See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/227860534 Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of nuclear ribosomal DNA and... Article in Flora - Morphology Distribution Functional Ecology of Plants · December 2010 DOI: 10.1016/j.flora.2009.12.028 CITATIONS READS 15 171 4 authors, including: Hamid Moazzeni Shahin Zarre 17 PUBLICATIONS 161 CITATIONS 84 PUBLICATIONS 834 CITATIONS Ferdowsi University Of Mashhad SEE PROFILE University of Tehran SEE PROFILE Klaus Mummenhoff Universität Osnabrück 168 PUBLICATIONS 3,291 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: http://www.home.uni-osnabrueck.de/kmummenh/Research_focus_Mummenhoff.pdf View project All content following this page was uploaded by Hamid Moazzeni on 01 April 2015. The user has requested enhancement of the downloaded file. 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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy ARTICLE IN PRESS Flora 205 (2010) 337–343 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.de/flora Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of nuclear ribosomal DNA and morphological characters Hamid Moazzeni a,n, Shahin Zarre a, Ihsan A. Al-Shehbaz b, Klaus Mummenhoff c a Department of Plant Sciences, School of Biology, College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri, 63166-0299, USA c Universität Osnabrück, Spezielle Botanik, Barbarastrasse 11, 49076 Osnabrück, Germany b a r t i c l e in fo abstract Article history: Received 22 January 2009 Accepted 14 May 2009 Systematics of the genus Isatis (Brassicaceae) is difficult and controversial, and previous studies were based solely on morphological characters. Sequence variation of the internal transcribed spacer (ITS) regions and the 5.8S gene of nuclear ribosomal DNA (nrDNA) were analyzed using parsimony and Bayesian methods. Twenty-eight taxa of Isatis and related genera of the tribe Isatideae were sampled, including 20 Isatis species representing almost all major morphological lineages, all three species of Pachypterygium, two of nine species of Sameraria, and monospecific Boreava, Myagrum, and Tauscheria. Two well-supported clades were resolved in the ITS tree, and they demonstrate the artificiality of the present delimitation of the tribe. One clade includes I. emarginata, I. minima, I. trachycarpa, P. brevipes, P. multicaule, P. stocksii, and T. lasiocarpa. The second clade includes I. buschiana, the polymorphic I. cappadocica with five subspecies, I. gaubae, I. kotschyana, I. leuconeura, I. pachycarpa, I. takhtajanii, I. tinctoria, and S. armena. Pachypterygium is polyphyletic and, together with Boreava, Sameraria, and Tauscheria, all are nested within Isatis. This study is a continuation of our recent systematic survey based on seed-coat microsculpturing (Moazzeni et al., 2007. Flora 202, 447–454) and reveals that fruit characters mapped onto the molecular tree show considerable convergence. The reliance on fruit characters alone in the delimitation of genera may well lead to erroneous phylogenetic results and thus to incorrect taxonomic conclusions. & 2009 Elsevier GmbH. All rights reserved. Keywords: Brassicaceae Isatideae Isatis ITS phylogeny Iran Introduction Isatis L. (Brassicaceae or Cruciferae), a Eurasian genus of 79 species (Al-Shehbaz et al., 2006), is distributed primarily in the Irano-Turanian region, where nearly 90% of its species grow (Appel and Al-Shehbaz, 2003; Davis, 1965). Some species (e.g., I. cappadocica) are highly polymorphic in fruit morphology, the structures that provide the most diagnostic characters in the genus (Davis, 1965; Hedge, 1968; Jafri, 1973). Intermediates have been reported even among the most morphologically distinct species (Davis, 