Flora 206 (2011) 957–973 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.de/flora Seed and gland morphology in Euphorbia (Euphorbiaceae) with focus on their systematic and phylogenetic importance, a case study in Iranian highlands Yasaman Salmaki a,b , Shahin Zarre a,∗ , Hans-Joachim Esser c , Günther Heubl b a Department of Plant Biology, School of Biology, College of Science, University of Tehran, PO Box 14155-6455, Tehran, Iran Department Biologie I, Systematische Botanik, Ludwig-Maximilians-Universität München, Menzinger Str. 67, 80638 München, Germany c Botanische Staatssammlung München, Menzinger Str. 67, 80638 München, Germany b a r t i c l e i n f o Article history: Received 5 December 2010 Accepted 19 April 2011 Keywords: Caruncle Euphorbia Euphorbiaceae Gland Phylogeny Seed micromorphology Subgen. Esula Systematics Iranian highlands a b s t r a c t A comprehensive study based on gland and seed micromorphology in Euphorbia (Euphorbiaceae) for species distributed in Iranian highlands is presented. A total of 86 species were studied. The gland structure was examined by direct field observations. Taxonomically important characters of glands were observed and measured: size, texture, shape, color, and horns. For species out of Iran herbarium materials were studied. Seed characteristics were examined using scanning electron microscopy (SEM) as well as dissecting light microscopy. Significant features are: seed size, seed shape, presence of caruncle, shape of caruncle, and seed surface ornamentation. A phylogenetic study using Maximum Parsimony (MP) and Bayesian Inference (BI) was performed based on sequences of nuclear DNA internal transcribed spacers (ITS) for selected species representing the main clades known in Euphorbia and with special focus on the species distributed in Iranian highlands. ITS sequences for 20 accessions representing 19 species are provided for the first time, and 48 accessions of 47 species were used from GenBank. The topologies of both analyses were congruent. The results indicate: (1) four main clades with high supports in subgen. Esula which are appropriate to be recognized at sectional rank. (2) E. larica is nested within clade A including few members of subgen. Rhizanthium and is closely related to sect. Balsamis, which is suggested here to be transferred from subgen. Esula into subgen. Rhizanthium. (3) E. osyridea of the monotypic subsect. Osyrideae is closely related to E. buhsei and to the members of sect. Esula. Tracing morphological characters on the phylogenetic tree shows that several morphological characters, such as seed ornamentation applied in previous subgeneric classification of the subgen. Esula, are homoplasious, but the gland structure and capsule surface characters are more reliable for classification purposes. © 2011 Elsevier GmbH. All rights reserved. Introduction Euphorbia L., one of the most diverse genera of flowering plants, and the largest genus of the Euphorbiaceae, includes about 2000 species (Govaerts et al., 2000; Radcliffe-Smith, 2001). The genus has a nearly cosmopolitan distribution and is extremely variable in life form consisting of annual as well as perennial herbs, shrubs, succulents, lianas and trees. The Iranian highlands, an area including Iran, Afghanistan, W Pakistan, N Iraq, Azerbaijan, and Turkmenistan, house about 95 Euphorbia species (Rechinger and Schiman-Czeika, 1964, and additions after that including: Nasseh and Joharchi, 2004; Nasseh et al., 2006; Pahlevani, 2006, 2007; Pahlevani and Riina, in press). These species grow under various habitats such as ∗ Corresponding author. E-mail address: zarre@khayam.ut.ac.ir (S. Zarre). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2011.07.005 deserts (e.g., E. petiolata1 and E. grossheimii), sand dunes (e.g., E. cheirolepis and E. turczinanowii), mountain steppes (E. teheranica and E. sahendi), gravelly calcareous or gypsaceous slopes (E. aucheri, E. bungei), banks of streams (E. boissieriana), open Quercus forests (E. erubescens and E. macrocarpa), Hyrcanian forests (E. macroceras and E. squamosa), or rarely rocky mountain slopes (E. cheiradenia and E. buhsei). Several Euphorbia species are known as ruderal elements growing in disturbed sites mainly due to road construction (E. malleata and E. connata) or mining (E. macroclada and E. seguieriana), as well as abandoned farming fields (e.g., E. heteradena and E. falcata), abandoned irrigation ditches (E. peplus and E. helioscopia) and overgrazed mountain slopes (E. decipiens and E. plebeia). Some species are known as hyper-accumulators of heavy metals and 1 The authorities of all species studied here are given in Table 1 and are adopted from Govaerts et al. (2000). For other species (for example outgroups) the authorities are mentioned in the text. 958 Y. Salmaki et al. / Flora 206 (2011) 957–973 are appropriate candidates in phytoremediation (Chehregani and Malayeri, 2007; Chehregani et al., 2009; Nouri et al., 2010). The infrageneric classification of Euphorbia is extremely difficult due to the species richness accompanied by a subcosmopolitan distribution, the extreme morphological plasticity among certain species (often relating to the habitat) and the convergent evolution of certain morphological characters. The last complete taxonomic monograph of Euphorbia was published by Boissier (1862). Currently the EuphORBia project aims at a virtual digital up-to-date monograph of the genus (Esser et al., 2009). Several authors discussed and erected infrageneric classification of this huge genus, in particular Bentham (1880), Pax and Hoffmann (1931), Wheeler (1943). Euphorbia was revised in several floras covering the Iranian highlands, namely Prokhanov (1949), Khan (1963), Rechinger and Schiman-Czeika (1964), and Geltman (2006). The classification of Boissier (1862), still in use, includes the 720 treated species in 27 sections, which were placed in turn into two series (his series are not valid, because he considered the rank of series higher than section which is against the rules of International Code for Botanical Nomenclature, Article 3.1: Vienna Code; McNeill et al., 2006) and several unranked subsectional groups (which he indicated by “§”). Prokhanov (1949) did not follow Boissier’s treatment and presented a new system accepting three subgenera: Chamaesyce (Rafin.) Gray, Cystidospermum Prokh. and Paralias (Rafin.) Prokh. (Prokhanov, 1933) which were recognized by other authors (Gray, 1821; Rafinesque, 1837; Wheeler, 1943; Webster, 1994) as separate genera. Prokhanov (1949) considered also 10 sections, several subsections and series for 159 species distributed in the area of the “Flora of the U.S.S.R”. His subgeneric classification has been refined and improved during a long run project on subgen. Esula by Geltman (2000a,b, 2001a,b, 2006, 2007). Currently, the majority of the species growing in Iranian highlands are attributed to subgen. Esula Pers. including 79 species (over 500 species worldwide: Bruyns et al., 2006), and subgen. Chamaesyce consisting of 15 species (about 250 species worldwide: Webster, 1994), respectively. Among these subgenera, subgen. Esula is taxonomically by far the most complex group and has been divided into 10 sections and 21 subsections (Geltman, 2007), of which six sections are present in the Iranian highlands. Despite considerable efforts to classify and understand the striking morphological diversity in Euphorbia, little is known about interspecific relationships within the taxon. Recent molecular phylogenetic studies using several chloroplast DNA (cpDNA) markers and nuclear (nr) internal transcribed spacer (ITS) sequences provided new insights into the delimitation of subgeneric taxa in Euphorbia (Bruyns et al., 2006; Park and Jansen, 2007; Steinmann and Porter, 2002; Zimmermann et al., 2010). However, re-evaluation of morphological characters formerly used for circumscription of subgeneric taxa was rarely applied to examine character evolution. Nowadays, the genus is accepted in its wide definition including several former genera such as Chamaesyce