Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 39-50, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1114 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Kooh- dar (2021) Molecular phylogeny and mor- phometric analyses in the genus Cous- inia Cass. (Family Asteraceae), sec- tions Cynaroideae Bunge and Platya- canthae Rech. f. Caryologia 74(4): 39-50. doi: 10.36253/caryologia-1114 Received: October 10, 2020 Accepted: November 27, 2021 Published: March 08, 2022 Copyright: © 2021 Neda Atazadeh, Masoud Sheidai, Farideh Attar, Fahimeh Koohdar. This is an open access, peer-reviewed article pub- lished 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. Molecular phylogeny and morphometric analyses in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f. Neda Atazadeh1,*, Masoud Sheidai1, Farideh Attar2, Fahimeh Koohdar1 1 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotech- nology, Shahid Beheshti University, 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. E-mail: Atazadeh_neda@yahoo.com Abstract. Taxonomy and molecular phylogeny of the genus Cousinia are complicated and unresolved mainly because of disagreement between morphological and molecular phylogenetic studies. The genus Cousinia has approximately 700 species, which makes it one of the most varied genera found in central and southwest Asia. Section Cynar- oideae, containing 89 species, is considered the largest section of the genus. Identifi- cation and delineation as well as classifying the section and the species’ relationships within the genus Cousinia generally remain debatable. Therefore, the present study aims to: 1) identify and delineate the species within the two sections Cynaroideae and Platyacanthae; 2) study the species relationships based on both morphological and molecular features (Internal Transcribed Spacer (ITS) marker); 3) study the sectional delimitation and its monophyly; and 4) estimate the divergence time of the studied sections. To this end, 50 Cousinia species occurring in Iran were investigated for the first time. A maximum parsimony tree of the morphological features separated the species of the two sections from each other. However, the ITS-based phylogenetic tree did not delimit the two studied sections. The relationships among the studied Cousinia species in the genetic trees were generally not congruent with the obtained morpholog- ical tree. The divergence time of the studied species within the Cynaroideae and Plat- yacanthae sections determined using Bayesian Evolutionary Analysis Sampling Trees (BEAST) was estimated to be around 3.5 million years ago (Mya). Keywords: Cousinia, Cynaroideae, ITS, Phylogeny, Platyacanthae. INTRODUCTION The genus Cousinia Cass. of the tribe Cardueae (family Asteraceae) has approximately 700 species, which makes it one of the most diverse genera fol- lowing Senecio L. (c. 1500 species) and Vernonia Schreb. (c. 1000 species) in central and southwest (SW) Asia (Tscherneva 1962; Rechinger 1972, 1979; Fro- din 2004; Attar and Ghahreman 2006; Susanna and Garcia-Jacas 2007; Attar 40 Neda Atazadeh et al. and Djavadi 2010; Mehregan and Assadi 2016; Minaeifar et al. 2016; Rastegar et al. 2017, 2018). This genus has the greatest prevalence in the Flora Iranica area, with more than 400 different species distributed in SW Asia, of which, 379 are considered endemic. These species are dis- tributed in mountainous areas of Iran, Afghanistan, and Turkmenistan (Rechinger 1986; Knapp 1987). The genus Cousinia is not considered to be mono- phyletic, and comprises the Arctium-Cousinia complex as well as the genus Arctium L. (Susanna et al. 2003; Lopez-Vinyallonga et al. 2009). Although the exact number of species in this genus in Iran is in dispute (Attar 2000; Mehregan 2008; Mehregan and Kadereit 2008; Assadi 2009; Attar and Mirtadzadini 2009; Mehregan and Assadi 2009; Attar and Djavadi 2010), approximately 270 Cousinia species have been reported of which, almost 200 species are con- sidered endemic (Djavadi et al. 2007; Zare et al. 2013). Cousinia species could be taxonomically categorized into 70 sections (Rechinger 