Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 3-12, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-275 Citation: N. Atazadeh, M. Sheidai, F. Attar, F. Koohdar (2020) Species delimita- tion in the genus Cousinia Cass. (Fam- ily Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f.: morphometry and molecular analysis. Caryologia 73(3): 3-12. doi: 10.13128/ caryologia-275 Received: May 30, 2019 Accepted: April 05, 2020 Published: December 31, 2020 Copyright: © 2020 N. Atazadeh, M. Sheidai, F. Attar, F. Koohdar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Species delimitation in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f.: morphometry and molecular analysis Neda Atazadeh1,*, Masoud Sheidai1, Farideh Attar2, Fahimeh Kooh- dar1 1 Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Evin, Tehran, Iran 2 Central Herbarium of Tehran University, School of Biology, College of Science, Univer- sity of Tehran, P.O. Box: 14155, Tehran, Iran * Corresponding author: Email: Atazadeh_neda@yahoo.com Abstract. The genus Cousinia of the tribe Cardueae with about 700 species is one of the most diverse genera in Central and southwest Asia. The section Cynaroides with 89 species is the largest section of the genus. Due to the controversy in the number of Cousinia species and their delineation, the first step in studying the genus is to iden- tify and delimit presumed species. Species delimitation is usually difficult in the spe- cies with overlaps in their morphological features. Therefore, we used a combination of morphological and molecular markers (ISSRs) to carry out delimitation in 204 taxa of 68 Cousinia species whitin the Cynaroideae and Platyacanthae sections. The spe- cies delineation based on morphometry and ISSR data were done by UPGMA cluster- ing. The samples of each species were placed close to each other and formed a single sub-cluster, separated from the other studied Cousinia species. In the present study the studied Cousinia species within Cynaroideae and Platyacanthae sections could be delimited from each other based on ISSR and morphological data. Therefore, using ISSR and morphological data can be useful in identifying and delineating crucial spe- cies. The Mantel test performed between morphological distance and Nei genetic dis- tance produced non-significant correlation. This result also supports distance analyses of the trees and reveals that the two dendrograms are not correlated. Some possible reasons for this incongruence are proposed: the high number of taxa in the genus Cousinia, morphological traits homoplasious, convergent evolution and incomplete lin- eage sorting. Keywords: Cousinia, Cynaroideae, ISSR, morphometry. INTRODUCTION The genus Cousinia Cass. of the tribe Cardueae (Family Asteraceae) with about 700 species is one of the most diverse genera in Central and southwest (SW) Asia After Senecio L. (c. 1500 species) and Vernonia Schreb. (c. 1000 species) (Tscherneva 1962; Rechinger 1972, 1979; Frodin 2004; Attar and 4 Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Koohdar Ghahreman 2006; Susanna and Garcia-Jacas 2006; Attar and Djavadi 2010; Mehregan and Assadi 2016; Minaeifar et al. 2016; Rastegar et al. 2017, 2018). Due to the exten- sive morphological variability in the genus, Cousinia taxonomy is complicated and controversial (Mabber- ley 1990; Haffner 2000; Susanna et al. 2003). The genus Cousinia contains more than 400 species in SW Asia, with the highest number of species in the Flora Iranica area, out of which 379 are endemic. These species are distributed in mountainous regions of Iran, Afghani- stan and Turkmenistan (Rechinger 1986; Knapp 1987). Although the exact number of Cousinia species in Iran is still unknown, about 270 species have been reported till now (Assadi 2009; Attar and Djavadi 2010). Out of these, nearly 200 