Rafin. Gray and Tithymalus Gaertn. Based on molecular phylogenetic studies four well supported clades, currently called “A”−“D”, have been revealed within the genus (Bruyns et al., 2006; Park and Jansen, 2007; Steinmann and Porter, 2002; Zimmermann et al., 2010). Species belonging to Euphorbia subgen. Esula distributed in the Iranian highlands were recently included in a molecular phylogenetic study (Kryukov et al., 2010), indicating that the subgenus is composed of two major clades (clades B1 and B2). However, many species distributed in Iranian highlands have not been included in any molecular phylogenetic study yet. The taxonomic and phylogenetic significance of seed micromorphology in the systematics of Euphorbia have been emphasized in several publications (Ehler, 1976; Heubl and Wanner, 1996; Hügin, 1998; Morawetz et al., 2009, 2010; Park, 2000; Stuppy, 1996; Wagner et al., 2010). Khan (1963) even presented a well functional diagnostic key to the species distributed in Turkey based on seed characters. Moreover, seed characters have been applied in various floras for discrimination of species of Euphorbia (as for example: Hügin, 1998; Prokhanov, 1949; Rechinger and SchimanCzeika, 1964). Apart from the ultrastructure, seed morphology was used in the taxonomy of Euphorbia in various ways, mainly to separate certain sections or to delimit closely related species. The seed characteristics of Euphorbia have also been used to recognize subgenera (Carter and Radcliffe-Smith, 1988; Webster, 1967), sections or subsections (Ehler, 1976; Khan, 1963; Park et al., 1999), and to resolve the boundaries between closely related species (Hassall, 1977; Richardson, 1968; Simon et al., 1992). However, the systematic value of seed sculpturing patterns was only the subject of few comprehensive studies (Ehler, 1976; Heubl and Wanner, 1996; Park, 2000; Tokuoka and Tobe, 2002) including few species distributed in the Iranian highlands. The caruncle is a localized arilloid tissue developing near the micropyle of the seeds. Its ontogeny in particular in Euphorbia was studied in detail by Schweiger (1905) and Singh (1969), and also discussed by Stuppy (1996). Whereas Euphorbiaceae in humid tropical areas often develop fleshy diaspores (Esser, 2003), species of drier or temperate environments usually disperse their seeds by explosive dehiscence of the fruits and subsequent dispersal by ants (diplochory). These processes have been the subject of some intensive studies, in much detail for another Australian genus, Micrantheum (Berg, 1975), and for Euphorbia several times (Espadaler and Gómez, 1996; Gómez and Espadaler, 1994, 1998; Narbona et al., 2005; Pemberton, 1988; Wolff and Debussche, 1999). There is an enormous range of variation in the shape and size of the caruncle in Euphorbiaceae (illustrated by Baillon, 1858). Although it is known that the morphology of a caruncle is constant within a species and often can separate different species (Schweiger, 1905), comparative studies here are still lacking. Another unique and interesting feature of Euphorbia species is a ring of usually 4−5 glands alternating with the lobes of the cyathium involucre. Plants of Euphorbia are usually pollinated by a wide range of less specialized insects (e.g., Ehrenberg, 1979). These glandular organs have an ecological role by attracting pollinators to the reduced flowers. However, it has been shown that their shapes and structures may fit to certain morphological lineages in accordance with accepted taxonomical groups within the genus (Bruyns et al., 2006; Steinmann and Porter, 2002; Zimmermann et al., 2010). Although the importance of glands and the cyathium structure have been mentioned in most taxonomical treatments of the genus, they have not been used for the delimitation of large natural groups, especially at sectional or subsectional ranks, compared with seed characters. Therefore, the importance of these characters should be re-evaluated in a phylogenetic context. Paucity of taxonomical and morphological studies on the species distributed in the Iranian highlands is the main concern in assessing the subgeneric classification and interspecific relationships of Euphorbia in this area. The unique features of the inflorescence, the cyathium, and several characters of the seeds make Euphorbia attractive for more detailed studies. Despite several local studies indicating the importance of seed and gland characters in Euphorbia, detailed studies are missing for most species growing in our area. Furthermore, the phylogenetic position of several species distributed in Iranian highlands is surveyed in the present paper using ITS sequences of nuclear ribosomal DNA (nrDNA ITS). The main aim of the present study is to survey the patterns of homoplasy regarding selected morphological characters considered to be of taxonomic value in the genus. Y. Salmaki et al. / Flora 206 (2011) 957–973 Materials and methods Plant materials Samples and specimens for the present study were collected mainly during several field trips between March 2009 and July 2010 in different parts of Iran by the first (Y.S.) and second authors (S.Z.). Several photographs from different parts of the plants were taken for each specimen using a Canon Powershot S3IS digital Camera (photographs available upon request). The original herbarium materials are deposited in the Central Herbarium of Tehran University (TUH), and the duplicates are located in the herbarium of Botanische Staatssammlung München (M). For the species distributed outside of Iran as well as the specimens which were not available in TUH, the collections in the herbaria of the Komarov Botanical Institute in Saint Petersburg (LE) as well as Munich (M and MSB) were studied. Several species, particularly the type specimens of subgeneric groups of subgen. Esula, which are not distributed in the selected area were also studied. Seed morphology Seeds of 86 taxa of the genus Euphorbia were studied. As a rule, 20 different seeds belonging at least to five different specimens (when available) were measured or studied for color, length, width, shape, caruncle size, and caruncle shape for each species by means of dissecting microscope with magnifications of 80–240×. Only fully mature and normal seeds were used for measurement. However, for few species not more than three seeds could be examined, due to paucity of samples. For this reason no statistic measures, such as mean or standard deviation is given. The sizes presented and discussed here represents minimum and maximum only. For scanning electron microscopy (SEM) selected seeds were mounted directly on stubs using double-sided adhesive tape and coated with a thin layer (ca. 25 nm) of gold/palladium in a sputter coater. The SEM micrographs were taken in a Leo SEM-440I at an accelerating voltage of 10–15 kV. The terminology of seed coat surface sculpturing basically follows Ehler (1976), Heubl and Wanner (1996), and Park (2000). The features of anticlinal (occurring at right angles to the surface or circumference of the seed) and periclinal (occurring parallel to the surface or circumference of the seed) walls of the testa cells were also considered. The number of testa cells per 100 ␮m2 was also determined on the seed surface for each species. Gland morphology For several species growing in Iran the glands were observed and studied directly in the field. The structure of the glands was examined using images from field trips or from the herbarium materials after rehydrating them using hot water for few minutes. The following diagnostic characters were considered for each species: gland texture, gland shape, gland appendages and structure. To ensure constancy or variation of gland characters within species several specimens were observed, photographed and measured for each species (where available). Capsule surface The capsules of the species were photographed in the field or studied with aid of a dissecting microscope for surface ornamentation as well as indumentum. Phylogenetic reconstruction A total of 46 ITS sequences representing 