Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 45-56, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1236 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Ruonan Zheng, Shuhua Zhao, Majid Khayatnezhad, Sayed Afzal Shah (2021) Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers. Cary- ologia 74(2): 45-56. doi: 10.36253/caryo- logia-1236 Received: March 05, 2021 Accepted: April 28, 2021 Published: October 08, 2021 Copyright: © 2021 Ruonan Zheng, Shuhua Zhao, Majid Khayatnezhad, Sayed Afzal Shah. 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. Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers Ruonan Zheng1,*, Shuhua Zhao2, Majid Khayatnezhad3, Sayed Afzal Shah4 1 Zhengzhou Railway Vocational and Technical College,  Zhengzhou,Henan,  450052,  Chi- na 2 Zhengzhou central hospital, Zhengzhou, Henan, 450007, China 3 Department of Environmental Sciences and Engineering, Ardabil Branch, Islamic Azad University, Ardabil, Iran 4 Department of Biological Sciences, National University of Medical Sciences, The Mall, Abid Majeed Road, Rawalpindi, Punjab, 46000, Pakistan *Corresponding Author. E-mail: zhengruonan2021@163.com; majidkhayatnezhad126@ gmail.com Abstract. Salvia has a high degree of environmental compatibility and is widespread around the world, especially in tropical and temperate regions. It is represented by 61 species including 19 endemic species. Salvia species are mostly shrubs or subshrubs, occasionally herbs, typically perennial, sometimes biennial or annual, and often aro- matic. The genus has high medicinal, commercial and horticultural value. It is the larg- est and one of the taxonomically complicated genus of Lamiaceae. To determine the genetic diversity and understand the species, we produced both morphological and molecular data using 145 randomly collected plants representing 30 species from 18 provinces of Iran. A total of 107 reproducible bands were generated by 10 of 25 ran- dom amplified polymorphic DNA (RAPD) primers, with an average of 10.7 bands/ primer and 44% polymorphism. Largest number of effective alleles (Ne), genetic diver- sity (H), and Shannon Index (I) were shown by Salvia reuterana. Our data depicted highest similarity between S. suffruticosa and S. hydrangea and lowest between S. aristata and S. oligphylla. S. limbata showed relatively low level of genetic variation. Finally, the Neighbor Joining (NJ) trees based on RAPD markers data divided the populations into two different clusters, indicating their genetic difference which is dis- cussed in details. Keywords: Gene flow, Genetic Variation, Random Amplified Polymorphic DNA (RAPD), Salvia. INTRODUCTION One of the most important aspect of biological diversity for conservation strategies is the genetic diversity, particularly in rare, and narrow endem- ic species (Mills and Schwartz 2005; Tomasello et al. 2015). Most authors 46 Ruonan Zheng et al. agree that longstanding evolutionary potential of a spe- cies necessitates maintenance of genetic diversity (Falk and Holsinger 1991). Similarly, most geneticists regard population size as a significant factor in preserving genetic diversity (Ellegren and Galtier 2016; Turchetto et al. 2016). In fragmented populations, this is critical as they are more susceptible due to allelic resources’ loss and bigger population differentiation through genetic drift (reduces heterozygosity and subsequent allele fixa- tion) and inbreeding depression (develop homozygosity within populations) (Frankham 2005). Understanding of genetic variability and inter-, and intra-population diversity of rare and endemic species is therefore neces- sary for their conservation and management (e.g. Cires et al. 2012, 2013; Jing, et al. 2021). Salvia L. of family Lamiaceae (Mentheae-Salviinae) is recognized as the largest genus, comprising about 1000 species distributed in Central and South America (500 species), Western Asia (200 species), and Eastern Asia (100 species) (Walker et al. 2004; Will and Claßen- Bockhoff 2017). Iran is considered one of the major regions for diversity of Salvia in Southwest Asia repre- sented by 19 endemic species out of 61 (Jamzad 2012). The name of the genus ‘Salvia’ comes from the Latin name ‘Salvio’ which means to recover or save (Wang et al. 2011). Salvia is one of the groups of herbs most valued for its richness in essential oil and biologically active compounds (Erbano et al. 2015). In industries such as pharmaceuticals, the use of the Salvia has been widely increased since it has pharmacological potentials including anti-inflammatory, and antiplatelet proper- ties (Erbano et al. 2015). Salvia