1986), with section Cynar- oideae being the largest section of the genus contain- ing 89 species (Tscherneva 1962; Rechinger 1972, 1979; Huber- Morath 1975; Attar and Djavadi 2010; Rastegar et al. 2017, 2018). This section includes species consisting of decurrent leaves and appendiculate bracts (Tscher- neva 1962; Rechinger 1972, 1979; Huber- Morath 1975), which are recognized as the Irano-Turkestanian ele- ments (Djamali et al. 2012; Dehghani et al. 2017). Iran has 77 taxa, 66 of which are endemic, thus appearing to be the variety center of the section (Attar and Ghahre- man 2006; Attar and Djavadi 2010). The extensive morphological variability in the genus renders the taxonomy of Cousinia complicated and controversial (De Candolle 1837; Boissier 1875; Win- kler 1892, 1897; Tscherneva 1962; Rechinger 1972, 1979; Huber-Morath 1975; Haffner 2000; Susanna et al. 2003; Mehregan 2011; Mehregan and Assadi 2016; Mabberley 2018; Atazadeh et al. 2020). There is a controversy over the number of species within a single section, too; for instance, Mehregan and Kadereit (2008), in a taxonomic revision of the section Cynaroideae, reduced the number of species occurring in Iran to 31, while Attar and Djavadi (2010) reported 77 Cousinia species in this section as present within the country. The sect. Platyacanthae Rech. f. has six species in Flo- ra Iranica and is considered the sister group of the sect. Cynaroideae (Lopez-Vinyallonga et al. 2009), five of the six species of which are endemic in Iran (Rechinger 1972). Species identification and delineation as well as classification of the sections and the species relation- ships within the genus Cousinia generally remain debat- able, even after molecular investigations (Susanna et al. 2003; Ghaffari et al. 2006; Lopez-Vinyallonga et al. 2009; Mehregan and Assadi 2016; Galtier 2019). There- fore, the present study aims to: 1) identify and delineate the species based on differentiating taxonomical features within the two sections Cynaroideae and Platyacanthae; 2) study the species relationships based on morphologi- cal and molecular features (Internal Transcribed Spacer (ITS) marker); 3) study sectional delimitation and its monophyly, and 4) estimate the divergence time of the studied species within the Cynaroideae and Platyacan- thae sections for the first time. Molecular information has been commonly uti- lized to create a system for phylogenetic classification. Specifically, the ITS regions are considered as nuclear DNA regions described by parental inheritance pat- terns and can be changed faster compared with the cod- ing regions, which results in higher levels of disparity among those narrowly-related individuals. Therefore, the ITS regions were used herein to study the interspecific and intergeneric relationships along with developmental styles and patterns in genetic variation (Baldwin 1992; Alvarez and Wandel 2003; Felniner and Rossello 2007). Molecular studies in the two sections Cynaroideae and Platyacanthae have not been fully performed until now. Therefore, present study has attempted to investi- gate 50 species of both studied sections based on molec- ular features (ITS) for the first time and specifically col- lect new information on molecular phylogeny, evolution, divergence time, and species relationships. These find- ings can enhance our knowledge of the true evolution- ary pathway of the genus Cousinia. MATERIALS AND METHODS Plant material Morphological studies were conducted on 150 plant specimens, of which 138 belonged to 46 species of Cynaroideae and 12 belonged to 4 species of Platya- canthae sections (Table 1). One specimen of each spe- cies was used for the ITS marker. The voucher specimens were deposited in the Herbarium of Tehran University (TUH). Arctium umbrosum Bung (accession number: AY373745; AY373712) and Arctium lappa L. (accession number: EU923773; EU923887) were obtained as out groups based on studies by Susanna and Garcia-Jacas (2007) and Lopez-Vinyallonga et al. (2009). ITS sequenc- es for all of the species except for the out groups were newly generated. 