Molecular (ISSR, cp DNA, ITS) and Morphological Study of the Genus Tragopogon L. (Asteraceae) Hejraneh Azizi (  hejraneh.azizi@yahoo.com ) Shahid Beheshti University Masoud Sheidai Shahid Beheshti University Valiollah Mozaffarian Research Institute of Forests and Rangelands Zahra Noormohammadi Azad University: Islamic Azad University Research Article Keywords: Tragopogon, Morphometry, ISSR, ITS, cp DNA. Posted Date: January 10th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1160711/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 2 3 Molecular (ISSR, cp DNA, ITS) and Morphological study of the genus Tragopogon L. (Asteraceae) 4 5 6 Hejraneh Azizi 1*, Masoud Sheidai 1, Valiollah Mozaffarian 2, Zahra Noormohammadi 3 7 8 1Faculty 9 2Research of life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Institute of Forests and Rangelands, Tehran, Iran 10 3Department 11 University (SRBIAU), Tehran, Iran 12 *Corresponding 13 14 15 16 17 18 19 20 21 22 23 24 of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad author. E-mail: Hejraneh.azizi@yahoo.com 25 Abstract 26 27 Tragopogon L. (Cichorioideae, Lactuceae, Scorzonerinae) is an Old World genus with 150 species, Rechinger in 28 Flora Iranica divided this genus in 13 section and 37 species that 26 species of them are exist in Iran. Safavi et al. 29 divided it into 26 species without sections in flora Iran. Despite the anatomical and molecular studies done around the 30 world, the exact classification of this genus is not clear due to the high number of secret species, hybridization, 31 polyploidy and rapid diversification. The morphology studies of 32 species and Molecular studies (ISSR, ITS, cp 32 DNA) of 22 species of the genus Tragopogon was investigated. The purpose of these studies are classification and 33 determination of interspecific relationship in this genus. Sections of Rubriflori, Sosnowskya, Chromopappus, Majores, 34 Angustissimi, Krascheninnikovia in flora of Iranica are confirmed on the basis of morphometry and molecular data. 35 Section of Profundisulcati in flora Iranica is confirmed on the base of morphometry data. The Species of T. jesdianus, 36 T. porphyrocephalus, T. rezaiyensis and T. Stroterocarpus in the flora of Iranica are not classified inany sectionwhich 37 we classified in the Rubriflori section, Cp DNA dendrogram are not useful for classification in this genus and 38 Chloroplast sequences are very similar among Tragopogon species, Therefore, the use of cp DNA markers in the 39 classification of this genus is not recommended. 40 Keywords: Tragopogon, Morphometry, ISSR, ITS, cp DNA. 41 42 43 44 45 46 47 48 49 50 51 Introduction: 52 Tragopogon L. is an Old World genus of approximately 150 species that occurs across Eurasia from the Atlantic to 53 the Pacific Ocean, with a center of distribution in the Mediterranean region, the Middle East and Eastern Europe 54 (Mavrodiev et al. 2005; 2008a-c). The monophyly of the genus was strongly supported in a recent phylogenetic 55 analysis of Scorzonerinae based on internal transcribed spacer (ITS) sequence data (Mavrodiev et al. 2004). 56 Tragopogon includes biennial and perennial herbs with linear or linear-lanceolate leaves; solitary, simple, or sparingly 57 branched stems; one or only a few capitulum and receptacles without scales. The achenes of Tragopogon are usually 58 fusiform, with five to 10 more or less distinct ribs and a beak of varying length. The involucral bracts are always in 59 one row, ligulate flowers are yellow or purplish, and the pappus is in one row of mostly plumose hairs (Richardson 60 1976). It is almost impossible to identify the species without adequate knowledge of mature achene and ligule color. 61 Tragopogon is taxonomically difficult due to its high degree of species morphological variability, which in turn arises 62 due to frequent occurrence of inter-specific hybridization and different ploidy levels (Mavrodiev et al. 2008; Bell et 63 al. 2012). Occurrence of interspecific hybridization reported in the literature (Ownbey 1950; Ownbey & McCollum 64 1953; Pires et al. 2004; Buggs et al.2008; Mavrodiev et al. 2008) or facultative apomixis recently found in T. dubius 65 (Kashin et al. 2007)Relationships within Tragopogon are poorly understood. Many species of Tragopogon have not 66 been placed in a section; most of these are narrow endemics that have been recognized and named but not treated 67 taxonomically furthermore, the genus has never been the subject of a comprehensive monograph. Regional floras have 68 provided treatments for species only in those geographic areas. (Mavrodiev et al. 2005). (Blanca and De la guardia, 69 1997) Schishkin (1961) In Komarov Flora Russia divided it in 17 section and 79 species that 10 species of them are 70 share with flora Iran. Rechinger (1977) according to schishkin in flora Iranica divided this genus in 13 section and 37 71 species that 26 species (11 species endemic) of them are exist in Iran (Rechinger 1977). Safavi et al. (2014) divided 72 it into 26 species without sections in flora Iran. 73 In an initial effort to resolve relationships within Tragopogon, we conducted a phylogenetic analysis of Tragopogon 74 species in Iran, using ISSR (ISSRs: Inter simple sequenced repeats), ITS and cp DNA (non-coding region of 75 chloroplast DNA) sequence data. 76 77 78 Materials and methods: 79 Plant material 80 Tragopogon species were collected from natural habitats in Iran in 2015-2018.The voucher specimens are deposited 81 in the Shahid Beheshti University herbarium (HSBU), herbarium of Research Institute of Forests and Rangeland 82 (TARI) and herbarium of Museum of Natural History Vienna (W). (Table 1) 83 Morphological studies 84 26 species of this genus belonging to the flora of Iran and 6 species belonging to the flora of Iranica were studied. 