Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 57-65, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-680 Citation: S. Mehri, H. Shirafkanajir- lou, I. Kolbadi (2020) Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL.. Caryologia 73(1): 57-65. doi: 10.13128/caryolo- gia-680 Received: July, 2019 Accepted: October, 2019 Published: May 8, 2020 Copyright: © 2020 S. Mehri, H. Shi- rafkanajirlou, I. Kolbadi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. Shahram Mehri*, Hassan Shirafkanajirlou, Iman Kolbadi Department of Agronomy and Plant Breeding, ParsAbad Moghan Branch, Islamic Azad University, ParsAbad Moghan, Iran *Corresponding author. E-mail: sh.mehri2000@gmail.com Abstract. Stellaria media L. VILL., is known under the name of chickweed, it is an annu- al plant in the family Caryophyllaceae. Stellaria media is distributed in the all regions of Iran and has been introduced to many habitats of the world. S. pallida is very similar to S. media. This plant is considered to be as a herbal remedy and is used in folk medi- cine. Stellaria media is edible and nutritious. In the present study, we used morphologi- cal and ISSR data for this species. For this, 43 morphological characteristics, including 16 qualitative and 26 quantitative. AMOVA and Gst analyses showed that the populations of this species are genetically differentiated. Nm analysis revealed very low value of genetic diversity among the studied population and mantel test indicated isolation by distance occurred among them. The present study showed that the studied populations of S. media are differentiated in morphological characteristics and genetic content. In general, species relationships obtained from morphological and molecular data were largely congruent. Keywords. Genetic diversity, ISSR, Morphology, Species relationship, Stellaria media. INTRODUCTION The family Caryophyllaceae comprised about 81 genera and 2600 species (Bittrich 1993; Ullah et al. 2019a). Stellaria L. (Caryophyllaceae, Alsinoideae) includes both annual and perennial herbaceous plants that are widely dis- tributed in the temperate zones of Europe and Asia (Lu and Rabeler 2001; Keshavarzi and Esfandani –Bozchaloyi, 2014a, 2014b; Ullah et al. 2019b, 2019c) and about 120 species with worldwide distribution, mainly in the north temperate zone (Morton 2005; Ullah et al., 2018a, 2018b). In Flora Iranica this genus has 9 species and divided into 2 sections: sect. Pseudalsine Boiss. consist of one species S. alsinoides Boiss. & Buhse and sect. Stellaria with six species: S. holostea L., S. persica Boiss., S. gramin- ea L., S. nemorum L., S. media (L.) Vill., S. pallida (Dumort.) Pire (Rechinger 1988). Main center of diversification for Stellaria is Eurasia, with a center of distribution in the mountains of E. central Asia. Some species are also cos- mopolitan (Bittrich 1993; Ullah et al. 2018c). 58 Shahram Mehri, Hassan Shirafkanajirlou, Iman Kolbadi There are limited chromosome records for Stellaria in the world. Basic Chromosome numbers of x=10, 11, 12 and 13 have been reported for the genus (Federov 1969; Moore 1973; Goldblatt 1981). Stellaria media, chickweed, are annual and with slender stems, they have hairs on one side of the stem. The leaves are linear or oval, smooth or minutely, 13 to 17 × 1.5 to 7 mm. Flowers are hermaph- rodite and petals are white with 5 deeply. Sepals promi- nently 4 to 6-nerved, 4 to 7 mm. Stigmas are 3 and the stamens are 3. Stellaria media common in waste places, open areas, lawns, meadows, and widely distributed to temperate regions of Europe, Asia and Northern America. Stellaria media is edible and nutritious and has a history of herbal use and medicinal properties. This species has been used as to soothe severe itchiness even where all other remedies have failed (Slavokhotova et al. 2011). it is considered for rheumatic pains, skin diseases, and period pain as well as for bronchitis and arthritis (Slavokhotova et al. 2011). Stellaria media possess signifi- cant chemicals known as saponins, which can be cause saponin poisoning in cattle (Haragan 1991). There are many studies which are on taxonomy, pollen morpholog y, phylogeny, seed micromorphol- ogy, anatomy, trichome and cytology of stellaria species (Esfandani-Bozchaloyi and Keshavarzi 2014; Keshavarzi and Esfandani-Bozchaloyi 