1965; Hedge, 1968), and this suggests that hybridization may have played an important role in the evolution of the genus. The genera Pachypterygium Bunge (3 spp.), Tauscheria Fisch. ex DC. (1 sp.), Sameraria Desv. (9 spp.), and Chartoloma Bunge (monospecific, not included in this study) were placed with Isatis in the tribe Isatideae (Al-Shehbaz et al., 2006; Candolle, 1821), the Arabideae subtribe Isatidinae (Hayek, 1911), and Lepidieae subtribe Isatidinae (Schulz, 1936). These genera are delimited n Corresponding author. Tel.: + 98 2161112482; fax: +98 2166405141. E-mail address: moazzeni@khayam.ut.ac.ir (H. Moazzeni). 0367-2530/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2009.12.028 solely on the basis of differences in single fruit characters. For example, Sameraria differs from Isatis by its distinct (vs. obsolete) style, and Pachypterygium is separated from Isatis by the presence of thickened (vs. thin) fruit margin (Hedge 1968). Indeed, some authors (e.g., Jafri, 1973; Rechinger, 1958; Sajedi et al., 2005) reduced Pachypterygium to synonymy of Isatis and considered the thickened fruit margin to be unreliable for the separation of these genera. Based strictly on the overall morphology, Al-Shehbaz et al. (2006) suggested that the Isatideae include the genera Pachypterygium, Sameraria, Boreava Jaub. & Spach, Chartoloma, Glastaria Boiss., Schimpera Hochst. & Steud. ex Endl., Spirorrhynchus Kar. & Kir., and Tauscheria. Both Myagrum L. and Tauscheria were placed by Candolle (1821) with Isatis (including Sameraria) in the Isatideae. This morphologically well-defined and primarily central and southwestern Asian tribe is characterized by having indehiscent, 1- or rarely 2-seeded angustiseptate fruits, yellow or rarely whitish flowers, sessile and often auriculate cauline leaves, and simple or no trichomes (Appel and Al-Shehbaz, 2003; Al-Shehbaz et al., 2006). ITS sequence data were shown to be useful in evaluating relationships among several genera of the Brassicaceae, including Arabidopsis (DC.) Heynh. (O’Kane and Al-Shehbaz, 2003), Cardamine L. Author's personal copy ARTICLE IN PRESS 338 H. Moazzeni et al. / Flora 205 (2010) 337–343 (Franzke et al., 1998), Cochlearia L. (Koch et al., 1999b), Crambe L. (Francisco-Ortega et al., 1999), Vella L. (Crespo et al., 2000), Yinshania Ma & Y. Z. Zhao (Koch and Al-Shehbaz, 2000), Sisymbrium L. (Warwick et al., 2002), Thlaspi (Koch and Mummenhoff, 2001), and others (see Al-Shehbaz et al., 2006). Convergence is quite widespread in almost every morphological character in the Brassicaceae (Al-Shehbaz et al., 2006; Dvorák, 1971; Hedge, 1976; Koch et al., 2003; Meyer 1973; Mummenhoff et al., 1997b). Therefore, assessing relationships based solely on morphology will very likely lead to erroneous conclusions (Al-Shehbaz et al., 2006; Koch et al., 1999a; Meyer, 1991). The principal goal of the present study is to investigate the phylogenetic relationships within the complex and widespread genus Isatis and to determine if the genera Boreava, Pachypterygium, Sameraria, and Tauscheria are sufficiently distinct from it. The study also focuses on the evolution of characters within the tribe Isatideae and on their value in the delimitation of genera. Material and methods Plant material DNAs were extracted from 28 Iranian taxa of Isatideae sensu Al-Shehbaz et al. (2006), including Isatis (20 taxa representing the major groups), Boreava (1 sp.), Pachypterygium (3 spp.), Sameraria (2 spp.), and Tauscheria (1 sp.). Myagrum perfoliatum was sister to Isatis in the molecular analyses of Beilstein et al. (2006) and Bailey et al. (2006) and served herein as the outgroup. Because no sequence differences were found among the subspecies of I. cappadocica, they were