85 Morphological characters studied were: Flower color, Anther tube color, Collar type, Investigation of branching in 86 plant, presence of indumentums in plant, indumentums of involucre bracts, Number of flowers per pile, status of 87 involucre bracts and ligule, Ratio length of bract to papus, shape of base leaves, Presence or absence of sheath in leaf, 88 condition of tips of stem leaves, Leaf margin type, Leaf type, Condition of middle vein thickness, Central achene 89 surface, Marginal achene surface, papus status, condition of papus and achene, papus type, Condition of beak to 90 achene, beak type, condition of tip of beak, Surface on the beak, diameter of inflorescence, length of involucre, Width 91 of involucre, length of bract, width of bract, number of involucre bracts, length of achene, width of achene, length of 92 papus, length of beak, Number of vine or wings on the achene, length of the base leaves, width of the base leaves, 93 Stem leaf length, stem leaf width, Number of basal leaf veins, ratio of length to width of the involucre, ratio of length 94 to width of the bract, ratio of length to width of the achene, ratio of length to width of the papus, ratio of length to 95 width of the beak, ratio of length to width of the base leaf, ratio of length to width of the stem leaf. 96 DNA extraction and molecular assays 97 Fresh leaves were collected from randomly selected plants and dried in silica gel powder. Genomic DNA was extracted 98 using a CTAB activated charcoal protocol (Sheidai et al. 2013). The quality of extracted DNA was examined by 99 electrophoresis on 0.8% agarose gels. four ISSR primers custom synthesized by UBC (the University of British 100 Columbia) were used: UBC807, UBC810, UBC 834, (AGC) 5GA. PCR reactions were performed in a 25μl volume 101 containing at final concentrations 10 mM Tris-HCl buffer at pH 8, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each 102 dNTP (Bioron, Germany), 0.2 μM of a single primer, 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, 103 Germany). The DNA amplification reactions were performed in a thermocycler (Germany) with the following 104 program: 5 min initial denaturation step at 94°C, 45s at 94°C; 1min at 50°C and 1:30 min at 72°C. The reaction was 105 completed with a 7 min extension step at 72°C. The amplification products were visualized by electrophoresis on 2% 106 agarose gels, followed by the ethidium bromide staining. The fragment size was estimated by using a 100 bp molecular 107 size ladder (Fermentas, Germany). 108 The ITS5 region was amplified with 0.2 μM primer ITS5 (5’-GGA AGT AAA AGTCGT AAC AAG G-3’; Bioron, 109 Germany), and primer ITS4 (5’-TCC TCC GCT TATTGA TAT GC -3’) (White et al. 1990). PCR reactions were 110 performed in a 25μl volume containing at final concentration 10 mM Tris-HCl buffer at pH 8, 50 mM KCl, 1.5 mM 111 MgCl2, 0.2 mM of each dNTP (Bioron, Germany), 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, 112 Germany). The following thermocycler parameters were used: 95°C for 2 min, followed by 33 cycles at 95°C for 30s, 113 56°C for 60s, and 72°C for 2 min, followed by one final extension step at 72°C for 7 min. 114 The plastid intergenic spacer psbA-trnH(GUG) was amplified and sequenced with universal primers following the 115 methodology of Shaw et al. (2005) and Timme et al. (2007). The psbA-trnHGUG forward primer was trnHGUG (5´- 116 CGC GCA TGG TGG ATT CAC AAT CC-3´) and, the reverse primer was psbA (5´- GTT ATG CAT GAA CGT 117 AAT GCT C-3´) (Table 2). Each 20 μl PCR reaction contained 10 μl of 2x PCR buffer, 0.5 mM of each primer, 200 118 mM of each dNTP, 1 Unit of Taq DNA polymerase (Bioron, Germany), and 1 μl of template genomic DNA at 20 ng 119 μl-1. The amplifications were performed in a Techne thermocycler (Germany) with the following program: 2 min 120 initial denaturation step 94°C, 1 min at 94°C; 1 min at 58°C and 1 min at 72°C. The reaction was completed by a final 121 extension step of 6 min at 72°C. 122 The PCR products were electrophoresed on 2.5% agarose gels and visualized through GelRed™ Nucleic Acid Gel 123 Staining. Fragment size was estimated using a 100 bp size ladder (Thermo- Fisher Scientific, Waltham, MA USA). 124 Data analysis 125 Morphological analyses 126 Grouping of the species was obtained by using UPGMA (Unweighted Paired Group Method with Arithmetic mean) 127 and Ward (Minimum spherical cluster method) as well as PCoA (Principal Coordinate Analysis) (Podani, 2000). 128 Morphological characters were first standardized (Mean = 0, Variance = 1) and used to establish Euclidean distances 129 among pairs of taxa (Podani, 2000; Sheidai et al., 2014). The obtained distances were used for clustering. 130 Morphological difference of the studied species was investigated by ANOVA (Analysis of Variance) and CVA 131 (Canonical Variance Analysis). PCA (Principal Components Analysis) was performed to identify the most variable 132 morphological characters (Podani, 2000). Morphometric analyses were performed by PAST ver. 2.17 (Hammer et 133 al., 2012). 