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

Hossein Akhani; Maryam Malekmohammadi; Parastoo Mahdavi; Arootin Gharibiyan; Mark W. Chase

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...

The names Statice cumana (currently Limonium cumanum), S. inarimensis (≡ L. inarimensis), and S. remotispicula (≡ L. remotispiculum) are lectotypified on specimens preserved at NAP and FI. These species are endemic to the south Tyrrhenian coasts (SW Italy), two of them (L. cumanum, and L. inarimensis) being narrow endemic to Campania region. A nomenclatural change (Limonium cumanum var. glabrescens, comb. nov.) is also proposed.

The absence of the cosmopolitan genus Limonium Miller (1754: no pagination) (Plumbaginaceae) in the mega-diverse flora of the Malesian region (comprising Brunei Darussalam, Indonesia, Malaysia, Papua New Guinea, the Philippines, and Singapore; Merrill 1923, Ridley 1923, Steenis 1949, Backer & Bakhuizen van den Brink Jr. 1965, Balgooy 1993, Coode et al. 1996, Balgooy 2001, Conn et al. 2004, Chong et al. 2009, Pelser et al. 2011) has intrigued taxonomists and biogeographers since Steenis (1949) noted this. During a field survey as part of the project on floristic and phylogenetic biogeography in the island chain of the Philippines, Taiwan, and southern Japan, we found a population of Limonium in the northern Philippines (Batan Islands) that represents the first record of the genus in the Malesian region. Batan Islands, comprising 10 small (≤ 83.1 km2) oceanic islands, is the northernmost tip of the Malesian region, being ca. 190 km north of Luzon Island of the Philippines and ca. 140 ...

bs_bs_banner Botanical Journal of the Linnean Society, 2013, 171, 519–550. With 46 figures HOSSEIN AKHANI1*, MARYAM MALEKMOHAMMADI1, PARASTOO MAHDAVI1, AROOTIN GHARIBIYAN1 and MARK W. CHASE2 1 Department 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 2 Jodrell 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. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550. 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 & Doğru-Koca, 2006; Dogan, Duman & Akaydin, 2008) and in the Flora Iranica area (including Iran, Afghanistan, western *Corresponding author. E-mail: akhani@khayam.ut.ac.ir Pakistan and adjacent areas of Iraq and Turkmenistan) 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 Mediterranean area. There are also several genera related to Limonium that have representatives in the IranoTuranian area, but these have been subject to different generic interpretations in taxonomic treatments; © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 519 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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 520 H. AKHANI ET AL. MATERIAL AND METHODS MORPHOLOGICAL 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 dehydrated through an ethanol series and xylene. The dehydrated samples were permanently mounted in Euparal. Epidermal characters were studied on herbarium 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 H2O2 for 2 h. They were then thoroughly washed with water and placed in ‘Eau de Javelle’ for bleaching. 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. AND TAXONOMICAL STUDIES Natural populations of these species have been investigated 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 following Index Herbariorum, (Thiers, 2009) and the private herbarium of the first author (Hb. Akhani, currently housed in the Halophytes and C4 Plants Research 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) © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 these include: Cephalorhizum Popov & Korovin, Dictyolimon Rech.f., Aeoniopsis Rech.f., Chaetolimon Lincz., Eremolimon Lincz., Goniolimon Boiss., Ikonnikovia Lincz. Popoviolimon Lincz., Neogontscharovia Lincz., Bamiania Lincz., Bukiniczia Lincz. and Vassilczenkova 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 distribution ranging from Turkmenistan, through Kazakhstan, Kyrgyzstan and Tajikistan, to northern China in Gansu and northern Xinjiang (Linczevski, 1952; Pen & Kamelin, 1996). This species has been introduced into North America (Hickman, 1993; Smith, 2005). Limonium reniforme in its former circumscription has a similar range but extends into southcentral 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 transcribed 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 identification key and distribution maps. Sunken Sunken At surface Deeply sunken At surface At surface At surface At surface At surface At surface At surface Sunken Deeply sunken Sunken Sunken At surface Sunken At surface At surface At surface At surface At surface At surface At surface Sunken Sunken Entire Entire Wavy Entire Entire Entire Wavy Sinuous Sinuous Sinuous Wavy Entire Entire (39) (20) (21) (22) (23) (22) (26) (16) (18) (19) (27) (21) (16) 