41Molecular phylogeny and morphometric analyses in the genus Cousinia Ta bl e 1. In ve st ig at ed C ou si ni a sp ec ie s an d th ei r vo uc he r in fo rm at io n as w el l a s th e ac ce ss io n nu m be rs o f t ax a in p hy lo ge ny s tu di es . R . Ta xa Se ct io n Lo ca lit y V ou ch er n um be r A cc es si on n um be r A bb re vi at io n 1 C ou si ni a ca ro lih en ri ci A tt ar & G ha hr em an C yn ar oi de ae B un ge K ur de st an 22 45 5 (T U H ) M H 99 27 48 ca ro lih en ri ci 2 C ou si ni a fu rs ei R ec h. f. C yn ar oi de ae B un ge K ur de st an -M ar iv an 18 31 4( T U H ) M H 99 27 34 fu rs ei 3 C ou si ni a m ill ef on ta na R ec h. f. C yn ar oi de ae B un ge K ur de st an -M ar iv an 20 22 7( T U H ) M H 97 12 23 m ill ef on ta na 4 C ou si ni a co nc in na B oi ss . & H au ss kn . C yn ar oi de ae B un ge K ur de st an 20 56 2( T U H ) M H 99 27 35 co nc in na 5 C ou si ni a su bi nfl at a B or nm . C yn ar oi de ae B un ge K er m an sh ah (T U H ) M K 00 51 81 su bi nfl at a 6 C ou si ni a ha m ad an en si s R ec h. f. C yn ar oi de ae B un ge H am ad an - M al ay er 46 29 0( T U H ) M K 00 51 82 ha m ad an en si s 7 C ou si ni a ba rb ey i C . W in kl . C yn ar oi de ae B un ge B oy er -A hm ad 22 49 4( T U H ) M K 00 51 64 ba rb ey i 8 C ou si ni a de na en si s A tt ar & D ja va di C yn ar oi de ae B un ge B oy er -A hm ad 22 49 5( T U H ) M H 99 27 39 de na en si s 9 C ou si ni a sa rd as ht en si s R ec h. f. C yn ar oi de ae B un ge C ha ha r M ah al & B ak ht ia ri 20 07 3( T U H ) M K 00 51 84 sa rd as ht en si s 10 C ou si ni a da la hu en si s A tt ar & G ha hr em an C yn ar oi de ae B un ge K er m an sh ah - M ah id as ht 19 92 9( T U H ) M H 99 27 47 da la hu en si s 11 C ou si ni a gr an di s C . A . M ey . C yn ar oi de ae B un ge A za rb ai ja n 21 34 3( T U H ) M H 99 27 38 gr an di s 12 C ou si ni a gr an tii R ec h. f. C yn ar oi de ae B un ge A za rb ai ja n 22 49 0( T U H ) M K 00 51 83 gr an tii 13 C ou si ni a ga ha re ns is A tt ar & D ja va di C yn ar oi de ae B un ge Lo re st an - Sh ul ab ad 38 25 9( T U H ) M K 00 51 66 ga ha re ns is 14 C ou si ni a ke re dj en si s B or nm . & G au ba C yn ar oi de ae B un ge Te hr an 21 80 7( T U H ) M H 99 27 32 ke re dj en si s 15 C ou si ni a za rd ku he ns is A tt ar & G ha hr em an C yn ar oi de ae B un ge C ha ha r M ah al & B ak ht ia ri 21 88 7( T U H ) M H 99 07 88 za rd ku he ns is 16 C ou si ni a lo rd eg an en si s M eh re ga n C yn ar oi de ae B un ge C ha ha r M ah al & B ak ht ia ri 46 30 1( T U H ) M K 00 51 73 lo rd eg an en si s 17 C ou si ni a el w en de ns is B or nm . C yn ar oi de ae B un ge H am ad an -A lv an d M ou nt ai ns 20 56 6( T U H ) M H 99 27 41 el w en de ns is 18 C ou si ni a kh or ra m ab ad en si s B or nm . C yn ar oi de ae B un ge Lo re st an 21 85 1( T U H ) M H 99 27 37 kh or ra m ab ad en si s 19 C ou si ni a ph yl lo ce ph al a B or nm . & G au ba C yn ar oi de ae B un ge Lo re st an - K ho rr am A ba d 46 29 2( T U H ) M K 00 51 68 ph yl lo ce ph al a 20 C ou si ni a m ac ro ce ph al a C . A . M ey . C yn ar oi de ae B un ge A rd eb il- M es hk in s ha hr 42 92 5( T U H ) M H 99 03 19 m ac ro ce ph al a 21 C ou si ni a lu re st an ic a A tt ar & D ja va di C yn ar oi de ae B un ge Lo re st an 21 82 4( T U H ) M H 99 27 46 lu re st an ic a 22 C ou si ni a ir an ic a C . W in kl . & S tr au ss . C yn ar oi de ae B un ge A ra k 21 88 1( T U H ) M K 00 51 74 ir an ic a 23 C ou si ni a pa rs an a G ha hr em an C yn ar oi de ae B un ge H am ad an 20 55 3( T U H ) M K 00 51 69 pa rs an a 24 C ou si ni a ko rn hu be ri H ei m er l C yn ar oi de ae B un ge H am ad an 22 37 2( T U H ) M K 00 51 85 ko rn hu be ri 25 C ou si ni a ec ba ta ne ns is B or nm . C yn ar oi de ae B un ge H am ad an 22 37 1( T U H ) M H 98 87 70 ec ba ta ne ns is 26 C ou si ni a ve rb as ci fo lia B un ge C yn ar oi de ae B un ge K ho ra sa n- M as hh ad 43 01 3( T U H ) M K 00 51 79 ve rb as ci fo lia 27 C ou si ni a di sf ul en si s B or nm . C yn ar oi de ae B un ge Lo re st an - K ho rr am A ba d 27 58 9( T U H ) M H 99 27 42 di sf ul en si s 28 C ou si ni a sh ul ab ad en si s A tt