merged in the analyses as I. cappadocica. The same approach was used for the species pairs I. koeiei and I. raphanifolia (the former was reduced to synonymy of the latter recently, Moazzeni et al., 2008), I. koelzii and I. tinctoria, I. glauca and I. kotschyana, and S. armena and S. elegans. The nomenclature of taxa, collection data, and vouchers are given in Table 1. ITS amplification and sequencing Leaves from herbarium specimens or dried in silica gel were taken from individual plants. Total DNA was isolated following Doyle and Doyle (1987) as modified in Mummenhoff and Koch (1994). Double-stranded DNA of the ITS-1 and ITS-2 regions were amplified using the polymerase chain reaction (PCR) protocol of Mummenhoff et al. (1997a). PCR products were purified using the purification kit (Roche Molecular Biochemicals). The four primers used for sequencing both strands of the ITS-1 and ITS-2 regions were 18 F, 5.8 F, 5.8 R and 25 R (for details, including modification of the 18 F primer, see Mummenhoff et al., 1997a). Sequencing reactions were run on an ABI 377XL automated sequencer. Boundaries of the coding and spacer regions were determined by comparison of our sequences to that of Sinapis alba L. (Rathgeber and Capesius, 1989). DNA sequences were aligned Table 1 Origin, collection data and GenBank accession numbers of taxa used in the current study Taxon Collection data and collector Voucher number and herbarium Genbank accession Borevea orientalis Isatis buschiana I. cappadocica subsp. besseri W. Azarbaijan; Chaldoran to Khoy, 57 km to Khoy, Moazzeni E Azarbaijan; Mianeh to Tabriz, 20 km to Tabriz, Moazzeni Ardebil; 5 km from Khalkhal to Rasht. Above Aznav spring, Moazzeni E Azarbaijan; Mianeh to Qareh Chaman, 26 km to Qareh Chaman, Zarre & Moazzeni Kordistan; Sanandaj to Kamiaran, Noshor village, Awalan mountain, Maroofi Esfahan; Semirom, Vanak, Dalan kuh, Norouzi 35798-TUH 35800-TUH 35756-TUH GQ131309 GQ131310 GQ131311 35797-TUH GQ131312 2108-Hb. Kordistan GQ131333 14157-Esfahana GQ131334 6663-Hb. Kordistan 24858-FUMH GQ131335 GQ131313 35781-TUH 15687-IRAN 20309-TUH 35803-TUH 35777-TUH GQ131314 GQ375458 GQ131315 GQ131316 GQ131317 35759-TUH 12340-TUH 35765-TUH 35785-TUH 29346-TUH 1941-Hb. Kordistan GQ131318 GQ131319 GQ131320 GQ131321 GQ131322 GQ131332 35779-TUH 28466-TUH GQ131323 GQ131324 6946-Hb. Kordistan 35788-TUH 35788-TUH 15060-FUMH 28450-TUH GQ131325 GQ131326 GQ131327 GQ131328 GQ131329 14266-FUMH 16774-FUMH GQ131330 GQ131331 I. cappadocica subsp. cappadocica I. cappadocica subsp. macrocarpa I. cappadocica subsp. stenophylla I. cappadocica subsp. subradiata I. emarginata I. I. I. I. I. gaubae glauca koeiei koelzii kotschyana I. I. I. I. I. I. leuconeura lusitanica minima pachycarpa raphanifolia takhtajanii I. tinctoria I. trachycarpa Myagrum perfoliatum Pachypterygium brevipes P. multicaule P. stocksii Sameraria armena S. elegans Tauscheria lasiocarpa a Kordistan; SW Sanandaj, Dulab pass, Norouzi Khorasan; SE Tabas, road of Nayband to Ali Abad, 47 km to Nistan, Zangooie & Ayatollahi Gorgan; Azadshahr to Khosh Yelagh, Moazzeni Hamedan; Aq-Bolaq, Palat Kohgiluyeh; Yassuj, 5 km from Dehdasht to Behbahan Khorasan; NE Bojnord, Tazeh kand to Gifan, Moazzeni Tehran; N Tehran, road of Firuzkuh, 20 km after Emmamzadeh Hashem, Moazzeni Semnan; 32 km to Firuzkuh from Semnan, Moazzeni Markazi, Arak, Emarat, Ghahreman & Attar Kerman, NE Bazman mt., Moazzeni Kerman; Jebale-Barez, Moazzeni. Tehran, Darake, Naqinezhad Kordistan; Saqez to Baneh, Piramaran village, Nacaroz mountain, Maroofi Khorasan; NE Bojnord, Tazeh kand to Gifan. Moazzeni Khorasan; Torbat-e Jam to Afghanistan border, Cheshmeh-Zakani, Ghahreman & Attar Kordistan; Sanandaj, near Qods hospital, Maroofi Kerman; Jupar mts, Mirtajedini