40.66 ± 1.52 (42) 21.5 ± 1.29 (23) 24.62 ± 2.87 (29) 24.66 ± 2.59 (29) 26.85 ± 2.47 (31) 26.16 ± 5.78 (41) 31.89 ± 2.65 (35) 22.75 ± 3.77 (27) 20.2 ± 2.4 (24) 22.25 ± 2.81 (24) 29 ± 1.41 (31) 21.6 ± 0.89 (23) 25.3 ± 8.73 (36) (36) (24) (18) (21) (20) (15) (26) (13) (20) (27) (38) (15) (13) Entire Entire Wavy Entire Entire Entire Wavy Wavy Sinuous Sinuous Wavy Entire Entire L. axillare L. carnosum L. caspium L. iranicum L. lobatum L. gmelinii s.l. L. nudum L. otolepis L. perfoliatum L. reniforme L. sogdianum L. stocksii L. suffruticosum Adaxial Adaxial Abaxial 39.6 ± 3.04 (43) 25.75 ± 2.21 (29) 19.8 ± 1.78 (22) 26.28 ± 3.4 (31) 21 ± 1.01 (23) 24.5 ± 7.27 (36) 27.33 ± 1.15 (28) 17 ± 4.61 (21) 21.77 ± 1.56 (25) 27.5 ± 0.7 (28) 42.4 ± 3.04 (46) 16 ± 0.81 (17) 20 ± 6.48 (32) Similar Similar Similar Similar Smaller Smaller Similar Similar Similar Similar Similar Similar Similar Similar Similar Longer Similar Longer Longer Longer Longer Longer Longer Longer Similar Similar Salt gland level Stomata level Cells around salt glands/ epidermal cells Subsidiary cells/epidermal cells Abaxial Stomata and salt glands: Stomata in all species are anisocytic with three subsidiary cells; rarely in Species DESCRIPTIONS Stomatal density (500 ¥ 500 mm) ANATOMICAL 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. sogdianum (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. lobatum (L.f.) Kuntze (Table 1, Figs 5–9). Epidermal cells wall RESULTS 521 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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 Terminator Kit ver. 3.1 (Applied Biosystems Inc., ABI, Warrington, 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) following the manufacturer’s protocols. Cleaned products were then sequenced on an ABI 3730 Genetic Analyser following the manufacturer’s protocols. Sequencing output files were edited and contigs assembled either by using Sequencher 4.0 (Genecodes, 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 parsimony method was used; otherwise the BIONJ method with Markov linkage clustering (MCL) distance 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. 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 PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 522 H. AKHANI ET AL. L. axillare, L. carnosum, L. iranicum, L. perfoliatum (C.A.Mey.ex Boiss.) Kuntze, L. reniforme, L. sogdianum 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, stomatal 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Figures 1–9. 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. PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 523 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 Palisade tissue layers Position of sclerenchyma cells Abaxial Adaxial L. axillare L. carnosum L. caspium L. iranicum 4 2 (midvein) 3 (blade) Absent 2 (midvein), 3 (blade) 3 2 (midvein), 3 (blade) 1 2 (midvein), 3 (blade) L. lobatum L. gmelinii L. nudum L. otolepis L. perfoliatum L. reniforme L. sogdianum L. stocksii L. suffruticosum Absent Absent Absent Absent Absent Absent 0 (midvein), 3 (blade) 3–4 layers 2 (midvein), 3 (blade) 2 1–2 (midvein*), 3–4 (blade) 1 (midvein), 2 (blade) 0 (midvein), 1 (blade) 1–2 1–2 3 (midvein), 4 (blade) 3 layers 2 layers P (+), SP (±), S (-) P (±), PS (+) Absent PS (+), penetrating sometimes to palisade Absent P (+), midvein (very rare) Absent Absent Absent Absent P (±), PS (+) PS (+) P (±), PS (+) Sclereid type Fusiform Fusiform Absent Fusiform Absent Ramiform Absent Absent Absent Absent Fusiform Fusiform Fusiform *Short palisade-like cells in midvein. 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 occurring 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 isobilateral 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. carnosum, 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 complete 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. perfoliatum 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 discontinuous 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 welldeveloped 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- © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Species 524 H. AKHANI ET AL. 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. carnosum, 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 classified into two main groups using terminology suggested 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 vertically 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. suffruticosum (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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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. PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 525 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 characterized 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). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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. 526 Palisade tissue layers Species Abaxial Adaxial Arrangement of vascular bundles (VB) L. axillare L. carnosum 3–4 2 (midvein), 3 (blade) L. caspium 4 2 (midvein), 3 (blade) 0 L. gmelinii 0 L. iranicum L. otolepis 2 (midvein), 3 (blade) 0 0 (midvein), 2 (blade edges) 0 0 L. perfoliatum 0 0 L. reniforme 0 0 L. sogdianum 0 2–3 L. stocksii L. suffruticosum 3 3 2 (midvein), 3 (blade) 2 3 large VB in centre, some smaller VB in spongy tissue 1 large VB, smaller VB in line with the main and dispersed in spongy tissue 3 large VB, small intercalary VB occur between them on the same line Irregularly distributed in central part, secondary veins are horizontally derived from central bundles 1 large VB, smaller VB in line with the main and dispersed in spongy tissue 1 large, small VB dispersed in spongy tissue 3 large VB, small intercalary VB occur between them on the same line 3 large VB, small intercalary VB occur between them on the same line 3 large VB, small intercalary VB occur between them on the same line 3 large VB, small intercalary VB occur between them on the same line 1 large VB, smaller VB occur on the same line and are distributed on adaxial side of spongy tissue 3 large VB in centre, some smaller VB in spongy tissue 1 large VB, smaller VB in line with the main and dispersed in spongy tissue 0 (midvein), 2 (blade), 3 (edges) 2 (midvein), 3 (blade) 2 PL 2 P (+), PS (±), S (–) P (±), PS (+) Absent P (+), PS (rare), S (+) PS (+), penetrating sometimes to palisade Absent Mostly absent, rarely occur in S Mostly absent, rarely occur in S Mostly absent, rarely occur in S Mostly absent, rarely occur in S P (–), PS (±), S (+) (only on adaxial side) P (–), PS (+), S (±) P (–), PS (+), S (–) Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 L. lobatum L. nudum 0 Position of sclerenchyma cells H. AKHANI ET AL. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 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 PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 527 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Figures 25–30. See caption on next page. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 528 H. AKHANI ET AL. 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. PHYLOGENETICS New ITS sequences were provided in this study for 74 accessions belonging to 19 Limonium spp., one Cephalorhizum sp. and two Psylliostachys spp. Sequences of eight species were included from GenBank (accession numbers are provided in the Appendix). After trimming the ends of the matrix, the analysis involved 847 characters including 397 variable sites, among which 300 sites were potentially parsimony informative. 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 Limonium) form a strongly supported clade (BP 99). The genera Cephalorhizum and Psylliostachys (outgroups) form a well-supported clade (BP 95). Internal relationships are largely resolved with five clades: (1) the L. axillare clade, sister to all other clades; (2) a moderately supported clade (BS 81) comprising species of section Limonium subsections Dissitiflorae Boiss. and Steirocladae Boiss. and L. echioides (subgenus Limonium section Schizhymenium) marked as ‘Mediterranean 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 Limonium subsection Hyalolepidae, L. sogdianum (section Siphonocalyx) and L. nudum (section Platyhymenium), 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 subclade (BP 90). DISCUSSION PHYLOGENY Our ITS tree includes almost all Limonium spp. in Iran, except L. stocksii (Rechinger & SchimanCzeika, 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 southwestern 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 splitting the genus into smaller genera, such as Eremolimon, as proposed by Linczevski (1979). Additionally, the independent status of Cephalorhizum and Psylliostachys as distinct from Limonium is strongly supported. However, additional sequences are required to clarify the relationships of some monotypic and oligotypic genera from Central Asia and Afghanistan, such as Dictyolimon, Aeoniopsis, Chaetolimon, Ikonnikovia, 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 distinctive winged stems comprises c. 15 species of © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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). Ramiform 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 OF IRANO-TURANIAN LIMONIUM L iranicum 3986 L iranicum 3826 L. iranicum L iranicum 15760 L iranicum 18851 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 90 L iranicum 58 L iranicum 16499 L iranicum 4813 L iranicum 3975 L. suffruticosum L iranicum 20820 L carnosum 18997 68 subclade L carnosum 18891 96 L carnosum 15625 71 L carnosum 18894 67 L carnosum 