species have been used against different ailments including diabetes, acquired immunodeficiency syndrome (AIDS), liver disease, and Alzheimer’s disease (Sepehry Javan et al. 2012). Members of the genus have economical value in the perfumery industry, cosmetics, spices, and flavoring agents (Wang et al. 2011). Plant genetic diversity is a crucial feature regarding their breeding and domestication. Some researcher have thus attempted to evaluate the variability in various Sal- via species using ISSR and RAPD techniques (Song et al. 2010; Wang et al. 2011; Sepehry Javan et al. 2012; Zhang et al., 2013; Peng et al. 2014; Erbano et al. 2015). They have documented high polymorphism in markers data and reported the utility of these techniques for assessing the genetic diversity in Salvia (Song et al. 2010; Javan et al. 2012). Kharazian (2010) studied the taxonomy and mor- phology of 42 Salvia spinosa L. accessions (Lamiaceae) from Iran. The hair frequency and indumentum of the base and surface of the stem, the shape of the leaf, the leaf margin, and the leaf apex were all linked to the morpho- logical diversity of this species. Cluster analysis revealed that there was variation among the accessions. Hence, its morphological diversity  may be attributed to polymor- phism, hybridization, and new varieties. (Kharazian 2010, Zou et al. 2019; Niu et al. 2021; Sun et al. 2021). ISSR and RAPD molecular techniques were used to assess the genetic relationships among twenty-one ecotypes of eight Salvia species in Iran (Yousefiazar- Khanian et al. 2016). The findings of their marker parameter analysis revealed no significant differences between the two marker systems. ISSR and RAPD mark- ers were shown to have identical efficiency in identifying genetic polymorphisms and a strong ability to distin- guish closely related Salvia ecotypes. For genetic diver- sity and relationship study of nine Salvia species in Iran, Etminan et al., (2018) used inter-simple sequence repeats (ISSR) and start codon targeted (SCoT) markers. Twen- ty-one ISSR and twenty SCoT primers, amplified  350 and 329 loci, respectively,  all of which were polymor- phic. The average polymorphism information quality (ISSR, 0.38; SCoT, 0.40), average band informativeness (ISSR, 16.67; SCoT, 16.45), and resolving power (ISSR, 9.75; SCoT, 12.52) found within Salvia accessions dem- onstrated a high level of genetic diversity. Their find- ings suggested that SCoT markers can be reliably used to assess genetic diversity and relationships among Salvia species. ISSR is a simple and efficient marker system for identification of genetic diversity for plant germplasm collection (Peng et al. 2014). ISSR molecular markers have been used to show polymorphism and distinguish germplasms of Salvia miltiorrhiza Bunge by incorporat- ing phenotypic characters (Zhang et al. 2013). Molecular markers are commonly used in genetic analysis, fingerprinting, linkage mapping, germplasm characterization, and molecular breeding. RAPD analy- sis using PCR along with short arbitrary sequence prim- ers has been reported sensitive to detecting variation at level of individuals (Williams et al. 1990). The benefits of this method are: a) a large number of samples are test- ed easily and efficiently using less quantity of material; b) the DNA amplicons are independent of ontogenetic expression; c) several genomic regions may be sampled with likely infinite numbers of markers (Soniya et al. 2001; Esfandani-Bozchaloyi et al. 2017 a, 2017b, 2017c, 2017d). This study has been carried out to evaluate the genetic diversity and relationships among the Iranian Salvia species based on RAPD data. This is the first step towards using RAPD markers on a broader sampling of Iranian Salvia and aims at answering the follow- ing questions: 1) is there infra- and interspecific genetic diversity among Salvia species? 2) Is genetic distance 47Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers correlated with distribution of these species? 3) What is the populations’ genetic structure? 