Kerman; Kuh Payeh, Mirtajedini. Khorasan; Sarakhs to Sangar, 21 km to Sangar, Joharchi. Khorasan; Torbat-e Jam to Afghanistan border, Cheshmeh-Zakani, Ghahreman & Attar Khorasan, Birjand, Shokra mountain. Joharchi & Zangooie Prov. Khorasan; NE Mashhad, Akhlamad, Joharchi & Zangooie Research Institute of Forest and Rangelands, Esfahan. Author's personal copy ARTICLE IN PRESS H. Moazzeni et al. / Flora 205 (2010) 337–343 visually by sequential pairwise comparison (Swofford and Olsen, 1990). The alignments required the introduction of seven indels of one-bp length scattered among ITS-1, ITS-2, and 5.8S gene. Empirical studies have shown that different approaches of gap coding have only minimal, if any, effects on ITS tree topologies (reviewed in Baldwin et al., 1995), and indels were coded as missing data in parsimony analysis. Phylogenetic analysis Both maximum parsimony and Bayesian approaches to phylogenetic estimation were used. The data matrix was analyzed by assuming character states unordered and unweighted (i.e., Fitch parsimony) using the heuristic search strategy in PAUPn v. 4.0b10 (Swofford, 2002) with MULPARS, TBR (Tree BisectionReconnection) branch swapping, and random taxon addition. Sets of equally parsimonious trees were summarized by the strict consensus approach. Bootstrap analyses (Felsenstein, 1985) with 1000 replicates were performed to obtain estimates of reliability for each monophyletic group. Pairwise nucleotide differences of unambiguously aligned positions were determined by the DISTANCE MATRIX option in PAUP. Bayesian inference of phylogeny using MrBayes 3.1 (Huelsenbeck and Ronquist, 2003) was performed on the ITS alignment 339 using settings derived from MrModelTest 2.2 analysis (Nylander, 2004) and the Akaike information criterion (AIC). Following MrModeltest, the symmetrical model of sequence evolution (SYM) was employed in MrBayes, with an allowance for a gamma (G) distribution of rates. The Markov chain Monte Carlo search was run with 4 chains, one of which ‘‘cold,’’ for 1,000,000 generations, with trees being sampled every 100 generations. After discarding the first 25% of trees as ‘‘burnin,’’ Bayesian search results were summarized by 50% majority rule consensus and posterior probability values (‘‘clade credibility’’) are indicated at the branches (Fig. 1). Morphological data Patterns of morphological evolution were assessed for 28 characters emphasized in earlier taxonomic treatments of Isatideae (e.g., Davis, 1965; Hedge, 1968; Sajedi et al., 2005). The characters (Table 2) were compiled from original observations on field and herbarium material and further discussed in Moazzeni (2006). The polarity of character states was determined following Maddison et al. (1984). The cladistic analysis of morphological characters is not presented here, but four characters (Fig. 2, Table 2) previously considered as taxonomically important in Isatis and allied genera (see Davis, 1965; Hedge, 1968) were optimized onto the Bayesian Fig. 1. Bayesian 50% majority rule consensus tree inferred from nuclear ITS sequences of selected Isatis species and related genera Boreava, Pachypterygium, Tauscheria and Sameraria. Posterior probability values (clade credibility) are shown below branches. The sectional classification is that of Hedge (1968). Myagrum perfoliatum served as the outgroup. Author's personal copy ARTICLE IN PRESS 340 H. Moazzeni et al. / Flora 205 (2010) 337–343 Table 2 Morphological characters used for the optimization onto the Bayesian tree. Characters 1, 11, 17, and 25 are mapped onto the Bayesian 50% majority rule consensus tree inferred from nuclear ITS sequence data (Fig. 2). Habit 1. Annual or biennial (0), perennial (1) 2. Height: Z40 (0), o 