors Voucher and/or origin and [DNA accession no.]

L. iranicum

(Bornm.) Lincz.

L. subsect.

Sarcophyllae

Sarcophyllum† Arak: c. 18 km north-east of Arak, saline lands near the

southern margin of Arak salt lake, 34°12′10″N,

49°55′8″E, 1686 m, 27.viii.2007, Akhani &

Malekmohammadi 18851 [JX983704]; Bushehr:

Kangan–Asalooyeh road, 45 km before Asalooyeh, after

Akhtar village, 27°41′32″N, 52°15′16.5″E, 13 m, 7.

xii.2007, Malekmohammadi, Khoshravesh & Mahdavi

3972 [JX983707]. Esfahan: 26 km south–south-east of

Delijan, 22.ix.2001, Akhani 15760 [JX983705]; 25 km

from Delijan towards Esfahan, Rude Shur bridge,

33°47′33″N 50°50′42″E, 1861 m, 3.x.2007,

Malekmohammadi et al. 3826 [JX983706]; Fars: 30 km

south-east of Arsanjan, near Tashk Lake, 27.ix.1991,

Khademian 58 (Shiraz University Herbarium)

[JX983696]; Hormozgan: Bandar-Abbas towards

Haji-Abad, between Sarchahan and Gahkom, 28°5′35″N,

55°52′10″E, 663 m, 9.xii.2007, Malekmohammadi et al.

3977 [JX983700]; Bandar Lengeh–Bandar Khamir road,

63 km after Bandar Lengeh, 22 km before Dejgan

village, left side of the road, sandy soil, 29°48′59″N,

55°21′05″E, 6 m, 8.xii.2007, Malekmohammadi,

Khoshravesh, Mahdavi 3976 [JX983701]; Bandar-e

Emam Hasan, behind Emam Hasan police station,

coastal area, 29°50′36″N, 50°15′18″E, 3 m, 30.ix.2007,

Malekmohammadi & Kazemi 3823 [JX983709]. Ilam:

c. 5 km south-west Dehloran, 6.viii.1989, Akhani 5563

[JX983699]; Tehran, east of Hassan Abad, after Key

Ghobad village, 13.ix.1987, Akhani 4813 [JX983695];

Khorassan: 10 km from Ghaen to Gonabad, 1450 m,

5.x.1991, Joharchi & Zangui 21058 [JX983702];

Khuzestan: Persian Gulf, Khore Musa, 20 km south-west

of Bandare Khomeini, Ghabre Nakhoda Island,

30°18′21″N, 48°54′41″E, 10.iii.2010, Akhani & Pahlevani

20723 [JX983711]; north-west of Persian Gulf, Dayreh

Island, sandy and saline soils, 30°6′N, 49°6′E,

7–8.v.2010, Akhani & Pahlevani 20820 [JX983712];

Semnan: 37 km north-east Torud, along a river 11 km

north-east of Razeh, Cheshmeh Morrah, 35°35′38″N,

55°20′2″E, 1218 m, 9.xi.2007, Akhani et al. 19133

[JX983710]; 18 km north of Damghan towards

Cheshmeh Ali, saline and gypsum hills, 36°13′22″N,

54°12′28″E, 1402 m, 6.vii.2007, Akhani et al. 18805

[JX983697]. Bushehr: Bidkhon, Nayband National Park,

27°27′28″N, 52°40′31″E, 7.xii.2007, Malekmohammadi

et al. 3975 [JX983708]; Semnan: 28 km east of Khors

towards Chajam, south of Kavire Haji Ali Gholi,

14.xi.2002, Akhani 16499 [JX983698]; Hamadan: east of

Ghahavand, 3 km from Ghahavand towards Boyaghchi,

left side of the road, beginning of the road of Haji Abad,

34°51′N, 49°0′E, 1637 m, 31.viii.2008, Malekmohammadi

et al. 3986 [JX983703].

548 H. AKHANI ET AL.

APPENDIX Continued

Name

Boissier’s sections

and subsections

(sub Statice)

Sections or

subsections of

later authors Voucher and/or origin and [DNA accession no.]

L. lilacinum (Boiss.

& Bal.) Wagenitz

Sphaerostachys‡ Turkey: Ankara: 28 km north of S¸ ereﬂikochisar

towards Ankara, north of Tzu Gölü Lake, saline

soils around Mogan Gol Lake, 39°8′50″N,

33°19′4″E, 895 m. 27.viii.2004, Akhani et al. 17930

[JX983693].