ar & G ha hr em an C yn ar oi de ae B un ge Lo re st an - Sh ul A ba d 21 87 4( T U H ) M H 99 27 44 sh ul ab ad en si s 29 C ou si ni a sa ha nd ic a A tt ar & D ja va di C yn ar oi de ae B un ge A za rb ai ja n 46 27 2( T U H ) M K 00 51 75 sa ha nd ic a 30 C ou si ni a gi lli at ii R ec h. f. C yn ar oi de ae B un ge A za rb ai ja n 21 96 7( T U H ) M K 00 51 70 gi lli at ii 31 C ou si ni a al gu rd in a R ec h. f. C yn ar oi de ae B un ge A za rb ai ja n- T ab ri z 30 53 3( T U H ) M K 00 51 65 al gu rd in a 32 C ou si ni a cy na ro id es C . A . M ey C yn ar oi de ae B un ge A rd eb il 22 58 1( T U H ) M K 00 51 67 cy na ro id es 33 C ou si ni a ko ts ch yi B oi ss . C yn ar oi de ae B un ge A za rb ai ja n 46 24 4( T U H ) M K 00 51 71 ko ts ch yi 34 C ou si ni a na na A tt ar C yn ar oi de ae B un ge A ra k 14 34 7( T U H ) M K 00 51 72 na na 35 C ou si ni a sh eb lie ns is G ha hr em an C yn ar oi de ae B un ge A za rb ai ja n- T ab ri z 20 58 0( T U H ) M K 00 51 77 sh eb lie ns is 36 C ou si ni a ca lo ce ph al a Ja ub . & S pa ch C yn ar oi de ae B un ge A za rb ai ja n- M ia ne h 46 27 6( T U H ) M H 99 27 49 ca lo ce ph al a 42 Neda Atazadeh et al. DNA extraction, amplification and sequencing Garden-fresh leaves were dried in powder of silica gel. Cetyltrimethyl-ammonium bromide (CTAB) with activated charcoal protocol was used to extract genomic DNA (Murray and Thompson 1980). The quality of the extracted DNA was examined by running on 0.8% aga- rose (Sheidai et al. 2013). ITS region (ITS1, 5.8S, ITS2) was amplified using 0.2 μM primer ITS1 (5 -́ TCCGTAGGTGAACCTGCGG-3 ;́ Bioron, Germany), and primer ITS4 (5´- TCC GCT TATTGA TAT GC -3´) (Chen et al. 2010). PCR reactions were accomplished in a 25 μLvolume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 20 ng genom- ic DNA; and 3 U of Taq DNA polymerase (Bioron, Ger- many). The mentioned reactions were amplified in a Techne thermocycler (Germany) by applying the process as follows: 5 min at 94 °C, followed by 35 cycles for 30 s at 94 °C, 1 min at 52 °C, and 1 min at 72 °C, followed by one ultimate extension for 10 min at 72 °C. The process was run on 2% agarose gel to picture the amplification result, followed by the staining with ethidium bromide. A 100-base pair (bp) molecular-sized ladder (Fermentas, Germany) was used to determine fragment size. Data analysis Morphological analysis In all, 19 morphological characteristics were inves- tigated (quantitative and qualitative) and coded (Table 2). After, the obtained data was standardized (mean = 0, variance = 1) and applied to perform multivariate analy- ses. The species categorization was performed using the WARD (minimum spherical cluster method), UPGMA (unweighted paired group using average), PCoA (prin- cipal coordinate analysis), and MDS (multidimensional scaling) methods (Podani 2000). Paleontological Statis- tics software (PAST), ver. 2.17 (Hammer et al. 2012) was used for analysis. Moreover, morphological characteris- tics were coded for the maximum parsimony (MP) tree, and then created after tree bisection reconnection (TBR) branch swapping by applying them along with boot- strapping 1000 times in PAUP (phylogenetic analysis using parsimony) software, ver. 4 (Swofford 2002). Molecular analysis To assess homology, ITS sequences were aligned with MUSCLE implemented in MEGA 7 (Tamura et al. R . Ta xa Se ct io n Lo ca lit y V ou ch er n um be r A cc es si on n um be r A bb re vi at io n 37 C ou si ni a be hb ou di an a R ec h. f. & E sf an d. C yn ar oi de ae B un ge G ha zv in 27 62 9( T U H ) M H 99 27 36 be hb ou di an a 38 C ou si ni a ki rr in di ca B or nm . & R ec h. f. C yn ar oi de ae B un ge Il am 19 71 1( T U H ) M K 00 51 63 ki rr in di ca 39 C ou si ni a m ob ay en ii G ha hr em an & A tt ar C yn ar oi de ae B un ge K er m an sh ah - Es la m ab ad 20 56 9( T U H ) M K 00 51 80 m ob ay en ii 40 C ou si ni a sa na nd aj en si s R ec h. f. C yn ar oi de ae B un ge H am ad an 46 28 7( T U H ) M H 99 27 43 sa na nd aj en si s 41 C ou si ni a lu ro ru m B or nm . C yn ar oi de ae B un ge K er m an sh ah - M ah id as ht 20 56 