40 (1) Basal leaves 3. Shape of blade: oblong (0), obovate to rounded (1) 4. Apex: obtuse to rounded (0), acute (1) 5. Blade length: 47 cm (0), r 7 cm (1) 6. Petiole length: r 7 mm (0), 47 mm (1) Cauline leaves 7. Limb length: r 40 mm (0), 440 mm (1) 8. Base: auriculate (0), not auriculate (1) 9. Apex of auricle: acute (0), obtuse to rounded (1) 10. Length of auricle: 45 mm (0), r5 mm (1) Flower 11. Petal shape: oblong (0), obovate (1) 12. Size of petals: 43 mm (0), r 3 mm (1) 13. Length of the longest filament:r 3 mm (0), 4 3 mm (1) Pedicel 14. Orientation: erect (0), patent to reflexed (1) 15. Apex: not thickened (1), thickened (0) 16. Size: r 5 mm (0), 45 mm (1) Fruit 17. Type: silicle (0), silique (1) 18. Length: r 10 mm (0), 410 mm (1) 19. Width: r 6 mm (0), 46 mm (1) 20. Locule: non-spongy (0), spongy (1) 21. Position of locule: apex or base (0), middle (1) 22. Base: cuneate (0), cordate or obtuse (1) 23. Apex: beaked (0), beakless (1) 24. Wing position: apical or basal (0), all around (1) 25. Rim of fruit: non thickened (0), thickened (1) Seed 26. Length: 43 mm (0), r3 mm (1) 27. Seed shape: oblong (0), elliptic (1) 28. Reticulate to reticulate–areolate (0), lineate (1), ocellate (2) tree using Mesquite version 1.11 (Maddison and Maddison, 2006). Fruit shape is also outlined on the Bayesian tree (Fig. 1). Results The length of ITS-1 and ITS-2 regions and the 5.8S gene within the Isatideae (sensu Al-Shehbaz et al., 2006) varied from 268 to 269 bp for ITS-1, 167 to 169 bp for ITS-2, and 173 to 174 bp for 5.8S gene. Proper alignments of ITS sequences resulted in a matrix of 622 characters. Of these, 531 base positions were constant and uninformative, 47 were variable but not parsimony informative, and 44 were potentially parsimony informative. The sequence alignments required the introduction of gaps in ITS-1 and ITS-2. No alignment ambiguities were found. The sequence divergences among the studied species varied between 3.3% in the species pairs Isatis tinctoria-I. gaubae and P. stocksii-P. multicaule to as high as 6.4% in the species pair I. takhtajanii–I. emarginata. Fitch parsimony analysis (heuristic search) resulted in three maximally parsimonious topologies of 135 steps with a consistency index (CI) of 0.793 and retention index (RI) of 0.818. The Bayesian analysis of the ITS sequence data set is consistent with that of maximum parsimony (MP) analysis. The tree topology of the parsimony strict consensus tree (not shown) is very similar to Bayesian tree (BT) in Fig. 1. The two differences observed are: 1-Tauscheria is not sister to the rest as appeared in the parsimony tree (PT), and this node, with 57% bootstrap support, collapsed in the BT (see clade C in Fig. 1); 2-Boreava is not sister to the remaining species (clade I in Fig. 1), and the node, with bootstrap below 50%, also collapsed in the BT. Both molecular analyses suggest that analyzed taxa of the genus Isatis may be divided into two main lineages (Fig. 1). Clade I This well-supported clade (98% posterior probability in BT and 87% bootstrap in MP) includes 12 species in the genera Isatis, Boreava, and Sameraria (Fig. 1). It consists of two subclades (A and B), and B. orientalis. Subclade A is well supported (100% posterior probabilities in BT and 88% bootstrap in MP) and includes two groups, of which one forms a polytomy of perennial species with silicle fruits, and the other includes the sister species I. lusitanica and I. raphanifolia that are annuals with silique fruits and unique ocellate microsculpturing of seed surface (Moazzeni et al., 2007). Subclade B, which is moderately supported (79% posterior probabilities in BT and 57% bootstrap in MP), includes four species of Isatis and S. armena and is characterized