L. latifolium (Sm.)

Kuntze

Mediterranean coast of France: Provence–Alpes–Cote

d’Azur, 10 km west of Fos-sur-Mer, Etange de Caban,

43°25′16″N, 04°48′38″E, sea level, 12.xii.2008, Akhani

20504 [JX983694].

L. lobatum (L.f.)

Kuntze

Pteroclados subs.

Odontolepidae

Pteroclados†‡ Fars: 39 km south-west of Jahrom, Mobarak

Abad village, 1.iv.2008, Hekmatara 3978

[JX983659].

L. lobatum (L.f.)

Kuntze

Pteroclados Pteroclados†‡ Palacios et al., 2000: Almería (AJ132333)

L. minutum (L.)

Chaz.

L. subsect.

Steirocladae

Subs.

Steirocladae§

Palacios et al., 2000; Spain: Baleares (AJ132332)

L. narbonense Mill. L. subsect.

Limonium

sub. Limonium Palacios et al., 2000: Spain: Valencia (AJ222838)

L. nudum (Boiss.

& Buhse)

Kuntze

Platyhymenium† Semnan: 18 km north of Damghan towards

Cheshme Ali, saline and gypsum hills, 36°13′22″N,

54°12′28″E, 1402 m, 6.vii.2007, Akhani et al. 18804

[JX983672]

L. otolepis

(Schrenk)

Kuntze

L. subsect.

Hyalolepidae

Nephrophyllum†Malekmohammadi 3897* [JX983682]; Akhani

10185-T* [JX983683]; Uzbekistan: Kyzylkum,

c. 15 km north-east of Chingil’dy (Shingeldi), near

Karaktau station, near Noorbulak village, 41°2′2″N,

64°29′39″E, 100 m, 26.x.2009, H. Akhani 20367

[JX983684].

L. perfoliatum

(C.A.Mey ex

Boiss.) Kuntze

L. subsect.

Hyalolepidae

Nephrophyllum†Akhani 10169T* [JX983680]; Akhani et al. 18808*

[JX983679]; 18815 [JX983678]*; Akhani & Zangui

10130 [JX983681]*; 24567 [JX983677]*; 10057

[JX983676]*

L. reniforme

(Girard) Lincz.

L. subsect.

Hyalolepidae

Nephrophyllum†Malekmohammadi et al. 3970 [JX983674]*; Khosravi &

Tahari s.n. (30.iii.2004) [JX983675]*; Zehzad 66965

[JX983673]*

L. sogdianum

(M.Pop.)

Ikonn.-Gal.

–Eremolimon¶ Khorassan: Between Neyshabur and Kashmar, 70 km

south of Neyshabur, gypsum hills near Chelpu,

35°37′23″N, 58°31′48″E, 1846 m, 24.x.2007, Akhani &

Memariani 19055* [JX983723]

PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 549

APPENDIX Continued

Name

Boissier’s sections

and subsections

(sub Statice)

Sections or

subsections of

later authors Voucher and/or origin and [DNA accession no.]

L. suffruticosum

Kuntze

L. subsect.

Sarcophyllae

Sarcophyllum† IRAN: Khorassan: Petergan, Cheshme shirin, 21.x.1996,

Rafei & Zangui 28088 [JX983666]; 143 km east of Qaen

towards Afghanistan border, west of Daqe Petergan,

33°31′29″N, 60°39′12″E, 644 m, 31.viii.2003, Akhani &

Joharchi 17282 [JX983667]; 45 km east of Torbategan,

between Gale-Hamam and mallu-Olia, 29.iv.2003,

collector? 41 (FUMH) [JX983668]; Golestan National

Park, south Mirza-Baylu ﬂats, near Armadlu village,

1200 m, 15.xi.1996, Akhani 12277 [JX983669]; Yazd:

50 km from Tabas to Yazd, 1000 m, 20.x.1982, Assadi &

Abouhamzeh 40226 (TARI) [JX983662]; Khorassan:

Birjand, between Mahirud and Estakhr deraz, 1100 m,

3.ix.2003, Joharchi 35188 [JX983665]; Sistan va

Baluchestan: Kavire Lut, Deh-e Salm, 9.xi.1972, 1100 m,

Mobayen 8108 (TUH) [JX983663]. TURKMENISTAN:

Balkhan Province: c. 14 km south-east of Nebet-Dagh,

12.ix.1994, Akhani 10160T [JX983664]. UZBEKISTAN:

Kyzylkum, c. 15 km north-east of Chingil’dy (Shingeldi),

near Karaktau station, near Noorbulak village, 41°2′2″N,

64°29′39″E, 100 m, 26.x.2009, Akhani 20364 [JX983671];

c. 100 km north of Bukhara, Ayagakitma saline lake,

highly saline soils near the lake marshes, 40°39′30″N,

64°29′37″E, 148 m, 26.x.2009, H. Akhani 20382

[JX983670].

L. vulgare Mill. L. subsect.

Limonium

Palacios et al., 2000: Spain: Cantabria (AJ222839)

Psylliostachys

leptostachya

(Boiss.) Roshk

Psylliostachys – Fars: Maharlu, 15.iv.1993, Elmi 57 (Shiraz University

Herbarium) [JX983657].

Psylliostachys

spicata (Willd.)

Nevski

Psylliostachys – Khuzestan: W Shoosh, along Karkhe River, 1.iv.2001,

Akhani 14878 [JX983656]

†Rechinger & Schiman-Czeika, 1974.

‡Bokhari, 1973.

§Palacios et al., 2000.

¶Linczevski, 1985.

550 H. AKHANI ET AL.

A taxonomic backbone for the Plumbaginaceae (Caryophyllales)

Article

Full-text available

Jun 2024

A taxonomic backbone of the Plumbaginaceae is presented and the current state of knowledge on phylogenetic relationships and taxon limits is reviewed as a basis for the accepted taxon concepts. In total, 4,476 scientific names and designations are treated of which 30 are not in the family Plumbaginaceae. The Plumbaginaceae are subdivided in three tribes with 26 genera and 1,179 accepted species. Two subgenera, 17 sections, two subsections and 187 infraspecific taxa are accepted. At the species and infraspecific level 2,782 synonyms were assigned to accepted taxa, whereas 194 names were excluded from the core checklist (i.e., unplaced taxa, infrageneric subdivisions with still uncertain application, names of verified uncertain application, invalid horticultural names, excluded names from other families, other excluded designations, and unresolved names). The EDIT Platform for Cybertaxonomy was utilized as the tool to compile and manage the names and further taxonomic data under explicit taxon concepts. Secundum references are given in case taxon concepts were taken from the literature, whereas this study serves as reference for newly circumscribed taxa. The family’s division into the tribes Aegialitideae, Limonieae, and Plumbagineae departs from earlier two-subfamily classifications, prompted by recent phylogenetic findings that challenge the subfamilial affinity of Aegialitis . The genus Acantholimon was extended to include Gladiolimon , as currently available phylogenetic and morphological data support this merger. In Limonium , all accepted species could be assigned to sections and subsections or the “Mediterranean lineage”, respectively, making use of the phylogenetic distribution of their morphological characters and states. A new combination and/or status is proposed for Dyerophytum socotranum , Limonium thymoides , Limonium × fraternum , Limonium × rossmaessleri , and Limonium sect. Jovibarba. Special attention is given to nomenclatural issues, particularly for Statice nomen ambiguum to resolve the names under accepted names. The use of artificial groupings like “aggregates”, “complexes” and “species groups” in alpha-taxonomic treatments is discussed. The taxonomic backbone will receive continued updates and through the Caryophyllales Taxonomic Expert Network, it contributes the treatment of the Plumbaginaceae for the World Flora Online.

Analysis of the taxonomic status of Limonium caspium (Willd.) P. Fourn. using the ITS1-5.8S-ITS2 region