endemic Cousinia species occur in Iran (Djavadi et al. 2007; Zare et al. 2013). The Cousinia spe- cies are distributed in 70 sections (Rechinger 1986). The section Cynaroideae Bunge with 89 species is the larg- est section of the genus and contains Irano-Turkestan- ian elements (Tscherneva 1962; Rechinger 1972, 1979; Huber- Morath 1975; Attar and Djavadi 2010; Rastegar et al. 2017, 2018). This sect. includes those species con- sisting of decurrent leaves and appendiculate bracts (Tscherneva 1962; Rechinger 1972, 1979; Huber- Morath 1975). Iran with 77 taxa, of which 66 are endemic, seems to be the centre of diversity of the section Cynaroideae (Attar and Ghahreman 2006). The section Platyacanthae Rech. f. has 6 species in Flora Iranica of which 5 species are endemic in Iran (Rechinger 1972). Due to the controversy in the number of Cousinia species and their delineation, the first step in studying the genus is to identify and delimit presumed species. Species delimitation is usually difficult in the species with overlaps in their morphological features (Wiens 2007). In such cases, combined morphological and molecular data have been used to delimit these taxonom- ic identities (Duminil and Di Michele 2009; Minaeifar et al. 2016; Hassanpour et al. 2018; Eftekharian et al. 2018). Different molecular markers have been used in plant taxonomy and phylogeny, but some of them such as inter- simple sequence repeats (ISSRs) seems to be very effi- cient in delineating species, varieties, ecotype and even genotypes of a single species (See for example, Sheidai et al. 2012, 2013; Safaei et al. 2016; Eftekharian et al. 2018). Therefore, we used a combination of morphological and molecular markers (ISSRs) to carry out Cousinia species delimitation in sections Cynaroideae and Platyacanthae. MATERIALS AND METHODS Plant material The data investigated and discussed in the present study are based on 204 samples of 68 species in of the sections Cynaroideae and Platyacanthae. Sixty-three spe- cies (189 specimens) of Cynaroideae and five species (15 specimens) of Platyacanthae were selected. The plant samples were collected from Iran (Table 1). The vouch- Table 1. Investigated Cousinia species and their voucher information. No Taxa Section Locality Voucher no. 1 C. keredjensis Bornm. & Gauba Cynaroides Bunge Tehran 21807(TUH) 2 C. elwendensis Bornm. Cynaroides Bunge Hamadan-Alvand Mountains 20566(TUH) 3 C. grandis C. A. Mey. Cynaroides Bunge Azarbaijan 21343(TUH) 4 C. disfulensis Bornm. Cynaroides Bunge Lorestan- Khorram Abad 27589(TUH) 5 C. bornmulleri C. Winkl. Cynaroides Bunge Esfahan 22532(TUH) 6 C. behboudiana Rech. f. & Esfand. Cynaroides Bunge Ghazvin 27629(TUH) 7 C. inflata Boiss. & Hausskn. Cynaroides Bunge Kurdestan 39552(TUH) 8 C. eriocephala Boiss. & Hausskn. Cynaroides Bunge Azarbaijan 22442(TUH) 9 C. calocephala Jaub. & Spach Cynaroides Bunge Azarbaijan-Mianeh 46276(TUH) 10 C. farsistanica Bornm. Cynaroides Bunge Kerman 28636(TUH) 11 C. jaccobsii Rech. f. Cynaroides Bunge Ilam 22370(TUH) 12 C. denaensis Attar & Djavadi Cynaroides Bunge Boyer-Ahmad 22495(TUH) 13 C. concinna Boiss. & Hausskn. Cynaroides Bunge Kurdestan 20562(TUH) 14 C. grantii Rech. f. Cynaroides Bunge Azarbaijan 22490(TUH) 15 C. bobeckii Rech. f. Cynaroides Bunge Ardabil 46221(TUH) 16 C. barbeyi C. Winkl. Cynaroides Bunge Boyer-Ahmad 22494(TUH) 17 C. kirrindica Bornm. & Rech. f. Cynaroides Bunge Ilam 19711(TUH) 5Species delimitation in the genus Cousinia Cass. No Taxa Section Locality Voucher no. 18 C. khorramabadensis Bornm. Cynaroides Bunge Lorestan 21851(TUH) 19 C. lactiflora Rech. f. Cynaroides Bunge Lorestan 46299(TUH) 20 C. phyllocephala Bornm. & Gauba Cynaroides Bunge Lorestan- Khorram Abad 46292(TUH) 21 C. lurorum Bornm. Cynaroides Bunge Kermanshah- Mahidasht 20568(TUH) 22 C. verbascifolia Bunge Cynaroides Bunge