by the annual or biennial habit and often siliquose fruits (S. armena has silicles). Clade II This clade of seven species is also well supported (100% posterior probabilities in BT and 91% bootstrap in MP) and includes Tauscheria lasiocarpa and three species each of Isatis and Pachypterygium. Species of this clade are annual herbs with either siliques (Isatis) or silicles (Pachypterygium and Tauscheria). Patterns of morphological evolution The evolution of morphological characters previously emphasized in taxonomic treatments of Isatideae genera (e.g., Davis, 1965; Hedge, 1968; Sajedi et al., 2005; Table 2) was investigated by optimizing character-state changes onto the BT tree. The BT tree (Fig. 1) shows a moderate congruency to the morphological findings in the above studies. Among the 28 characters studied, the optimization of four taxonomically important characters onto the Bayesian tree is shown in Fig. 2A D and further discussed below. These characters include habit (Fig. 2A), petal shape (Fig. 2B), and fruit type (Fig. 2C and D). Discussion The major classification systems of the Brassicaceae (e.g., Hayek, 1911; Janchen, 1942; Prantl, 1891; Schulz, 1936) variously divided the family into 4–19 tribes based on a limited number of morphological characters, and they did not pay enough attention to convergence as a factor in the family evolution. However, recent studies (e.g., Appel and Al-Shehbaz, 2003; Al-Shehbaz et al., 2006; Koch et al., 2003; Price et al., 1994; Zunk et al., 1996) have demonstrated the polyphyly and artificiality of almost all tribes recognized in those earlier systems. Such artificiality was also elucidated at the generic level, especially by molecular studies on Cochlearia (Koch et al., 1999b), Thlaspi (Mummenhoff et al., 1997a, b), Arabis (Koch et al., 1999a, 2001), and Arabidopsis (O’Kane and Al-Shehbaz, 2003). The Isatideae was suspected to be monophyletic based strictly on morphology (Al-Shehbaz et al., 2006; Koch et al., 2003), but prior to the present study no molecular phylogenetic analysis was conducted on the tribe. The ITS data presented herein show that Isatis sensu Schulz (1936) is not monophyletic because its species form two major clades, within one of which (clade I, Fig. 1) the genera Pachypterygium and Tauscheria are nested, and within the other (clade II, Fig. 1) both Sameraria and Boreava are nested. Author's personal copy ARTICLE IN PRESS H. Moazzeni et al. / Flora 205 (2010) 337–343 341 Fig. 2. A D. Overlay of selected morphological characters on the Bayesian tree inferred from nuclear ITS sequences of selected Isatis taxa and related genera. A. Habit: annual to biennial (open lines), perennial (black lines); B. petal shape: oblong (open lines), obovate (black lines); C. fruit type: silicle (open lines), silique (black lines) and D. rim of fruit: not thickened (open lines), thickened (black lines). Previous molecular analyses (e.g., Beilstein et al., 2006, 2008; Bailey et al., 2006) show that Isatis and Myagrum form a monophyletic group (93% bootstrap support) sister to a clade including the tribes Brassiceae, Schizopetaleae, and Sisymbrieae. However, the phylogenetic relationships and delimitation of genera within the Isatideae were not analyzed. According to the present ITS sequence data (Fig. 1), Isatis should be broadly delimited to include the genera Tauscheria, Boreava, Pachypterygium, and Sameraria. The systematic position of various genera of the Isatideae is discussed below in the light of morphological and molecular data. Boreava Schulz (1936) placed Boreava and Tauscheria in the tribe Euclidieae, whereas Koch et al. (2003) and Al-Shehbaz et al. (2006) suggested that they belong to the Isatideae. Our molecular data support