45 species of Euphorbia as well as Calycopeplus casuarinoides L.S.Sm. and Neoguillauminia 959 cleopatra (Baill.) Croizat as outgroups, were downloaded from GenBank. Furthermore, 20 sequences representing 19 species (two accessions for E. plebeia) have been generated for selected species with uncertain phylogenetic position. The sampling should cover: (1) the majority of Euphorbia sections and subsections distributed in the Iranian highlands, and (2) representative species of the known phylogenetic lineages. In general, 66 sequences of Euphorbia representing 64 species along with the two outgroups were included in order to develop a molecular phylogenetic framework which could be used to evaluate and discuss character evolution. For sequencing the ITS-region total genomic DNA was extracted using NucleoSpin Plant-Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol with an additional phenol/chloroform extraction to remove contaminants. The DNA was dissolved in 30 ␮l elution buffer (10 mM Tris/HCl) and checked for quality on a 1% agarose-gel. A standard amount of 1 ␮l of the dissolved DNA was used for amplification. The nuclear internal transcribed spacer (ITS1, 5.8S rDNA, ITS2) regions were amplified through polymerase chain reaction (PCR) with Taq polymerase (Boehringer) and the primer pair aITS1 and aITS4 used (Meimberg, 2002). PCR reactions were performed in volumes of 50 ␮l (or rarely 100 ␮l) containing a dNTP solution of 2.5 mM, Taq-polymerase with 1 U/␮l, primer solutions with a concentration of 100 pmol/␮l, and varying amounts of unquantified genomic DNA. When necessary, an alternative preparation containing 0.05% bovine serum albumin (BSA) and 100% dimethyl sulfoxide (DMSO) was used. The PCR amplifications were carried out in a MWG thermocycler (Primus). The following program was chosen: (1) 94 ◦ C for 2 min 30 s, (2) 40 cycles at 94 ◦ C for 30 s, 53 ◦ C for 30 s, 72 ◦ C for 1 min 15 s and (3) a terminal extension phase at 72 ◦ C for 10 min. The PCR products were then cleaned using the QIAquickQiagen PCR Purification Kit (Qiagen GmbH, Hilden, Germany). Cycle Sequencing was carried out using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in a final volume of 20 ␮l. Runs were performed on an ABI 3730 48 capillary sequencer (Applied Biosystems). In all cases, the ITS marker was sequenced bidirectionally using the same primers as in PCR reactions. All sequences generated in this study and obtained from GenBank were assembled, edited and aligned manually using BioEdit 7.0.5.1 (Hall, 1999). The alignment is available from the corresponding author upon request. A list of voucher specimens and accession numbers as submitted to GenBank is provided in Appendix 1. A maximum parsimony analysis (MP) was conducted on the ITS dataset using PAUP* 4.0b10 (Swofford, 2003) with all parsimonyuninformative characters excluded and the included characters unordered and equally weighted. Indel characters were not treated as separate data partition. A heuristic search was conducted using random stepwise taxon addition with tree-bisection-reconnection (TBR) branch swapping, MULTREES and steepest descent on. To assess support of clades the dataset was analyzed using the “bootstrap” option in PAUP* for 100 replicates. A 50% majority rule consensus tree was then obtained. Bayesian Inference (BI) was also performed using the Markov–Chain–Monte-Carlo algorithm of MrBayes 3.1.4 (Ronquist and Huelsenbeck, 2003) for four million generations under the GTR model (Rodríguez et al., 1990), assuming invariable positions and a gamma distributed substitution rate heterogeneity (GTR + G + I). This model had been determined as best-fit model by the likelihood ratio test implemented in jModelTest 0.1.1 (Posada, 2008). Four chains were run simultaneously for the dataset, according to MrBayes’ default setting, with every 1000th tree sampled. After completion of four million generations the average standard deviation of split frequencies had dropped below 0.01 (0.007351). After discarding trees yielded before likelihood stationary (burnin = 1000), the remaining trees were summarized in a 50% majority rule consensus tree, using posterior probabilities (PP) as a measure of clade support. TreeGraph 2.0.45-197 beta (Stöver & Müller, 2010) was used for tree presentation. 960 Y. Salmaki et al. / Flora 206 (2011) 957–973 Analysis of morphological data Patterns of character evolution were assessed for four characters emphasized in earlier taxonomic treatments of Euphorbia (Boissier, 1862; Geltman, 2007; Prokhanov, 1949) to infer history and interpret processes of change. Parsimony mapping was performed in Mesquite v. 1.12 (Maddison and Maddison, 2006) by using one of the most parsimonious trees. The following traits were coded in a binary matrix and traced on the tree: plant habit (perennial/annual), bract gland structure (gland hornless/gland variously horned/gland pectinate), caruncle size proportional to seed (tiny, i.e. its diameter is less than 1/20 of seed length; small, i.e. its diameter ranges from 1/20 to 1/10 of seed length; medium, i.e. its diameter ranges from 1/10 to 1/3 of seed length; large, i.e. its diameter is larger than 1/3 of seed length; absent) and seed surface (variously ornamented or sculptured/smooth). Results Gross morphology of seeds Light microscopic micrographs of seeds from both dorsal and ventral sides in selected species distributed in Iranian highlands are presented in Figs. 1–18. Characters of taxonomic importance are summarized in Table 1, columns 2–6. Some data and measurements, such as caruncle shape, were not presented either due to their low importance and low applicability, or due to limited range of variation. The colors of seeds vary from whitish (E. aserbajdzhanica), yellowish (E. malleata), light brown (E. myrsinites), to dark brown (E. stricta). The seed color is age dependent and mostly is white when immature, but becomes brown when ripen. The seed shape is described for the dorsal view, as the ventral and lateral views are influenced strongly by the ornamentations or by the raphe. The seeds of studied species vary in shape from ovoid (E. bossieriana, Fig. 7), tetrahedral (E. phymatosperma, Fig. 14) to almost globose (E. macrocarpa, Fig. 13). The smallest seeds (0.7−1.1 mm long) were observed in E. maculata and the largest ones (4.0–6.0 mm) in E. megalocarpa. The caruncle may be absent (for example in the species of subgen. Chamaesyce, Figs. 1 and 2), or reduced in size and tiny (E. helioscopia, Fig. 12), or small in size (E. inderiensis, Figs. 14; and Figs. 17 and 18 in other species), or medium in size (in most species studied, for example Figs. 6 and 9) or large in size (E. heteradena, Fig. 3; E. myrsinites, Fig. 15). It is, when present, often conical and shallowly divided, but rarely deeply fringed at base (in E. connata, Fig. 4). The caruncle is in most species stipitate, but the stalk is hidden behind caruncle lobes and margins. However, in some species with small disciform caruncule a long stalk can be visible (E. arvalis, E. aserbajdzhanica, E. inderiensis, E. phymatosperma and E. szovitsii; Figs. 17−18). The seed surface as observable under dissecting light microscope is smooth (E. erubescens, Fig. 11), wrinkled (E. aucheri, Fig. 6), pitted (E. cheiradenia, Fig. 9), tuberculate (E. denticulata, Fig. 16), transversely furrowed (as some species of sect. Chamaesyce, for example E. indica, Fig. 2) or foveate (E. peplus, not shown), longitudinally furrowed (for example in E. inderiensis, Fig. 18), or rarely alveolate (E. helioscopia, Fig. 12), verrucate (E. petiolata, Fig. 5). Seed micromorphology Scanning electron micrographs (SEM) of the seed surfaces of selected species studied are shown in Figs. 19−45 and summarized in Table 1, column 7−8. The testa surface is composed of cells of various shapes and arrangement. They are in some species polygonal in lateral view (E. malleata, Fig. 32), but they may be rounded in other species (E. nutans, Fig. 21; E. petiolata, Fig. 25). The periclinal walls are variously cutinized: striate (E. allepica, Fig. 