Khorasan-Mashhad 43013(TUH) 23 C. monocephala Bunge Cynaroides Bunge Khorasan- Ghouchan 21931(TUH) 24 C. shebliensis Ghahreman Cynaroides Bunge Azarbaijan- Tabriz 20580(TUH) 25 C. millefontana Rech. f. Cynaroides Bunge Kurdestan-Marivan 20227(TUH) 26 C. sanandajensis Rech. f. Cynaroides Bunge Hamadan 46287(TUH) 27 C. zardkuhensis Attar & Ghahreman Cynaroides Bunge Chahar Mahal& Bakhtiari 21887(TUH) 28 C. pergamacea Boiss. & Hausskn. Cynaroides Bunge Kurdestan 22571(TUH) 29 C. macrocephala C. A. Mey. Cynaroides Bunge Ardebil- Meshkin shahr 42925(TUH) 30 C. onopordioides Ledeb. Cynaroides Bunge Khorasan: Kashmar 28685(TUH) 31 C. aligudarzensis Attar & Ghahreman Cynaroides Bunge Lorestan-Aligudarz 27613(TUH) 32 C. dalahuensis Attar & Ghahreman Cynaroides Bunge Kermanshah- Mahidasht 19929(TUH) 33 C. carolihenrici Attar & Ghahreman Cynaroides Bunge Kurdestan 22455 (TUH) 34 C. khansarica Attar & Ghahreman Cynaroides Bunge Esfahan: Khansar 20037(TUH) 35 C. lurestanica Attar & Djavadi Cynaroides Bunge Lorestan 21824(TUH) 36 C. parsana Ghahreman Cynaroides Bunge Hamadan 20553(TUH) 37 C. pasargadensis Attar Cynaroides Bunge Fars: Dashte Arjan 36294(TUH) 38 C. perspolitana Attar & Ghahreman Cynaroides Bunge Fars: Abadeh 22509(TUH) 39 C. silvanica Attar Cynaroides Bunge W Azarbaijan: Urmie 24064(TUH) 40 C. shulabadensis Attar & Ghahreman Cynaroides Bunge Lorestan- Shul Abad 21874(TUH) 41 C. algurdina Rech. f. Cynaroides Bunge Azarbaijan- Tabriz 30533(TUH) 42 C. mobayenii Ghahreman & Attar Cynaroides Bunge Kermanshah- Eslamabad 20569(TUH) 43 C. sabalanica Attar Cynaroides Bunge Ardebil 22570(TUH) 44 C. kurdistanica Attar Cynaroides Bunge Kurdestan- Maryvan 3232(TUH) 45 C. gaharensis Attar & Djavadi Cynaroides Bunge Lorestan- Shulabad 38259(TUH) 46 C. kermanshahensis Attar Cynaroides Bunge Kermanshah: Eslam-Abad 19810(TUH) 47 C. fursei Rech. f. Cynaroides Bunge Kurdestan-Marivan 18314(TUH) 48 C. chlorosphaera Bornm. Cynaroides Bunge Chahar Mahal& Bakhtiari: Soreshjan 26244(TUH) 49 C. cynaroides C. A. Mey Cynaroides Bunge Ardebil 22581(TUH) 50 C. gilliatii Rech. f. Cynaroides Bunge Azarbaijan 21967(TUH) 51 C. iranica C. Winkl. & Strauss. Cynaroides Bunge Arak 21881(TUH) 52 C. kotschyi Boiss. Cynaroides Bunge Azarbaijan 46244(TUH) 53 C. kopikaradaghensis Rech. f. Cynaroides Bunge Kurdestan: Saqqez (TUH) 54 C. sagittata C. Winkl. & Strauss. Cynaroides Bunge Arak 21822(TUH) 55 C. nana Attar Cynaroides Bunge Arak 14347(TUH) 56 C. sahandica Attar & Djavadi Cynaroides Bunge Azarbaijan 46272(TUH) 57 C. lordeganensis Mehregan Cynaroides Bunge Chahar Mahal& Bakhtiari 46301(TUH) 58 C. hamadanensis Rech. f. Cynaroides Bunge Hamadan- Malayer 46290(TUH) 59 C. subinflata Bornm. Cynaroides Bunge Kermanshah (TUH) 60 C. kornhuberi Heimerl Cynaroides Bunge Hamadan 22372(TUH) 61 C. sardashtensis Rech. f. Cynaroides Bunge Chahar Mahal& Bakhtiari 20073(TUH) 62 C. sefidiana Rech. f. Cynaroides Bunge Lorestan 21861(TUH) 63 C. platyacantha Bunge Platyacanthae Rech. f. Khorasan 43212(TUH) 64 C. freynii Bornm. Platyacanthae Rech. f. Semnan- Shahrud 27675(TUH) 65 C. reshingerorum Bornm. Platyacanthae Rech. f. Khorasan-Torbate Jam 39729(TUH) 66 C. bienerti Bunge Platyacanthae Rech. f. Khorasan-Neyshabur 28682(TUH) 67 C. trachyphyllaria Bornm. & Rech. f. Platyacanthae Rech. f. Khorasan- Ghouchan 21932(TUH) 68 C. ecbatanensis Bornm. Cynaroides Bunge Hamadan 22371(TUH) 6 Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Koohdar er specimens have been deposited in The Herbarium of Tehran University (TUH) (Table 1). DNA extraction and PCR amplification Total genomic DNA was extracted from leaf tis- sue using protocol of the CTAB-activated charcoal and Poly venyl Pyrrolidone (PVP) method (Murray and Thompson 1980). Quality of extracted DNA was exam- ined by running on 0.8% Agarose gels. Each 20 ml PCR mixture contained 10 ml of 2_ PCR