317 Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Abstract Helichrysum oocephalum is a medicinal plant of the genus Helichrysum that have limited distribution in Iran. Local geographical populations may differ in their genetic content and form different gene pools. Therefore, we carried out population genetic investigation and morphological studies in five geographical populations of Helichrysum oocephalum by using ISSR molecular markers. AMOVA produced the significant genetic differences. The mean Nm value revealed some degree of gene flow among Helichrysum oocephalum 8. Molecular and morphological analysis indicated that we have 2 groups in the studied populations. The present findings may be of use in the conservation of this medicinal plant in Iran. Iz vleček Helichrysum oocephalum je zdravilna rastlina iz rodu Helichrysum, ki ima v Iranu omejeno razširjenost. Lokalne geografske populacije se lahko genetsko razlikujejo in vzpostavljajo različne genske nabore, zato smo zastavili populacijsko genetsko raziskavo in morfološko študijo petih geografskih populacij vrste Helichrysum ooce- phalum z ISSR molekularnimi markerji. Z analizo AMOVA smo dokazali značilne genetske razlike. Povprečna vrednost Nm je razkrila genski pretok med vrstami Helichrysum oocephalum. Z molekularno in morfološko analizo smo dokazali dve skupini med obravnavanimi populacijami. Predstavljeni rezultati bodo uporabni pri ohranjanju te zdravilne vrste v Iranu. Key words: Gene flow, Helichrysum oocephalum, ISSR, morphology. Ključne besede: genski tok, Helichrysum oocephalum, ISSR, morfologija. Received: 16. 9. 2018 Revision received: 24. 1. 2020 Accepted: 24. 2. 2020 1 Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran. 2 Forest and Rangeland Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center. AREEO. Mashhad, Iran. * Corresponding author: E-mail: msheidai @yahoo.com Mobina Abbaszadeh1 , Masoud Sheidai1 , *  , Narges Azizi2 & Fahimeh Koohdar1  DOI: 10.2478/hacq-2020-0002 19/2 • 2020, 317–324 19/2 • 2020, 317–324 318 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Introduction The genus Helichrysum Mill., (Gnaphalieae) is a genus in the family Asteraceae that contains 500 to 600 annual, herbaceous perennials or shrub species (Galbany-Casals et al. 2009, Azizi et al. 2019). Some of these species have ornamental and medicinal values. For example, Heli- chrysum italicum, H. leucocephalum and H. artemisioides contain essential oils (Javidnia et al. 2009), while H. compactum and H. italicum contain flavonoids with an- tioxidant and antibacterial activity (Facino et al. 1990). There are 18 Helichrysum species in Iran (Azizi et al. 2019). According to this study H. persicum Ghahremani & Noori, is a member of H. oocephalum Boiss., species (Salehi et al. 2014). Helichrysum oocephalum grows in limited regions lo- cated in the North-East of Iran, is a medicinal plant and extensively used by locals for its anti-inflammatory, an- tiallergic, antipsoriatic and diuretic effects (Firouznia et al. 2007, Azizi et al. 2014). To our knowledge there has been no detailed investigation on the genetic variability and population genetic structure of this rare medicinal plant in Iran and the present study is the first report on the subject. Such investigations can provide information about potential gene pools which then might be used in conservation and breeding of medicinal plants (Chen 2000, Ellis & Burke 2007, Sheidai et al. 2012). It has been shown that many plant species which are distributed in different geographical regions, differ in their genetic structure and morphological characteristics too (see for example Sheidai et al. 2012; Azizi et al. 2014, Minaeifar et al. 2015, Azizi et al. 2019 ). Therefore, we may also encounter new infra-specific taxonomic forms like, varieties, or ecotypes within a single species (Sheidai et al. 2012, Koohdar et al. 2015). Molecular markers have been used extensively in popu- lation genetic investigations (Sheidai et al. 2012, Koohdar et.al. 2015, Mosaferi et al. 2015). Multilocus molecular markers including simple sequence repeat markers (SSRs) and inter simple sequence repeat markers (ISSR) are good genetic markers to identify hybrid plants and plan genetic diversity (Gaskin & Kazmer 2009, Noormohammadi et al. 2012). These molecular markers are known to reveal genetic diversity in