26), ring shaped (E. indica, Fig. 22), formed of irregular prominent edges (E. coniosperma, not shown), or ±smooth (E. larica, Fig. 23). The anticlinal walls are sunken in few species (E. hebecarpa, Fig. 36; E. seguieriana, Fig. 39), but they may more or less be at same height as the periclinal wall (Fig. 45, E. grossheimii), or raised in most species (E. kopetdaghi, Fig. 31; E. densa, Fig. 41). The intercellular spaces are small in size (E. heteradena, Fig. 24; E. malleata, Fig. 32) or large in size (E. amygdaloides, Fig. 27; E. craspedia, Fig. 40). However, their size is not constant among different populations of certain species. Furthermore, the size of intercellular spaces is age dependent, increasing during maturation. Spherical particles of 1–2 ␮m diameter emerging during maturation through intercellular spaces are present in most species studied (E. buhsei, Fig. 28; E. plebeia, Fig. 29; E. gypsicola, Fig. 30; E. microsciadia, Fig. 33; E. decipiens, Fig. 35), but they are not easily visible in the members of subgen. Chamaesyse (Figs. 19−22). The results of counting the number of testa cells per 100 ␮m2 are not presented here, as it shows high variability among different species of certain known natural groups. Gland structure As interpreted by most specialists, the cyathium is a cuplike structure of fused bracts (involucre), with distinct extrafloral nectar glands and sometimes also additional showy petaloid appendages, enclosing a single female flower surrounded by four or five groups of extremely reduced male flowers in our species. Photographs of the glands of selected species of the genus Euphorbia growing in Iranian highlands are presented in Figs. 46−66 and summarized in Table 1, column 9. Many species of subgen. Chamaesyce or at least all species of the subgenus which are growing in the Iranian highlands possess showy petaloid appendages variously colored (from white to pink, Figs. 46−47) and attached to their elliptic glands, while the involucre of other species show variously shaped, often conspicuous nectar glands which, at least for the Iranian species, usually lack petaloid appendages (Figs. 48−66). Glands vary in color, shape and size. In most species in the area of Iranian highlands they are shiny yellowish green or greenish. In few species (E. decipiens, Fig. 55; E. bungei, Fig. 58; E. denticulata, Fig. 63) they may be red or purple in color. Red glands were also observed among individuals of few species which have normally yellowish green glands (for example in E. szovitsii). The size of the glands depends normally on plant habit. In annual species the glands are generally small in size. The largest glands with about 8 mm diam. were observed in E. erubescens (Fig. 61). An exception in this regard was observed in E. larica (Fig. 49) with relatively small glands (4 mm in diam.) but shrubby habit. Gland shape provides the most valuable characters useful in discrimination of species. In several species the glands are elliptic and without any appendage or horn (E. connata, Fig. 50; E. condylocarpa, Fig. 59). In some species they are elliptic but horned (E. aucheri, Fig. 52; E. gypsicola, 56; E. densa, 64; E. aserbajdzhanica, Fig. 65). The number of gland horns may be variable in certain species. For example we observed individuals of E. cheiradenia (Fig. 54) and E. malleata (Fig. 57) with two horns on glands, while some individuals may have up to four horns. Euphorbia grossheimii (Fig. 48) and E. petiolata (Fig. 51) have deeply fringed pectinate glands. Some species are characterized by fleshy glands equipped with two horns, which are in turn more branched or club-shaped (E. marschalliana, Fig. 62). In E. denticulata (Fig. 63) the glands are large and denticulate at outer margins. The denticulate glands are observed also in E. buhsei (Fig. 53) and E. osyridea (Fig. 66). Euphorbia macroceras (Fig. 60) has the longest horns among the studied species which give the gland a horse-shoe appearance. Y. Salmaki et al. / Flora 206 (2011) 957–973 961 Figs. 1–15. Stereomicroscope micrographs of seeds in Euphorbia: (1) E. humifusa, (2) E. indica, (3) E. heteradena, (4) E. connata, (5) E. petiolata, (6) E. aucheri, (7) E. boissieriana, (8) E. buhsei, (9) E. cheiradenia. (10) E. bungei, (11) E. erubescens, (12) E. helioscopia, (13) E. macrocarpa, (14) E. phymatosperma, (15) E. myrsinites. (1–2) scale bar = 0.5 mm, (3–15) scale bar = 1 mm. Capsule surface Characteristics of capsule surface are summarized in Table 1, columns 10−11. Few micrographs of capsule surface are given in Figs. 67−75. The capsules are smooth in most species studied (E. larica, Fig. 69; E. heteradena, Fig. 71; E. macroclada, Fig. 72). Papillose capsules are also observed in few species (E. macroceras, Fig. 60). However, distinguishing these two types of capsules with aid of stereomicroscope is mostly difficult. Moreover, both types of smooth and papillose capsules were observed in different populations of certain species. In few cases the capsules are covered with tuberculate (E. macrocarpa, Fig. 74; E. orientalis, Fig. 75) or Species A. Subgen. Rhizanthium (Boiss.) Wheeler Sect. Balsamis Webb & Berthel. 1. E. larica Boiss. B. Subgen. Esula Pers. B1. Sect. Helioscopia (Roeper) Godron 2. E. cashmeriana Royle Seed shape Seed length (mm) Seed width (mm) Caruncle size Seed ornamentation Surface sculpturing Periclinal wall Gland Capsule surface Capsule indumentum 2.4–2.8 1.8–2.2 Medium sm ? ? ell-round; hornless sm gla Ovoid±globose Ovoid Ovoid-ell Globose Ovoid-ell Ovoid-globose Ovoid Globose Ovoid Ovoid Ovoid-globose Ell-ovoid 2.2–2.7 1.7–2.0 Tiny sm ret Flat ell; hornless tub ±gla 2.5–3.5 1.5–2.5 2.2–3.0 0.9–1.5 1.5–2.2 3.0–5.0 4.0–6.0 1.8–2.3 2.5–3.2 2.5–3.0 1.2–1.5 1.8–2.0 1.2–1.5 1.5–2.7 0.6–0.9 1.1–1.8 2.0–3.0 3.0–4.5 1.2–1.5 1.7–2.5 1.5–2.0 0.7–1.0 Tiny – – – Tiny – Tiny Tiny Tiny Tiny Tiny sm tub sm ± wri alv sm sm sm sm sm sm col col col col col col ret ret ret ret ret Flat ± raised ± raised ± raised Raised Sunken Flat ± raised Flat Sunken Flat ell-round; hornless ell-obl; hornless ell-obl; hornless broad ovate; hornless ell; hornless broad ell; hornless ell; hornless broad ovate; hornless ell-obl; hornless broad ell; hornless ell; hornless tub ves-tub ves sm sm tub sm ±tub tub-verm verm tub-verm gla gla vil gla gla gla spr gla sspr gla gla Ovoid-tet Ovoid-tet Ell-tet Ell-tet Ovoid-globose Ell-tet Ell-tet 4.5–5.5 4.2–4.7 2.5–3.5 3.5–4.0 3.0–3.5 2.6–3.4 3.2–3.5 2.7–3.2 2.3–2.5 1.5–1.9 1.7–2.0 1.8–2.2 1.7–1.9 1.6–1.8 Large Large Large Large Medium Medium Medium ±wri tub ±wri sm ver sm ver rug rug ret ret col-ret ret ret Raised Raised Raised Raised Raised Raised Raised ell; den ell; den ell-obl; two horned ell-obl; two horned ell-obl; two horned ell-obl; two horned ell-obl; two horned sm sm pap sm sm sm sm ves ves ves ves ves ves ves Ovoid 2.5–3.1 1.5–1.8 Medium pitted ret Flat sm gla 22. E. decipiens Boiss. & Buhse 23. E. erythradenia Boiss. 24. E. falcata L. 25. E. gedrosiaca Rech.f., Aellen & Esfand. 26. E. gypscicola Rech.f. & Aellen. 27. E. humilis C. A. Mey. ex Ledeb. 28. E. kopetdaghi (Prokh.) Prokh. Ovoid Broadly ell ell-tet Ovoid Ovoid Ovoid-ell Ovoid-ell 2.2–2.9 2.5–2.8 1.7–1.9 2.0–2.8 2.5–3.1 1.7–2.6 2.0–2.7 1.5–1.9 1.6–2.0 0.9–1.1 1.2–1.8 1.6–1.9 1.1–1.5 1.2–1.4 Medium Small Tiny Medium Medium Medium Small pitted pitted fov pitted ± pitted ±pitted pitted ret ret ret ret ret ret ±col Flat Flat Sunken Flat Flat ± flat Raised pap-rug pap sm sm pap pap pap gla gla gla gla spr gla gla 29. E. macroclada Boiss. 30. E. malleata Boiss. ell Ovoid-globose 2.8–3.5 2.2–2.6 1.7–2.2 1.3–1.5 Medium Medium-large sm sm ret ret Flat Flat sm sm ± spr gla 31. E. malurensis Rech.f. 32. E. microciadia Boiss. 33. E. plebeia Boiss. 