8( T U H ) M K 00 51 76 lu ro ru m 42 C ou si ni a ku rd is ta ni ca A tt ar C yn ar oi de ae B un ge K ur de st an - M ar yv an 32 32 (T U H ) M K 00 51 78 ku rd is ta ni ca 43 C ou si ni a bo rn m ul le ri C . W in kl . C yn ar oi de ae B un ge Es fa ha n 22 53 2( T U H ) M H 99 27 50 bo rn m ul le ri 44 C ou si ni a fa rs is ta ni ca B or nm . C yn ar oi de ae B un ge K er m an 28 63 6( T U H ) M H 99 27 33 fa rs is ta ni ca 45 C ou si ni a la ct ifl or a R ec h. f. C yn ar oi de ae B un ge Lo re st an 46 29 9( T U H ) M K 00 51 56 la ct ifl or a 46 C ou si ni a al ig ud ar ze ns is A tt ar & G ha hr em an C yn ar oi de ae B un ge Lo re st an -A lig ud ar z 27 61 3( T U H ) M H 99 27 45 al ig ud ar ze ns is 47 C ou si ni a pl at ya ca nt ha B un ge Pl at ya ca nt ha e R ec h. f. K ho ra sa n 43 21 2( T U H ) M K 00 51 87 pl at ya ca nt ha 48 C ou si ni a fr ey ni i B or nm . Pl at ya ca nt ha e R ec h. f. Se m na n- S ha hr ud 27 67 5( T U H ) M K 00 51 86 fr ey ni i 49 C ou si ni a bi en er ti Bu ng e Pl at ya ca nt ha e R ec h. f. K ho ra sa n- N ey sh ab ur 28 68 2( T U H ) M H 99 27 40 bi en er ti 50 C ou si ni a re sh in ge ro ru m B or nm . Pl at ya ca nt ha e R ec h. f. K ho ra sa n- To rb at e Ja m 39 72 9( T U H ) M H 99 70 00 re sh in ge ro ru m 43Molecular phylogeny and morphometric analyses in the genus Cousinia 2012). The test was conducted by comparing the maxi- mum likelihood (ML) values for the known topology in the presence and absence of the molecular clock con- straints under Tamura and Nei (1993). The similar rate of evolution of the investigated sequences was rejected by setting the significance level at 5%, and consequently, the relaxed molecular clock model was utilized in fur- ther analyses (Minaeifar et al. 2016). The HKY model was identified as the best substitution model as imple- mented in MEGA 7 (Tamura et al. 2011). Bootstrap anal- ysis (BS) (Felsenstein 1985) was completed to attain sup- port estimates for the nodes in the ML tree. ITS sequences were also analyzed by TCS Network- ing as implemented in the PopART (Population Analy- sis with Reticulate Trees) program (http://popart.otago. ac.nz). Estimation of species time of divergence BEAST v1.6.1 (Drummond et al. 2012a, b) was applied for the Bayesian MCMC inferred analyses of the nucleotide sequence data. BEAUti (Bayesian Evo- lutionary Analysis Utility version) v1.6.1 (Drummond et al. 2012a, b) was used to generate initial xml files for BEAST. A Yule speciation process (‘a pure birth’ process) was utilized as a tree prior for all tree model analyses. For the MCMC posterior analyses, the length of chain was 10,000,000. After 100 trees burn-in pro- cessing, 10,000 trees were utilized for the analyses. The BEAUti xml file was run in the BEAST v1.6.1 program, and the maximum clade credibility (MCC) chain gen- erations were repeated five times for each molecular clock model with separate runs to confirm suitable con- vergence and sufficient mixing. The MCC tree was gen- erated under the relaxed clock model (HKY substitu- tion). ITS substitution rates were applied between 1.72 × 10–9 to 8.34 × 10–9 (mean = 4.13 × 10–9), according to Lopez-Vinyallonga et al. (2009). Tracer v1.5 software (Rambaut and Drummond 2007) was utilized for the production of the model parameters to assay the sampling and convergence results obtained from BEAST. TreeAnnotator v1.6.1 software (Drummond et al. 2012a, b) was utilized to annotate the phylogenetic results generated by BEAST as a form of single ‘target’ tree. On the target trees are shown summary statistics of posterior probabilities of the nodes: the 95% highest posterior density (HPD) lim- its of the node heights, rates, and the posterior estimates. Table 2. Morphological characters and their codes. Characters Codes 1 2 3 4 5 Head diameter x<3 3≤x≤6 x>6 - - Flowers 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 - - Corolla length x< 20 20≤x≤25 x>25 - - Habitat Woodland Alpine Steppe - - Leaves indumentum Present Absent - - - Stem leaves Interruptedly decurrent Continuously decurrent Nondecurrent - - 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 