134 ISSR analysis 135 Significances of genetic differences among the studied species was determined by AMOVA (Analysis of Molecular 136 Variance) with 1000 permutations for dominant molecular markers as implemented in GenAlex v.6.4 (Peakall and 137 Smouse 2006). Nei’s genetic distances were determined among the studied species and used for clustering. For 138 grouping of the plant specimens, Neighbor Joining (NJ) clustering and PCoA were used (Podani 2000). GenAlex 6.4, 139 and PAST v.2.17 (Hammer et al. 2012), programs were used for these analyses. The genetic structure of species was 140 studied by STRUCTURE (Pritchard et al. 2000), for dominant markers (Falush et al. 2007), using the admixture 141 model. The Markov chain Monte Carlo simulation was run 20 times for each value of K for 106 iterations after a burn- 142 in period of 105. All other parameters were set at their default values. 143 Cp-DNA and nuclear gene sequence analyses 144 The intergenic chloroplast spacer psbA-trnHGUG as well as the ITS region nuclear sequences obtained for all the 145 studied species. The sequences obtained were aligned by MUSCLE program implemented in MEGA ver. 5 (Tamura 146 et al. 2011) and used to study the species relationship by different phylogenetic reconstruction methods like: Neighbor 147 Joining, UPGMA clustering (Unweighted paired group using average), and maximum parsimony and maximum 148 likelihood using MEGA v. 5 (Tamura et al. 2011) The proper model for sequence evolution was determined by the 149 same option provided in MEGA program. 150 151 152 153 154 155 Table 1. Tragopogon species studied, their locality information, code and voucher number Species code Locality Voucher number 1 T. capitatus Cap Iran: East Mazandaran W-2000/06412 2 T. vaginatus Vag1 Iran: West Azerbaijan: Sardasht- Piranshahr(IS) TARI-7748 2 T. vaginatus Vag2 Iran: West Azerbaijan: Ardalan village (IS) TARI-65501 2 T. vaginatus Vag3 Iran: Khalkhal to Ardebil TARI-86534 2 T. vaginatus Vag4 Iran: East Azerbaijan: Sahand (IS) TARI-30669 2 T. vaginatus Vag5 Iran: Kurdistan: Karabad (IS) TARI-644 2 T. vaginatus Vag6 Iraq: Sulaimaniya, inte Sulaimaniya et Dokan W-1991/4989 2 T. vaginatus Vag7 Iraq: Sulaimaniya, inter Kirkuk to ward Sulaimaniya W- 1991/19529 3 T. afghanicus Afg1 Afghanestan: qala salcan W- 1956/3032 3 T. afghanicus Afg2 afghanestan: Khurak W-1956/3122 4 T. paradoxus Par1 Afghanestan: N Darai suf, between qala sarkari and W-1987/4287 kupruk 4 T. paradoxus Par2 Afghanestan: nordl. Pul e khomrie W-1965/15880 5 T. kemulariae Kem1 Iran: West Azerbaijan: Khoy, Qoture (IS) TARI-84101 5 T. kemulariae Kem2 Iran: Hamadan: Fakhr abad (IS) TARI-4254 5 T. kemulariae Kem3 Iran: Kurdistan: 20 km toward Hamadan (IS) TARI-84907 5 T. kemulariae Kem4 Iran: Weast Azerbaijan: Khoy, Grees village (IS) TARI-84132 5 T. kemulariae Kem5 Iran: Ardebil: Arasbaran (IS) TARI-24712 5 T. kemulariae Kem6 Iran: Ardebil: Arasbaran (IS) TARI-24651 6 T. sosnowsky Sos Armenia,Sevan W-2004/2395 7 T. vvedenskyi Vve1 Afghanestan: Farakulum W-1980/3311 7 T. vvedenskyi Vv2 Iran: Tehran: Haraz valley TARI-85712 7 T. vvedenskyi Vv3 Iran: West Azerbaigan: Silvana to Salmas road (IS) TARI-85193 7 T. vvedenskyi Vv4 Iran: Semnan TARI-40684 7 T. vvedenskyi Vv5 Iran: Isfahan: Golestan koh (IS) TARI-83087 7 T. vvedenskyi Vv6 Iran: Gorgan: Almeh TARI-12618 7 T. vvedenskyi Vv7 Iran: Gorgan: Almeh TARI-12619 7 T. vvedenskyi Vv8 Iran: Mazandaran: Plur to Rineh ( 4 IS)(cp)(ITS) 8 T. longirostris Long1 Iran: Tehran: solghan valley TARI-32617 8 T. longirostris Lon2 Iran: Khorasan: Tandureh,Chehel mehr TARI-50839 8 T. longirostris Long3 Iran: Chahar mahale bakhtiari:Shalam zar,Agha saner TARI-54586 8 T. longirostris Long4 Iran: West Azerbaigan: Urmieh lake,Islamic island TARI-86916 8 T. longirostris Long5 Iran: Kermanshah TARI-60769 8 T. longirostris Long6 Iran: Kurdistan: Baneh to Marivan TARI-29317 8 T. longirostris Long7 Iran: Gorgan: Almeh TARI-12620 8 T. longirostris Long8 Iran: Fars:Nor abad TARI-45696 8 T. longirostris Long9 Iran: Alborz: Karaj- Chalus (IS) HSBU-2018801 8 T. longirostris Long1 Iran: Kurdistan: Baneh HSBU-2018802 Iran: Kurdistan: Saqez, Zagheh (2 IS) HSBU-2018803 Syria * W-1980/296324 HSBU-2018800 0 8 T. longirostris Long1 1 8 T. longirostris Long1 2 8 T. longirostris Long1 Iraq: Sulaimaniya, inter Kirkuk to ward Sulaimaniya W-1980/19526 3 8 T. longirostris Long1 E- Afghanestan: Kabul, Band-I Kharghak W-1980/619 4 9 T. badakhshanicus Bad Afghanestan: Wakhan, Ab-Gaj W-1965/18843 10 T. gracilis Grac1 Afghanestan: Paktya, Kotgay, Mandaher wald W-1980/1725 10 T. gracilis Grac2 East Afghanestan: Khost, chakmanni W-1980/777 11 T. gongylorrhizus Gon1 Iran: West Azerbaigan: Urmieh lake, Spireh island TARI-15052 11 T. gongylorrhizus Gon2 Iran: Gorgan # TARI-15053 11 T. gongylorrhizus Gon3 Iran: Gorgan: Golestan Park (IS) W-1980/5141 11 T. gongylorrhizus Gon4 Iran: Gorgan 12 T. buphthalmoides subsp. Bupb1 Iraq: Suleimaniya to Ghala Daze Bupb2 Iran: Tehran: Dehnar(IS) HSBU -4006 Bupb3 Iran: West Azerbaijan: Sardasht, Gerzhal(IS) HSBU -4007 Bupb4 Iran: West Azerbaijan: Jaldian HSBU -4008 Bupb5 Iran: Mazandaran: Siah bisheh(IS) HSBU -4009 Bupb6 Iran: Mazandaran: Rineh HSBU -4010 Bupb7 Iran: Alborez: Gach sar (IS) HSBU -4011 Bupb8 Iran: Kurdistan: Kani bard HSBU -4012 Bupb9 Iran: Isfahan: Naein (IS) HSBU -4013 Bupb1 Iran: Semnan HSBU -4014 TARI-5123 W-1980/19531 buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. buphthalmoides 12 T. buphthalmoides subsp. Latifolius 0 Buphb11 Iran: Kurdistan: Saqez HSBU -4015 12 T. Bupl1 Iran: Kurdistan: Boein (IS) HSBU -4016 