4) Is there any genet- ic exchange within Salvia species? MATERIALS AND METHODS Plant sampling A total of 145 individuals were collected from eco- geographically different populations representing 30 Sal- via species in East Azerbaijan, Lorestan, Kermanshah, Guilan, Mazandaran, Golestan, Yazd, Esfahan, Tehran, Arak, Hamadan, Kurdistan, Ilam, Bandar Abbas, Ghaz- vin, Khorasan and Ardabil Provinces of Iran during July–August 2017–2019 (Table 1; Figure 1). All of these samples were used during RAPD and morphometric analysis and stored for further use in -20°C. Morphological studies Three to twelve samples from each species were used for morphometric analysis. A total of 22 morphological (9 qualitative, 13 quantitative) characters were examined. The obtained data were standardized (Mean= 0, vari- ance= 1) and used to assess Euclidean distance for clus- tering and ordination analyses (Podani 2000). Morpho- logical characters studied were: basal leaf shape, basal leaf length, basal leaf width, stem leaf length, stem leaf width leaf surface, bract shape, bract length, bract color, pedicel length, calyx length. Table 1. Voucher details of Salvia species in this study from Iran by khayatnezhad. No Sp. Locality Latitude Longitude Altitude (m) Sp1 Salvia aristata Aucher ex Benth. East Azerbaijan, kaleybar, Shojabad 38°52’37” 47°23’92” 1144 Sp2 Salvia eremophila Boiss. Esfahan, Ghameshlou, Sanjab 32°50’03” 51°24’28” 1990 Sp3 Salvia santolinifolia Boiss. Fars, Jahrom 29°20’07” 51°52’08” 1610 Sp4 Salvia tebesana Bunge Khorasan, Tabas 38°52’373 47°23’92” 2234 Sp5 Salvia bracteata Banks & Sol Lorestan, Oshtorankuh, above Tihun village 33°57’12” 47°57’32” 2500 Sp6 Salvia suffruticosa Montb. & Aucher Hamedan, Nahavand 34°52’373 48°23’92” 2200 Sp7 Salvia dracocephaloides Boiss. East Azerbaijan, kaleybar, Cheshme Ali Akbar 38°52’373 47°23’92” 1144 Sp8 Salvia hydrangea DC. ex Benth. Arak, Komayjan, Pass of Chehregan village, the margin road 35°50’03” 51°24’28” 1700 Sp9 Salvia multicaulis Vahl. Mazandaran, Haraz road, Emam Zad-e-Hashem 36°14’14” 51°18’07” 1807 Sp10 Salvia syriaca L. Esfahan, Fereydunshahr 32°36’93” 51°27’90” 2500 Sp11 Salvia viridis L. Guilan, Sangar, Road sid 37°07’02” 49°44’32” 48 Sp12 Salvia mirzayanii Rech. f. & Esfand. Boushehr, Dashtestan 28°57’22” 51°28’31” 430 Sp13 Salvia macrosiphon Boiss. Yazd, Khatam 30°07’24” 53°59’06” 2178 Sp14 S. sharifii Rech. f. & Esfand. Bandar Abbas, Hormozgan 28°57’22” 51°28’31” 288 Sp15 Salvia reuterana Boiss. Hamedan, Alvand 34°46’10” 48°30’00” 1870 Sp16 Salvia palaestina Benth. Kermanshah, Islamabad 35°37’77” 46°20’25” 1888 Sp17 Salvia sclareopsis Bornm. ex Hedge Ilam, Ilam 33°47’60” 46°07’58” 1250 Sp18 Salvia spinose L. Guilan, Lahijan 37°07’02” 49°44’32” 48 Sp19 Salvia compressa Vent. Bandar Abbas, Hormozgan 28°57’22” 51°28’31” 288 Sp20 Salvia sclarea L. Esfahan:, Ghameshlou, Sanjab 32°36’93” 51°27’90” 2500 Sp21 Salvia aethiopis L. Azerbaijan, 78 km from Mianeh to Khalkhl. 37°38’53” 48°36’11” 1500 Sp22 Salvia microstegia Boiss. & Bal. Tehran, Darband 35°36’93” 51°27’90” 1700 Sp23 Salvia xanthocheila Boiss. ex Benth. Ardabil, Khalkhal 37°38’53” 48°36’11” 1958 Sp24 Salvia limbata C. A. Mey. Guilan,Gole rodbar, Road sid 37°09’45” 49°55’39” 15 Sp25 Salvia chloroleuca Rech. f. & Aell. Golestan, Ramian 37°09’45” 55°55’39” 1320 Sp26 Salvia virgate Jacq. Golestan, Ramian 37°09’45” 55°55’39” 1320 Sp27 Salvia nemorosa L. Mazandaran, Chalos 36°14’14” 51°18’07” 1807 Sp28 Salvia urmiensis Bunge Kurdistan, Sanandaj 35°20’53” 53°30’20” 2344 Sp29 Salvia oligphylla Aucher ex Benth. Ghazvin to Hamedan just after Avaj 35°36’93” 51°27’90” 2100 Sp30 Salvia verticillata L. Mazandaran Jadeh Chalous 36°14’14” 51°18’07” 1807 48 Ruonan Zheng et al. DNA Extraction and RAPD Assay In each of the populations studied, fresh leaves from one to twelve plants were used randomly. Leaves were dried with silica gel prior to DNA extraction (Esfandani- Bozchaloyi et al. 2019). By running on 0.8 percent aga- rose gel, the quality of extracted DNA was examined. A total of 25 Operon Technology Decamer RAPD Primers (Alameda, Canada) belonging to OPA, OPB, OPC, OPD sets were used. Among them, ten primers were selected with simple, enlarged and rich bands of polymorphism (Table 2). PCR reactions were performed in a 25μl vol- ume mixture containing the following component: Tris-HCl buffer (10 mM) at pH 8; KCl (50 mM); MgCl2 (1.5 mM); dNTPs (0.2 mM); primer (0.2 μM); genom- ic DNA (20 ng) and of Taq DNA polymerase (3 U). In Techne thermocycler (Germany), the amplification reac- tions were carried out with the following PCR settings: 5 min initial denaturation at 94 °C; 40 cycles of 1 min at 94 °C; 1 min at 52-57 °C and 2 min at 72 °C. The reac- tion was completed by 7-10 min extension at 72 °C. The PCR  amplified  products were detected by  running  on 1% agarose gel, preceded by staining with ethidium bro- mide. The size of fragments was measured using a ladder with a molecular size of 100 bp (Fermentas, Germany). DATA ANALYSES Morphological studies Morphological characters (Mean = 0, Variance = 1) were first standardized and used to determine the Euclidean distance between taxa pairs (Podani 2000). The ordination methods