18904 L anatolicum 17999 L suffruticosum 8108 91 Limonium Type L suffruticosum 40226 L suffruticosum 35188 clade 54 L suffruticosum 10160T L suffruticosum 12277 L suffruticosum 20364 88 L suffruticosum 20382 L suffruticosum 28088 L suffruticosum 17282 66 L suffruticosum 41 L lilacinum 17930 L gmelnii3 1792 L gmelimi 10637 69 L gmelinii 12229 Core Limonium L gmelinii 18948 58 L narbonense AJ222838 78 L vulgare AJ222839 76 L latifolium 20504 L. vulgare subclade 57 L gmelinii 19013 L gmelinii 18996 L gmelinii 18887 61 L gmelinii1 5636 L gmelinii 18852 L gmelinii 15740 54 L gmelinii 3827 L gmelinii 3825 L sogdianum 19055 L nudum 18804 53 L caspium 18888 62 L bellidifolium 20511 99 L iconium 17961 73 L iconium 17921 61 L iconium 18011 77 L reniforme 3970 97 L reniforme 30.3.2004 97 L reniforme 66965 L. reniforme clade 51 L otolepis 20367 L otolepis 3897 75 L otolepis 10185 80 50 L perfoliatum 10057 66 L perfoliatum 10130 99 L perfoliatum 24567 79 L perfoliatum 18815 L perfoliatum 18808 62 L perfoliatum 10169T L echioides AJ222861 L delicatulum AJ222851 81 L furfuraceum AJ222856 Mediterranean clade 96 L cf.cumanum 16815 96 L minutum AJ132332 L axillare 3974A L. axillare clade 96 L axillare 3974 L arborescens EU410356 Pteroclados clade 100 L lobatum AJ132333 56 L lobatum 3978 Cephalorhizum turcomanicum 28978 Psylliostachys spicata 14878 Outgroup 100 Psylliostachys leptostachys 57 529 51 0,001 Figure 31. See caption on next page. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 94 530 H. AKHANI ET AL. 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. 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. bellidifolium 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 epidermal 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. perfoliatum 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 anatomically 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) transferred 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 IranoTuranian/Mediterranean species (L. caspium, L. bellidifolium 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). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 annual herbaceous to shrubby species distributed from the Macaronesian to the Mediterranean and the Saharo-Sindian floristic regions (Karis, 2004). Anatomically, 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 phylogenetics 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 anatomical feature separating L. axillare and L. stocksii from other species already classified in section Sarcophyllum 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). Furthermore, the leaf lamina has three main veins and petioles are flattened. In L. iranicum, L. suffruticosum 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. Frequent apomixis and hybridization in this clade are further complications for resolving their phylogenetic relationships (Palacios et al., 2000; Lledó et al., 2005). Species/characters L. otolepis L. reniforme L. perfoliatum Height Sterile branches Cauline leaves position 40–50 (–90) cm Sterile branches numerous Only occurring on the lower nodes of main stem, rarely 1–2 small leaves present on nodes of lateral branches 0.5–1.5 ¥ 0.8–1.5 cm Entire or slightly undulate 17–30 cm 4–9 1(–2) 0.5–1.0 ¥ 1.0–1.5 mm (0–)0.5 ¥ 0–1 mm 35–70 (–90) cm Some lower branches sterile Present on all nodes of main and lateral branches 50–95 (–150) cm Without sterile branches Present on all nodes of main and lateral branches 0.5–2.5(–3.5) ¥ 0.5–2.0(–3.0) cm Entire 10–25 cm (1–)4–6 (1–)2 0.5–1.0 ¥ 1.0–1.5(–1.7) mm (0–) 0.5–0.7 mm ¥ (0–) 0.5–0.7 (–1.2)mm (0.7-)1.0–1.5 ¥ 1.0–1.2 mm Membranous with one or two brown nerves (1.5-)2.0–2.5 ¥ 2.0–2.5 mm (1.0–) 1.5-2 mm Elliptic or orbicular rarely rhombic 1.8–3.4 ¥ 1.2–2.2 cm Entire and undulate 24–70(–130) cm (5–)7–18 (2–)3 1.0–1.5(–1.7) ¥ 1.0–1.5(–1.7) mm 1.0–1.5 mm ¥ (0.2) 0.5–0.7 (1.0) mm 1.0–1.5 ¥ 1.0–1.5 mm Membranous usually with 1–2 narrow brown nerves 2.5–3.0(-3.5) ¥ 2.0–2.5 mm (1.7–)2.0–2.5(–2.7) mm Elliptic or broadly elliptic, rarely obovate (1–)3–5(–8) Funnel form (2.5–)3.5 mm Dense or sometimes lax long hairs on two or three of nerves, other nerves glabrous Cauline leaf size Cauline leaf margin Length of inflorescence No. of spikes per cyme No. of flowers per spike Outer bract size Length of herbaceous part of outer bract Middle bract length Middle bract colour 0.5–1.0 mm ¥ 0.5–1.0 Membranous, without nerve Inner bract length Inner bract length of herbaceous part Bracteole shape 1.5–2.0 ¥ 2.0–2.5 mm 1 mm Obovate No. of bracteoles per spike Calyx shape Calyx length Calyx indumentum (0–)1 Obconical 2.0 mm Mostly with short or sometimes sparse long hairs on the basal 0.7 mm, hairs often confined to one side Calyx diameter at apex Calyx nerve Length of calyx lobes Achene shape Epidermal cells of leaf abaxial side SD* (abaxial) SD* (adaxial) SG* (abaxial) SG* (adaxial) 1.2–1.7 mm 1.5 mm 0.5 mm Obovate Wavy 30.28 ± 5.28 22.80 ± 5.77 3.16 ± 0.4 3.00 ± 0.87 2–5(–7) Obconical 2.5–3.0 mm 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 (1.2–)1.5(–1.7) mm 2.0–2.5 mm 0.5–0.7 mm Fusiform Sinuous 16.33 ± 2.88 20.33 ± 1.15 3.66 ± 0.57 3.33 ± 0.57 2.0–3.0 mm (2.0–)2.2–2.5(–3.0) mm (0.5–)0.7–1.0 mm Obovate Sinuous 25.77 ± 1.39 29.50 ± 2.25 4.55 ± 0.52 6.16 ± 1.47 PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 531 * 500 ¥ 500 mm. Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 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 532 H. AKHANI ET AL. 32. L. lobatum 33. L. axillare 34. L. stocksii 40N 40N 40N 35N 35N 35N 30N 30N 30N 50E 55E 60E 65E 45E 50E 55E 60E 65E 45E 36. L. nudum 35. L. sogdianum 40N 40N 35N 35N 35N 30N 30N 30N 50E 55E 60E 65E 45E 38. L. otolepis 50E 55E 45E 60E 39. L. perfoliatum (dot) and L. reniforme (triangle) 40N 40N 35N 35N 35N 30N 30N 30N 50E 55E 60E 65E 45E 41. L. iranicum 50E 55E 60E 65E 45E 42. L. suffruticosum 40N 40N 35N 35N 35N 30N 30N 30N 50E 55E 60E 65E 45E 50E 55E 65E 50E 55E 60E 65E 50E 55E 60E 65E 60E 65E 43. L. gmelinii s.l. 40N 45E 60E 40. L. carnosum 40N 45E 55E 37. L. caspium 40N 45E 50E 60E 65E 45E 50E 55E Figures 32–43. Distributions of Limonium species in Iran. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 45E PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM subclade (BP 69) includes broad-leaved hemicryptophyte and hygrohalophytic species, with a distribution 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 characterized 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 dispersed 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 distinguished by minor differences: the former with congested 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 morphologically 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 microspecies 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 distinguished: one includes all samples of L. suffruticosum, 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 In one cluster, all six samples of L. perfoliatum are grouped into two smaller sets each having three representatives. 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 northeasternmost 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 compatible 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. perfoliata) 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 synonymized both names (Boissier, 1879). Ledebour (1849) also supported recognition of S. reniformis. In the standard floras of this area, these names have variously been used. Boissier (1879) accepted the name Statice perfoliata C.A.Mey. and considered S. reniformis 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 populations 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 533 534 H. AKHANI ET AL. BIOGEOGRAPHICAL REMARKS The sampling of Limonium from the Middle East in this paper is not sufficient to give a detailed phylogeographical 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 conspecific 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 distribution 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 representative of section Pteroclados subsection Odontolepidae (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 distribution. 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 salviifolius L., Caucalis platycarpos L., Convolvulus cantabrica 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 trilobum (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. stocksii 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 L. carnosum are widespread in Iran. They occur in almost all saline depressions and salt marshes of the Persian Gulf. Morphologically, these species are separable based on the treatment of Flora Iranica (see key below). Two other previously described species belonging 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 morphological features, L. gabrielii could merit subspecific rank. Limonium failachicum is close and morphologically 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 Limoniumtype clade, L. sogdianum belonging to the L. reniforme 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 examples (Figs 27, 29, 30). Isobilateral leaf anatomy is another homoplastic character occurring in three clades of Iranian Limonium (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). PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM KEY 1. 1. 2. 2. 3. 3. 4. 4. 5. 5. 6. 6. 7. 7. 8. 8. 9. 9. 10. 10. 11. 11. 12. 