Bupl2 Iran: Mazandaran: Rineh (IS) HSBU -4017 Bupl3 Iran: Alborez: Gachsar (IS) HSBU -4018 Bupl4 Iran: Tehran: Dehnar )IS( HSBU -4019 Bupl5 Iran: West Azerbaijan: Mosalan HSBU-4020 Bupl6 Iran: West Azerbaijan: Sardasht,Gerzhal (IS)(ITS) HSBU-4021 buphthalmoides subsp. Latifolius 12 T. buphthalmoides subsp. Latifolius 12 T. buphthalmoides subsp. Latifolius 12 T. buphthalmoides subsp. Latifolius 12 T. buphthalmoides subsp. Latifolius 12 T. buphthalmoides subsp. Latifolius 13 T. rechingeri Rec1 Iran:)Hamadan: Agh blagh (IS) TARI-3392 13 T. rechingeri Rec2 Iran: Hamadan: 20 km Asad abad to Akhtachi )IS( TARI-3443 village 13 T. rechingeri Rec3 Iran: Hamadan: 20 km Asad abad to Akhtachi TARI-3444 village(IS) 13 T. rechingeri Rec4 Iran: Hamadan:8 km East Ganj nameh )IS( TARI-36848 13 T. rechingeri Rec5 Iraq: Kurdistan: Erbil, Haji omran * W-1980/5240 14 T. bornmuelleri Borb1 Iran: Kurdistan: 36 km Sanandaj to Kamiaran, )2IS( TARI-60592 Noshor Valley var. bornmuelleri 14 T. bornmuelleri Borb2 Iran: Kurdistan: Maran village )2IS( Borb3 Iran: Kurdistan: Saqez to Baneh )2 IS( TARI-60418 var. bornmuelleri 14 T. bornmuelleri var. bornmuelleri TARI-2951 14 T. bornmuelleri Borb4 Iran: Kurdistan: Ravandoz W-1986/1645 Borb5 Iran: Kurdistan: Riwandous in m. Sakrisakran W-1966/5248 Borb6 Iraq: Kurdistan:Erbil Borl1 Iran: Kermanshah to Kamiaran (IS)(ITS) TARI-87735 Borl2 Iran: Kermanshah:Mansure agha village, Shahoo TARI-89153 var. bornmuelleri 14 T. bornmuelleri var. bornmuellerVai 14 T. bornmuelleri W-1980/11190 var. bornmuelleri 14 T. bornmuelleri var. latifolius 14 T. bornmuelleri mountain var. latifolius 15 T. acanthocarpus Aca1 Iran: Mazandaran: Rineh, Abgarm TARI-5486 15 T. acanthocarpus Aca2 Iran: Zanjan: 10 km Mah neshan, Ghezal ozan river TARI-61228 15 T. acanthocarpus Aca3 Iran: East Azerbaijan: Marand to Jolfa, Zenoz, 22 km TARI-84034 Zenoz to Kohe Kamar 15 T. acanthocarpus Aca4 Iran: West Azerbaijan: Urmieh University (IS) TARI-17743 15 T. acanthocarpus Aca5 Iran: Alborz: Karaj, Research center to Alborz TARI-1340 15 T. acanthocarpus Aca6 Iran: Hamadan: Absard (IS) TARI-5464 15 T. acanthocarpus Aca7 Iran: Markazi: Arak, Miran khaneh, Sefid khani (IS) 15 T. acanthocarpus Aca8 Iran: Mazandaran: Dashte Nazir (IS) HSBU-2018804 15 T. acanthocarpus Aca9 Iran: Kurdistan: Salavat abad village (IS) HSBU-2018805 15 T. acanthocarpus Aca10 Iran: Tehran: Firuz koh (IS) HSBU-2018806 16 T. graminifolius Gra1 Iran: Tehran: Chitgar (IS) HSBU-14466 16 T. graminifolius Gra2 Iran: North Khorasan:Ziarat, Shirvan (IS) HSBU-14466 16 T. graminifolius Gra3 Iran: East Azarbaijan: Ilkhchi (IS) HSBU-14469 16 T. graminifolius Gra4 Iran: West Azarbaijan: Orumieh HSBU-14470 16 T. graminifolius Gra5 Iran: Markazi: Arak, Gavkhaneh)IS( HSBU-14471 16 T. graminifolius Gra6 Iran: Isfahan: Semirom (IS) HSBU-14472 TARI-47745 17 T. reticulatus Ret1 Iran: West Azarbaijan: Orumieh lake, Kabodan island TARI-24916 17 T. reticulatus Ret2 Iran: Khozestan: Deh dez )IS( TARI-63121 17 T. reticulatus Ret3 Iran: West Azarbaijan: Ghasemloo)IS( TARI-4184 17 T. reticulatus Ret4 Iran: Hamedan: Abas abad)IS( TARI-4252 17 T. reticulatus Ret5 Iran: kurdistan:baneh,boeen)IS( TARI-2081 17 T. reticulatus Ret6 Iran: Mazandaran:Polor,Haraz Road TARI-4461 18 T. kotschyi Kot1 # 18 T. kotschyi Kot2 Iran: Mazandaran:91 Km Karaj to Chalous )IS( TARI-5430 18 T. kotschyi Kot3 Iran: East Azerbayjan :Kandovan)IS( (cp) TARI-4504 18 T. kotschyi Kot4 Iran: Tehran:gajereh)IS( TARI-24193 18 T. kotschyi Kot5 Iran: Tehran:Gajereh)IS( TARI-5473 18 T. kotschyi Kot6 Iran: Mazandaran:8 km to polor TARI-86047 18 T. kotschyi Kot7 Iran: Gorgan:Aalmeh TARI-12619 18 T. kotschyi Kot8 Iran: Lorestan:Khoram Abad)IS( 19 T. marginatus Mar1 Iran: West Azarbayejan:20 Km to Seyah Rod TARI-86729 19 T. marginatus Mar2 Iran: West Azarbayjan :Arasbaran,Topkhaneh TARI-81373 19 T. marginatus Mar3 Iran: Karaj:Kondar)IS( TARI-4374 19 T. marginatus Mar4 Iran: Yazd:Tarzjan Mountain,South Eastern Shirkoh TARI-1452 19 T. marginatus Mar5 Iran: West Azarbayjan: Uromieh,Sero Road)IS( TARI-7708 19 T. marginatus Mar6 Iran: Ardabil:Arasbaran)IS( TARI-20954 19 T. marginatus Mar7 Iran: West Azarbayjan:Sahand Mountain)IS( TARI-99404 19 T. marginatus Mar8 Iran: Gilan:Damash to Jirandeh TARI-13515 20 T. maturatus Mat Iran: Golestan:Golestan jungle,Tangeh Rah W-1976/10148 21 T. coloratus Colo1 Iran: Gorgan TARI-296322 W-1986/32097 HSBU-2018813 21 T. coloratus Colo2 Iran: Gorgan:18 Km from Maraveh Tapeh to Ash TARI-55598 Khaneh 21 T. coloratus Colo3 Iran: Gorgan:43 Km Rodbar to Gilan TARI-60180 21 T. coloratus Colo4 Iran: Azarbayjan:30 Km Southern Khalkhal)IS( TARI-36251 21 T. coloratus Colo5 Iran: Zanjan:25 Km from Gilvan to Zanjan TARI-60280 21 T. coloratus Colo6 Iran: Tehran:Sorkheh Hesar)IS( TARI-5476 21 T. coloratus Colo7 Iran: Tehran:Hesarak TARI-5481 21 T. coloratus Colo8 Iran: Arak:Miran house,Kooh Sefid Khani)IS( TARI-47748 21 T. coloratus Colo9 Iran: Kermanshah:Kamyaran,Varmanjeh,Padegan )IS( TARI-87737 Shahid Rajaee 21 T. coloratus Colo10 Iran: Kermanshah:Tagh Bostan,Tangeh Konesht)IS( TARI-87738 21 T. coloratus Colo11 Iran: Ardabil:Arasbaran protect region,Shib )IS( TARI-81348 Jonobi Jangal Tahghighati 22 T. pterocarpus Pte1 Iran: Chahar Mahal Bakhtyari:Shahr Kord,Shams Abad TARI-96641 22 T. pterocarpus Pte2 Arak:Arak to Khomeyn,Koh Vercheh to Istgah TARI-48044 Macrowave Iran: 22 T. pterocarpus Pte3 Iran: Ghazvin:Almot Region,Balaye Rostaye Moalem TARI-50992 Kalateh 22 T. pterocarpus Pte4 Iran: Alborz:40 Km Karaj