Article

Full-text available

Dec 2023

There are 15 representatives of the genus Limonium Mill in Ukraine. The taxonomic status of some of them requires clarification. In particular, some taxonomists consider L. caspium (Willd.) P. Fourn. to be a separate species, while others tend to consider this name as a synonym for L. bellidifolium Dumort. Until now, only one sample of L. caspium from eastern Iran has been analyzed using molecular genetic approaches: for this accession, the ITS1-5.8S-ITS2 of 35S rDNA region was sequenced. For both species, L. caspium and L. bellidifolium, samples from the territory of Ukraine have not yet been examined using molecular taxonomy methods. In this work, we amplified and sequenced ITS1-5.8S-ITS2 region for two L. caspium and two L. bellidifolium samples from southern Ukraine. All four samples appeared to be identical in this region. They also do not differ from previously sequenced samples of L. bellidifolium from France, Turkey and Greece. On the other hand, the sequence of L. caspium from Iran has five nucleotide substitu-tions and four indels compared to the mentioned samples. The phylogenetic analysis revealed that representatives of the so-called "L. bellidifolium complex", which, in addition to the two species mentioned, also includes L. iconium and L. tamaricoides analyzed in this work, are grouped together with representatives of the Nephrophyllum section. A similar result was obtained in the studies of other authors using the ITS1-5.8S-ITS2 sequence and regions of chloro-plast DNA. However, according to the IGS 5S rDNA sequence, representatives of the "L. bellidifolium complex" in our previous work were grouped together with the species of the Limonium section, to which they were previously as-signed based on morphological characters. Such a discrepancy between phylogenies using different genomic regions indicates the reticular nature of the evolution of the genomes of representatives of this group and indicates probable hybridization events in their past. Within the clade of the "L. bellidifolium complex" that we obtained in our analysis, the sample of L. caspium from Iran forms a sister branch to all its other representatives. Thus, the comparison of the ITS1-5.8S-ITS2 region shows that the Ukrainian samples identified as L. caspium do not differ from L. bellidifolium. However, to finally clarify the taxonomic status of L. caspium, it is necessary to analyze more samples from different geographic origins.

A first look at sea-lavenders genomics – can genome wide SNP information tip the scales of controversy in the Limonium vulgare species complex?

Article

Full-text available

Jan 2023
BMC PLANT BIOL

Background Sea-lavenders (Limonium Mill., Plumbaginaceae) are a cosmopolitan group of diploid and polyploid plants often adapted to extreme saline environments, with a mostly Tethyan distribution, occurring in the Mediterranean, Irano-Turanian, Euro-Siberian and in the New World. The halophylic Limonium vulgare polyploid complex in particular, presents a large distribution throughout extreme salt-marsh habitats and shows little morphological but high taximetric variation, frequently blurring species delimitation. In this work we pursue three main goals: assert whether SNP data from polyploid individuals has the resolution to distinguish the seven sampled species, to better understand how genetically structured Limonium vulgare is, and attempt to identify specific molecular mechanisms for the differentiation between L. maritimum and L. vulgare. For this purpose, 95 individuals were genotyped using Genotyping by Sequencing (GBS), which were assembled as two independent datasets using ipyrad. All analyses performed downstream of assembly were fully automated. Phylogenetic inference, PCA, and admixture plots were used to infer answers to the study’s main goals. Results Close to 10,000 SNPs were obtained for each dataset. Phylogenetic analyses reveal that polyploid data can be used to infer species relationships. Population structure analyses suggest a genetically structured L. vulgare. A set of 34 SNPs were found to be fully segregated between L. vulgare and L. maritimum, two of which are potentially linked to proteins that might be involved in the speciation process. Conclusion Despite polyploid data analyses shortcomings, GBS generated SNPs have the resolution to discern all seven included species. Limonium vulgare revealed pronounced genetic structure along a geographical north-south cline. L. maritimum always appears as a distinct genetic entity. Segregated SNPs between L. vulgare and L. maritimum indicate salinity response and morphological trait control genes as potentially interesting to follow up for studying these species’ divergence process.

Leaf anatomical investigations in Acantholimon (Plumbaginaceae)

Article

Full-text available

Apr 2022
Rev Bras Botânica

In this study, we examined leaf anatomy of 24 species belonging to Acantholimon from flora of Iran. This paper, therefore, describes the foliar anatomical characters in details and discusses the extent to which leaf anatomical features might be developed in response to arid, semi-arid and mountainous condition of the Irano-Turanian region. The results revealed that the leaf anatomy of Acantholimon is highly consistent with the species habitat. The leaf anatomical characters such as the presence of salt glands and trichomes, isobilateral leaves, and sclerophylly are the most predominant responses to the environmental conditions. This paper is the first report of the existence of sclereids in Acantholimon species. The survey of the secreted elements from salt glands on the leaves epidermis of Acantholimon species were performed using SEM energy-dispersive X-ray (EDS) analysis for the first time. Based on these results, the crystals were determined to be truncated conical in shape, composed largely of Ca, O and C elements. Taxonomic implication of the anatomical characters in Acantholimon has been also discussed.