12. TO SPECIES OF 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 southwestern 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 recircumscribed in this paper. LIMONIUM IN IRAN Annual herbs; stem and flowering branches with wing...........................................................1. L. lobatum Perennial herbs or shrublets; stem and branches without wing................................................................2 Perennial herbs; leaves in a rosette, with or without cauline leaves..........................................................3 Shrublets; leaves mostly cauline.........................................................................................................9 Plants with many well-developed cauline, ± fleshy, rotundate–reniform and amplexicaul leaves ..................... 4 Plants without cauline leaves, often with scale-like reduced leaves on nodes..............................................6 Stem without sterile branches; cauline leaves undulate at margins, basal cauline leaves auriculate................. ................................................................................................................................8. L. perfoliatum Stem with sterile branches; cauline leaves entire, not auriculate..............................................................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 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 Sterile branches numerous, slender, occurring along the lower 2/3 of the plant height; inflorescence corymbosepaniculate; spikelets up to 5.5 mm long...............................................................................6. L. caspium Plants without sterile branches; combination of characters not as above....................................................7 Basal leaves small, spathulate, up to 7 cm long; inflorescence subcapitate or spicate ................................... 8 Basal leaves large, obovate or spathulate–obovate, 8–38 cm long; inflorescence much branched, paniculate ....... ...............................................................................................................................13 L. gmelinii s.l. 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 Inflorescence laxly spiciform, spikelets at intervals along spike; calyx tubular, scarious parts of calyx whitish; bracts glabrous............................................................................................................4. L. sogdianum Flowering branches elongate, often longer than half of the plant height; leaves with single midrib...............10 Flowering branches short, often shorter than one third of plant height; leaves with three main veins...........12 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 Leaves without auricles; inflorescence paniculate; spikelets occurring on lateral and terminal cymes ............ 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 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 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 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 cultivation indicate that these species have a continuous life cycle over the year. The presence of thick cuticle and sunken chalk glands are morphological traits that occur in L. axillare and L. stocksii 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. 535 536 H. AKHANI ET AL. 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. 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 Hormozgan and Bushehr Provinces (Fig. 33). Based on available 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. wendelboi 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 Salsuginosis Prov. Scinde Indiae Borealis, Stocks (K!, G, W!). 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, Turkmenistan), 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 [basionym]. SYNTYPES: Iran: Albursgebirge bei Radkann, Juli 1848; bei der Mineralquelle Gendäb auf steinigem Kalkboden in grosser Menge, 28 Juli 1848. Montibus 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Distribution: Southern Spain, southern Greece, northern Africa, Syria, Palestine, Saudi Arabia, Iraq, southern Iran (Bushehr, Fars, Hormozgan, Khuzestan provinces, Fig. 32). Distribution: Afghanistan, western Pakistan and south-eastern Iran (Sistan-o Baluchestan Province, Fig. 34). See notes under L. axillare. PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 537 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.). 