Chalos,Seyeda Chal TARI-5447 22 T. pterocarpus Pte5 Iran: Ardabil:8 Km north Khlkhal,Kelar Abad Road TARI-34091 22 T. pterocarpus Pte6 Iran: Isfahan:Ghamishlo protect region,Khersak)IS( TARI-1074 22 T. pterocarpus Pte7 Iran: Kurdistan:36km Sanandaj to Kamyaran, )IS( TARI-60624 noshur valley 22 T. pterocarpus Pte8 Iran: West Azarbaijan: Khoy, Razy valley)IS( 22 T. pterocarpus Pte9 Iran: Semnan: Abr jungle )IS( HSBU-2018814 22 T. pterocarpus Pte10 Iran: Tehran: Chaloos road, Moroud )IS( HSBU-2018815 23 T. collinus Coli1 Iran: Gilan: between Gazvin and Rudbar TARI-1392 TARI-27662 23 T. collinus Coli2 Iran: Khorasan: Birjand, Western part, Asfaraz village TARI-83349 23 T. collinus Coli3 Iran: Khorasan: 42 km Birjand TARI-84745 23 T. collinus Coli4 Iran: Khorasan: Birjand, around Sar bishe, )IS( TARI-83340 Salmabad 23 T. collinus Coli4 Iran: Tehran: Saveh, Rud shor )IS( TARI-9632 23 T. collinus Coli5 Iran: Tehran: Saveh, Rud shor)IS( TARI-9633 23 T. collinus Coli6 Iran: Isfahan: Semirom, Abshar )IS( TARI-88600 23 T. collinus Coli7 Iran: Isfahan: Semirom, Abshar)IS( TARI-88601 24 T.caricifolius Car1 Iran: Tehran: Homand, Absard TARI-5465 24 T.caricifolius Car2 Iran: Mazandaran: Chalus, Dashte Nazir)IS( TARI-1377 24 T.caricifolius Car3 Iran: Karaj: 12 km northwest Karaj TARI-5470 24 T.caricifolius Car4 Iran: East Azarbaijan: 20 km Tabriz to Tehran TARI-28013 24 T.caricifolius Car5 Iran: Yazd: 50 km east Bafegh, Hamsuk village )IS( TARI-56083 24 T.caricifolius Car6 Iran: Bandar Abas: Geno mountain TARI-16083 24 T.caricifolius Car7 Iran: Kerman: 50 kmWest of Khajeh Mountain )IS( TARI-56206 24 T.caricifolius Car8 Iran: Baluchestan: Taftan mountain, Tamndan )IS( TARI-53202 region 24 T.caricifolius Car9 Iran: Tehran: Darband sar)IS( TARI-49230 25 T. bakhtiaricus Bak1 Iran: Chahar mahale Bakhtiari: Brojen, Research TARI-54377 Institute of Forests and Rangelands (IS)(cp) 25 T. bakhtiaricus Bak2 Iran: Chahar rmahale Bakhtiari: Brojen, Bar aftab )IS( TARI-54767 mountain 25 T. bakhtiaricus Bak3 Iran: Fars: Kharman mountain)IS( TARI-46922 25 T. bakhtiaricus Bak4 Iran: Chahar mahale Bakhtiari:Vardanjan to )IS( TARI-54126 Kakolak 25 T. bakhtiaricus Bak5 Iran: Gilan: Loshan to Amar loo)IS( TARI-5445 26 T. gaudanicus Gau1 Iran: Fars: Kazeron, ketel (pir zan))IS( TARI-9145 26 T. gaudanicus Gau2 Iran: North Khorasan: Birjand, Kharashad)IS( 26 T. gaudanicus Gau3 Iran: Kurdistan: around Salavat abad river, East )IS( TARI-12137 TARI-297 Sanandaj 26 T. gaudanicus Gau4 Iran: Khorasan: Hezar masjed mountain)IS( 26 T. gaudanicus Gau5 Afghanestan: Abe chist 27 T. montanus Mon1 Iran: Tehran: Firoz koh, Veresk bridge )IS( 27 T. montanus Mon2 Iran: Golestan: Gorgan,Golestan jungle park, near )IS( TARI-4842 W-1980/3031 TARI-1342 TARI-11056 Bojnord 27 T. montanus Mon3 Iran: Golestan: Gorgan,Golestan jungle park, near )IS( TARI-11057 Bojnord 27 T. montanus Mon4 Iran: Golestan: Gorgan,Golestan jungle park, near TARI-11058 Bojnord 27 T. montanus Mon5 Iran: Golestan: Almeh jungle )IS( TARI-4284 27 T. montanus Mon6 Iran: Golestan: Almeh jungle )IS( TARI-4285 27 T. montanus Mon7 Iran: Golestan: Almeh jungle )IS( TARI-4286 27 T. montanus Mon8 Afghanestan: 65 km north west Harat 28 T. erostris Ero Iran: Kurdistan: Sanandaj to Kamyaran, Avalan W-1980/4276 TARI-9439 mountain 29 T. jesdianus Jez1 Iran: Yazd: Taft to Nier, Sakhoid neck )IS( TARI-77549 29 T. jesdianus Jez2 Iran: Semnan: Shahrood, Shish mountain)IS( TARI-28673 29 T. jesdianus Jez3 Iran: Yazd: Nodoushan, between Sadr abad and Hemat TARI- 77777 abad 29 T. jesdianus Jez4 Iran: Bandar abas: Geno mountain)IS( TARI-39783 29 T. jesdianus Jez5 Iran: Semnan: Turan protect region, western Shotor koh TARI-28457 29 T. jesdianus Jez6 Iran: Semnan: Turan protect region, 4 km to Delbar TARI-28953 29 T. jesdianus Jez7 Iran: Semnan: Turan protect region, 4 km to Delbar TARI-28954 29 T. jesdianus Jez8 Iran: Semnan: Turan protect region, southern part )IS( TARI-28803 Kalateh mountain 29 T. jesdianus Jez9 Iran: Semnan: 30 km northern west Shahrud, )IS( TARI-21160 between Tash and Chahar bagh 30 T. Pro1 Iran: West Azarbaijan: Rezaeieh lake W-1964/4029 Pro2 Iran: Chahar mahale Bakhtiari: Shahre kord, )IS( TARI-62060 porphyrocephalus 30 T. Harchegan porphyrocephalus 30 T. Pro3 T. rezaiyensis TARI-370 Sanandaj, Faghieh Soleiman porphyrocephalus 31 Iran: Kurdistan: 40 km north Kamyaran to )IS( Rez1 Iran: Khorasan: Torbate Heidarieh to Mashhad, 5 km TARI-84842 Kameh Sofla 31 T. rezaiyensis Rez2 Iran: Tehran: Gilavand HSBU-2018816 31 T. rezaiyensis Rez3 Iran: Tehran: Shahid Beheshti university HSBU-2018817 31 T. rezaiyensis Rez4 Iran: Tehran: Abali HSBU-2018818 31 T. rezaiyensis Rez5 Iran: West Azarbaijan: Rabus valley to Rezaeieh W-2000/5182 32 T. stroterocarpus Str Iran: West Azarbaijan: Sardasht W-1980/5183 156 157 158 Results 159 Morphometry 160 161 UPGMA, NJ dendrogram, Ward and PCA plot and Parsimony produced similar results therefore, only UPGMA 162 dendrogram is presented here (Fig 1). species delimitation between the studied species are determined from eachother, 163 T. capitatus ،T. vaginatus ،T. afghanicus ،T. paradoxus located in one cluster Which T. capitatus, T. afghanicus and 164 T. paradoxus belong to the flora of Iranica and do not exist in Iran. Populations belonging to T. kemulariae together, 165 populations belonging to T. longirostris together and populations belong to T. gngylorhizus to gether have formed 166 cluster. 