of UPGMA (Unweighted paired group using average) were used for clustering the sam- ples (Podani 2000). In order to demonstrate morphologi- Figure 1. Map of Iran showing sampling localities for Salvia. sp1= Salvia aristata; sp2= S. eremophila; sp3= S. santolinifolia; sp4= S. tebesana; sp5= S. bracteata ; sp 6= S. suffruticosa; sp7= S. draco- cephaloides; sp8= S. hydrangea; sp9= S. multicaulis; sp10: S. syriaca; sp11: S. viridis; sp12= S. mirzayanii; sp13= S. macrosiphon; sp14= S. sharifii; sp15= S. reuterana; sp16= S. palaestina; sp17= S. sclare- opsis; sp18= S. spinose; sp19= S. compressa; sp20= S. sclarea; sp21= S. aethiopis; sp22= S. microstegia; sp23= S. xanthocheila; sp24= S. limbata; sp25= S. chloroleuca; sp26= S. virgate; sp27= S. nemorosa; sp28= S. urmiensis; sp29= S. oligphylla; sp30= S. verticillata. Table 2. RAPD primers used for this study and the extent of polymorphism. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI OPA-05 5’-AGGGGTCTTG-3’ 12 12 100.00% 0.46 3.86 10.55 4.45 OPA-06 5’-GGTCCCTGAC-3’ 9 8 84.99% 0.33 4.51 8.43 2.85 OPB-01 5’-GTTTCGCTCC-3’ 9 9 100.00% 0.44 3.34 10.55 4.44 OPB-02 5’-TGATCCCTGG-3’ 10 10 100.00% 0.47 3.18 9.56 2.65 OPC-04 5’-CCGCATCTAC-3’ 11 11 100.00% 0.35 5.23 8.23 5.47 OPD-02 5’-GGACCCAACC-3’ 14 13 93.74% 0.47 4.66 8.56 3.67 OPD-03 5’-GTCGCCGTCA-3’ 13 12 92.31% 0.44 4.21 6.60 3.55 OPD-05 5’ -TGAGCGGACA-3’ 13 13 100.00% 0.47 4.32 9.55 2.45 OPD-08 5’-GTGTGCCCCA-3’ 11 9 82.89% 0.33 6.56 9.34 2.11 OPD-11 5’-AGCGCCATTG-3’ 10 10 100.00% 0.49 4.25 11.11 3.87 Mean 11.2 10.7 93.68% 0.44 4.6 9.3 3.5 Total 112 107 Note: TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: poly- morphism index, EMR, effective multiplex ratio; MI, marker index; PIC, polymorphism information content for each of CBDP primers. 49Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers cal variation between populations, ANOVA (Analysis of variance) was performed, while PCA (Principal Com- ponents Analysis) bi-plot was employed to identify the most variable characters (Podani 2000). PAST software version 2.17 (Hammer et al. 2012) was used for multivar- iate statistical analyses of morphological data. Molecular analyses The obtained RAPD bands were coded as binary characters (absence = 0, presence = 1) and used for the study of genetic diversity (Powell et al. 1996; Heikrujam et al. 2015). For each primer, the number of polymorphic bands (NPB) was determined followed by the effective multiplex ratio (EMR). Other parameters such as Nei’s gene diversity (H), Shannon information index (I), num- ber of effective alleles, and percentage of polymorphism (P% = number of polymorphic loci/number of total loci) were also determined (Weising et al, 2005; Freeland et al. 2011). The formula for calculation of Shannon’s index was: H’ = -Σpiln pi. Rp is defined per primer as: Rp = ∑ Ib, where “Ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the propor- tion of each genotype containing the band. The percent- age of polymorphic loci, UHe, H’ and PCA were deter- mined by GenAlEx 6.4 software (Peakall and Smouse 2006). For generating Neighbor Joining (NJ) clusters and Neighbor-Net networking, Nei’s genetic distance between populations was employed (Freeland et al. 2011; Huson and Bryant 2006). The Mantel test determined the correlation between the geographical and genetic distances of the populations (Podani 2000). These tests were performed in PAST ver. 2.17 (Hammer et al. 2012) and DARwin software ver. 5 (2012). As implemented in GenAlex 6.4 (Peakall & Smouse, 2006), the AMOVA (Analysis of Molecular Variance) test (with 1000 per- mutations) was used to evaluate population genetic dif- ferences. Gene flow was estimated by calculating Nm, an estimate of gene flow from Gst in PopGene ver. 1.32 (1997) as: Nm = 0.5 (1 - Gst)/Gst. This method consid- ers the equal amount of gene flow among all populations (Yeh et al. 1999). RESULTS Species identity and relationships – Morphometry ANOVA test showed substantial differences (P <0.01) between the studied species in quantitative mor- phological characteristics. PCA analysis was conducted to determine the most variable characters among the taxa analysed. It showed that over 77 % of the over- all variance was composed of the first three variables. Characters such as seed shape, calyx shape, calyx length, bract length and basal leaf shape have shown the highest association (>0.7) in the first PCA axis with 55 per cent of total variance. Characters affecting PCA axis 2 and 3 respectively were seed colour, leaf surface, corolla length, filament length, nut width, basal leaf length. Different ordination and clustering