Shape of appendages of median bracts Sagitate Triangular Rhombic Ovate Lanceolate Margin of appendages 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 - 44 Neda Atazadeh et al. FigTree v1.3.1 (Rambaut 2009) program was also applied for the annotated BEAST MCC tree production analyses. The posterior probability was fix to 0.5, which is equal to the bootstrapping value in PAUP (Phylogenetic Analy- sis Using Parsimony analysis) analyses (Hong and Jury 2011). RESULTS Morphometry PCA analysis of morphological features showed that the first two PCA components included about 79% gen- eral alteration. Morphological features like the shape and length of the appendages of the median bracts, diameter of the heads, number of the flowers, and length of the corolla were the most variable morphological fea- tures among the investigated plants. In fact, these mor- phological features are of taxonomic value in the two sections Cynaroideae and Platyacanthae. An MP tree (Figure 1) of morphological character- istics can delimit the two studied sections Cynaroideae and Platyacanthae because of difference in traits, like stem leaves (sect. Cynaroideae: decurrent; sect. Platya- canthae: nondecurrent) and the appendages of the medi- an bracts (sect. Cynaroideae: present; sect. Platyacan- thae: absent). In the MP tree, within the sect. Platyacanthae, C. Platyacantha Bunge and C. freynii Bornm. were locat- ed close to each other due to similarity in all charac- ters except for the color of the corolla (C. Platyacantha: white; C. freynii: purple). Likewise, C. reshingerorum Bornm. and C. bienerti Bunge exhibited morphological similarity in traits like number of flowers, length of the corolla, color of the anther tube, median and the inner bracts, receptacle bristles, and the diameter of the head and ratio of the limb/tube. In the sect. Cynaroideae, C. grandis C. A. Mey. and C. grantii Rech. f. were located close to each other because of similar morphological traits such as the shape of the appendages of the median bracts (ovate) and leaves indumentum (glabrous). The same applies for C. ecbatanensis Bornm. C. kornhuberi Heimerl, C. elwen- densis Bornm., C. parsana Ghahreman, C. denaensis Attar & Djavadi and C. khorramabadensis Bornm.; these species have similar morphological characters like the color of the corolla (white) and the position of the medi- an bracts (spreading). C. millefontana Rech. f., C. fursei Rech. f., C. sardashtensis Rech., C. carolihenrici Attar & Ghahreman, C. dalahuensis Attar & Ghahreman and C. concinna Boiss. & Hausskn. were also close to each other because of the similarity in morphological features such as the position of the median bracts (imbricated). The same applies for C. calocephala Jaub. & Spach and C. behboudiana Rech. f. & Esfand. because of their simi- lar morphological traits, like the position of the median bracts (recurved) and the color of the corolla (yellow). ITS sequence analysis The pair-wise genetic distances determined for the studied Cousinia species arranged from 0.01 (the low- est value between C. ecbatanensis and C. kotschyi Boiss.) to 0.50 (the highest value between C. shebliensis Ghah- reman and C. behboudiana). These values showed the degree of sequence variability within species. The ML tree (Figure 2) and TCS network (Figure 3) of the studied species based on ITS sequences produced similar results. In these trees, the outgroups (A. umbrosum and A. lappa) were basically separated from the other species. Based on these results, the ITS marker did not delimit the two studied sections. In the obtained genetic trees, the species of the sect. Platyacanthae, such as C. platyacantha, C. reshingerorum, C. bienerti and C. freynii, were placed among the species of the sect. Cynaroideae. These trees exhibited a close genetic affinity between C. platyacantha, C. reshingerorum and C. bienerti, which is in agreement with their morphological similarities. How- ever, C. freynii is located far from the others in the genet- ic tree but close to them in the morphological tree. Figure 1. Maximum Parsimony tree of the studied Cousinia species based on morphological data. (species are according to Table 1). Values above branches are bootstrap value. 