167 T. badachschanicus and T. gracilis species and T. sosnovsky and T. vvedenskyi species show the closest relationship 168 with each other. The species T. badachschanicus, T. gracilis and T. sosnovsky do not exist in Iran and belong to the 169 flora of Iranica. 170 T. kotschy, T. marginatus and one population of T. reticulatus together in a cluster and populations of T. collinus, T. 171 caricifolius, T. bakhtiaricus, T. gaudanicus and one population of T. montanus are located in the same cluster. The 172 populations of T. buphthalmoides, T. bornmuelleri, T. rechingeri, T. acanthocarpus, T. graminifolius with five 173 populations of T. reticulatus are in the same cluster. The populations of T. pterocarpus and T. coloratus are locatedin 174 two separated clusters and are located close to each other. 175 PCA analysis of morphological characters revealed that the firs 3 PCA components comprise about 76% of total 176 variability. Diameter of inflorescence, flower color, number of bract and length of beak showed the highest level of 177 correlation with the first PCA component (40), while character length of papus, length of achene, status of involucre 178 bracts and ligule were highly correlated with the second PCA component (12.81). Therefore, these are the most 179 variable morphological characters among the studied species. 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 Fig 1. UPGMA dendrogram of morphological characters among Tragopogon species (Species code are according to Table 1). 196 197 ISSR markers 198 This study was performed on 110 individuals belonging to 102 populations of 22 species of this genus. We obtained 199 153 reproducible ISSR bands from almost all ISSR primers used. These bands formed our initial data matrix. AMOVA 200 test revealed the presence of significant molecular difference among the studied populations (P = 0.01). It also revealed 201 that 60% of total variance occurred due within species genetic variability, while 40% was due to among species genetic 202 difference. UPGMA, NJ dendrogram and PCOA, PCA, PCO plot produced similar results Therefore, only NJ 203 dendrogram is presented here (Fig. 2). Almost populations of each species were located close to each other. This 204 indicates that the ISSR molecular marker can be used to determine the species delimitation of Tragopogon. ISSR 205 determine delimitation of species and show relationship between species in this genus. ISSR can determine 206 delimitation of species and relationship between species in this genus. Two populations of T. gaudanicus with species 207 of T. jesdianus, T. porphyrocephalus, T. bakhtiaricus, T. montanus and T. caricifolius are in one main cluster. 208 Populations of T. vvedenskyi, T. vaginatus, T. graminifolius are located in separated cluster. Species of T. kotschy and 209 T. marginatus are showed close relationship. Populations of T. longirostris are located in one cluster and are closely 210 related to T. gongylorrhizus. A number of T. buphthalmoides populations and T. bornmuelleri populations are show 211 close relationship. All populations of T. kemulariae were placed in a cluster. The Evanno test produced delta k = 7 as 212 the best number of genetic groups. The STRUCTIRE plot based on k = 7 (Fig. 3) identified seven genetic groups/gene 213 pools. The genetic affinity revealed by STRUCTURE analysis was almost in agreement with the NJ tree result. 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 Fig 2. UPGMA tree of Tragopogon species based on ISSR data (Species code are according to Table 1). 237 238 239 240 241 242 243 244 Fig 3. STRUCTURE plot of Tragopogon species based on ISSR 1:T. graminifolius,2: vaginatus, 3: T. gongylorrhizus, 4: T. vvedenskyi, 5: T. kemulariae, 6: T. acanthocarpus, 7: T. bornmuelleri var. bornmuelleri, 8: T. bornmuelleri var. latifolius, 9: T. rechingeri, 10: T. buphthalmoides var. buphthalmoides, 11: T. buphthalmoides var. latifolius, 12: T. longirostris, 13: T. kotshy, 14: T. marginatus, 15: T. reticulatus, 16: gaudanicus, 17: T. bakhtiariccus, 18: T. montanus, 19: T. collinus, 20: T. caricifolius, 21: T. coloratus, 22: T. pterocarpus, 23: T. jesdianus, 24: T. porphyrocephalus. 245 246 ITS and Cp-DNA sequences 247 NJ, Maximum parsimony and maximum likelihood trees produced similar results and therefore ML tree is presented 248 and discussed (Fig 4). The ML tree based on ITS has two main clusters, T. bornmuelleri var. latifolius is separated 249 from the rest of the species and second cluster is divided into two sub-clusters, One of the sub-clusters includes T. 250 buphthalmoides and T. acanthocarpus. The rest of the species are arranged in sub clusters. T.gaudanicus, T. montanus, 251 T. collinus, T. caricifolius, T. jesdianus, T. stroterocarpus, T. porphyrocephalus, T. bakhtiaricus, T. graminifolius, T. 252 rechingeri, T. bornmuelleri, T. kotschy and T. marginatus are in one sub-cluster and T. rezaiyensis, T. longirostris, T. 253 pterocarpus, T. coloratus, T. vvedenskyi, T. kemulariae are in one sub-cluster. The tree based on cp DNA datashowed 254 poor clustering (Fig 5). T. marginatus, T. colinus and T. rechingeri are arranged in one cluster. Cp DNA data are not 255 suitable for classifying and examining relationship between species in this genus, The results based on ITS sequences 256 are more efficient than the results based on cp DNA for species delimitation and relationship between species. 257 258 259 260 261 262 263 264 265 266 267 268 269 270 Fig 4. ML tree based on ITS sequence data in Tragopogon genus (Lactuca sp: out group, Species code are according to Table 1). 