34. E. sahendi Bornm. Ovoid-ell Ovoid Ovoid Ovoid 3.2–4.3 2.5–3.2 2.5–3.0 2.5–2.9 1.7–2.1 1.2–2.1 1.0–1.3 1.6–2.0 Medium Medium Medium Medium pitted ±pitted pitted ±pitted ret ret ret ret Flat Flat ± flat Flat sm sm sm pap gla gla gla spr, pub Ovoid-globose Ovoid-ell ell-tet 1.7–2.5 2.2–2.9 2.4–2.9 1.2–1.5 1.3–1.8 1.5–1.8 Tiny Medium Medium ±pitted pitted ±pitted ret ret ret Sunken Raised Raised ell; two- or multi-horned ell; hornless ell; ±two horned ell; hornless ell; two horned ell; two horned ell; hornless ell; two horned or multi-horned ell; two horned ell; two horned or multi-horned ell; two horned ell; two horned ell; two horned ell; hornless or two horned ell; hornless ell; two horned ell; two horned pap sm sm gla sparsely hairy gla Ovoid-globose Ovoid-ell Ovoid ell-ovoid to globose 3.0–3.9 2.9–3.2 3.0–4.5 2.5–3.2 1.8–2.2 1.7–1.9 1.8–2.2 1.8–2.7 Small-Medium Medium Large Large ±wri ±wri sm sm ret ret ret ret Flat Flat Flat Flat ell; hornless ell; hornless broad ell; hornless ell; hornless sm sm sm sm spr gla spr gla 3. E. condylocarpa M. Bieb. 4. E. coniosperma Boiss. & Buhse 5. E. eriophora Boiss. 6. E. gaillardotii Boiss. & Blanche 7. E. helioscopia L. 8. E. macrocarpa Boiss. & Buhse 9. E. megalocarpa Rech.f. 10. E. microsphaera Boiss. 11. E. orientalis L. 12. E. squamosa Willd. 13. E. stricta L. B2. Sect. Myrsiniteae Boiss. ex Pojero 14. E. craspedia Boiss. 15. E. denticulata Lam. 16. E. marschalliana Boiss. 17. E. monostyla Prokh. 18. E. myrsinites L. 19. E. rigida M. Bieb. 20. E. spinidens Bornm. ex Prokh. B3. Subsect. Conicocarpae Prokh.a 21. E. cheiradenia Boiss. & Hohen. 35. E. seguieriana Neck. 36. E. teheranica Boiss. 37. E. volkii Rech.f. B4. Sect. Chylogala (Fourr.) Prokh. 38. E. bungei Boiss. 39. E. caeladenia Boiss. 40. E. connata Boiss. 41. E. grossheimii (Prokh.) Prokh. Y. Salmaki et al. / Flora 206 (2011) 957–973 Ovoid-tet 962 Table 1 Characteristic features of seeds, glands and capsules of the examined Euphorbia species distributed in the Iranian highlands. Classification follows Geltman (2007) with some modifications suggested here. − = absent; ± = more or less; alv = alveolate, col = colliculate, den = denticulate, ell = ellipsoid, fur = transversely furrowed, l. fur = longitudinally furrowed; gla = glabrous; pap = papilose, pet app = petaloid appendage, rug = rugose, sm = small, spr = spreading; tet = tetrahedral, ret = reticulate, tuberculate = tub, ver = verrucate; verm = vermiculate, ves = vesiculate, vil = villous; wri = wrinkled. 42. E. heteradena Jaub. & Spach 43. E. multifurcata Rech.f., Aellen & Esfandiari 44. E. turkestanica Regel B4. Sect. Esula Dumort. Subsect. Osyrideae Boiss. ex Pax 45. E. buhsei Boiss. 3.5–4.2 2.5–2.8 3.2–3.6 1.7–2.6 1.6–1.9 1.8–2.5 Large Large Medium sm ±wri ±wri ret ret ret Flat Flat Flat broad ell; hornless ell; hornless ell-obl; hornless sm sm sm gla gla gla; pub Broad ovoid 2.0–2.5 1.4–2.0 Medium sm-±wri col Raised pap gla 46. E. osyridea Boiss. Subsect. Esula 47. E. boissieriana (Woronow) Prokh. 48. E. cyrtophylla (Prokh.) Prokh. 49. E. hebecarpa Boiss. 50. E. iberica Boiss. 51. E. leptocaula Boiss. Subsect. Patellares Prokh. 52. E. amygdaloides L. 53. E. erubescens Boiss. 54. E. macroceras Fisch. & C.A. Mey. Broad ovoid 2.4–2.6 1.8–2.2 Tiny sm ret Flat triang; two horned, den triang; den pap spr Ovoid Ovoid Ovoid-ell Ovoid-ell Ovoid-ell 1.7–3.0 1.8–2.2 2.0–2.8 1.8–2.2 1.9–2.4 1.2–2.2 1.2–1.7 1.3–1.7 1.0–1.5 1.2–1.5 Small Medium Medium Medium Tiny sm sm sm sm sm ret ret ret ret ret Flat Flat Flat Flat Flat ell; two horned cres; two horned ell; two horned ell; two horned cres; two horned pap-rug pap-rug sm sm sm gla gla spr gla gla Small Small Small sm sm sm ret ret ret Flat Flat Flat sm sm pap gla gla gla 55. E. oblongifolia (K. Koch) K. Koch Subsect. Pachycladae Boiss. ex Pax 56. E. aucheri Boiss. 57. E. deltobracteata Prokh. Broad ovoid 2.9–3.4 2.1–2.5 Tiny sm ret Flat broad ell; two horned triang; ±two horned horse-shoe shaped; two horned cres; two horned pap gla tet-ovoid Ovoid-ell 2.0–3.0 2.9–3.4 1.1–1.6 2.1–2.5 Medium Tiny ±l.fur sm ret ret ± sunken Flat pap pap gla gla Ovoid Broad ovoid ell 2.5–2.7 3.1–3.4 2.2–2.6 1.4–1.6 2.6–2.9 1.0–1.3 Tiny Medium Tiny tub-wri sm ±l.fur col ret ret Raised Flat Raised ell; two horned ell-reniform; two horned ell-obl; two horned reniform; hornless ell; two horned sm sm sm gla gla gla Broad ovoid Ovoid-tet Ovoid-tet 1.6–2.0 1.2–1.5 1.0–1.3 1.0–1.2 0.6–0.8 0.5–0.7 – Tiny Tiny tub tub-ver fov col col ret Raised Raised Raised ell; two horned ell; two horned ell; two horned pap pap rugose gla gla gla Broad ovoid ell-tet ell-tet Ovoid-tet tet ell-tet tet ell-tet tet 1.4–1.6 1.9–2.5 2.1–2.4 1.8–2.2 1.5–1.9 1.7–2.0 2.4–2.7 1.8–2.3 1.6–1.9 0.8–1.1 0.6–0.8 1.0–1.2 0.8–1.0 0.8–0.9 0.7–0.9 1.3–1.5 1.2–1.4 0.7–1.0 Small Small – Small Small Small Small Small Small wri-ver ver ver ver-l.fur l.fur l.fur fur-tub ver ver ret ret ret col col ret col ret col Raised Sunken Sunken Raised Raised Raised Raised Sunken Raised ell; two horned ell; two horned ell; two horned ell; two horned ell; two horned ell; two horned ell; hornless ell; hornless ell; hornless pap sm sm sm pap sm sm pap pap gla gla gla gla gla gla gla gla gla tet Ovoid-tet Ovoid-tet ell-tet tet ell-tet Broad ovoid ell-tet Ovoid-tet Ovoid-conical Ovoid-tet ell-tet 0.9–1.5 1.0–1.5 1.1–1.6 0.9–1.1 0.8–1.4 1.3–1.5 1.0–1.2 0.7–1.1 1.0–1.2 2.6–3.2 0.9–1.3 0.9–1.3 0.4–0.6 0.7–0.8 0.5–0.7 0.5–0.6 0.4–0.6 0.6–0.8 0.8–0.9 0.2–0.7 0.5–0.6 1.5–2.3 0.5–0.6 0.6–0.7 – – – – – – – – – – – – fur fur fur fur sm fur sm-±wri sm-±wri sm-±wri sm fur sm-±wri col col col col col col col col col col col col Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app ell; pet app sm sm sm sm sm sm sm sm sm sm sm sm ± spr app pilose app pilose spr gla spr app pilose app pilose app pilose gla app pilose gla tet ell 3.3–4.0 4.2–4.5 1.2–1.8 1.8–2.1 Tiny Small tub ver ver ver Raised Raised ell; pectinate ell; pectinate sm sm vil vil 58. E. dracunculoides Lam. 59. E. rosularis A. Theod. 60. E. turczaninowii Kar. & Kir. Subsect. Oleraceae Prokh. 61. E. aleppica L. 62. E. exigua L. 63. E. peplus L. Uncertain subsection 64. E. arvalis Boiss. & Heldr. 65. E. aserbajdzhanica Bordz. 66. E. consanguinea Schrenk 67. E. densa Schrenk 68. E. franchetii B. Fedtsch. 69. E. inderiensis Less. ex Kar. & Kir. 70. E. phymatosperma Boiss. 71. E. sororia Schrenk 72. E. szovitsii Fisch. & C.A.Mey. C-No native representative in Iran D. Subgen. Chamaesyce (Rafin.) Gray D1. Sect. Chamaesyce (Gray) Rchb. 73. E. anisopetala (Prokh.) Prokh. 74. E. chamaesyce L. 75. E. granulata Forssk. 76. E. hispida Boiss. 77. E. humifusa Willd. 78. E. inaequilatera Sond. 79. E. indica Lam. 80. E. maculata L. 81. E. nutans Lag. 82. E. peplis L. 83. E. prostrata Aiton 84. E. serpens Kunth D2. Subgen. Cystidospermum Prokh. 85. E. cheirolepis Fisch. & C.A.Mey. ex Karelin 86. E. petiolata Banks & Sol. This subsection should probably be elevated to section rank. 1.7–3.0 3.0–3.5 2.6–3.0 1.2–1.8 1.6–2.2 1.4–2.0 963 a Ovoid ell Ovoid Y. Salmaki et al. / Flora 206 (2011) 957–973 ell-ovoid Ovoid-ell Ovoid-ell 964 Y. Salmaki et al. / Flora 206 (2011) 957–973 Figs. 16–30. Stereomicroscope micrographs of seeds in Euphorbia (16–18): (16) E. denticulata, (17) E. aserbajdzhanica, (18) E. inderiensis. (16) scale bar = 1 mm, (17–18) scale bar = 0.5 mm. SEM micrographs of seeds in Euphorbia spp. (19) E. inaequalitera, (20) E. maculata, (21) E. nutans, (22) E. indica, (23) E. larica, (24) E. heteradena, (25) E. petiolata, (26) E. allepica, (27) E. amygdaloides, (28) E. buhsei, (29) E. plebeia, (30) E. gypsicola. vermiculate (E. squamosa, Fig. 73) projections. Type of capsule indumentum varies from glabrous (E. larica, Fig. 69; E. heteradena, Fig. 71) to villous (E. petiolata, Fig. 70). Some species of subgen. Chamaesyce are characterized by an appressed indumentum (E. granulata, Fig. 67; E. indica, Fig. 68). Analysis of ITS sequences Sequences generated for this study and those obtained from Genbank ranged from 581 bp (E. exigua) to 690 bp (E. primulifolia). The final aligned matrix was 692 bp long after the exclusion of the Y. Salmaki et al. / Flora 206 (2011) 957–973 965 Figs. 31–45. SEM micrographs of seeds (31–36, 38–45) and capsule (37) in Euphorbia spp. (31) E. kopetdaghei, (32) E. malleata, (33) E. microsciadia, (34) E. dracunculoides, (35) E. decipiens, (36) E. hebecarpa, (37–38) E. helioscopia, (39) E. seguieriana, (40) E. craspedia, (41) E. densa, (42–43) E. osyridea, (44–45) E. grossheimii. last 33 bp of the original matrix due to a high number of missing data. Large deletions of 69 and 34 bp were revealed in E. exigua and E. peplus, respectively. Maximum Parsimony (MP) analysis yielded 1297 trees with length (L) = 2294, CI = 0.3251 and RI = 0.6622. The general topology of the strict consensus tree obtained from MP analysis (not shown) was similar to the Bayesian tree topology (Fig. 76). Main clades of the trees were labeled as “A”–“D” as suggested in previous phylogenetic studies of Euphorbia. Both analyses show E. larica as a member of clade “A” sister to E. balsamifera. Clade “B” includes the species of subgen. Esula containing most of the species sampled in the present study. Few species selected from subgen. Euphorbia are grouped in clade “C”. The species of subgen. 