2014 a, b; Ullah et al. 2018a, 2018b, 2018c). However, genetic diversity of stellaria spe- cies have been reported in a few studies (Verkleij et al. 1980; Chinnappa and Morton 1984), also outcrossing or inbreeding, genetic structure, genetic variability within/ between populations and ecological adaptation on Stel- laria of Iran have not been investigated yet. According to Ellis and Burke (2007) genetic diversi- ty are essential in the adaptability and survival of popu- lation, because it is as a way for adapt to changing envi- ronments in populations. The adapt of the population to the changing environment will depend on the presence of the genetic diversity. Large populations have higher genetic diversity due to more to maintain genetic materi- al and small populations have the loss of diversity which is called genetic drift. Mating or inbreeding between individuals with similar genetic occur in small popula- tion sizes, thus decreasing genetic diversity and finally we have more common alleles. Hence, the used of markers will depend on the type of the species. We have been used DNA marker based tech- niques such as Inter-Simple Sequence Repeats (ISSRs), due to easy, highly reproducible, stable and useful in species delimitation, gene tagging, gene flow, breeding programs and evolutionary biology (Ellis and Burke 2007; Esfandani- Bozchaloyi et al. 2018a, 2018b, 2018c). Therefore, we stud- ied morphological and molecular study of 11 geographical populations of S. media for the first time in Iran. MATERIALS AND METHODS Morphological studies 85 plant sample were selected from eleven popula- tions located in three provinces of Iran. Identification of species Stellaria media were based on the descriptions provided by Flora Iranica (Rechinger 1988). The sam- pling sites and herbarium number are provided in Table 1, Figure 1. Vouchers were deposited at the herbarium of Islamic Azad University, Science and Research Branch, Tehran, Iran (IAUH). DNA extraction Fresh leaves of 85 individuals following a modified CTAB protocol. The quality was checked on a 1 % agarose Table 1. Location addresses and ecological characters of the Stellaria media Population Locality Latitude Longitude Altitude (m) Voucher no. 1 Guilan, Road to Sangar 37°06’ 57” 49°11’06” 47 IAUH 201600 2 Guilan, Bandar Anzali, Pine artificial woodland 37°27’34” 49°42’40” -25 IAUH 201701 3 Guilan, Loleman 37°28’59” 49°33’45” -29 IAUH 201702 4 Guilan, Siahkal, Sangar 37°09’08” 49°55’02” 27 IAUH 201603 5 Guilan, Gole rodbar river 37°10’05” 49°56’38” 15 IAUH 201604 6 Guilan , Sheytan kouh hill side 37°12’04” 50°03’12” 9 IAUH 201605 7 Guilan , Lahijan , Highlands of Sheytan Kouh 37°11’52” 50°03’17” 159 IAUH 201606 8 Guilan, Bandar Anzali, Road side 37°27’48” 49°22’30” -11 IAUH 201707 9 Mazandaran, Chalos Neamat abad 36°49’02” 50°52’20” -16 IAUH 201608 10 Mazandaran, Shirodi Ring Road 36°51’10” 50°32’11” -18 IAUH 201709 11 Mazandaran, Noshahr 36°35’04” 51°35’14” -20 IAUH 201710 59Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. gel and spectrophotometry. A set of ten primers; (GG) 5GT, (AA) 7GT, (AAA) 5Gt, UBC 234, (GG) 7AT, (AG) 7G, UBC 825, UBC 823, (GG) 5T and (GC) 8GG were used for ISSR analysis. PCR were carried out in 25 μl reactions contain- ing 20 ng of template DNA, 0.3 mM dNTPs, 1μM prim- ers, 1.0 μl of 20 ×PCR buffer (Cinnagen, Iran),1.8 mM of MgCl2, and 5 units of Taq polymerase (Cinnagen, Iran). The amplif ication was carried out, with pro- grammed as initial pre-denaturation at 95°C for 5 min followed by 36 cycles of denaturation at 94°C for 45 s, annealing at temperature (52-55°C) for 40 s, and exten- sion at 72°C for 1min. A final 5 min extension at 72°C followed the completion of 38 cycles. Karyological study For somatic chromosome study, the seeds were soaked for 24 hours in running water and germinated in the laboratory (ca. 21º-24º). The root tips were cut between 9-11 AM and pretreated in 0.002M 8- hydrox- yquinoline (4hours) and fixed in a cold mixture of eth- anol and acetic acid (3:1) for 24 hours. Root tips were macerated in 1N HCl for 10 minutes (Cold Hydrolysis) at room temperature. The slides were staining in 2% Fe- acetocarmin for 10 hours. Data analyses Morphological studies For morphological studies 43 morphological char- acters including 16 qualitative