Ecologica Montenegrina 26: 127-136 (2019) This journal is available online at: www.biotaxa.org/em Infraspecific genetic variation and population structure of Salvia nemorosa L. (Lamiaceae) in Iran SEYED M. TALEBI1, REZA REZAKHANLOU2, ALEX V. MATSYURA*3,4 1 Department of Biology, Faculty of Sciences, Arak University, Arak, Iran E-mail: [email protected] 2 Department of Agriculture, Islamic Azad University, Saveh, Iran. *3 Altai State University, Barnaul, Russian Federation 4 Tomsk State University, Tomsk, Russian Federation, E-mail: [email protected] Received 23 November 2019 │ Accepted by V. Pešić: 30 December 2019 │ Published online 27 December 2019. Abstract Salvia nemorosa is widely distributed in different parts of Iran, while, there is no information about its population genetic structure and genetic diversity. The current information of its potential protection or conservation status in Iran is almost absent and unclear. Our investigation was the first molecular study of this medicinal species. We performed analysis of genetic variability and population structure of 11 populations of S. nemorosa in Iran using ISSR technique. We revealed intra and inter-population genetic diversity in the studied populations. Genetic parameters widely varied among the studied populations and confirmed their high genetic diversity. Moreover, AMOVA test showed significant molecular variation among and within the populations. The arrangement of populations and their individuals in NJ tree, PCA and MDS plots was in agreement with AMOVA results and individuals of five groups were overlapped. The Nm value showed low amount of gene flow among the populations. Based on STRUCTURE analysis and UPGMA tree of genetic distance, six genetic groups were identified among the studied populations, while two populations had significant differences and could be definite as ecotypes. Key words: woodland sage, ISSR technique, molecular study, genetic parameters, genetic groups. Introduction Infraspecific variations provide the material regards the long and short-term evolutionary adaptation to seasonal and other rapid fluctuations in environmental conditions. Diminishing populations triggered the inbreeding, genetic drift and loss of genetic difference that make the organisms vulnerable to ecological changes (Ramel 1998). Myers (1997) has believed that populations’ loss can be more fatal than loss of species. Hughes et al. (1997) estimated that about of 1800 populations per hour get extinct in tropical forests. Several studies (Chen 2000; Ellis & Burke 2007) confirmed that population genetics provides important data on genetic variation, inbreeding, self-pollination versus out-crossing, gene flow, the partitioning of genetic variability intra and inter populations, and on effective size of population. In addition, the results of population genetics investigation are useful in conservation management of important medicinal plants. Ecologica Montenegrina, 26, 2019, 127-136 GENETIC VARIATION AND POPULATION STRUCTURE OF SALVIA NEMOROSA IN IRAN Different investigations (Sheidai et al. 2014; Noormohammadi et al. 2015; Koohdar et al. 2016) have proved that ISSR molecular markers are informative for genetic diversity and population structure studies. For example, ISSR polymorphism has been widely used all over the world to characterize plant genetic variation at the infraspecific level in different species such Hypericum perforatum (Mohammad et al. 2015), Ginkgo biloba (Zhiqiang et al. 2014), Elettaria cardamomum (Anjali et al. 2016), Pistacia lentiscus (Turhan–Serttaş & Özcan 2018), and Capsicum annuum (Nagy et al. 2007). Salvia with over 1000 taxa is one of the largest and important genera of Lamiaceae family (Farimani et al. 2015). These species are traditionally used in various parts of the world, from Mexico to South Africa and from China to Europe. Many species of this genus have economic importance as food, spices and flavors (Bahadori et al. 2016). Salvia nemorosa (syn. S. pseudosylvestris), is commonly known as wood sage and growing in central Europe, Caucasia, Turkey, and Iran (Skala & Wysokinska 2004; Jamzad 2012). Salvia nemorosa is a perennial, herbaceous plant, drought resistant, native of Central and West Asia and grows in large groups. The 4-edged stem is 30-100 cm tall, erect, hairy, with sessile shoots at the axil of the leaves and branched to the tip. The lower lanceolate leaves are petiolate (1-9 cm long), hairy, pointed to the apex, with a cordate base, unequally crenate margins and reticulate venation. The lower leaves are 2.5-11 cm long, 1.5-4.5 cm wide and the middle and upper leaves are smaller. The green leaves are often rough, slightly hairy ventrally and dorsally almost glabrous, with short hairs along the veins. The species forms long inflorescence (13-40 cm), purple bracts are 7-13 cm long and 5-7 mm width. The violet lowers with a bilabiate corolla, are 8-16 cm long (Rechinger 1982; Jamzad 2012). Takeda et al. (1997) reported that leaves of this plant are traditionally used