the latter conclusion. Boreava is readily separated from the other genera of Isatideae by having a distinct style and quadrangular, 4-winged fruits. Molecular data (Fig. 1) show that Boreava clearly belongs to clade I. Tauscheria This genus clusters with Pachypterygium and three species of Isatis (Fig. 1). The main differences between Boreava, Tauscheria and Pachypterygium are fruit characters (e.g., presence vs. absence of fruit wings or thickened margin). It was recently suggested that differences in only a few genes can cause substantial alterations in fruit shape, size, and dehiscence in the Brassicaceae (see Al-Shehbaz et al., 2006 and references therein). Therefore, the use of fruit characters alone for the delimitation of genera must be critically evaluated. Author's personal copy ARTICLE IN PRESS 342 H. Moazzeni et al. / Flora 205 (2010) 337–343 Pachypterygium The present molecular analysis demonstrates that Pachypterygium is polyphyletic, and its three species group with Tauscheria and three Isatis species in clade II (Fig. 1). The genus is traditionally distinguished from other members of the Isatideae by having thickened (vs. thin) fruit margins. As shown in Fig. 2D, fruit thickening is clearly homoplasious in the tribe and therefore is an unreliable phylogenetic character. Rechinger (1958) and Jafri (1973) suggested that Pachypterygium should be united with Isatis, but this position was not followed by other authors (e.g., Appel and Al-Shehbaz, 2003; Hedge, 1968). Based on our molecular analysis, Pachypterygium cannot be maintained as distinct from Isatis. homoplasy and can be considered as synapomorphies of certain monophyletic groups. Fifteen additional characters provided at least some support for grouping of taxa. The remaining six characters did not provide support for any grouping. These are: apex of basal leaves (4), base of cauline leaves (8), length of auricles (10), pedicel apex (15), pedicel length (16), and fruit base (22). Only a few characters previously considered as taxonomically important in Isatis and allied genera (see Davis, 1965; Hedge, 1968) are discussed below in connection with the molecular data. Habit The distribution of this character is strikingly congruent with the Bayesian tree (Fig. 2A). Most species of the Isatideae are annuals, as in the outgroup, and the perennial habit is apomorphic. Sameraria Sameraria is nested within Isatis (Fig. 1), and the two genera differ solely by the presence in Sameraria of a distinct (vs. obsolete) style (Davis, 1965; Hedge, 1968; Jafri, 1973). However, field studies by one of us (H. M.) demonstrated that the style can be highly reduced in some populations of S. armena. The separation of these genera was highly questioned by Jafri (1973) and Al-Shehbaz et al. (2006) who suggested uniting them under Isatis, the earlier name. Our molecular data support this view, and we recommend the reduction of Sameraria to synonymy of Isatis. The recognition of mono- or oligospecific genera in the Isatideae mirrors many other cases in the Brassicaceae where the delimitation of such genera is based on only minor differences in fruit characters that are overemphasized at the expense of potentially more useful aspects of other structures. Examples include Twisselmannia Al-Shehbaz vs. Tropidocarpum Hook., Lepidium vs. Coronopus Zinn., Heliophila L. versus the endemicrelated genera of the Cape region of South Africa Cycloptychis E. Mey., Thlaspeocarpa C. A. Sm., Schlechteria Bolus, Silicularia Compton, and Brachycarpaea DC. (Al-Shehbaz et al., 2006; Koch et al., 2003; Mummenhoff et al., 2005). Infrageneric classification of Isatis Boissier (1867) divided Isatis into the four sections Eremoglaston, Apterolobus, Sameraroides, and