966 Y. Salmaki et al. / Flora 206 (2011) 957–973 Figs. 46–60. Photographs of glands in Euphorbia spp. (46) E. granulata, (47) E. indica, (48) E. grossheimii, (49) E. larica, (50) E. connata, (51) E. petiolata, (52) E. aucheri, (53) E. buhsei, (54) E. cheiradenia, (55) E. decipiens, (56) E. gypsicola, (57) E. malleata, (58) E. bungei, (59) E. condylocarpa, (60) E. macroceras. Chamaesyce as well as two species (E. petiolata and E. cheirolepis) traditionally treated as members of subgen. Cystidospermum are part of clade “D”. Subclade “B1” includes mainly the species of sect. Paralias subsect. Myrsiniteae, subclade “B2” consists of members of sect. Chamaebuxus subsect. Lutescentes and subsect. Apios as well as E. helioscopia, subclade “B3” comprises the representatives of sect. Paralias subsect. Conicocarpae as well as E. falcata, and subclade “B4” is a miscellaneous group including the representatives of sect. Y. Salmaki et al. / Flora 206 (2011) 957–973 967 Figs. 61–75. Photographs of glands (61–66) and capsules (67–75) in Euphorbia spp. (61) E. erubescens, (62) E. marschalliana, (63) E. denticulata, (64) E. densa, (65) E. aserbajdzhanica, (66) E. osyridea, (67) E. granulata, (68) E. indica, (69) E. larica, (70) E. petiolata, (71) E. heteradena, (72) E. macroclada, (73) E. squamosa, (74) E. macrocarpa, (75) E. orientalis. 968 Y. Salmaki et al. / Flora 206 (2011) 957–973 Fig. 76. Phylogeny resulting from Bayesian analysis of the nrITS dataset including sequences of 66 species of Euphorbia. Posterior probabilities (PP) are shown above branches and the bootstrap supports ≥50% are given below the branches. Clades “A–D” refer to main clades indicated in Euphorbia by Steinmann and Porter (2002). “B1−B4” represent main subclades of subgen. Esula (see under Results). The subgeneric classification (right hand) follows mainly Geltman (2007). Where no classification is mentioned, no subgeneric group has been proposed in earlier studies. Taxa highlighted with bold font are sequenced here for the first time. Y. Salmaki et al. / Flora 206 (2011) 957–973 969 Fig. 77. A–D. Evolutionary histories of four selected morphological characters mapped on one of the most parsimonious trees obtained from heuristic search of nrITS sequences of 66 species of Euphorbia. 970 Y. Salmaki et al. / Flora 206 (2011) 957–973 Chylogala, sect. Esula, and sect. Peplus. The majority of species belong to subsect. Esula (PP = 1.00, BS = 79) and subsect. Patellares (PP = 1.00, BS = 100). The main differences between the BI and MP tree topologies were in the position of few taxa summarized as follow: (1) Subclade “B3”, including the representatives of sect. Chamaebuxus, nested within subclade “B4”, as sister to members of sects. Esula and Peplus; (2) E. peplus was sister to all taxa of subclade “B4” except for sect. Chylogala. The results of tracing some morphological characters on one of the MP trees are presented in Fig. 77. Discussion ITS phylogeny The general topology that resulted from the Bayesian Inference (BI, Fig. 76) and Maximum Parsimony (MP, not shown) analyses of ITS sequences was in most parts in accordance with previous studies (Bruyns et al., 2006; Steinmann and Porter, 2002; Zimmermann et al., 2010). Minor differences between our results and the previous studies concern mainly the more basal position of clades “C” + “D” compared with the clades “A” + “B”, which might be correlated with the unbalanced and low sampling from the former clades. As the main goal of the present paper is the evaluation of selected morphological characters of taxonomical importance based on previous studies, we refrain from performing a detailed discussion on all clades here, but emphasize new findings of the phylogenetic study. Previous studies suggested that few species such as E. balsamifera, distributed from Canary Islands to Arabian Peninsula (Steinmann and Porter, 2002) should be transferred from subgen. Esula into clade “A”. Our results show a close relationship between E. larica distributed in SE Iran and E. balsamifera (PP = 1.00, BS = 71). Euphorbia larica is also very similar to the latter in habitat (arid vegetation of the subtropical zone), growth habit (shrubby) as well as gland shape (hornless elliptic, Fig. 49, Table 1), seed characters (caruncle of medium size and smooth seed surface, Fig. 23), capsule shape and surface (subglobose, non-sulcate and glabrous, Fig. 69). This species is a well-known Saharo-Sindian element confirming the extension of this phytochory into South of Iran (White and Leonard, 1991; Zohary, 1973). The data presented corroborate the treatment presented by Bruyns et al. (2006) treating sect. Balsamis (with E. balsamifera as type, see Wheeler, 1943: p. 489) as a member of clade “A” or subgen. Rhizanthium. The tree topologies resulting from our analyses oppose Gilbert’s (1987) and Holmes (1993) view who assigned sect. Balsamis to a widely circumscribed subgen. Esula, as well as Rechinger and Schiman-Czeika’s treatment (1964) that attributed E. larica to sect. Tirucalli (of clade “C”). Euphorbia tirucalli as the type of sect. Tirucalli belongs to clade “C” (subgen. Euphorbia) in all previously published phylogenies using ITS sequences (Bruyns et al., 2006; Steinmann and Porter, 2002; Zimmermann et al., 2010). Clade “B”: As one of the largest subgenera of Euphorbia, subgen. Esula has been divided into several sections, subsections and series in previous taxonomical studies (Geltman, 2000a, 2001b, 2007; Prokhanov, 1949). Four major subclades, indicated as B1−B42 (Fig. 76), correspond partly with the major sections and subsections known currently in the subgenus. Two main subsections of sect. Paralias do not form a common clade indicating their paraphyly (subclades “B1” and “B3”). Large seed caruncles, fleshy and large glands as well as grayish leaves are some synapomorphies of subsect. Myrsiniteae. The results of the present study corroborate previous molecular analyses of subgen. Esula and suggest to elevate the rank of this group from subsection 2 The labels B1–B4 do not correspond to those indicated by Kryukov et al. (2010). to section. The same should be applied to subsect. Conicocarpae with a similarly high support (PP = 1.00, BS = 100) and characterized by pitted or foveate seed surface. The phylogenetic position of the unsampled taxa E. macroclada and E. malleata, two members of subsect. Conicocarpae, could not be examined, but they do not share their smooth seed surfaces with other species of this subsection. It was surprising that the annual E. falcata was nested within this subclade, as no other annual species was known in subsect. Conicocarpae. However, the gland shape (elliptic and hornless) in this species is very similar to E. seguieriana. Although a close relationship between E. falcata and E. seguieriana is also suggested by Kryukov et al. (2010), the subsect. Conicocarpae is poorly sampled there. So, the monophyly of this group could not be surveyed based on the tree performed there. The members of subclade “B2” including mainly sect. Chamaebuxus as well as E. helioscopia as the only sampled member of sect. Helioscopia are characterized by reduced caruncles, hornless glands and reticulate or colliculate seed surface. In subclade “B4” subsect. Esula includes