Helichrysum species and (Azizi et al. 2014, Taban et al. 2015). We used ISSR (Inter-simple se- quence repeats) to study genetic diversity of populations in Helichrysum oocephalum, since these markers are repro- ducible, cheap, easy to work and are known to be efficient in population genetic diversity studies (Azizi et al. 2014, Sheidai et al. 2014). Therefore, the aim of present study was population genetic analysis of 5 geographical populations in Heli- chrysum oocephalum by using ISSR molecular markers for the first time. These informations may be of use for future conservation and breeding of this medicinally important plant species. Material and Methods Plant material Sixty-eight plant specimens were collected from 5 popu- lations of Helichrysum oocephalum. Details of the studied populations are provided in Table 1 and Figure 1. Table 1: Populations studied, their locality and ecological features. Tabela 1: Obravnavane populacije, njihove lokacije in ekološke lastnosti. Pop Province Locality Altitude (m) Longitude Latitude Voucher number 1 North Khorasan Gholaman village 1226 3803 5708 HSBU68 2 North Khorasan Raz city 1294 3756 5706 HSBU69 3 Razavi Khorasan Khanroodvillage 1656 3608 5924 HSBU70 4 Razavi Khorasan Boghmech village 1740 3650 5914 HSBU71 5 Razavi khorasan Abqad village 1660 3629 5959 HSBU72 Figure 1: Loctions of the studied populations in Helichrysum oocephalum. Slika 1: Lokacije obravnavanih populacij vrste Helichrysum oocephalum.                         19/2 • 2020, 317–324 319 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Morphological study At first, 50 morphological characters were studied in the randomly selected plants of these 5 populations. Prelimi- nary analysis revealed that 9 morphological characters dif- fer among the studied populations. These characters are involucre length, sporangia width, stem length, involucre color, involucre form, involucre overlap, leaf color and, stem color. They were coded as binary (1 = presence, or 2 = absence), or multistate characters and used for further multivariate analysis (Podani 2000). DNA extraction and ISSR assay Fresh leaves were collected randomly in each of the stud- ied populations and dried in silica gel powder. Genomic DNA was extracted using CTAB with activated charcoal protocol (Sheidai et al. 2014, Koohdar et al. 2015). The quality of extracted DNA was examined by run- ning on 0.8% agarose gel. T en ISSR primers; (AGC)5GT , (CA)7GT, (AGC)5GG, UBC810, (CA)7AT, (GA)9C, UBC807, UBC811, (GA)9A and (GT)7CA commercial- ized by UBC (the University of British Columbia) were used (Sheidai et al. 2014, Koohdar et al. 2015). PCR reactions were carried out according to our previ- ous reports (Sheidai et al. 2014, Koohdar et al. 2015). For this we used 25μl volume mixture containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany), 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of T aq DNA polymerase (Bioron, Germany). The amplifications’ reactions used also were accord - ing to our previous reports (Sheidai et al. 2014, Koohdar et al. 2015). They were performed in Techne thermocy- cler (Germany) with the following program: 5 min initial denaturation step 94 °C, 30 S at 94 °C; 1 min at 50 °C and 1min at 72 °C. The reaction was completed by fi - nal extension step of 7 min at 72 °C. The amplification products were visualized by running on 2% agarose gel, followed by the ethidium bromide staining. The fragment size was estimated by using a 100 bp molecular size ladder (Fermentas, Germany). Morphological analysis For morphological grouping of the studied populations, coded characters were used to determine Gower distance. This was then used in UPGMA (Unweighted paired group method using average) and Ward (minimum spherical cluster) clustering (Podani 2000, Koohdar et al. 2015). Principal components analysis (PCA) biplot was performed to identify the most variable morphologi- cal characters differentiating the studied populations. Genetic diversity and population structure For genetic diversity analysis we used ISSR bands. These bands were coded as binary characters (presence = 1, ab- sence = 0). Data obtained were analyzed for the genetic diversity parameters like,, Nei’s gene diversity (H), Shan- non information index (I), number of effective alleles, and percentage of polymorphism (Weising et al. 2005, Freeland et