Plastome analysis unveils Inverted Repeat (IR) expansion and positive selection in Sea Lavenders (Limonium, Plumbaginaceae, Limonioideae, Limonieae)

Article

Full-text available

Apr 2021

The genus Limonium , commonly known as Sea Lavenders, is one of the most species-rich genera of the family Plumbaginaceae. In this study, two new plastomes for the genus Limonium , viz. L. tetragonum and L. bicolor , were sequenced and compared to available Limonium plastomes, viz. L. aureum and L. tenellum , to understand the gene content and structural variations within the family. The loss of the rpl16 intron and pseudogenisation of rpl23 was observed. This study reports, for the first time, expansion of the IRs to include the ycf1 gene in Limonium plastomes, incongruent with previous studies. Two positively selected genes, viz. ndhF and ycf2 , were identified. Furthermore, putative barcodes are proposed for the genus, based on the nucleotide diversity of four Limonium plastomes.

Taxonomy Complexity of Some Tyrrhenian Endemic Limonium Species Belonging to L. multiforme Group (Plumbaginaceae): New Insights from Molecular and Morphometric Analyses

Article

Full-text available

Nov 2022

The delimitation of Limonium taxa is highly complicated due to hybridization, polyploidy, and apomixis. Many “microspecies” were described and aggregated into groups, most of which are still poorly known from both molecular and morphological points of view. The aim of this study is to investigate four endemic species from the Tyrrhenian coast of central Italy and the Ponziane Archipelago belonging to the L. multiforme group (L. amynclaeum, L. circaei, L. pandatariae, and L. pontium) by means of molecular and morphometric analyses. Molecular data by sequencing ITS and three plastid markers and morphometric data highlight new information about the taxonomy of these taxa so as to reduce them into a single specific entity. In fact, the better taxonomic choice is to consider the populations studied as part of a single species, i.e., Limonium pontium. Three subspecies are recognized, i.e., subsp. pontium [= L. circaei = L. amynclaeum; from Circeo to Gianola localities (excluding Terracina) and from islands Ponza, Palmarola, Zannone, and Santo Stefano], subsp. pandatariae comb. et stat. nov. (from island of Ventotene), and subsp. terracinense subsp. nov. (from Terracina).

Remarks on the types of three accepted names in Limonium (Plumbaginaceae) described from the eastern Iberian Peninsula

Article

Feb 2021

The typification of three names in the genus Limonium published by Sandro Pignatti from the eastern Iberian Peninsula (L. confusum subsp. densissimum, L. lucentinum, and L. parvibracteatum) is discussed. The personal herbarium of Prof. Sandro Pignatti was originally deposited at TSB (Herbarium of the University of Trieste-Herbarium Universitatis Tergestinae, Friuli-Venezia region, NE Italy). However, all holotypes of the genus Limonium are no longer kept at the herbarium TSB, since S. Pignatti transferred them to the herbarium RO ("La Sapienza" University of Rome, Lazio region, C Italy) in the early 1980s. However, at RO there is only the holotype (or perhaps the lectotype) of L. confusum subsp. densissimum. Threfore, the names L. lucentinum and L. parvibracteatum are lectotypified using Pignatti's original specimens from GOET and RO, respectively.

The genus Limonium (Plumbaginaceae) in Tunisia: taxonomy, bio- geography and conservation Abstract

Article

Dec 2022

Plumbaginaceae) in Tunisia: taxonomy, biogeography and conservation.-Fl. Medit. 32: 261-277. 2022.-ISSN: 1120-4052 printed, 2240-4538 online. The recent increase in the known richness of Limonium in Tunisia, mainly due to the description of 25 new endemic species in the last decades, has made the only existing identification key for the country obsolete. Based on an exhaustive review of the literature dealing with Limonium from Tunisia, consultation of herbarium specimens preserved in Tunisia and Sicily, and a number of new collections, we propose here a new complete key, accompanied by an updated mapping for all taxa. The obtained results reveal that most Tunisian Limonium are perennial herbs (81 %), halophytes (79 %), endemic to the country (61 %), and mainly distributed along coasts and around chotts. A significant number of taxa (51 %) present important conservation issues, mainly related to microendemism (especially on islands and peninsulas) and to the alarming acceleration of habitat degradation. Djerba Island is currently the subject of a project involving local authorities, residents and environmental protection associations aimed at conserving the microendemic Limonium formosum, whose only location is threatened in the short term by a project to turn it into a tourist attraction.