and 26 quantitative char- acters were studied following the protocols of (Ashfaq et al. 2019; Attar et al. 2019; Gul et al. 2019a; Gul et al. 2019b; Kandemir et al. 2019; Shah et al. 2018a, 2018b; Zaman et al. 2019) (Table 2). Figure 1. Distribution map of the studied populations. Table 2. List of selected characters and their codes in morphologi- cal studies. No. Characters Numerical code 1 Plant height mm 2 Length of basal leaves mm 3 Width of basal leaves mm 4 Length of stem leaves mm 5 Width of stem leaves mm 6 Bract length mm 7 Width bract mm 8 Length pedicel mm 9 Number of seeds per capsule 10 Number of flowers per inflorescence 11 Number of calyx 12 Length calyx mm 13 Width calyx mm 14 Number of petal 15 petal length mm 16 Petal width mm 17 Cleft size of petals mm 18 Inter node length mm 19 Number of stamen 20 Number of stigma 21 Capsule length mm 22 Seed length mm 23 Seed width mm 24 Cleft size of capsule mm 25 Number suture capsules 26 Veins number sepals 27 Growth period 0-annual 1- perennial 28 Bract apex 0-acute 1- narrow 2- absence 29 State of stem 0-unbranched 1- branched 30 State of stem strength 0-thin 1- strong 31 Hairs of stem 1-unilateral hair 2- multilateral hair 32 Cross-section of stem 0-round1- rectangular 2- elliptical 33 Shape of basal leaves 0-linear 1- linear- lanceolate 34 Basal leaves apex 0-acute 1- narrow 35 Basal leaves petiole 0-absence 1- presence 36 Hair of basal leaves petiole 0-absence 1- presence 37 Shape caulin leaves 0- linear 1- linear- lanceolate 38 Caulin leaves apex 0- acute 1- narrow 39 Caulin leaves petiole 0-absence 1- presence 40 Hair of caulin leaves petiole 0-absence 1- presence 41 Hair of caulin leaves margin 0-absence 1- presence 42 Hair of caulin leaves lamina 0-absence 1- presence 43 Shape of bract 0-linear 1- linear- lanceolate 60 Shahram Mehri, Hassan Shirafkanajirlou, Iman Kolbadi Morphological traits were standardized (Mean = 0, Variance = 1) and used to estimate Euclidean distance for ordination analyses (Podani 2000). PCA (Principal components analysis) biplot and MDS (Multidimen- sional scaling) were applied for grouping and identify the most variable morphological traits of among the populations (Podani 2000). We used from PAST ver- sion 2.17 (Hammer et al. 2012) for multivariate statisti- cal analyses. Molecular analyses ISSR bands scored as present (1) or absent (0). Genetic polymorphism was determined by genetic diversity parameters: Shannon information index (I), percentage of polymorphism, the number of effective alleles and Nei’s gene diversity (H) (Freeland et al. 2011). Neighbor-Net networking was used for Nei’s genetic identity among studied populations (Huson and Bry- ant 2006; Weising et al. 2005). We used from PAST ver. 2.17 (Hammer et al. 2012), SplitsTree4 V4.13.1 (2013) and DARwin ver. 5 (2012) software for analysis data. For AMOVA (Analysis of molecular variance) we used of GenAlex 6.4 software (Peakall and Smouse 2006; Meirmans and Van Tienderen 2004) that was deter- mined Genetic differentiation of the species and Nei,s Gst analysis in GenoDive ver.2 (2013) (Hedrick 2005; Jost 2008) were used to revealed genetic distance of the species. First data were scored as dominant markers (ISSR) so we used from STRUCTURE analysis for estimate the parameters that related to gene flow among studied pop- ulation. Burn-in = 10000, and 10 runs were performed for relationship between Genetic structure and distance of geographical. Maximum likelihood method and Bayesian Information Criterion (BIC) was studied by structure analysis (Falush et al. 2007; Evanno et al. 2005; Meirmans 2012). Gene flow was determined by Calculat- ing Nm from Gst by PopGene ver. 1.32 (1997). (Pritchard et al. 2000). RESULTS In this study 11 populations of Stellaria media were selected from northern regions of Iran. Genetic diversity parameters revealed that the highest percent of genetic polymorphism (48.89%) and gene diversity (0.179) exist in Guilan, Bandar Anzali, (population No.5), while the lowest amount of genetic polymorphism (13.33%) showed in population Guilan, Road side Bandar Anzali (No.8) Table 3. AMOVA test showed that, 40% of total genetic diversity was within population and 60% was among population. Hedrick standardized fixation index makes of genetic distance among the studied populations. We have