al. 2011). For grouping of the studied populations, we used Nei’s genetic distance (Weising 2005, Freeland et al. 2011). Neighbor Joining (NJ) clustering and principal coordi- nate analyses (PCoA) were performed after 100 times bootstrapping/ permutations (Sheidai et al. 2016, Kooh- dar et al. 2015). The correlation between genetic and geographical dis- tance in the studied populations was determined by us- ing Mantel test as performed in PAST ver. 2.17 (Podani 2000, Hammer et al. 2001). DARwin ver. 5 (2012) pro- grams were used for cluster analyses. Two approaches were used to determine genetic differ- entiation of the studied populations, 1 – AMOVA (Anal- ysis of molecular variance) (with 1000 permutations) as implemented in GenAlex 6.4 (Peakall & Smouse 2006), and 2 – Nei’s Gst analysis of GenoDive ver. 2 (2013) (Meirmans & Van tienderen 2004, Sheidai et al. 2016). The new genetic differentiation parameters like G’st_est = standardized measure of genetic differentiation (Hedrick 2005), and D_ est = Jost measure of differentiation (Jost 2008), were also determined. The potential problems caused by the dominance of ISSR markers, were resolved by using the Bayesian pro- gram, Hickory (ver. 1.0) (Holsinger & Lewis 2003). It was used to estimate parameters related to genetic struc- ture (theta B value) (Tero et al. 2003). The genetic structure of the studied populations was also determined by two approaches: 1 – Bayesian based model STRUCTURE analysis (Pritchard et al. 2000), and 2 – maximum likelihood-based method of K-Means clustering (Sheidai et al. 2014). For STRUCTURE analysis of dominant ISSR molecu- lar markers, we followed the instructions of Falush et al. (2007). This was followed by performing the Evanno test to find out the proper number of genetic groups (K) by us- ing delta K value (Evanno et al. 2005). We performed K- Means clustering according to GenoDive ver. 2. (2013), which produces two summary statistics of 1 – pseudo-F and 2 – Bayesian Information Criterion (BIC). These sta- tistics provide the best fit for k (Çaliskan 2012). Finally, the correlation coefficient was determined between gene diversity/ genetic polymorphism and the studied envi- 19/2 • 2020, 317–324 320 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran ronmental features i.e. altitude, longitude, and, latitude (Sheidai et al. 2014, 2016). Gene flow Gene flow among populations was determined by two different approaches: 1 – Calculating Nm an estimate of gene flow from Gst by PopGen ver. 1.32 (1997) as: Nm = 0.5(1 - Gst)/Gst. This approach considers equal amount of gene flow among all populations. 2 – Popu- lation assignment test based on maximum likelihood as performed in Genodive ver. in Genodive ver. 2. (Sheidai et al. 2014, 2016). Results Genetic diversity The Genetic diversity parameters estimated are provided in Table 2. The highest value for gene diversity occurred in populations 1 and 4 (0.133 and 0.129, respectively). The highest level of genetic polymorphism (15.79) oc- curred in population 2, while the lowest value of the same parameter occurred in population 4 (35.62). tiation parameters also support AMOVA results, as Gst (0.218, P = 0.001), Hedrick , standardised fixation index (G’st = 0.233, P = 0.001) and Jost’ differentiation index (D-est = 0.06, P = 0.001), revealed that the studied popu- lations are genetically differentiated. Neighbor Joining (NJ) tree and PCoA plot produced similar results. Therefore, only PCoA plot is presented (Figure 2). The PCoA plot separated some of the studied populations from each other due to their genetic differ- ence. This is in agreement with AMOVA. However, in some cases, plants of different populations were inter- mixed. This happened due to within-population genetic variability and gene flow/ shared alleles in those popula- tions. Table 2: Genetic diversity parameters in the studied popula- tions of Helichrysum oocephalum. (Population numbers are according to Table 1). Tabela 2: Genetska raznolikost obravnavanih populacij vrste Helichrysum oocephalum. (Oznake populacij so enake kot v Tabeli 1). Pop N Na Ne I He UHe %P Pop1 9.0 0.895 1.214 0.206 0.133 0.141 44.74 Pop2 3.0 0.421 1.109 0.091 0.062 0.075 15.79 Pop3 5.0 0.763 1.167 0.172 0.109 0.122 36.84 Pop4 8.0 1.053 1.184 0.212 0.129 0.138 52.63 Pop5 9.0 1.053 1.154 0.184 0.108 0.114 52.63 Abbreviations : N = Number of populations, Na = No. of Different Alleles, Ne = No. of Effective Alleles, I = Shannon’s Information Index, He = Gene diversity, UHe = Unbiased gene diversity, and %P = Percentage of polymorphism. Population genetic structure AMOVA revealed significant genetic difference among the studied population (PhiPT = 0.196, P = 0.010). It also revealed that, 80% of total genetic variation was due to within population diversity, while 20% was due to among population genetic differentiation. Moreo- ver, Hickory test also produced high Theta B value (0.40) supporting AMOVA. The new genetic differen - Figure 2: PCoA plot in Helichrysum oocephalum populations based on ISSR marker. Slika 2: Graf PCoA populacij vrste Helichrysum oocephalum na podlagi ISSR markerjev. Evanno test performed on STRUCTURE analysis and pseudo-F index of K-Means clustering produced optimum number of k = 2. These results indicated that we have 2 genetic groups in the studied populations. STRUCTURE plot (Figure 3) based on k = 2, identified these two genetic groups (potential gene pools).          Figure 3: STRUCTURE plot based on k = 2 in Helichrysum oocephalum populations. Slika 3: Graf STRUCTURE populacij vrste Helichrysum oocephalum s k = 2.                                         19/2 • 2020, 317–324 321 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran The STRUCTURE plot revealed that some plants in the population 1 are genetically different from the other plants within this population. They also differed greatly from the other studied populations. Plants in population 2–5 also had some alleles shared with these plants (red colored seg- ments). Therefore, limited gene flow among these plants may explain genetic difference observed in population 1. Correlation coefficient determined between gene diversity and environmental features was not signifi- cant (r = 0.29 with altitude, r = 0.32 with latitude, and, r = -0.10 with longitude, P .0.1). Similarly, ge- netic polymorphism was not also correlated with lati- tude (r= -0.3974), altitude (r = 0.56), or the longitude (r = -0.3974) as well. Gene flow and genetic admixture The reticulogram obtained revealed some degree of ge- netic admixture in the studied populations (Figure 4). For example, plant No. 17 of population 3 had shared alleles with plants in population 5. Similarly, plants in popula- tion 1, had shared alleles with plants in population 5. More detailed information was obtained by popula- tion assignment test (Table 3). It revealed the occurrence of gene flow or ancestral shared alleles between plants in populations 1 and 4, 3 and 4, as well as 1 and 6. Table 3: Population assignment test result showing plants inferred to be from another population. Tabela 3: Določitev pripadnosti posameznih primerkov določeni populaciji. Individual Current Inferred Lik_max Lik_home Lik_ratio 6 Pop001 Pop004 -18.283 -20.724 4.881 7 Pop001 Pop004 -11.304 -20.765 18.922 8 Pop001 Pop004 -9.869 -12.016 4.295 9 Pop001 Pop004 -14.315 -18.057 7.484 13 Pop003 Pop005 -19.529 -25.196 11.335 14 Pop003 Pop004 -15.490 -18.106 5.232 17 Pop003 Pop005 -12.022 -15.868 7.691 22 Pop004 Pop003 -8.554 -12.833 8.558 24 Pop004 Pop003 -12.055 -16.591 9.071 29 Pop005 Pop001 -8.624 -13.213 9.177 30 Pop005 Pop004 -15.239 -15.67 0.861 34 Pop005 Pop001 -10.59 -11.632 2.082 Figure 4: The reticulogram in Helichrysum oocephalum populations. Slika 4: Retikulogram populacij vrste Helichrysum oocephalum. The mean Nm value = 1.25 was obtained for these pop- ulations that is showing high degree of gene flow among them. Therefore, all these results revealed some degree of gene flow among Helichrysum oocephalum. The Mantel test produced significant correlation be - tween genetic distance and geographical distance of the studied populations (r = 0.22, P = 0.01). This indicated the occurrence of isolation by distance (IBD) in Heli- chrysum oocephalum populations. Therefore, gene flow mainly occurred between the neighboring populations. Morphological variability UPGMA and Ward clustering of morphological char- acters produced similar results. Therefore, only Ward dendrogram is presented (Figure 5). This dendrogram grouped the studied populations in two major clusters. The populations 1 and 2 formed the first cluster, while populations 3–5 comprised the second.                                    