methods generated similar results. Therefore, PCoA plot of morphological charac- ters are presented here (Fig. 2). Samples of each species were separately grouped. This finding indicates that the studied species were divided into different classes by both quantitative and qualitative morphological features. Among the studies sample we did not find any  interme- diate forms. Species identification and genetic diversity Ten RAPD primers were screened in order to study genetic relationships within Salvia. All primers gener- ated reproducible polymorphic bands in 30 Salvia spe- cies. Figure 3 shows an image of the amplification of the RAPD created by the OPD-03 primer. In total, 107 amplified polymorphic bands were produced across 30 species. The size range of the amplified fragments was 150 to 3000 bp. The highest and lowest numbers of poly- morphic bands were 13 for OPD-02, OPD-05 and 8 for OPA-06, with an average of 10.7 polymorphic bands per primer. The PIC of the 10 RAPD primers ranged from 0.32 (OPD-08) to 0.48 (OPD-011) with an average of 0.44 per primer. MI of the primers ranged from 2.11 (OPD- 08) to 5.47 (OPC-04) with an average of 3.5 per primer. EMR of the RAPD primers ranged from 6.60 (OPD-03) to 11.11 (OPD-011) with an average of 9.3 per primer (Table 2). The primers with the high EMR values were considered to be more informative in distinguishing the genotypes. Genetic parameters were determined for all the 30 Salvia species amplified with RAPD primers (Table 3). The range of Unbiased expected heterozygosity (H) was 0.099 (Salvia limbata) to 0.31 (S. reuterana) (mean: 0.18). A similar trend was depicted by Shannon’s information index (I), with the highest value of 0.39 found in S. reu- terana and the lowest value of 0.13 found in S. limbata (mean: 0.27). The observed number of alleles (Na) varied between 0.201 in S. nemorosa and 1.28 in S. eremophila. The range of effective number of alleles (Ne) was 1.00 (S. nemorosa) to 1.670 (S. santolinifolia). AMOVA test revealed substantial genetic varia- tion (P = 0.01). It showed that 81% of total variation was interspecific and 19% was intra-specific (Table 4). In 50 Ruonan Zheng et al. addition, genetic differentiation of was demonstrated by significant Nei’s GST (0.29, P = 0.001) and D_est values (0.137, P = 0.01). Compared to intra-species, these results revealed a greater distribution of interspecific genetic diversity. Two main clusters were produced in the NJ tree (Fig. 4). The first main cluster comprised two sub-clusters: S. xanthocheila and S. verticillata were separated from the rest of the species and join the others with a great distance and comprised the first sub-cluster. The sec- ond sub-cluster comprised of S. limbata, S. aethiopis, S. sclarea and S. virgata. The second main cluster also com- prised two sub-clusters: three species including S. sclare- opsis, S. macrosiphon and S. sharifii were placed close Figure 2. PCoA plots of morphological characters revealing species delimitation in the Salvia. sp1= Salvia aristata; sp2= S. eremophila; sp3= S. santolinifolia; sp4= S. tebesana; sp5= S. bracteata ; sp 6= S. suffruticosa; sp7= S. dracocephaloides; sp8= S. hydrangea; sp9= S. multicaulis; sp10: S. syriaca; sp11: S. viridis; sp12= S. mirzayanii; sp13= S. macrosiphon; sp14= S. sharifii; sp15= S. reuterana; sp16= S. palaestina; sp17= S. sclareopsis; sp18= S. spinose; sp19= S. compressa; sp20= S. sclarea; sp21= S. aethiopis; sp22= S. microstegia; sp23= S. xanthocheila; sp24= S. limbata; sp25= S. chloroleuca; sp26= S. virgate; sp27= S. nemorosa; sp28= S. urmiensis; sp29= S. oligphylla; sp30= S. verticillata. Figure 3. Electrophoresis gel of studied ecotypes from DNA fragments produced by OPD-03. 1= Salvia aristata; 2= S. eremophila; 3= S. santolinifolia; 4= S. tebesana; 5= S. bracteata ; 6= S. suffruticosa; 7= S. dracocephaloides; 8= S. hydrangea; 9= S. multicaulis; 10: S. syriaca; 11: S. viridis; 12= S. mirzayanii; 13= S. macrosiphon; 14= S. sharifii; 15= S. reuterana; 16= S. palaestina; 17= S. sclareopsis; 18= S. spinose; 19= S. compressa; 20= S. sclarea; 21= S. aethiopis; 22= S. microstegia; 23= S. xanthocheila; 24= S. limbata; 25= S. chloroleuca; 26= S. virgate; 27= S. nemorosa; 28= S. urmiensis; 29= S. oligphylla; 30= S. verticillata;L = Ladder 100 bp, Arrows are representative of polymorphic bands. 51Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers to each other, while close genetic affinity between other species. Relationships obtained from RAPD data usu- ally agree well with the relationship of species obtained from morphological data. This is in accordance with the parameters of AMOVA and genetic diversity previ- ously reported. Salvia species are genetically well dis- tinguished from each other. The species are well dis- tinguished from each other genetically. These findings show that RAPD molecular markers can be used in the taxonomy of Salvia. The Nm analysis by Popgene soft- ware also produced mean Nm= 0.288, that is deemed a low value of gene flow. A strong association (r = 0.16, p=0.0002) between genetic- and geographical distance was demonstrated by Mantel test with 5000 permuta- tions. It indicates that  isolation by distance  (IBD) has occurred among these species. The results of Nei’s genetic identity and the genetic distance (Table 5) show the highest genetic similarity Table 3. Genetic diversity parameters in the studied Salvia species. SP N Na Ne I He UHe %P S. aristata 8.000 0.333 1.016 0.19 0.12 0.22 48.23% S. eremophila 12.000 1.287 1.233 0.271 0.184 0.192 51.91% S. santolinifolia 5.000 0.358 1.670 0.18 0.20 0.29 43.50% S. tebesana 6.000 0.299 1.029 0.231 0.18 0.23 44.38% S. bracteata 5.000 0.462 1.095 0.288 0.25 0.22 62.05% S. suffruticosa 8.000 0.399 1.167 0.259 0.234 0.133 32.88% S. dracocephaloides 8.000 0.477 1.187 0.256 0.233 0.148 31.26% S. hydrangea 8.000 0.313 1.026 0.144 0.13 0.26 49.23% S. multicaulis 12.000 1.144 1.322 0.28 0.284 0.192 50.91% S. syriaca 5.000 0.358 1.117 0.28 0.15 0.12 44.30% S. viridis 6.000 0.458 1.039 0.28 0.18 0.23 49.38% S. mirzayanii 5.000 0.455 1.077 0.277 0.24 0.22 55.05% S. macrosiphon 8.000 0.499 1.067 0.14 0.13 0.14 49.26% S. sharifii 9.000 0.261 1.014 0.142 0.23 0.23 43.15% S. reuterana 6.000 0.555 1.021 0.39 0.35 0.31 68.53% S. palaestina 4.000 0.344 1.042 0.20 0.23 0.20 57.53% S. sclareopsis 5.000 0.369 1.011 0.15 0.18 0.12 42.15% S. spinose 9.000 0.261 1.014 0.142 0.33 0.23 43.15% S. compressa 6.000 0.555 1.021 0.29 0.25 0.28 43.53% S. sclarea 10.000 0.431 1.088 0.33 0.22 0.13 57.53% S. aethiopis 3.000 0.255 1.021 0.15 0.18 0.12 42.15% S. microstegia 3.000 0.288 1.024 0.23 0.15 0.17 44.30% S. xanthocheila 9.000 0.352 1.083 0.23 0.22 0.14 45.05% S. limbata 5.000 0.369 1.011 0.13 0.11 0.099 29.15% S. chloroleuca 6.000 0.244 1.032 0.26 0.23 0.18 55.53% S. virgata 4.000 0.314 1.044 0.16 0.18 0.23 43.38% S. nemorosa 8.000 0.201 1.00 0.33 0.17 0.12 42.23% S. urmiensis 5.000 0.341 1.058 0.24 0.27 0.20 53.75% S. oligphylla 3.000 0.567 1.062 0.24 0.224 0.113 44.73% S. verticillata 5.000 0.336 1.034 0.23 0.25 0.19 51.83% Abbreviations: N: number of samples; Na: number of different alleles; Ne: number of effective alleles, I: Shannon’s information index, He: gene diversity, UHe: unbiased gene diversity, P%: percentage of polymorphism, populations. Table 4. Analysis of molecular variance (AMOVA) of the studied species. Source df SS MS Est. Var. % ΦPT Among Pops 28 1601.364 79.789 12.154 81% 81% Within Pops 130 234.443 4.777 2.888 19% Total 158 1955.777 14.060 100% df: degree of freedom; SS: sum of squared observations; MS: mean of squared observations; EV: estimated variance; ΦPT: proportion of the total genetic variance among individuals within an accession, (P < 0.001). 52 Ruonan Zheng et al. Ta bl e 5. Th e m at ri x of N ei g en et ic s im ila ri ty ( G s) e st im at es u si ng R A PD m ol ec ul ar m ar ke rs a m on g 30 S al vi a sp ec ie s. s p1 = Sa lv ia a ri st at a; s p2 = S. e re m op hi la ; s p3 = S. s an to lin ifo lia ; sp 4= S . t eb es an a; s p5 = S. b ra ct ea ta ; sp 6 = S. s uff ru tic os a; s p7 = S. d ra co ce ph al oi de s; sp 8= S . h yd ra ng ea ; s p9 = S. m ul tic au lis ; s p1 0: S . s yr ia ca ; s p1 1: S . v ir id is ; s p1 2= S . m ir za ya ni i; sp 13 = S. m ac ro si ph on ; s p1 4= S . s ha ri fii ; s p1 5= S . r eu te ra na ; s p1 6= S . p al ae st in a; s p1 7= S . s cl ar eo ps is ; s p1 8= S . s pi no se ; s p1 9= S . c om pr es sa ; s p2 0= S . s cl ar ea ; s p2 1= S . a et hi op is ; s p2 2= S . m ic ro st e- gi a; s p2 3= S . x an th oc he ila ; s p2 4= S . l im ba ta ; s p2 5= S . c hl or ol eu ca ; s p2 6= S . v ir ga te ; s p2 7= S . n em or os a; s p2 8= S . u rm ie ns is ; s p2 9= S . o lig ph yl la ; s p3 0= S . v er tic ill at a. sp 1 sp 2 sp 3 sp 4 sp 5 sp 6 sp 7 sp 8 sp 9 sp 10 sp 11 sp 12 sp 13 sp 14 sp 15 sp 16 sp 17 sp 18 sp 19 sp 20 sp 21 sp 22 sp 23 sp 24 sp 25 sp 26 sp 27 sp 28 sp 29 sp 30 sp 1 1. 00 0 sp 2 0. 70 8 1. 00 0 sp 3 0. 66 7 0. 71 2 1. 00 0 sp 4 0. 66 6 0. 73 7 0. 84 2 1. 00 0 sp 5 0. 64 9 0. 80 7 0. 78 6 0. 75 4 1. 00 0 sp 6 0. 61 7 0. 78 2 0. 84 6 0. 92 8 0. 79 3 1. 00 0 sp 7 0. 59 9 0. 70 2 0. 80 8 0. 87 5 0. 83 6 0. 86 2 1. 00 0 sp 8 0. 73 5 0. 70 6 0. 61 8 0. 70 8 0. 82 3 0. 93 6 0. 76 4 1. 