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. bellidifolium auct. non (Guan) Dumort. Additional specimens examined from outside Iran: KAZAKHSTAN: Golodnaja Steppe, 9.1915, Dimo and Popov (TARI). TURKMENISTAN: Charshanga District, 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.). Distribution: Central Asia, Dsungaro-Kashgar, Mongolia, Turkey and Iran (restricted to the surrounding area of the Uromieh Lake in eastern Azerbaijan, Fig. 37). Note: This species is known in Iran only from cultivated 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). Note: L. bellidifolium and L. caspium have been variously interpreted in many taxonomic treatments of the Irano-Turanian and Mediterranean areas. Several authors considered L. caspium as a synonym of L. bellidifolium 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. bellidifolium 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 locality 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 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 diameter, 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 cavities, 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 including 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, © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 unlikely that two specimens bear the same number. Based on personal communication by Fernand Jacquemoud (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 Azerbaijan, near Jolfa is documented by a specimen collected by T. Heideman & H. Alive on 27.5.1932 (HAM). 538 H. AKHANI ET AL. Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 539 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 540 H. AKHANI ET AL. 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). Khorassan: 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 (northeast 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). General distribution: Afghanistan, Iran (Golestan and Khorassan Provinces, Fig. 39), Turkmenistan and Uzbekistan. 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) 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- Additional specimens examined outside Iran: TURKMENISTAN: 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, Sejfulin s.n. (ASH, Hb. Akh.). Note: L. reniforme, L. perfoliatum and L. otolepis are related Irano-Turanian species characterized by perfoliate leaves. See Table 4 for distinguishing characters 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, AucherEloy 5246 (LECTOTYPE: P!, ISOLECTOTYPE G!, K!, W). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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, glabrous, 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 membranous 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, glabrous, 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. PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 541 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 542 H. AKHANI ET AL. 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 Fengnnek near Schiraz, 29.6.1885, Stapf 2172 (B); Bakhtegan 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 Ghalatooyeh, 55°14′N, 28°14′E, 750 m, 30.3.2004, Khosravi & 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 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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; semiamplexicaul 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–) twoflowered. Outer bracts triangular, or rarely semicircular, 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 membranous band, sometimes without a fleshy part and completely membranous, brown, glabrous, entire, acute, rarely truncate at apex, semiamplexicaul, or amplexicaul 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, semiamplexicaul 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 truncate 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 PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM Frankenia L. (Olson, Gaskin & Ghahremani-nejad, 2003). Salicornia persica Akhani and S. perspolitana Akhani are other species described from the same area (Akhani, 2008). 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 funnelform 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 distinguish L. carnosum from L. iranicum by Assadi (2005) cannot be confirmed here. Several specimens, including 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 Ghahavand in Hamedan and Semnan Provinces (Assadi 61097, Assadi & Hamdi 85415) were misidentified as L. carnosum by Assadi (2005). Specimens with a glabrous 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. Provinces, 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. suffruticosum. 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. Khorassan 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 inflorescence and longer life cycle. Because of the arid tropical climate of southern Iran and absence of low winter temperatures, many plant species including L. iranicum 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. iranicum, 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, southeastern Russia, Siberia, Turkmenistan. Note: Limonium suffruticosum extends from Central Asia through eastern parts of Iran (Fig. 42). The southernmost populations of L. suffruticosum collected 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, © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Limonium carnosum (Boiss.) Kuntze Rev. Gen. Pl. 2: 395 (1891). Statice carnosa Boiss. in DC. Prod. 12: 663. 