buffer, 0.5 mM of each primer, 200 mM of each dNTP, 1 Unit of Taq DNA polymerase (Bioron, Ludwigschafen, Germany), and 1 ml of template genomic DNA at 20 ng mle1. The PCR amplification program was performed in a Techne thermocycler (Germany) with the following program: 5 min at 94 °C, followed by 45 cycles of 30 s at 94 °C, 30 s at 54.6 °C, and 2 min at 72 °C, with a final extension step of 10 min at 72 C. The amplification products were visualized by running on 2% agarose gel, followed by ethidium bromide staining. The fragments size was esti- mated by using a 100-bp molecular size ladder (Fermen- tas, Germany). The experiment was replicated 3 times and constant ISSR bands were used for further analyses. Ten ISSR primers, UBC 807, UBC 810, UBC 811, UBC 834, CAG(GA)7, (CA)7AC, (CA)7AT, (CA)7GT (GA)9A, and (GA)9T, commercialized by the University of British Columbia, were used (Godwin et al. 1997). Morphological analysis In total, 19 morphological characters (quantitative and qualitative) were studied (Table 2). Morphological characters were coded accordingly. Data were standard- ized (mean = 0, variance = 1) and used for multivariate analyses. UPGMA (Unweighted paired group using aver- age), and Ward (Minimum spherical variance) clustering based on Euclidean distance and Gower distances as well as principal coordinate analysis (PCoA) and multidimen- sional scaling (MDS) methods were used for grouping of the species. Principal components analysis (PCA) was used to identify the most variable morphological charac- ters. (Podani 2000; Safaei et al. 2016). Data analyses were performed by PAST ver. 2.17 (Hammer et al. 2012). Molecular analysis The obtained ISSR bands were treated as binary characters (presence = 1, absence = 0). The number of Table 2. Morphological characters and their code. Character Code Head diameter x<3 3≤x≤6 x>6 Flower number x<80 80≤x≤150 x>150 Bracts number x<80 80≤x≤120 x>120 Appendages length of median bracts x<9 9≤x≤15 x>15 Appendages width of median bracts x< 5 5≤x≤15 x>15 Crolla length x< 20 20≤x≤25 x>25 Habitate Woodland Alpine Stepp Leaves indumentum Present Absent Stem leaves Interruptedly decurrent Countinuously decurrent Undecurrent Uppermost leaves Distant from the head Close to the head Surrounding the head Appendages Present Absent Inner bracts indumentum Smooth Scabrous Position of median bracts Imbricated Spreading Recurved Spreading-recurved Imbricated- spreading Appendages shape of median bracts Sagitate Triangular Rhombic Ovate Lanceolate Appendages margin of median bracts Smooth 1-2 spins Spinose Receptacle bristles Smooth Scabrous Corolla color Yellow Pink Purple White Ratio limb to Anther tube Longer Shorter As long as Anther tube color Yellow Pink Purple White 7Species delimitation in the genus Cousinia Cass. private bands versus common bands was determined. The genetic diversity parameters like Nei’s gene diver- sity (H), Shannon information index (I), number of ef fective a lleles, and percentage of poly morphism (Freeland et al. 2011) were determined for each popu- lation. Nei’s genetic distance was used for clustering (Weising et al. 2005). Neighbor Joining (NJ) and UPG- MA (Unweighted paired group using average) cluster- ing were used for the species grouping after 100 times bootstrapping/permutations (Freeland et al. 2011). The consensus tree was constructed from the obtained morphological and ISSR trees. Similarly, tree distance was estimated accordingly. The Mantel test between dendrograms was performed to check their agreement. PAST ver. 2.17 (Hammer et al. 2012) and DARwin ver. 5 (Perrier & Jacquemoud-Collet 2006) programs were used for these analyses. AMOVA (analysis of molecu- lar variance) (with 1000 permutations) as implemented in GenAlex 6.4 (Peakall and Smouse 2006) was used to determine species genetic differentiation. Gene flow