moderate level for AMOVA produced after 999 permutations (G’st = 0.515, P = 0.001) and Hedrick dif- ferentiation index (D-est = 0.331, P = 0.001). Our results showed that the populations of S. media are differenti- ated from each other. Populations, genetic affinity Neighbor-Net network and Nj tree revealed identity results but here only Neighbor-Net network is discussed (Figure 2). In the network showed that the populations 1 and 4, as well as populations 7 and 8 show are placed close to each other and they have closer genetic affinity. The populations 3 and 5, 6, 11 are differentiated from the other populations. The studied specimen in MDS plot revealed that they were stay in different groups, which this results were in agreement with the AMOVA results (Figure 3). The relationship between altitude distance and genetic distance by Mantel test after 5000 permutations makes significant in these populations (r = 0.38, P = 0.001). We have isolation in Stellaria media occurred that we have low amount of gene flow due to geographically more dis- tant of populations. Populations genetic structure The result carried out on STRUCTURE analyses by Evanno test which makes a peak at k = 9 (Figure 4). Fur- Table 3. Genetic diversity parameters in the studied populations. (N = number of samples, Ne = number of effective alleles, I= Shan- non’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism, populations). Pop N Na Ne I He uHe %P pop1 6.000 0.633 1.136 0.120 0.080 0.087 23.33% pop2 8.000 0.644 1.119 0.121 0.077 0.083 25.56% pop3 23.000 0.756 1.140 0.138 0.088 0.090 32.22% pop4 5.000 0.511 1.123 0.109 0.073 0.081 20.00% pop5 10.000 1.011 1.312 0.265 0.179 0.188 48.89% pop6 6.000 0.944 1.279 0.231 0.158 0.172 40.00% pop7 5.000 0.533 1.118 0.101 0.068 0.075 18.89% pop8 4.000 0.422 1.099 0.078 0.054 0.061 13.33% pop9 6.000 0.678 1.140 0.120 0.081 0.089 21.11% pop10 6.000 0.922 1.260 0.227 0.152 0.166 42.22% pop11 6.000 0.878 1.217 0.198 0.130 0.142 40.00% 61Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. thermore, STRUCTURE analyses shown genetic identity between populations 1 and 4 (similarly colored), popula- tions 7 and 8, like populations 9-10. But also it indicat- ed genetic difference of populations 3 and 5 (differently colored), likes 6 and 11. The results of Reticulogram (Figure 5), indicated some of shared alleles that is based on the least square method among populations 10 and 4, 6 and between 7 and 4 and 10, also between 3, 11, 1 and 2 and 8. The mean Nm = 0.29 that is very low level of genetic diver- sity and supports genetic stratification as showed by STRUCTURE analyses and K-Means. Nm result agreed with population assignment test and cannot showed gene flow among these populations. In total ten ISSR primers produced 90 bands, fragment size ranged from 100 to 2800 bp. Morphometric analyses ANOVA test for 85 plant specimen were examined from 11 populations. Our results indicated significant difference in compare with the studied populations (P < 0.05). Ordination plot and other analyses produced similar result among populations (Figure 6). Our result revealed that among of the studied populations exist of morphological divergence and this divergence was due to quantitative traits. For example, length of stem leaves character separated population No. 9, but the popula- tions 3 and 5 separated from the other populations due to character calyx length. Figure 2. Neighbor-Net network of populations in S. media based on ISSR data. Figure 3. MDS plot of populations in S. media based on ISSR data. Figure 4. STRUCTURE plot of S. media populations based on k = 9 of ISSR data. Figure 5. Reticulogram of S. media populations based on least square method analysis of ISSR data. (Population numbers are according to Table 1. Figure 6. PCA plot of S. media populations based on morphologi- cal characters. 62 Shahram Mehri, Hassan Shirafkanajirlou, Iman Kolbadi We performed for both morphological and ISSR data a consensus tree (Figure 7). It indicated that some population are differenced from other population based on both morphological and molecular characters. Karyological characteristics In this study three populations of S. media show a tetraploid level, 2n=40 (Figure 8a), six populations show a tetraploid level, 2n=42 (Figure. 