19/2 • 2020, 317–324 322 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Representative plants of each population were almost grouped together and formed a separate cluster. This is particularly true for populations 1 and 2. This result indi- cated that each population differed in its morphological features from the other populations. However, in populations 3–5, some of the plants were intermixed with plants of the other populations due to morphological similarities. These morphological similari- ties might be consequence of gene flow or genetic admix- ture of these populations as presented before. PCA analysis of morphological characters revealed that the first 3 PCA axes comprised about 70% of total mor- phological variations. It also showed that morphological characters like, Involucre length, involucre color, involu- cre form, involucre overlap, leaf color and stem color, are the most variable morphological characters among the studied Helichrysum oocephalum populations. PCA biplot showed that the involucre form separated populations 1 and 2 from the others. Discussion Medicinal plants such as Helichrysum oocephalum are ex- tensively used by locals and therefore are subject to nega- tive selection pressure which reduces the in number of plants or their complete elimination from the natural habitat. We encountered high degree of population ge- netic differentiation. Environmental disturbances causing disappearance and fragmentation of natural populations reduce the rate of gene flow among populations, which in turn increases the population genetic differentiation. In this situation, the genetic drift acts strongly and reduces within population genetic variability (Setsuko et al. 2007, Hou & Lou 2011). However, we obtained a high degree of within population genetic variability in the studied populations of Helichrysum oocephalum. AMOVA indi- cated that, out of total genetic variation, 80% was due to within population. This was also indicated by high Nm values obtained. The observed within population genetic variability may be related to out crossing nature of this species. The presence of high within population genetic variability helps the population to cope with local envi- ronmental changes. Both STRUCTURE analysis and population assign- ment test, along with reticulogram obtained revealed the occurrence of some degree of gene flow among Heli- chrysum oocephalum populations. Gene flow brings about adequate genetic diversity for the studied populations. Ge- netic diversity is important for continuity of plant species and adaptation to environmental conditions (Çalişkan 2012). The occurrence of gene flow between different geo - graphical populations introduces new genes to the local populations and adds to the genetic variability of these populations (Hou & Lou 2011, Sheidai et al. 2014). Significant AMOVA, Gst and differentiation param- eters obtained for the studied populations, indicate that, in spite of limited gene flow, the local populations have acquired their specific genetic structure. This may be due to genetic drift, isolation by distance or local adaptations (Hou & Lou 2011, Sheidai et al. 2014). This has also been reported by Galbany‐Casals et al. (2011) in the Mediterranean Helichrysum italicum. Sabetta et al. (2006) investigated the genetic diversity in populations of H. italicum from Corsica and Italy by AFLP (Amplified fragments length polymorphism) mo- lecular markers and obtained a dendrogram that grouped these populations in three primary clusters without any cases of homonymy. The plants collected from different geographical regions showed different genetic structure. Smissen et al. (2006) investigated the genetic diversi- ty of the endemic complex species of H. lanceolatum in New Zealand and reported a weak geographic structure. However, they observed that the populations followed isolation by distance model. Azizi et al. (2014) reported within-population genetic variability and the occurrence of gene flow among geographical populations in Helic- rysum leucocephalum. These populations did not reveal isolation by distance. Mantel test revealed a pattern of isolation-by distance across the distribution range of the studied Helichrysum oocephalum populations. This means that the dispersal of populations might be constrained by distance, and gene flow occurs mostly between neighboring populations (Sheidai et al. 2014). As a result, more closely situated populations tend to be more genetically similar to one another (Slatkin 1993, Hutchison & Templeton 1999, Medrano & Herrera 2008). Figure 5: WARD dendrogram of Helichrysum oocephalum populations based on morphological data. Slika 5: Dendrogram WARD populacij vrste Helichrysum oocephalum na podlagi morfoloških podatkov.                      