1848 [basionym]. TYPE: Aderbidjan [Azerabaijan], Persiae bor. Prope Khoi, AucherEloy 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. 543 544 H. AKHANI ET AL. 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. 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, as in the Flora 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). Furthermore, younger plants have looser inflorescences than older plants. Boissier (1848), who originally described L. meyeri, later reduced it in Flora Orientalis (Boissier, 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 characterized by hairs on the calyx only on the basalmost 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 characterized 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 IranoTuranian area using detailed morphological and 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 University 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 Humboldt 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. REFERENCES Akhani H. 2008. Taxonomic revision of the genus Salicornia L. (Chenopodiaceae) in central and southern Iran. Pakistan Journal of Botany 40: 1635–1655. Akhani H, Deil U. 2012. First observations of the flora and vegetation of three islands in the NW Persian Gulf (Iran). Phyton-Annales Rei Botanicae 52: 73–99. Akhani H, Ghorbanli M. 1993. 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Khan MA, Böer B, Kust GS, Barth H-JE. 2006. Sabkha ecosystems, volume II: west and Central Asia. Berlin: Springer. Kubitzki K. 1993. Plumbaginaceae. In: Kubitzki K, ed. The families and genera of vascular plants. Berlin: Springer, 523–530. Ledebour CF. 1849. Flora Rossica. Stuttgart: E. Schweizerbart. 545 546 H. AKHANI ET AL. Van den Berg C, Higgins WE, Dressler RL, Whitten WM, Soto Arenas MA, Culham A, Chase MW. 2000. A phylogenetic analysis of Laeliinae (Orchidaceae) based on sequence data from nuclear internal transcribed spacers (ITS) of ribosomal DNA. Lindleyana 15: 96–114. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 315–322. Yıldırımlı Ş, Doğru-Koca A. 2006. A new species Limonium adilguneri Yıld. & Doğru-Koca (Plumbaginaceae) from edge of Tuzgülü, Inner Anatolia Turkey. Ot Sistematik Botanik Dergisi 13: 11–16. Zohary M. 1973. Geobotanic foundation of the Middle East. Amsterdam: Gustav Fische. 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) Cephalorhizum turcomanicum Pop. Sections or subsections of later authors Voucher and/or origin and [DNA accession no.] 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]. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 APPENDIX PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 547 APPENDIX Continued Name Boissier’s sections and subsections (sub Statice) Sections or subsections of later authors Limonium cf. cumanum (Ten.) Kuntze Voucher and/or origin and [DNA accession no.] Italy: Naples (Napoli), south-east of Naples, c. 10 km south-west of Salerno, Amalfi, 11.v.2003, Akhani 16815 [JX983717]. 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 Şereflikochisar 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]. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 L. delicatulum (Girard.) Kuntze 548 H. AKHANI ET AL. APPENDIX Continued Name Boissier’s sections and subsections (sub Statice) Sections or subsections of later authors L. iranicum (Bornm.) Lincz. L. subsect. Sarcophyllae Sarcophyllum† Voucher and/or origin and [DNA accession no.] © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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]. PHYLOGENETICS OF IRANO-TURANIAN LIMONIUM 549 APPENDIX Continued Name Boissier’s sections and subsections (sub Statice) L. lilacinum (Boiss. & Bal.) Wagenitz Sections or subsections of later authors Voucher and/or origin and [DNA accession no.] 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] © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 Sphaerostachys‡ Turkey: Ankara: 28 km north of Şereflikochisar 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]. 550 H. AKHANI ET AL. APPENDIX Continued Name Boissier’s sections and subsections (sub Statice) Sections or subsections of later authors L. suffruticosum Kuntze L. subsect. Sarcophyllae Sarcophyllum† L. vulgare Mill. L. subsect. Limonium 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] Voucher and/or origin and [DNA accession no.] Palacios et al., 2000: Spain: Cantabria (AJ222839) †Rechinger & Schiman-Czeika, 1974. ‡Bokhari, 1973. §Palacios et al., 2000. ¶Linczevski, 1985. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 519–550 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/3/519/2416255 by guest on 04 June 2020 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].

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