Phylogenetic Analysis

Chapter

Sep 2021

In this chapter, the authors attempt to understand the underlying phylogeny principle and how researchers implement diverse methods to discover the appropriate phylogeny. Results obtained revealed that phylogenetic trees reflect evolutionary past as a canonical framework. Phylogenetic tree building step essentially comprises of five steps: (a) selecting molecular markers; (b) multiple sequence alignment; (c) determining the best evolutionary model; (d) determination of tree building method; and (e) assessment of tree reliability. Phylogenetic trees have various functional uses in different biological fields, such as conservation biology, epidemiology, forensics, cancer evolution, HIV transmission, gene expression prediction, protein structure prediction, and drug design. However, researchers face different challenges for generating a more accurate tree, like memory efficiency and implementation and optimization of the likelihood function. The authors believe, in the near future, the development of exciting new algorithms, which dramatically reduce the necessary amount of likeliness assessment, combined with enhanced knowledge of previously described high-performance machine problems in the group, is likely to detect more accurate phylogenetic tree that include 10,000–20,000 sequences. Additionally, it will also permit the tree inferences on medium-sized PC.

An integrated morpho‐molecular study of Salicornia ( Amaranthaceae‐Chenopodiaceae ) in Iran proves Irano‐Turanian region the major center of diversity of annual glasswort species

Article

Oct 2021

Salicornia (Chenopodiaceae) with ca. 25–30 annual, hyperhalophytic succulent species is a taxonomically difficult sub‐cosmopolitan genus. The taxonomic complexity arose from succulent shoots causing a lack of taxonomically useful characters, scarcity and variability of characters, and poor genetic differentiation. The Irano‐Turanian region, a major center for several lineages of chenopod diversification, is also an important diversity center and more likely the center of origin of annual Salicornia. This paper presents long‐term comprehensive morpho‐molecular and cytological studies of the genus in Iran, as the most species‐rich country in the Irano‐Turanian region. We originally generated 250 sequences of one nuclear (ETS) and two plastid markers (atpB‐rbcL spacer, intron rpl16). We analyzed a global ETS dataset including 257 sequences of putative 28 taxa using Bayesian inference and maximum likelihood analysis. In order to evaluate the application of plastid data in species taxonomy in annual Salicornia, we performed numerical morphological analysis (principal component analysis and agglomerative hierarchical clustering) of 186 Iranian accessions using 18 characters. Our results (1) confirm the efficiency of plastid sequences comparing to ETS sequences for clarifying species‐level phylogeny of Salicornia; (2) identify the S. persica clade as a monophyletic Irano‐Turanian endemic lineage; (3) recognize nine origins of the Irano‐Turanian Salicornia based on nuclear ETS sequences; (4) approve the monophyly of tetraploid species using plastid sequences; (5) provide a synopsis and diagnostic key for 10 Iranian taxa of Salicornia; (6) describe S. turanica sp. nov.; (7) classify S. iranica at infraspecific level (S. iranica subsp. iranica, S. iranica subsp. rudshurensis comb. nov., S. iranica subsp. sinus‐persica comb. & stat. nov.); (8) suggest three informal taxa to be studied in the future; and (9) discuss the threatened status of Salicornia‐dominated ecosystems in Iran.

Tasks for vegetation science

Chapter

Full-text available

Jan 1993

An account is given of the halophytic vegetation and flora of Iran. With respect to field studies the halophytic vegetation of Iran is classified into 10 vegetational units. Also, a total of 165 halophytic and salt tolerant species within 73 genera of flowering plants are recognized as a first provisional list of the halophytic flora of Iran. Some data on the life form, distribution, chorotype, halotype and photosynthetic pathway (if available) are given. Finally the role of man in destroying the halophytic vegetation is briefly discussed and a proposal is made to use some of the halophytes for soil improvement.