19/2 • 2020, 317–324 323 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Morphometric studies also revealed that the studied populations differed from each other. Therefore, geo - graphical populations in Helichrysum oocephalum differed in both genetic and morphological features. Azizi et al. (2014) reported morphological, cytological and genetic differences among geographical populations of Helicrysum leucocephalum and considered different forms as potential ecotypes within this species. Therefore, we conclude that, a combination of genetic and morphological divergence, limited gene flow and local adaptation have played role in diversification of Helichrysum oocephalum populations in Iran. The use of molecular markers has revolutionized the techniques for characterizing genetic variation and vali- dates genetic selection. However, molecular markers are the most reliable source for the analysis of genome struc- ture and behavior in medicinal plant (Chen et al. 2016). Therefore, these findings may be of use in conservation this medicinal plant in the country Masoud Sheidai , https://orcid.org/0000-0003-3983-6852 Fahimeh Koohdar , https://orcid.org/0000-0002-7878-1906 References Azizi, N., Sheidai, M., Mozaffarian, V., Arman, M. & Noormohammadi Z. 2019: Assessment of relationships among and within Helichrysum Mill. (Asteraceae) species by using ISSR markers and morphological traits. Hacquetia18: 105–118. Azizi, N., Sheidai, M., Mozafarian, V. & Noormohammadi, Z. 2014: Genetic, cytogenetic and morphological diversity in Helicrysum leucocephalum (Asteraceae) populations. Biologia 69: 566–573. Çalişkan, M. 2012: Genetic Diversity in Plants Edited by, ISBN 978- 953-51-0185-7, 510 pages, Publisher, in: Tech, Chapters published under CC BY 3.0 license. Chen, X. Y. 2000: Effects of fragmentation on genetic structure of plant populations and implications for the biodiversity conservation. Acta Ecologica Sinica 20: 884–892. Chen, Sh. M., Yu, H., Luo, H. M., Wu, Q., Li, Ch. F . & Steinmetz. A. 2016: conservation and sustainable use of medicinal plants: problems, progress, and prospects. Chin Med 11: 37. Ellis, J. R. & Burke, J. M. 2007: EST-SSRs as a resource for population genetic analyses. Heredity 99: 125–132. Evanno, G., Regnaut, S. & Goudet, J. 2005: Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14: 2611–2620. Facino, R. Carini, M. M., Franzoi, L., Pirola, O. & Bosisio, E. 1990: Phytochemical characterization and radical scavenger activity of flavonoids from Helichrysum italicum G. Don (Compositae). Pharmacological research 22: 709–721. Falush, D., Stephens, M. & Pritchard, J. K. 2007: Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Resources 7: 574–578. Firouznia, A., Akbari, M. T., Rustaiyan, A., Masoudi, S., Bigdeli, M. & Anaraki, M. T. 2007: Composition of the essential oils of Artemisia turanica Krasch., Helichrysum oocephalum Boiss. and Centaurea ispahanica Boiss. three asteraceae herbs growing wild in Iran. Journal of Essential Oil Bearing Plants 10: 88–93. Freeland, J. R., Kirkland, S. & Petersen, D. 2011: Molecular Ecology, 2nd ed. Willy-Blackwell, London, 464 p. Galbany‐Casals, M., Blanco‐Moreno, J., Garcia‐Jacas, N., Breitwieser, I. & Smissen, R. 2011: Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin? Plant Biology 13: 678–687. Gaskin, J. F . & Kazmer D. J. 2009: Introgression between invasive saltcedars (T amarix chinensis and T. ramosissima) in the USA. Biological Invasions 11: 1121–1130. Hammer, Ø., Harper, D. A.T. & Ryan, P . D. 2012: PAST: Paleontological Statistics software package for education and data analysis. Palaeontologia Electronica 4: 9. Hedrick, P . W. 2005: A standardized genetic differentiation measure. Evolution 59: 1633–1638. Holsinger, K. E. & Lewis, P . O. 2003: Hickory: a package for analysis of population genetic data V1. 0. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, USA. Hou, Y. & Lou, A. 2011: Population Genetic Diversity and Structure of a Naturally Isolated Plant Species, Rhodiola dumulosa (Crassulaceae). PLoS ONE6 e24497. Hutchison, D. W. & Templeton, A. R.1999: Correlation of pair wise genetic and geographic distance measures: inferring the relative influences of gene flow and drift on the distribution of genetic variability. Evolution 53: 1898–1914. Javidnia, K., Miri., R., Soltani, M. & Khosravi, A. 2009: Essential oil composition of two Iranian endemic Helichrysum Miller. species (H. leucocephalum Boiss. and H. artemisioides Boiss. et Hausskn.). Journal of Essential Oil Research 21: 54–56. Jost, L. 2008: GST and its relatives do not measure differentiation. Molecular ecology 17: 4015–4026. Koohdar, F ., Sheidai1. M., Talebi, S. M. & Noormohammadi, Z. 2015: Population genetic structure in medicinal plant Lallemantia iberica (Lamiaceae). Biodaiversitas 16: 139–14. Medrano, M. & Herrera, C. M. 2008: Geographical structuring of genetic diversity across the whole distribution range of Narcissus longispathus, a habitat-specialist, Mediterranean narrow endemic. Annals of Botany 102: 183–194. Meirmans, P . G. & Van Tienderen P . H. 2004: GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Molecular Ecology 4: 792–794. Minaeifar, A. A., Sheidai, M., Attar, F ., Noormohammadi, Z. & Ghasemzadeh-Baraki, S. 2015: Genetic and morphological diversity in Cousinia tabrisiana (Asteraceae) populations. Biologia 70: 328–338. Mosaferi, S., Sheidai, M., Keshavarzi M. & Noormohammadi. Z. 2015: Genetic diversity and morphological variability in Polygonum aviculare s.l. (Polygonaceae) of Iran. Phytotaxa 233: 166–178. 19/2 • 2020, 317–324 324 Mobina Abbaszadeh, Masoud Sheidai, Narges Azizi & Fahimeh Koohdar Genetic and morphological variability in medicinal plant Helichrysum oocephalum Boiss. (Asteraceae) in Iran Noormohammadi, Z., Samadi-Molayoosefi H. & Sheidai M. 2012: Intra-specific genetic diversity in O. cuspidate subsp. cuspidata in Hormozgan province. Genetics and Molecular Research 11: 707–716. Peakall, R. & Smouse, P . E. 2006: GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288–295. Podani, J. 2000: Introduction to the Exploration of Multivariate Data [English translation], Leiden, Netherlands. Backhuyes, 455 p. Pritchard, J. K., Step hens, M. & Donnelly, P . 2000: Inference of population structure using multilocus genotype Data. Genetics 155: 945–959. Sabetta, W. Montemurro, C., Perrini, R., Morone Fortunato, A. Blanco, A., 2006: Genetic diversity assessment of Helichrysum italicum (Roth.) Mediterranean germplasm by AFLP markers. Proceedings of the 50th Italian Society of Agricultural Genetics Annual Congress. Ischia, Italy. Setsuko, S., Ishida, K., Ueno, S., Tsu Mura, Y. & Tomaru, N. 2007: Population differentiation and gene flow within a metapopulation of a threatened tree, Magnolia stellata (Magnoliaceae). American Journal of Botany 94: 128–136. Sheidai, M., Seif, E., Nouroozi, M. & Noormohammadi, Z. 2012: Cytogenetic and molecular diversity of Cirsium arvense (Asteraceae) populations in Iran. Journal of Japanese Botany 87: 193–205. Sheidai, M., Ziaee, S., Farahani, F ., Talebi, S. M., Noormohammadi, Z. & Hasheminejad-Ahangarani Farahani, Y.2014: Infra-specific genetic and morphological diversity in Linum album (Linaceae). Biologia 69: 32e39. Sheidai, M., Taban. F ., Talebi, S. M. & Noormohammadi, Z. 2016: Genetic and morphological diversity in Stachys lavandulifolia (Lamiaceae) populations. Biologija 62: 9–24. Slatkin, M. 1993: Isolation by distance in equilibrium and non- equilibrium populations. Evolution 47: 264–279. Smissen, R., Breitwieser, I. &Ward, J. 2006: Genetic diversity in the New Zealand endemic species Helichrysum lanceolatum (Asteraceae: Gnaphalieae). New Zealand Journal of Botany 44: 237–247. Taban, M., Sheidai, M., Noormohammadi, Z., Azizi, N., Ghasemzadeh-Baraki, S. & Koohdar, F . 2015: Population genetic analysis and evidence of inter-specific introgression in Helichrysum Armenium and H. Rubicundum (Asteraceae). GENETIKA 47: 451–468. Tero, N., Aspi, J., Siikamäki, P ., Jäkäläniemi, A. & T uomi, J .2003: Genetic structure and gene flow in a metapopulation of an endangered plant species, Silene tatarica. Molecular ecology 12: 2073–2085. Weising, K., Nybom, H., Wolf, K. & Kahl, G. 2005: DNA Fingerprinting in Plants. Principles, Methods, and Applications. (2nd ed.), Boca Raton, Fl., USA: CRC Press, 444 p.