relatively tall and erect plants preferring wet conditions and characterized by shiny dark brown seeds with reduced caruncles as well as two horned glands. The present study is the first phylogenetic work indicating the position of subsect. Osyrideae. Euphorbia buhsei and E. osyridea are indicated as sister taxa and, together with E. franchetii (formerly attributed to sect. Peplus; Geltman, 2007), are closely related to members of subsect. Esula (PP = 1.00, BS = 98). The results presented here corroborate Kryukov et al. (2010) findings on a possible polyphyly of sect. Peplus. Furthermore, E. aucheri (sect. Herpetorrhiza) is probably related to E. dendroides (subsect. Pachycladeae as defined here, see Table 1) and some species formerly attributed to sect. Peplus, but a broader taxon and molecular sampling are still needed here. A close relationship between the species of sect. Herpetorrhiza and sect. Peplus (= sect. Cymatospermum Prokh.) was already mentioned by Prokhanov (1949). He (Prokhanov, 1949) stated that: “sect. Herpetorrhiza is apparently the origin of the annuals of sect. Cymatospermum”. Patterns of homoplasy for selected morphological characters in Euphorbia species distributed in Iranian highlands One of the main applications of molecular plant systematics is the evaluation of morphological characters in classification of plant groups and finding morphological synapomorphies for natural taxonomic groups. The species of Euphorbia have been variously classified since Boissier (1862). The differences among numerous classification systems are mainly due to subjective evaluation of morphological characters by different authors (for example Boissier, 1862; Gilbert, 1987; Geltman, 2007; Prokhanov, 1949). There are a large number of morphological characters which could be used for separation of species or certain lineages in Euphorbia. But we refrain to provide extensive discussion on all of them here and will focus by preference on seed and gland features. The patterns of homoplasy for certain morphological characters of high importance in previous classifications of this genus are discussed below. Growth habit In Euphorbia subgen. Esula the annual versus perennial habit has been always highly weighted. As an example, the most important characteristic feature of sect. Peplus is the annual habit. However, the present study as well as the recently published phylogeny on the subgenus (Kryukov et al., 2010) show that annual species of this section are dispersed among several branches of the phylogenetic tree (Fig. 77A). The annual habit seems to be a synapomorphy for clade “D” (with exception of E. gradyi) including the members Y. Salmaki et al. / Flora 206 (2011) 957–973 of subgen. Chamaesyce (predominantly annual in the Iranian highlands) and subgen. Cystidospermum. However, the importance of this character would decrease, if more species of this subgenus with biennial or perennial habit would be added to the analysis. The species of subgen. Chamaecyse included here, are only few representatives of this clade. Therefore, the phylogeny presented here is not adequately sampled for detailed discussion on this clade. In general, we suggest lower value for the character state annual versus perennial in future classification proposals of subgen. Esula. We suspect that if more species out of the Iranian highlands would be added to the analysis, the homoplasy for this character would increase considerably. Glands and involucral appendage structures (Fig. 77B) The conspicuous glands of Euphorbia can be of various shapes and with variously shaped horns. Pectinate glands (Figs. 48 and 51) were observed in E. cheirolepis, E. petiolata and E. gradyi in clade “D”. Although the latter (known from Mexico) is geographically far from the distribution area of both former species, its glands are pectinate (Steinmann and Ramírez-Roa, 1998) and with similar structure as E. cheirolepis and E. petiolata. Transversely elliptic glands without any horns are apparently the plesiomorphic state and the most common type in Euphorbia (Fig. 77B: white branches, Figs. 49 and 59). The glands in both E. buhsei and E. osyridea are dentate at margins, and the horns are not very distinct. However, in some species, such as E. cheiradenia (Fig. 54) the number of horns and the gland margin are variable among different populations. Therefore, before scoring the character states for this feature, it is important to study the intraspecific variation. The gland margin provides probably a useful tool in recognition of natural groups in the complex sect. Peplus. Euphorbia falcata, E. franchetii and E. szovitsii which are separated from other species of sect. Peplus in the ITS phylogeny (Fig. 76), have hornless glands, while in most other species the glands are two-horned. The texture of glands is also very important. For example, all species of subsect. Myrsiniteae are characterized by fleshy and thick glands which are variously colored and two-horned (mostly with clavate horns, Fig. 62) or dentate (Fig. 63). Seed morphology Members of sect. Chamaebuxus and subsect. Chylogala are characterized by smooth seed surfaces comprising often reticulate testa cells. However, these character states can be observed in species of various other sections too (Fig. 77D). The verrucate, tuberculate and foveate seeds are also dispersed throughout the phylogenetic tree. The foveate and verrucate seed patterns are common among the members of sect. Peplus as circumscribed by Geltman (2007). It seems that such ornamentation is correlated with annual habit among the studied species. Most annual species independent of the clade to which they belong possess verrucate or foveate seed surfaces. Therefore, we suggest low value for the character state verrucate or foveate seed surfaces in determining large natural groups in subgen. Esula. Testa surface with colliculate periclinal cell walls is also more common among the annual species and mostly accompanied with foveate and verrucate ornamentations. The size of testa cells (expressed often as the number of cells in each 100 ␮m2 ) is another feature of seed micromorphology mentioned to be of systematic value in Euphorbia (Ehler, 1976). However, our results as well as others (Heubl and Wanner, 1996) show that this character can be maximally applicable at species rank and not for determining natural groups of species. Furthermore, this character is correlated with seed size as well as to seed area. It means that the size of testa cells is highly variable on different positions even of one seed. 971 Seed caruncles aid dispersal of seeds in Euphorbia. It has been shown that seeds with distinct and large caruncle can be dispersed more distantly from the bearing plants. The role of caruncles in myrmechochory is well known in the genus (Espadaler and Gómez, 1996; Gómez and Espadaler, 1998; Narbona et al., 2005; Pemberton, 1988; Wolff and Debussche, 1999). Surprisingly, species with large caruncle (for example members of sect. Chylogala, Fig. 77C) are most common in deserts or arid subalpine areas. In contrast, we suppose that the ants play a minor role in dispersal of seeds in members of sect. Chamaebuxus (adapted to more humid conditions) characterized by smooth surface and reduced caruncles (Fig. 13). It seems that the size of caruncle proportional to seed is of high systematic value in Euphorbia. However, low importance should be assigned to caruncle shape, which is mostly species specific. Although the shape of the caruncle shows a wide range of variation (see Baillon, 1858; for a detailed terminology see also Heubl and Wanner, 1996), it is applicable for separation of taxa at species level. Capsule surface In most species studied the capsule surface is smooth or ±papillose. In members of sect. Chamaebuxus the capsules are tuberculate or vermiculate on surface (Figs. 73−75). Euphorbia helioscopia, which is sister to a clade with taxa of sect. Chamaebuxus, shows prominent papillae on capsules (Fig. 37). The capsule surface in subsect. Myrsiniteae is uniquely vesiculate. Based on specimens