45Molecular phylogeny and morphometric analyses in the genus Cousinia In the ML tree based on ITS sequences, C. verbasci- folia was placed far from the other species. It differs mor- phologically from the other studied species in the color of corolla (light pink), bracts (numerous), the appendage of the median bracts (triangular), presence of long spine found at the apex, and the spinulose at the margin. Both MP and ML trees exhibited an affinity between C. kotschyi and C. ecbatanensis. These species are simi- lar in morphological traits such as shape of the append- ages in the median bracts (Lanceolate) and position of the median bracts (Spreading). Similarly, C. elwenden- sis and C. denaensis are related to C. ecbatanensis. They have morphological similarities in the color of the corolla (white) and the position of the median bracts (spreading). The ITS-based phylogenetic tree showed a close affin- ity between C. millefontana and C. concinna, which have similar morphological characters such as the position of the median bracts (Imbricated). C. keredjensis Bornm. & Gauba is well separated from the two species mentioned above (C. millefontana and C. concinna) and also differs in its morphological traits, like the color of the corolla (white), the number of flowers (ca. 125), and the number of bracts (ca. 130). The same applies between C. sahandi- ca Attar & Djavadi and C. sanandajensis Rech. f., as well as for C. nana Attar and C. cynaroides C. A. Mey. Disagreement was observed between the other stud- ied species of Cousinia based on morphological and genetic characters. The TCS network exhibited the process of specia- tion and the number of nucleotide substitution in ITS sequences among the studied species. The highest num- ber of nucleotide substitutions in ITS occurred in C. khorramabadensis (9). The Bayesian tree (Figure 4) obtained with BEAST based on ITS sequences estimated the divergence times of the studied species within the Cynaroideae and Platy- acanthae sections to be approximately 3.5 mya. DISCUSSION Taxonomy, molecular phylogeny, and the species relationships of the genus Cousinia are complicated and unresolved, mainly because of disagreement between morphological and molecular phylogenetic studies (Sau- sana et al. 2003; Lopez-Vinyallonga et al. 2009; Mehregan and Assadi 2016). Moreover, several overlapping morpho- logical characteristics at the species level hinder species identification and delineation (Attar and Djavadi 2010; Minaeifar et al. 2016; Atazadeh et al. 2020). Susanna et al. (2003), performed an extensive inves- tigation on the evolution and generic delineation in the Arctium-Cousinia complex, based on two very important characters: pollen type and chromosome number. They divided all studied species into two major lineages: The Arctioid clade (including: Arctium, Sehmalhallsenia) with Arctiastrum pollen type and x= 18 and the Cous- inioid clade (including: Cousinia subg. Cousinia) with Cousinia pollen type and x= 11, 12, 13. They also showed that the palynological and chromosome number results Figure 2. Maximum likelihood tree of the studied Cousinia species based on ITS data. (species are according to Table 1). 46 Neda Atazadeh et al. are incongruent with morphological and molecular data in the Arctium-Cousinia complex and considered to be homoplasious morphological characters. These results were later confirmed by Lopez-Vinyallonga et al. (2009). In the present study, the studied Cousinia species were delimited and useful taxonomic features, including the shape and length of the appendages of the median bracts, position of the median bracts, diameter of the heads, number of flowers and bracts, and the color and length of the corolla were identified. The species relationships obtained based on the morphological features within sect. Platyacanthae are in agreement with those reported in previous stud- ies (Asaadi and Mehregan 2017). For instance, with- in sect. Platyacanthae, C. Platyacantha