Stem Anatomy Is Congruent with Molecular Phylogenies Placing Hypericopsis persica in Frankenia (Frankeniaceae): Comments on Vasicentric Tracheids

Article

Aug 2003

Stem and root anatomy of Hypericopsis persica is evaluated in light of molecular data reconstructing Hypericopsis within a clade of Asian Frankenia. No anatomical information contradicts the idea that Hypericopsis should be subsumed within Frankenia. Anatomy in the two genera is comparable, taking into account the unusual habit of Hypericopsis, which consists of slender, short-lived shoots from a long-lived caudex, whereas most species of Frankenia are small shrubs with long-lived shoots. Wood of the slender stems of Hypericopsis is similar to twig wood of the related Frankenia hirsuta in qualitative and quantitative features but differs from mature wood of other species of Frankenia described in previous studies in having smaller cells and little storying. Wood of Hypericopsis is rayless and is made up mostly of libriform fibers and vessel elements associated with vasicentric tracheids. Axial parenchyma is occasional at the margins of growth rings. We briefly evaluate wood characters in the context of Caryophyllales s.l. and suggest characters that may be synapomorphies of clades within this group, e.g., minute lateral wall pits apparently characterize the clade comprising Plumbaginaceae, Polygonaceae, Frankeniaceae, and Tamaricaceae. That vasicentric tracheids elongate considerably beyond the lengths of the vessel elements with which they are associated is cited as a distinct ontogenetic difference between these cell types. Likewise, numerous vessel elements comparable in diameter to vasicentric tracheids were observed. These observations are offered as reasons to reject the concept of vasicentric tracheids as simply vessel elements that are too narrow for the formation of perforation plates.

The families and genera of vascular plants

Book

Jan 2014

K. Kubitzki

This volume presents systematic treatments for the families and genera of the Malpighiales, which more recently have been recognised as a new major group of the eudicots. Apart from several herbaceous lineages (already treated in Vol. IX of this series), the order consists mainly of rainforest trees, particularly those of the understorey. Accompanied by other early eudicot lineages, this reflects the well-documented origin of the group as invaders into the conifer-, cycad- and seed fern-dominated forests of the Cretaceous which, at that time, were transformed into the tropical rainforest biome. In this volume, 24 families with 429 genera comprising over 12,000 species are treated. Many of these belong to the vast family of the Euphorbiaceae (here conceived in a broader sense), followed by the Violaceae, whereas some of the remaining families are very small and even relictual. The revised classification includes a complete inventory of the genera belonging to the families treated in this volume, along with their diagnostic features and keys for their identification. References to the latest taxonomic literature and links to many different disciplines important to modern plant systematics make the volume a valuable source of information on the manifold aspects of plant diversity.

CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP

Article

Jul 1985
EVOLUTION

Joseph Felsenstein

The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.

Limonium Miller

Article

Jan 1982

A new species Limonium adilgunerii Yıldırımlı & A. Doğru-Koca (Plumbaginaceae) from edge of Tuzgölü, Inner Anatolia, Turkey

Article

Jan 2006

New species and interesting plant records from Iran

Article

Jan 1992

V. Mozaffarian

Sabkha Ecosystems Vol.II: West and Central Asia

Book

Jan 2006

Sabkha Ecosystems Volume I: The Arabian Peninsula and Adjacent Countries was published in 2002. It was the first comprehensive volume dealing with the subject of sabkha research, and sabkha environmental management. Valuable new information was provided for the sabkha of numerous countries on and adjacent to the Arabian Peninsula. This new volume now follows up on this important process, and provides data and information on salt desert ecosystems of numerous West and Central Asian countries, including many of which are located in the Arabian Peninsula. The information provided assists the reader to better understand sabkha geology, hydrology, geomorphology, zoology, botany, ecology, ecosystem functioning, as well as sabkha conservation, utilisation, and development. The volume is paramount literature for anyone dealing with sabkha research and development.

Limonium gueneri (Plumbaginaceae), a New Species from Turkey

Article

Oct 2008

Limonium gueneri Dogan, Duman & Akaydın sp. nova (Plumbaginaceae) is described and illustrated from Patara (C2 Antalya, Turkey), where it grows on calcareous slopes on the coast. It is probably closely related to L. ocymifolium (Poir.) Kuntze, an East Mediterranean species. The diagnostic morphological characters are discussed. A taxonomic key for the coastline species of Turkish Limonium without sterile branches is also presented along with some notes on the ecology and biogeography of the new species.

Flora of Turkey and The East Aegean Islands

Article

May 1989

Phylogenetics of the Irano-Turanian taxa of Limonium (Plumbaginaceae) based on ITS nrDNA sequences and leaf anatomy provides evidence for species delimitation and relationships of lineages

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