00 0 sp 9 0. 75 0 0. 79 7 0. 81 6 0. 88 4 0. 78 5 0. 67 6 0. 69 9 0. 75 6 1. 00 0 sp 10 0. 77 9 0. 79 8 0. 75 2 0. 75 4 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 1. 00 0 sp 11 0. 71 9 0. 92 0 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 63 5 0. 81 6 0. 88 4 0. 72 1 1. 00 0 sp 12 0. 81 2 0. 77 4 0. 87 6 0. 72 2 0. 63 5 0. 81 6 0. 63 2 0. 75 2 0. 75 4 0. 63 5 0. 83 9 1. 00 0 sp 13 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 66 7 0. 71 2 0. 77 9 0. 75 0 0. 79 9 0. 64 2 1. 00 0 sp 14 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 66 6 0. 73 7 0. 67 5 0. 67 5 0. 72 7 0. 72 8 0. 68 4 1. 00 0 sp 15 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 73 7 0. 64 9 0. 80 7 0. 69 1 0. 68 1 0. 74 6 0. 79 6 0. 67 6 0. 72 2 1. 00 0 sp 16 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 61 7 0. 78 2 0. 73 4 0. 73 3 0. 80 0 0. 70 9 0. 77 0 0. 75 4 0. 77 0 1. 00 0 sp 17 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 59 9 0. 70 2 0. 74 4 0. 74 0 0. 78 5 0. 67 6 0. 69 9 0. 75 6 0. 73 5 0. 77 8 1. 00 0 sp 18 0. 73 0 0. 61 4 0. 84 3 0. 75 9 0. 59 9 0. 70 2 0. 73 5 0. 70 6 0. 71 9 0. 95 3 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 75 6 1. 00 0 sp 19 0. 70 1 0. 80 0 0. 75 1 0. 77 4 0. 73 2 0. 79 0 0. 75 0 0. 79 7 0. 81 2 0. 77 4 0. 99 0 0. 72 2 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 75 0 0. 79 9 1. 00 0 sp 20 0. 76 4 0. 72 3 0. 68 3 0. 65 9 0. 67 9 0. 75 4 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 67 5 0. 72 7 0. 75 5 1. 00 0 sp 21 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 84 3 0. 74 1 0. 69 0 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 68 1 0. 74 6 0. 68 4 0. 71 1 1. 00 0 sp 22 0. 84 3 0. 75 9 0. 59 9 0. 70 2 0. 74 4 0. 77 8 0. 77 4 0. 84 3 0. 77 8 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 73 7 0. 67 5 0. 80 8 0. 73 3 0. 80 0 0. 84 8 0. 77 4 0. 71 2 1. 00 0 sp 23 0. 82 5 0. 72 2 0. 64 1 0. 81 4 0. 73 5 0. 70 6 0. 75 0 0. 79 9 0. 71 0 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 74 0 0. 78 5 0. 84 6 0. 75 7 0. 70 7 0. 87 4 1. 00 0 sp 24 0. 86 0 0. 75 9 0. 73 2 0. 79 0 0. 75 0 0. 73 5 0. 70 6 0. 71 9 0. 75 4 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 79 9 0. 95 3 0. 74 1 0. 69 0 0. 65 7 0. 64 5 0. 72 6 0. 73 5 1. 00 0 sp 25 0. 72 6 0. 64 7 0. 67 9 0. 75 4 0. 77 9 0. 75 0 0. 79 7 0. 81 2 0. 77 4 0. 99 0 0. 72 2 0. 63 5 0. 81 6 0. 88 4 0. 81 2 0. 77 8 0. 77 4 0. 77 70 0. 77 8 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 75 7 1. 00 0 sp 26 0. 85 8 0. 70 3 0. 69 5 0. 68 1 0. 68 9 0. 77 9 0. 79 8 0. 83 4 0. 75 0 0. 79 9 0. 75 5 0. 63 2 0. 75 2 0. 75 4 0. 70 3 0. 70 6 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 69 0 0. 79 7 1. 00 0 sp 27 0. 83 6 0. 68 1 0. 68 6 0. 75 6 0. 70 1 0. 95 3 0. 74 1 0. 69 0 0. 69 1 0. 74 4 0. 63 6 0. 66 7 0. 71 2 0. 77 9 0. 79 8 0. 79 7 0. 69 1 0. 74 4 0. 82 9 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 67 3 0. 75 5 0. 76 8 1. 00 0 sp 28 0. 69 1 0. 74 4 0. 82 9 0. 74 0 0. 78 5 0. 62 4 0. 99 0 0. 77 8 0. 68 8 0. 75 7 0. 70 3 0. 66 6 0. 95 3 0. 74 1 0. 75 8 0. 74 6 0. 75 3 0. 79 5 0. 79 9 0. 79 9 0. 95 3 0. 74 1 0. 69 0 0. 65 7 0. 64 5 0. 77 9 0. 79 8 1. 00 0 sp 29 0. 53 8 0. 82 6 0. 78 6 0. 77 2 0. 68 6 0. 75 0 0. 79 9 0. 71 0 0. 73 3 0. 80 0 0. 68 1 0. 64 9 0. 80 7 0. 69 1 0. 66 5 0. 82 5 0. 73 3 0. 80 0 0. 73 0 0. 61 4 0. 84 3 0. 75 0 0. 79 9 0. 71 0 0. 68 8 0. 75 7 0. 70 3 0. 72 3 1. 00 0 sp 30 0. 72 1 0. 79 4 0. 75 4 0. 71 7 0. 79 5 0. 69 1 0. 74 4 0. 82 9 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 78 2 0. 73 4 0. 79 9 0. 67 6 0. 74 0 0. 78 5 0. 77 0 0. 64 1 0. 82 5 0. 72 2 0. 81 6 0. 74 0 0. 78 5 0. 62 4 0. 61 7 0. 65 6 0. 76 5 1. 00 0 53Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers (0.93) between S. suffruticosa and S. Hydrangea. Lowest of genetic similarity was shown between S. aristata and S. oligphylla (0.53). Lower Nm value (0.288) is an indi- cator of limited gene flow or ancestrally shared alleles between different species and indicating high genetic differentiation among and within Salvia species. DISCUSSION Genetic diversity is a fundamental element of bio- diversity and its conservation is indispensable for long- term survival of any species in unstable environments (Mills and Schwartz 2005; Tomasello et al. 2015). Genet- ic diversity is non-randomly distributed among different populations and is influenced by numerous factors such as geography, dispersal mechanisms, breeding systems, life span etc. Changes in environment often lead to vari- ation in genetic diversity levels among populations, and under adverse circumstances, populations with little