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 Fig 5. ML tree based on cp DNA data in Tragopogon genus (Lactuca sp: out group, Species code are according to Table 1). 290 291 292 DISCUSSION 293 Based on the morphological studies, the species related to each section were placed close to each other and the border 294 between the sections is almost clear. Species of section Majores (T. capitatus, T. vaginatus, T. afghanicus, T. 295 paradoxus) section of Kemulariae (T. kemulariae) section of Krascheninnikovia (T. longirostris) section of 296 Tuberosi(T. gongylorrhizus )section of Tragopogon ( T. gracilis, T. badachschanicus ) section of Angustissimi( (T. 297 sosnovsky , T. vvedenskyi) are clustered based on morphometric data and correspond to the sections of Iranica flora . 298 Marodive et al. 2005 have proven that Tragopogon and Majores sections are monophilic based on ITS-ETS data. 299 Sections include Kemulariae (T. kemulariae), Krascheninnikovia (T. longirostris) Tuberosi (T. gongylorrhizus) 300 confirmed based on ISSR data. Majores, Angustissimi, Krascheninnikovia and Tuberosi sections have been confirmed 301 based on morphometric and ISSR studies, which are consistent with studies by Azizi et al. 2021 and Iranica flora. 302 Species of T. kotschy, T. marginatus, T. reticulatus and T. maturatus are introduced according to the flora of Iranica 303 in Sosnowskya section, which in the present morphometric studies, except for T. maturatus, are arranged in a cluster. 304 Based on morphometric data of T. collinus, T. caricifolius, T. bakhtiaricus, T. gaudanicus and T. montanus are in the 305 same cluster and correspond to section Rubriflori in the flora of Iranica. 306 T. buphthalmoides, T. bornmuelleri, T. rechingeri, T. acanthocarpus, T. graminifolius, T. reticulatus are based on 307 morphometric data in a cluster, Species of T. buphthalmoides, T. bornmuelleri, T. rechingeri, T. acanthocarpus, are 308 classified in the profundisulcate section based on flora of Iranica. T. graminifolius is classified in the Brevirostres 309 section and T.reticulatus is classified in the Sosnowskya section in the flora of Iranica. Based on morphometric data, 310 two species T. graminifolius and T. reticulatus are in section profundisulcati. Subsequently, based on studies of seed 311 morphology and morphology by Sukhorukov and Nilova (2015) and molecular studies of ITS, ETS by Mavrodiev et 312 al. (2005) has also been confirmed. Two species of T. pterocarpus and T. coloratus are located in two separate clusters 313 close to each other based on morphometric data and ISSR data. According to the flora of Iranica, species of T. 314 coloratus and T. petrocarpus have many similarities to each other and are located in the section of Chromopapus 315 Pollen studies performed by Azizi et al. (2021) also confirm this section, therefore We confirm the chromopapus 316 section. 317 Rubriflori sections based on morphological, ISSR, ITS data confirms the flora of Iranica and pollen studies by Azizi 318 et al. (2021).Classification of Iranian endemic species such as T. jesdianus, T. erostris, T. porphyrocephalus, T. 319 rezaiyensis, T. stroterocarpus in the flora of Iranica is unknown, Marodive 2012 Based on 7 nuclear loci (Adh, GapC, 320 LFY, AP3, PI, ITS, ETS) studies species of T. rezaiyensis in B clade, species of T.jesdianus in C clade, species of T. 321 stroterocarpus in D clade, species of T. porphyrocephalus in F clade classified. Based on our morphometric studies, 322 these species belong to the Rubriflori section, ISSR data also proves that two species of T. prophyrocephalus and T. 323 jesdianus belong to the Rubriflori section. Rubriflori section introduced in Iranica flora has been proved by 324 morphometric and ISSR data. The species T.porphyrocephalus, T. stroterocarpus and T. jesdianus belongto Rubriflori 325 section based on ITS data results which is consistent with the results of pollen data by Azizi et al. (2021). 326 According to Marodive et al. (2005) studies, T. jesdianus species were classified in Collini (Rubriflori) section, which 327 this section has T. montanus, T. bornmuelleri, T. collinus, T. jesdianus, T. marginatus. 328 Populations of T. vaginatus species in one cluster as well as populations of T. vvedenskyi species in one cluster and 329 populations of T. graminifolius species in one cluster are arranged, Each of these species, according to the flora of 330 Iranica, are placed in separate sections under the names of Majores, Angustissimi, Brevirostris, respectively The ISSR 331 data results confirm these three sections. 332 According to the ISSR data, populations of T. longirostris are located in a cluster and confirm the Krascheninnikovi 333 section of the flora of Iranica. 334 According to ITS data, T. rezaiyensis, T. longirostris, T. ptrocarpus, T. coloratus, T. vvedenskyi, T. kemulariae are 335 related to each other Which is almost consistent with clade B by Marodive et al. 2012 studies based on 7 nuclear genes 336 (Adh, GapC, LFY, AP3, PI, ITS, ETS). 337 According to ITS data, T. buphthalmoides and T. acanthocarpus species are located in a cluster that confirms the 338 Profundisulcati section of Iranica flora. 339 Cp DNA dendrogram are not useful for classification in this genus and Chloroplast sequences are very similar among 340 Tragopogon species, Therefore, the use of cp DNA markers in the classification of this genus is not recommended. 