studied, the capsule surface provides apparently reliable characters for distinguishing large natural groups. Conclusion Although Euphorbia subgen. Esula, is probably one of the best studied groups within the genus from the taxonomical point of view, the data presented here suggest re-circumscribing some of its sections and subsections. Based on the data presented we suggest the classification presented in Table 1 in this subgenus for the species distributed in the area of Iranian highlands. The systematic position of several annual species such as E. franchetii, E. inderiensis and E. szovitsii could not be evaluated here and should be investigated more intensively using other DNA markers. Moreover, the analysis of homoplasy pattern regarding several morphological characters indicates a low reliability for most of them. Note added in proof During processing of the present paper for publication, a new paper (Barres et al., 2011) on the topic appeared. The results presented there could not be discussed in the present treatment. Acknowledgments We are grateful to DAAD “Deutscher Akademischer Austausch Dienst” for a grant to the first author as well as “Alexander von Humboldt Stiftung” for a grant to the corresponding author. The Research Council, University of Tehran partially provided financial support of this project. Kind assistance from Tanja Ernst (Munich) in Heubl’s lab of plant molecular systematics is appreciated. We would like to thank Prof. Vladimir Dorofeyev as well as other curators of LE for their helps in various respects. We want also to thank the curators of FUMH, M, MSB, TARI, and TUH for providing the herbarium materials used in this study. Dmitry Geltman (Saint Petersburg) and Ricarda Riina helped us in various ways, particularly with their valuable comments, providing some rare references as well as images of type specimens, for which we are very grateful. This study is part of the collaborative Planetary Biodiversity Inventory of the genus Euphorbia (NSF-PBI grant DEB-0616533). 972 Y. Salmaki et al. / Flora 206 (2011) 957–973 Appendix A. GenBank accession number of taxa For species whose ITS markers were sequenced during this study, complete voucher information is given; for species whose sequences were taken from GenBank, accession number and citation are listed. E. amygdaloides L., Kryukov et al. (2010), AF537544; E. antso Denis, Steinmann & Porter (2002), AF537579; E. acalyphoides Hochst. ex Boiss., Steinmann & Porter (2002), AF537576; E. aucheri Boiss., Iran, Prov. Khorassan: mountain above Rud-Baran, Dargaz (103 km to Dargaz from Ghouchan), 1770 m, 30.5.2009, Zarre et al. 38188 (TUH), JF732984; E. balsamifera Aiton subsp. adensis (Deflers) Bally, Steinmann & Porter (2002), AF537571; E. acalyphoides Hochst. ex Boiss., Steinmann & Porter (2002), AF537576; E. buhsei Boiss., Iran, Prov. Esfahan: ca. 5 km to Soh at the Soh deviation from Esfahan main road, 1500 m, 25.4.2009, Zarre et al. 39972 (TUH), JF732987; E. bungei Boiss., Iran, Prov. Golestan: Bojnurd to Azadshahr, Mirzabayloo to Solegerd, 10 km from Environment Protection Station, near the pass, 1600–1700 m, 1.6.2009, Zarre et al. 38084 (TUH), JF732980; E. characias L., Kryukov et al. (2010), GU984304; E. cheirolepis Fisch. & C.A.Mey., Steinmann & Porter (2002), AF537424; E. comosa Vell., Steinmann & Porter (2002), AF537503; E. condylocarpa M. Bieb., Kryukov et al. (2010), GU979426; E. connata Boiss., Iran, Prov. Kerman, Anar to Sirjan, ca. 20 km after Anar (76–77 km to Shahr-e Babak), 840 m, 14.3.2009, Zarre et al. 39940 (TUH), JF732982; E. craspedia Boiss., Kryukov et al. (2010), GU984326; E. cyparissias L., Zimmermann et al. (2010), AJ534823; E. decipiens Boiss. & Buhse (specimen 1), Iran, Prov. Esfahan: 15 km after Salafchegan toward Delijan, 1325 m, 13.3.2009, Zarre et al. 39933 (TUH), JF732972; E. dendroides L., Steinmann & Porter (2002), AF537539; E. erubescens Boiss, Iran, Prov. Shiraz: Dasht-e Arjan protected area, ca. 2 km after Kotal-e Pirzan (Dasht-e Arjan-Parishan), 1822 m, 30.4.2009, Zarre et al. 39986 (TUH), JF732985; E. esula L. (specimen 1), Steinmann & Porter (2002), AF537546; E. esula L. (specimen 2), Kryukov et al. (2010), GU984312; E. exigua L., Kryukov et al. (2010), GU984325; E. falcata L., Kryukov et al. (2010), GU984328; E. franchetii B.Fedtsch., Iran, Prov. Khorassan: Bajgiran, mountain East of Baba-Aslameh, DarBadam Protected area, 1670–1680 m, 31.5.2009, Zarre et al. 38194 (TUH), JF732986; E. gradyi V.W. Steinm. & Ram.-Rosa, Steinmann & Porter (2002), AF537407; E. grossheimii (Prokh.) Prokh., Iran, Prov. Markazi, 55 km to Saveh, on the road of Parand to Saveh, 1325 m, 29.4.2009, Zarre & Salmaki 41012 (TUH), JF732981; E. guerichiana Pax & Englm., Steinmann & Porter (2002), AF537415; E. gypsicola Rech.f. & Aellen, Iran, Prov. Semnan: 5 km toward North of Sorkheh, on the road Sorkhe-Firuzkuh, 840 m, 25.5.2009, Zarre et al. 38012 (TUH), JF732976; E. hebecarpa Boiss., Iran, Prov. Shiraz: Dasht-e Arjan protected area, ca. 2 km after Kotal-e Pirzan (Dasht-e ArjanParishan), 1822 m, 30.4.2009, Zarre et al. 39987 (TUH), JF732989; E. helioscopia L., Bruyns et al. (2006), AM040775; E. heteradena Jaub. & Spach, Kryukov et al. (2010), GU984327; E. hirta L. (as Chamaesyce hirta), Chen & Pang (2010), GU441814; E. humifusa Willd., Kim & Kim (2008), EU659774; E. humilis Ledeb., Kryukov et al. (2010), GU984329; E. iberica Boiss., Kryukov et al. (2010), GU984310; E. kopetdaghi (Prokh.) Prokh., Iran, Prov. Khorassan: 5 km after Bajrian, at the beginning of the Bajgiran pass to Imam-Gholi, 1640 m, 31.5.2009, Zarre et al. 38196 (TUH), JF732973; lamprocarpa Prokh., Kryukov et al. (2010), GU953745; E. larica Boiss., S Iran, Prov. Bandar Abbas: 5 km after Manoujan-Bandar-Abbas bifurcation, at the beginning of the first pass, 900 m, 16.3.2009, Zarre et al. 39951 (TUH), JF732969; E. leptocaula Boiss., Kryukov et al. (2010), GU984320; E. lucida Waldst. & Kit., Kryukov et al. (2010), GU984307; E. macroceras Fisch. & C.A.Mey., Kryukov et al. (2010), GU984306; E. maculata L., Zimmermann et al. (2010), AJ534801; E. marschallia Boiss., Iran, Prov. Tehran: on the road of Karaj-Chalous, Shahrestanak village, 1500 m, Date: 21.6.2009, Salmaki 39929 (TUH), JF732971; E. microsciadia Boiss., Iran, Prov. Qazvin: about 15 km after Qazvin, Mahmoud-Abad village, near the road, 1305 m, 16.6.2009, Salmaki et al. 39739 (TUH), JF732975; E. myrsinites L., Steinmann & Porter (2002), AF537551; E. oblongifolia (K.Koch) K.Koch, Kryukov et al. (2010), GU984306; E. orientalis L., Kim and Kim (2008), EU659764; E. peplus L., Steinmann & Porter (2002), AF537532; E. osyridea Boiss., Iran, Prov. Bandar Abbas: Genu mountain, ca. 4 km after Environmental Protection Station, 1810 m, 17.3.2009, Zarre et al. 39953 (TUH), JF732988; E. petiolata Banks & Soland., Steinmann & Porter (2002), AF537422; E. phymatosperma Boiss., Iran, Prov. Lorestan: Khorram-Abad, ca. 4 km to Sarab-e Doureh, 65 km to Kuh-Dasht, Kuh Sefid protected area, 1500 m, 1.5.2009, Zarre et al. 41004 (TUH), JF732970; E. plebeia Boiss. (specimen 1), Iran, Prov. Fars: ca. 4 km to Shiraz from MarvDasht, on the old road (Marvdasht-Abadeh), 1570 m, 29.4.2009, Zarre et al. 39982 (TUH), JF732978; E. plebeia Boiss. (specimen 2), Iran, Prov. Fars: ca. 40 km to Saadat-Shahr from Safa-Shahr, 1450 m, 27.4.2009, Zarre et al. 39979 (TUH), JF732979; E. primulifolia Baker, Steinmann & Porter (2002), AF537466; E. rigida M. Bieb., Kryukov et al. (2010), GU984327; E. sahendi Bornm., Iran, Prov. E Azerbaijan: Tabriz to Bostan-Abad, on the road of Iranagh to Matanagh, 11 km after bifurcation of Matanagh toward the peak of Sahand mountain, 2830–3050 m, 21.7.2009, Salmaki et al. 39891 (TUH), JF732974; E. sarawschanica Regel, Kryukov et al. (2010), GU979438; E. seguieriana Neck., Kryukov et al. (2010), GU984330; E. sinclairiana Benth. (as E. elata), Steinmann & Porter (2002), AF537495; E. squamosa Willd., Kryukov et al. (2010), GU937804; E. stenophylla Boiss., Steinmann & Porter (2002), AF537529; E. stricta L., Kryukov et al. 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