variability are generally deemed less adapted (Falk and Holsinger 1991; Olivieri et al. 2016). Most authors recog- nize that genetic diversity is fundamental to preserving the long-term evolutionary potential of a species (Falk and Holsinger 1991). Experimental and field research has shown that habitat fragmentation and population decline have reduced the effective population size in the last decade. Similarly, majority of geneticists regard pop- ulation size as a significant factor in preserving genetic variation (Turchetto et al. 2016). In fragmented popula- tions, this is very important because it is more vulner- able because of allelic richness  loss and increased pop- ulation differentiation via genetic drift and inbreeding depression (Frankham 2005). Information of inter- and intra-population genetic diversity is therefore important for their conservation and management (e.g. Esfandani- Bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). We used morphological and molecular R APD molecular data to test species relationships in Salvia in the current analysis. Morphological studies showed that both quantitative (the ANOVA test result) and qualita- tive characters are well distinguished from each other (The PCA plot result). Furthermore, PCA analysis sug- gests that morphological characters such as bract length, stipule length, bract shape, calyx shape, petal shape, stem-leaf length and width, petal length and width may be used in the delimitation of species groups. This mor- phological differentiation is attributed to quantitative and qualitative characters. Genetic structure and gene flow A primer’s PIC and MI characteristics assist in assessing its usefulness in the study of genetic diversity. Sivaprakash et al. (2004) asserted that the ability to over- come genetic diversity by a marker technique could be Figure 4. NJ tree of RAPD data revealing species delimitation in the Salvia. 54 Ruonan Zheng et al. more explicitly linked to the degree of polymorphism. In general, the PIC value 0 to 0.25 suggests a very low genetic diversity among genotypes, a mid-level of genet- ic diversity (Tams et al. 2005). In this study, the RAPD primers’ PIC values ranged from 0.33 to 0.49, with a mean value of 0.44, indicating a moderate level ability of RAPD primers in determining genetic diversity. Some- what similar but low PIC values have been reported for ISSR and RAPD in Salvia species (Yousefiazar-Khanian et al. 2016); RAPD and AFLP in African plantain (Ude et al. 2003), AFLP in wheat (Bohn et al. 1999) and SCoT markers (Etminan et al. 2018; Pour-Aboughadareh et al. 2017, 2018). In Heikrujam et al. (2015), CBDP mark- ers were shown to be more efficient than SCoT regard- ing the average PIC which was higher. In our analysis, the RAPD markers reflect success in estimating genetic diversity of Salvia species regarding average percentage polymorphism (93.68%), average PIC value of RAPD markers (0.44), average MI (3.5) and average EMR of RAPD markers (9.3), which were higher than other reported markers on Salvia (Wang et al. 2009; Song et al. 2010; Yousefiazar-Khanian et al. 2016; Etminan et al. 2018; Souframanien and Gopalakrishna 2004). Gene flow is inversely correlated with the gene differentia- tion but is very significant for population evolution, and occurs through pollen grains and seeds between popu- lations (Song et al. 2010). The observed gene flow (Nm) between Salvia species was 0.288 in the current study, indicating low genetic differentiation. Generally, the pollinators of Old World Salvia are insects (Claßen-Bockhoff et al. 2004). At the lower ele- vations, bees and at the higher altitudes insects such as flies are the major pollinators of bi-labiate flowers such as Salvia (Pellissier et al. 2010). According to Moein et al., (2019) SRAP marker’s genetic structure revealed that despite the existence of limited gene flow, two separate ecotypes were pro- duced which may be the result of reproductive isolation triggered by altitudinal gradient and dissimilar niches through parapatric speciation (Que et al. 2014). In con- clusion, the findings of this study showed that the prim- ers derived from RAPD were more efficient than the other molecular markers in assessing the genetic diversi- ty of Salvia in Iran. In addition, the Salvia species in the dendrogram and PCoA were clearly distinguished from each other, suggesting the greater efficiency of the RAPD technique in the identification of the genus. ACKNOWLEDGMENT The authors thank anonymous reviewers for valu- able comments on an earlier draft. 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