was determined by: (1) calculating Nm an estimate of gene flow from Gst by PopGene ver. 1.32 (1997) as: Nm 1 ⁄4 0.5(1 e Gst)/Gst, (2) reticulation analysis that is based on the least square method as performed in T-REX (Boc et al. 2012). RESULTS Morphometry UPGMA dendrogram of the studied Cousinia spe- cies based on morphological characters (Figure 1) placed the studied samples of most of the species together and in a separate sub-cluster. This indicates that Cousinia species can be differentiated by the used morphological features. UPGMA dendrogram also separated Cousinia species of the two sections Cynaroideae and Platyacan- thae.Therefore, the morphological characters studied can delimit these sections too. PCA analysis of morphological characters revealed that the first two PCA components comprised about 79% of total variation. Morphological characters like shape and length of the appendages of the median bracts, diameter of the heads, the No. of flowers and length of the corolla had the highest value of correla- tion with these components and are the most variable morphological features among the studied plants. In fact, these morphological features are of ta xonomic value in the two sections Cynaroideae and Platyacan- thae. ISSR assay The used ISSR primers produced 36 reproducible bands/loci, out of which only 1 band was monomor- phic, while the others were polymorphic bands. The highest number of ISSR bands occurred in C. keredjen- sis Bornm. & Gauba (20), while C. cynaroides C. A. Mey had the lowest number of bands (5). A single private ISSR band occurred in C. keredjensis, while the other bands were common among the Cousinia species. Discriminating power of ISSR loci as determined by Gst against Nm (migration) analysis (Table 3), revealed that almost all ISSR loci have excellent discriminating power (>0.95). Therefore, ISSR markers are efficient in differentiating Cousinia species studied. The highest value for Nei genetic distance (0.87) occurred between C. bienerti Bunge and C. elwendensis Bornm., followed by C. freynii Bornm. and C. elwenden- sis (0.81). Similarly, the lowest value for the same (0.02) was observed between C. reshingerorum Bornm. and C. bienerti. UPGMA dendrogram of the studied Cousinia spe- cies based on ISSR data (Figure 2) separated these spe- cies in distinct sub-clusters. Therefore, ISSR molecular markers can be used in taxonomy of the genus. These molecular markers can also differentiate two sections of Cynaroideae and Platyacanthae. AMOVA produced significant genetic difference among the studied Cousinia species (P = 0.001), which indicates that the studied species are genetically differ- entiated. AMOVA revealed that 99% of total genetic dif- ference was due to among species genetic differentiation, while 1% was due to within species genetic variability. The species relationship illustrated by UPGMA den- drograms based on morphological features and molecu- lar data were not congruent. It was also illustrated in the consensus tree of these dendrograms (Figure 3). This tree revealed that only in some cases the studied Cous- inia species show the same relationship in both morpho- logical and molecular trees. For instance, C. zardkuhen- sis Attar & Ghahreman (No. 27 in Figure 3) and C. chlorosphaera Bornm. (No. 48 in Figure 3) were placed close to each other. The same applied for for C. platya- cantha Bunge (No. 63 in Figure 3) and C. freynii (No. 64 in Figure 3). Similarly, three species of C. reshingero- rum, C. bienerti and C. trachyphyllaria Bornm. & Rech. f. (No. 65-67 in Figure 3) formed a distinct cluster in the obtained consensus tree. The rest of Cousinia species studied were placed together in an unresolved cluster. This means that, their relationship is differently pictured in the obtained morphological and molecular dendro- grams. Tree distance