in Turkish traditional medicine for stop bleeding. Moreover, in Bulgarian native medicine, this aromatic plant is used mainly for treatment of hemorrhages, diarrhea, abdominal pain, and furuncles (Daskalova, 2004). S. nemorosa has essential oil which riches in spathulenol, phytol, caryophyllene oxide, 14-Hydroxi-9-epi-(E) caryophyllene and p-Cymene (Mahdiyeh et al. 2018). This species grows in various parts of Iran and comprises several local populations. Our investigation of genetic diversity and structure of selected local populations was performed for the first time in Iran. The results of this research can be used in conservation strategies and breeding management of this aromatic herb. Material and methods Eleven natural populations were selected for this species from different parts of Iran (Table 1). Plant samples were identified according to Flora of Iran (Jamzad 2012). Table 1. Locality address and herbarium numbers of the studied populations of S. nemorosa. No Name Locality address Herbarium Numbers 1 Amir kabir Markazi province, Arak, Amir Kabir new town,1320 m. AUH 50601 2 Mahneshan Zanjan province, Mahneshan, 1675 m. AUH 50602 3 Sangak Markazi province, Saveh, Sangak village, 1950 m. AUH 50603 4 Varcheh Markazi province, Khomein, Varcheh, 1400 m. AUH 50604 5 Rabor Kerman province, Rabor, 1250 m. AUH 50605 6 Polor Mazandaran province, Amol, Polor, 2730 m. AUH 50606 7 Jajroud Tehran province, Jajroud, 1420 m. AUH 50607 8 Shahzand Markazi province, Shahzand, 1700 m. AUH 50608 9 Khalkhal Ardabil, Asalem to Khalkhal road, 1670m. AUH 50609 10 Mashhad Khorasan Razavi province, Mashhad,1700 m. AUH 50610 11 Azna Lorestan province, Azna, 2000 m. AUH 50611 128 TALEBI ET AL. Figure 1. AMOVA test of the studied S. nemorosa populations based on ISSR data. Fresh leaves were used randomly from five plants in each population. We used Cetrimonium bromide (CTAB, Sigma-Aldrich) activated charcoal protocol to extract genomic DNA; in addition, the quality of extracted DNA was examined by running on 0.8% agarose gel (Sigma-Aldrich). We tested 15 ISSR primers for PCR reactions: UBC807, UBC810, UBC811, UBC823, UBC834, UBC849 (University of British Columbia, Canada) and (GA)9A, (GA)9T, (CA)9GT, (AGC)5GG, (AGC)5GT, (CA)7AT, (CA)7GT, (GT)7CA and (GA)9C, while only six of them ( (AGC)5GG, (AGC)5GT, (CA)7AT, (CA)7GT, (GT)7CA and (GA)9C) produced scorable bands. We used a 25 μl volume containing 10 mM Tris-HCl buffer (Sigma- Aldrich) at pH 8, 50 mM KCl( Merck), 0.2 μM of a single primer, 1.5 mM MgCl2 ( Merck), 0.2 mM of each dNTP(Kawsar Biotech Company), 3 U of Taq DNA polymerase (Bioron, Germany) and 20 ng genomic DNA for PCR reactions. The used program for amplifications reactions were: 5 min for initial denaturation step at 94°C, 40 cycles of 1 min at 94°C; 1 min at 52—57°C, 2 min at 72°C and the final extension step of 7–10 min at 72°C. The PCR results were observed by running on 1% agarose gel, and staining with ethidium bromide. We used a 100 bp molecular size ladder in order to estimate fragment size. Molecular Analyses The obtained ISSR bands were coded as binary traits (absence = 0, presence = 1) and applied for the analyses of genetic diversity. We also calculated the number of effective alleles, Shannon information index (I), polymorphism percentage, and Nei’s gene diversity (H) (Freeland et al. 2011). We used Nei’s genetic distance among the studied populations and Neighbor-Net networking for UPGMA tree clustering (Freeland et al. 2011; Huson & Bryant 2006). Analysis of molecular variance (AMOVA) test with 1000 permutations and Nei’s Gst analysis were performed in GenAlex 6.4 and Geno- Dive ver.2, respectively (Peakall & Smouse 2006; Meirmans & Van Tienderen 2004). In addition, we investigated the population’s genetic variations by G’ST (standardized measure of genetic differentiation and D_est (Jost measure of differentiation) (Hedrick 2005; Jost 2008). We also analyzed populations’ genetic structure by Bayesian based model STRUCTURE (Pritchard et al., 2000). The data were scored as dominant markers for STRUCTURE analysis (Falush et al. 2007). The Evanno test was done on STRUCTURE results to determine correct number of K by using delta K value (Evanno et al. 2005). In K-Means clustering, two summary statistics, pseudo-F, and Bayesian Information Criterion, provide the best fit for k (Meirmans 2012). Results Parameters of S. nemorosa population’s genetic diversity determined in 55 plant individuals from 11 geographical populations are presented in Table 2. The highest value of polymorphism percentage (70.97%) was observed in Khalkhal (population no.9); moreover, Varcheh population (population no. 4) showed high value for gene diversity (0.26) and Shannon information index (0.23). Azna population (population no. 11) Ecologica Montenegrina, 26, 2019, 127-136 129 GENETIC VARIATION AND POPULATION STRUCTURE OF SALVIA NEMOROSA IN IRAN had the lowest value for percentage of polymorphism (22.58%) and the lowest value for Shannon information index (0.07) and He (0.06). Table 2.