Glastum based on minor differences in fruit size and nature of the seed locule (spongy vs. membranous). He included Pachypterygium and Sameraria in sections Sameraroides and Eremoglaston, respectively. Iran is the only country in which all of Boissier’s sections are represented by indigenous species, and taxa from all four sections were sampled in the present study. As shown in Fig. 1, the molecular data strongly suggest that all sections are polyphyletic and artificially delimited. For example, I. minima (fruits narrowly winged or wingless, locule spongy) and I. emarginata (fruits winged all around, locule membranous), representing the polyphyletic sections Apterolobus and Eremoglaston, respectively, are sister taxa with 100% bootstrap support (Fig. 1). Finally, we suggest considering a broadly defined genus Isatis which can be divided into two monophyletic lineages (lineage I and II in Fig. 1). However, formal taxonomic subdivision of Isatis should await further studies including additional species. Petal shape Both Myagrum perfoliatum and Boreava orientalis have oblong petals, and the rest of the Isatideae have obovate petals (Fig. 2B). It is not possible to determine the plesiomorphic state, but if the oblong petals are plesiomorphic, then a reversal took place in Boreava. On the other hand, if obovate petals are plesiomorphic, then oblong petals evolved independently in the two species above. The same can be said about pedicel orientation, which is erect in these two species and reflexed in the rest of the tribe. Fruit type As shown in Fig. 2C, species with siliques apparently evolved repeatedly from ancestors with silicles. The phylogenetic value of such difference is highly questionable (see conclusions). Fruit margin Although a thin margin appears to be plesiomorphic (Fig. 2D), it shows a reversal in the clade including I. minima, I. emarginata, and I. trachycarpa. Seed-coat microsculpturing The reticulate or reticulate–areolate sculpturing of seed coat is the plesiomorphic state in Isatideae. By contrast, the ocellate seeds are uniquely restricted to the clade consisting of I. raphanifolia and I. lusitanica, whereas lineate seeds evolved independently in P. multicaule and P. brevipes. Conclusions The molecular data presented herein demonstrate that the reliance on fruit characters alone in the delimitation of genera may well lead to erroneous taxonomic results. Fruit characters should be critically evaluated in light of molecular and other morphological data (Al-Shehbaz et al., 2006; Koch et al., 2003; Mummenhoff et al., 2005). Molecular data show that the smaller genera Boreava, Pachypterygium, Sameraria, and Tauscheria are nested within the larger Isatis. The maintenance of these four genera as distinct would make Isatis polyphyletic. Therefore, we suggest uniting them with Isatis. Acknowledgments Morphological character evolution Among 28 morphological characters selected (Table 2) seven characters including habit (character 1), shape of basal leaves (3), blade length (5), petal shape (11), length of the paired filaments (13), pedicel orientation (14), and fruit apex (23), do not show any The authors are grateful to anonymous referees for critical reviewing of the manuscript and for their useful suggestions and improvement of the manuscript. We thank the curators of the herbaria cited for providing plant material. We are grateful to U. Coja for technical help and to Dr. A. R. Khosravi (University of Author's personal copy ARTICLE IN PRESS H. Moazzeni et al. / Flora 205 (2010) 337–343 Shiraz, Iran), Dr. Sh. Kazempour Osaloo (Tarbiat Modarres University, Tehran) and M. Mehrnia (Potsdam University, Germany) for advice. 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