8b) and two popula- tions show a tetraploid level, 2n=44 (Figure. 8c) is in accordance with previous report (Morton 2005; Rune- mark 1996). There are high morphological variations in populations of S. media so that in some references subspecies have been defined for these taxa. The results show that such variations have chromosome number dif- ferences in Iran as most morphological variations were considered from different parts of Iran for this study. In S. media have been reported 2n=28, 36, 40, 42 and 44 from Eurasia with 2n=40 predominating (Federov 1974; Löve and Löve 1975; Moore 1973). This species shows a high phenotypic plasticity and genotypic flexibility. DISCUSSION According to Çalişkan (2012) genetic diversity pro- vides information about adapt to changing environ- ments, understanding of positive influence in the con- servation of endangered species, hybridization and gene flow among the populations. This study evaluates on the use of Inter simple sequence repeats markers for com- pare gene flow and relationships within the population of S. media in Iran. Verkleij, et al (1980) showed that Amylases isoenzymes could be successfully applied to assess interpopulational variation in Stellaria media. S. media has many medicinal properties and distrib- uted in our country, however, we provided information on current taxonomic, molecular study and geographical distance. The present study indicated data about gene flow and genetic structure in some part of Iran. Chickweed can any time of the year at all germinate and flower. Sys- tem pollination is mainly self-pollinating, but sometimes can occur cross-pollination by flies and insects. According to Chater and Heywood (1993) Stellaria media widespread weedy species and it is the accept- ed name. There are three subspecies;1- subsp. media, 2- subsp. Cupaniana and 3- subsp. postii but some peo- ple showed that subsp. cupaniana (Sinha 1965; Scholte 1978) and subsp. postii (Sinha 1965) should be included in S. neglecta. According to Fedorov (1969) chromosome numbers that have been reported for S. media included 2n = 24, 28, 36, 38, 40, 42 and 44 from many parts of the world. However, chromosome numbers 2n =40, 42 and 44 are the most commonly reported and this spe- cies revealed a high degree of genotypic variation that is highly correlated with its reproductive (Freeland et al. 2011; Verkleij unpublished). Figure 7. Consensus tree of morphological and molecular data in S. media populations. Figure 8. Micrographs of chromosomes of root tips in studied species. a) S. media (2n=40), b) S. media (2n=42), c) S. media (2n=44). 63Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. S. media is annual, characterized by the presence of five sepals and petals which are usually bifid; (Whitehe- ad and Sinha 1967). Generally, within family Caryophyl- laceae diversity of morphological features makes taxa complicated to be delineated and identified (Whitehead and Sinha 1967). S. media is occurring on abandoned fields and commonly sensitive to disturbance of its habi- tat. Between S. pallida and S. media there are crossing barrier and they are self-pollinating (Peterson 1936), this happened due to presence of polyploidy in S. media (2n=40-44) while observed the diploidy of S. pallida (2n = 22) (Scholte 1978; Slatkin 1993; Jolivet and Bernasconi 2007). Therefore, breeding systems plays role important in low level of gene flow in S. media (Hutchison and Templeton 1999; Medrano and Herrera 2008). Our results provided that the seed morphologies of Stellaria media and S. pallida are similar. Seed coat cells are rounded polygonal and V-shaped margin. Based on these characters, we decided that Stellaria media could be differenced from S. pallida. Seed coat morphology observed of 18 species of Stellaria by Chen (2010). They stated that there are differences between Myosoton and Stellaria. Rani et al. (2012) have studied some stem and leaf anatomical features through the pharmacognostical study for quality control of Stellaria media. Arora and Sharma (2012) did pharmacognostic and phytochemi- cal studies of Stellaria media and showed the presence of epidermis, palisade cells, trichomes and vascular bun- dles in leaf. CONFLICT OF INTERESTS The authors have not declared any conflict of inter- est. 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