between the obtained morpho- 8 Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Koohdar logical and ISSR dendrograms after adjusting the edges in each dendrogram was 0.36. Similarly, comparison of these two dendrograms based on Quartet tree distance method, produced 0.64 difference. Both these results indicate that morphological relationship of the stud- ied Cousinia species, differed in great extent with ISSR based species relationship. The performed Mantel test between morphological distance and Nei genetic distance produced non-signif- icant correlation (Correlation R = 0.06, p = 0.113). This indicates that morphological divergence in the studied species is not correlated with genetic distance. DISCUSSION As mentioned by the authors, taxonomy and molec- ular phylogeny of the genus Cousinia is complicated and unresolved mainly due to disagreement between the morphological and molecular phylogenetic studies (See Figure 1. UPGMA dendrogram of the studied Cousinia species based on morphological data. (The specie 1-68 are according to Table 1). 9Species delimitation in the genus Cousinia Cass. for example, Sausana et al. 2003; Lopez-Vinyallonga et al. 2009). Moreover, several overlapping morphologi- cal characteristics at the species level makes the species identification and delineation difficult (Attar and Dja- vadi 2010). In the present study, we could delimit the studied Cousinia species based on both the used mor- phological and molecular data. We suggest that certain morphological characters like shape and the length of the appendages of the median bracts, diameter of the heads, the No. of flowers and the length of the corolla are taxonomically useful at the species level. Interesting enough, the both sections Cynaroideae and Platyacanthae are separated from each other due to the difference in traits such as stem leaves and append- ages of median bracts. Therefore, these characters are of more practical utility, particularly in sectional level clas- sification in the genus Cousinia. The obtained species relationship based on morpho- logical features are in agreement with previous studies. For example, within the section Platyacanthae; C. Platy- acanthae and C. freynii were placed close each other due to the similarity in all features except color of corolla. Their close affinity to each other was also noticed by Asaadi and Mehregan (Flora of Iran 2017). Similarly, C. reshingerorum, C. bienerti and C. trachyphyllaria showed morphological resemblance due to the traits such as No. of the flowers, the length of the corolla, the color of the anther tube, inner bracts, receptacle bristles, diameter of head, ratio of limb/tube. Close morphological affinity among these species was also illustrated by Asaadi and Mehregan (Flora of Iran 2017). In the section Cynaroideae, ISSR data showed genet- ic affinity between C. grantii Rech. f. and C.grandis C. A. Mey., and also between C. verbascifolia Bunge and C. monocephala Bunge. These species also showed mor- phological similarities. The same holded true for C. per- gamaceae Boiss. & Hausskn., C. millefontana Rech. f., and C. carolihenrici Attar & Ghahreman; as well as for C. zardkuhensis and C. chlorosphaera. ISSR data revealed close affinity between C. disfulensis Bornm., C. jaccobsii Rech. f. and C.kermanshahensis Attar; which is almost in agreement with the morphological data. The same applied for C. nana Attar and C. kotschyi Boiss. These results are almost in agreement with the taxonomic treatment of the section Cynaroideae (Attar and Djavadi 2010). The other studied Cousinia species in the section Cynaroideae differed in their affinity in genetic tree ver- sus morphological tree. This is in agreement with results of Lopez-Vinyallonga et al. (2009), as they also indicated that morphological traits are highly incongruent with molecular