and C. freynii were placed close to each other because of the similar- ity found in all features except color of the corolla (C. Platyacantha: white; C. freynii: purple). Asaadi and Mehregan (2017) also noticed their close affinity to each other. Likewise, C. reshingerorum and C. bienerti exhib- ited morphological likenesses in traits such as number of flowers, length of the corolla, color of the anther tube, the median and inner bracts, receptacle bristles, diam- eter of the head, and the ratio of the limb/tube. Asaadi and Mehregan (2017) also showed the close morphologi- cal affinity among these species. The morphological results in sect. Cynaroideae, were in complete agreement with the obtained results by Attar and Djavadi (2010). The relationships among the studied Cousinia spe- cies in the genetic trees were generally incongruent with those in the obtained morphological tree. The present study revealed that the studied sections within the genus Cousinia, including Cynaroideae and Platyacanthae, are not monophyletic. This is fully con- sistent with the results reported by Susanna et al. (2003) and Lopez-Vinyallonga et al. (2009), who also showed that morphological traits are highly incongruent with molecular data in Arctium-Cousinia complex and con- sidered homoplasious morphological characters. Various reasons are suggested for the disagreement between “gene tree” and “species trees”, including the Figure 3. TCS Network of studied Cousinia species based on ITS sequences (species are according to Table 1). 47Molecular phylogeny and morphometric analyses in the genus Cousinia Figure 4. BEAST chronogram of studied Cousinia species based on ITS sequences (species are according to Table 1, numbers at the base of tree represents Mya). 48 Neda Atazadeh et al. high number of taxa in Cousinia (Susanna et al. 2003; Lopez-Vinyallong et al. 2009; Mehregan and Assadi 2016), interspecific hybridization, the occurrence of the intermediate forms (Mehregan and Kadereit 2009) and homoploid hybrid speciation, of which there is little proof to prove them (Lopez-Vinyallong et al. 2009), and incomplete lineage sorting (Zhang et al. 2015). The divergence time of the studied species within the Cynaroideae and Platyacanthae sections based on ITS sequences was estimated to be around 3.5 mya. This result is in agreement with Lopez-Vinyallonga et al. (2009), who also showed that the major radiation of the genus Cousinia has been estimated to have started ca. 8.7 mya. According to our findings and previous authors (Susanna et al. 2003; Lopez-Vinyallong et al. 2009) phy- logeny, evolutionary pathway and species relationships of the genus Cousinia are unclear and complicated. The genus Cousinia with its relatively young geological age (ca. 8.7 mya) and high number of taxa is unusual exposed to speciation. Djamali et al. (2012) showed that Cousinia consistently existed in the glacial age. They recorded an ~200,000-year pollen from Lake Urmia, northwest Iran. In contrast, the dispersal of its pollen grains was restricted. In the current results, all of the studied species except for C. calocephala had restricted geographical distributions and were isolated by geo- graphical boundaries, which reduced the genflow. There- fore, geographical processes can be determining factors in the speciation of this genus. This result 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. As a general conclusion, based on the molecular studies of the observed specimens, it is suggested that both Cynaroideae and Platyacanthae sections are syn- onymous. To make a definitive decision on this, further molecular studies are necessary. AUTHOR CONTRIBUTIONS All authors contributed to the study conception and design. Material preparation, data collection and analy- sis were performed by Neda Atazadeh, Masoud Sheidai and Farideh Attar. The first draft of the manuscript was written by Neda Atazadeh and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. REFERENCES Alvarez I, Wendel JF. (2003). Ribosomal ITS sequences and plant phylogenetic inference. 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