341 Compliance with ethical standards 342 Conflict of interest The authors declare that they have no conflict of interest. 343 344 References 345 346 347 Azizi H, Sheidai M, Mozaffarian V, Noormohammadi Z (2021), pollen morphology of the genus Tragopogon (Asteraceae). Acta Bot. Hung. 63(1–2): 31–43. 348 https://doi.org/ 10.1556/034.63.2021.1-2.2. 349 350 351 352 353 Bell CD, Mavrodiev EV, Soltis PS, Calaminus AK, Albach DC, Cellinese N, Garcia-Jacas N, Soltis DE (2012) Rapid diversification of Tragopogon and ecological associates in Eurasia. J. Evol. Biol. 25: 2470–2480. //doi.org/10. 10.1111/j.1420-9101.2012.02616.x 354 355 356 Blanca G, Diazdela Guardia C. (1997). Fruit morphology in Tragopogon L. (Compositae: Lactuceae) from the Iberian Peninsula. Bot. J. Linn. 125: 319 ‒ 329. 357 358 359 360 Buggs RJA, Soltis PS, Mavrodiev EV, Symonds VV, Soltis DE (2008). Does phylogenetic distance between parental genomes govern the success of polyploids Castanea 73(2): 74–93.http://dx.doi.org/10.2179/0008-7475 (2008)73[74: DPDBPG] 2.0.CO;2. 361 362 363 Falush D, Stephens M, Pritchard JK (2007). Inference of population structure using multilocus genotype data: dominant markers and null alleles. Mol. Ecol. Notes 7:574-578. 364 365 366 Hamer Q, Harper DA, Rayan PD (2012).PAST: Paleontological Statistics software package for education and data analysis. Palaeontol. Electron. 4: 9. 367 368 369 370 Kashin AS, Berezutsky MA, Kochanova IS, Dobrynicheva NV, Polyanskaya MV (2007). Pecularities of seed reproduction in populations of Asteraceae species under impact of anthropogenic factors. Botanicheskiy Zhurnal 92(9): 1408–1427. [In Russian] 371 372 373 Mavrodiev EV, Edwards CE, Albach DC, Gitzendanner A, Soltis PS, Soltis DE (2004)“Phylogenetic relationships in subtribe Scorzonerinae (Asteraceae: Cichorioideae: Cichorieae) based on ITS sequence data” Taxon 3: 699-712. 374 375 376 377 Mavrodiev EV, Tancig M, Sherwood AM, Gitzendanner MA, Rocca J, Soltis PS, Soltis DE (2005) Phylogeny of Tragopogon L. (Asteraceae) based on internal and external transcribed spacer sequencedata. International Journal of Plant Sciences 166: 117-133. 378 379 380 Mavrodiev EV, Soltis PS., Soltis DE (2008a), Putative parentage of six Old World polyploids in Tragopogon L. (Asteraceae: Scorzonerinae) based on ITS, ETS and plastid sequence data. Taxon 57: 1215-1232. 381 382 Mavrodiev EV, Nawchoo I, Soltis PS, Soltis DE (2008b), Molecular data reveal that the tetraploid 383 384 Tragopogon kashmirianus (Asteraceae: Lactuceae) is distinct from the North American T. mirus. Bot. J. Linn. 158: 391-398. 385 386 387 Mavrodiev EV, Albach DC, Speranza P (2008c) A new polyploid species of the genus Tragopogon (Asteraceae, Cichorieae) from Russia. Novon 18: 229-232. 388 389 390 Ownbey M (1950) Natural hybridization and amphiploidy in the genus Tragopogon. Am. J. Bot. 37(7): 487–499. //doi.org/10.2307/2438023. 391 392 393 Ownbey M, McCollum GD (1953) Cytoplasmic inheritance and reciprocal amphiploidy in Tragopogon. Am. J. Bot. 40: 788–796. //doi.org/10.2307/2438276 394 395 396 Peakall R, Smouse PE (2006). GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes. 6: 288-295. 397 398 399 400 Pires JC, Lim KY, Kovarik A, Matyasek R, Boyd A, Leitch AR, Leitch IJ, Bennett MD, Soltis PS, Soltis DE. (2004) Molecular cytogenetic analysis recently evolved Tragopogon (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors. Am. J. Bot. 91: 1022–1035. //doi.org/10.3732/ajb.91.7.1022. 401 402 Podani J (2000) Introduction to the Exploration of Multivariate Data. Backhuys Publ, Leiden. 403 404 405 Pritchard JK, Stephens M, Donnelly (2000) Inference of population structure using multilocus genotype Data. Genetics 155: 945-959. 406 407 408 Rechinger KH (1977) “Tragopogon” In: Rechinger KH (ed) Flora Iranica, Compositae II, Lactuceae. Osterreich: Akademische Druck- u. Verlagsanstalt. 409 410 411 Richardson I B K (1976) “Tragopogon” In: Tutin TG, Heywood V H, Burges NA, Moore DM, Valentine DH, Walters S A, Webb D A (eds) Flora Europaea, vol. 4. Cambridge Univ. Press, Cambridge. 412 413 414 Safavi S R, Naseh Y, Jafari E, Tavakoli Z, Heidar N (1992) Flora Iran, (Asteraceae Tribe cichorieae), Research Institute of Forests and Rangelands, Tehran, Iran. 415 416 417 Schishkin BK, Bobrov EG (1961) Compositae Tragopogon in Komarov Flora URSS. Doon Scientific Translation co. 318, Chukhuwala, Dehra Dun, India, 29: 131-196. 418 419 420 Shaw J, Lickey EB , Beck JB, Farmer SB, Lium W, Miller J, Siripun KC (2005) The tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am. J. Bot., 92: 142–166. 421 422 423 Sheidai M, Zanganeh S, Haji- Ramezani R, Nouroozi M, Noormohammadi Z, Ghasemzadeh- Baraki S (2013) Genetic diversity and population structure in four Cirsium (Asteraceae) species. Biologia 68: 384-397. 424 425 426 Sheidai M, Afshar M, Keshavarzi M, Talebi SM, Noormohammadi Z, Shafaf T (2014). Genetic diversity and genome size variability in Linum austriacum (Lineaceae) populations. Biochem. Sys. Ecol. 57: 20-26. 427 428 429 430 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011). MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 28: 2731-2739. 431 432 433 Timme R, Kuehl EJ, Boore JL, Jansen RK (2007) A comparative analysis of the Lactuca and Helianthus (Asteraceae) plastid genomes: identification of divergent regions and categorization of shared repeats. Am. J. Bot. 94: 302-313. 434 435 436 437 438 White TJ, Bruns S, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: 315–322 Academic Press, London.