data in Arctium‐Cousinia complex and con- sidered morphological characters homoplasious. In general, various reasons were suggested for this incongruence between molecular and morphological analyses: the high number of taxa in the genus Cousinia, homoplasious of the morphological traits, convergent evolution (Susanna et al. 2003; Lopez-Vinyallonga et al. 2009), incomplete lineage sorting (Zhang et al. 2015); as well as the occurrence of intermediate forms and Table 3. Discrimination power of ISSR loci in studied Cousinia species. Locus Sample Size Ht Hs Gst Nm* Locus1 204 0.1107 0.0000 1.0000 0.0000 Locus2 204 0.4027 0.0000 1.0000 0.0000 Locus3 204 0.4931 0.0000 1.0000 0.0000 Locus4 204 0.2712 0.0000 1.0000 0.0000 Locus5 204 0.3893 0.0000 1.0000 0.0000 Locus6 204 0.0843 0.0000 1.0000 0.0000 Locus7 204 0.0571 0.0000 1.0000 0.0000 Locus8 204 0.3599 0.0000 1.0000 0.0000 Locus9 204 0.3270 0.0000 1.0000 0.0000 Locus10 204 0.4961 0.0000 1.0000 0.0000 Locus11 204 0.3655 0.0088 0.9759 0.0124 Locus12 204 0.2297 0.0000 1.0000 0.0000 Locus13 204 0.4650 0.0000 1.0000 0.0000 Locus14 204 0.2297 0.0000 1.0000 0.0000 Locus15 204 0.3270 0.0000 1.0000 0.0000 Locus16 204 0.1454 0.0088 0.9394 0.0323 Locus17 204 0.2712 0.0000 1.0000 0.0000 Locus18 204 0.1499 0.0132 0.9118 0.0484 Locus19 204 0.0394 0.0088 0.7763 0.1441 Locus20 204 0.0107 0.0088 0.1791 2.2922 Locus21 204 0.0571 0.0000 1.0000 0.0000 Locus22 204 0.1107 0.0000 1.0000 0.0000 Locus23 204 0.2712 0.0000 1.0000 0.0000 Locus24 204 0.4377 0.0000 1.0000 0.0000 Locus25 204 0.4983 0.0000 1.0000 0.0000 Locus26 204 0.1609 0.0000 1.0000 0.0000 Locus27 204 0.2297 0.0000 1.0000 0.0000 Locus28 204 0.2712 0.0000 1.0000 0.0000 Locus29 204 0.0843 0.0000 1.0000 0.0000 Locus30 204 0.2297 0.0000 1.0000 0.0000 Locus31 204 0.2076 0.0000 1.0000 0.0000 Locus32 204 0.0843 0.0000 1.0000 0.0000 Locus33 204 0.0290 0.0000 1.0000 0.0000 Locus34 204 0.0571 0.0000 1.0000 0.0000 Locus35 204 0.1609 0.0000 1.0000 0.0000 Locus36 204 0.0571 0.0000 1.0000 0.0000 Mean 204 0.2270 0.0013 0.9941 0.0030 Nm = estimate of gene flow from Gst or Gcs. E.g., Nm = 0.5(1 - Gst)/Gst. 10 Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Koohdar homoploid hybrid speciation, which there is little proof to prove them (Mehregan & Kadereit 2009; Lopez-Vin- yallonga et al. 2009). Furthermore, the genus Cousinia with its relatively young geological age (ca. 8.7 mya) and high number of taxa is thoroughly unusually exposed to speciation (Lopez-Vinyallong et al. 2009). This is entirely consistent with the results reported by Lopez-Vinyallon- ga et al. (2009), as they also revealed that the dominant factor in speciation of the genus Cousinia is allopat- ric geographic speciation. These may partly justify the complexity and incongruence of the relationships in the studied species of the genus Cousinia. In conclusion, along with confirmation by the pub- lished literature, the current study proved that the mor- phological characters and ISSR molecular data are use- ful for the correct identification and species delimita- tion within the Cynaroideae and Platyacanthae sections. Both quantitative and qualitative morphological charac- teristics are important and suitable for the identification of species of the genus Cousinia. Figure 2. UPGMA dendrogram of the studied Cousinia species based on ISSR data. 11Species delimitation in the genus Cousinia Cass. REFERENCES Assadi M. 2009. Four new species of the genus Cousinia Cass. (Asteraceae) from Iran. Iranian Journal of Bot- any 15(1): 36-44. Asaadi M, Mehregan I. 2017. Flora of Iran. No. 125: (Asteraceae: Cousinia). Attar F, Ghahreman A. 2006. A synopsis of sect. Cynar- oides (Cousinia, Compositae), distribution patterns and diversity centers. Rostaniha 7 (Supplement 2): 315-342. Attar F, Djavadi SB. 2010. 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