Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 73-80, 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-135 Citation: S. Mohsenzadeh, M. Sheidai, F. Koohdar (2020) Populations genetic study of the medicinal species Planta- go afra L. (Plantaginaceae). Caryologia 73(2): 73-80. doi: 10.13128/caryolo- gia-135 Received: April 26, 2019 Accepted: February 23, 2020 Published: July 31, 2020 Copyright: © 2020 S. Mohsenza- deh, M. Sheidai, F. Koohdar. 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. Populations genetic study of the medicinal species Plantago afra L. (Plantaginaceae) Saeed Mohsenzadeh*, Masoud Sheidai, Fahimeh Koohdar Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran *Corresponding author. E-mail: s_mohsenzadeh@yahoo.com Abstract. Plantago afra (Plantaginaceae) is the most medicinally important species in genus Plantago and it is native to the western Mediterranean region, West Asia and North Africa, and cultivated extensively in Asia and Europe for seed husk known as black Psyllium. We have no data on the population genetic structure of this species in the world. Therefore a population genetic and morphological investigation was per- formed through light on genetic and morphological variability in this taxa. We used ISSR molecular markers for population genetic investigation. Genetic diversity analyses revealed a moderate genetic variability within Plantago afra, while PCoA showed some degree of genetic admixture among populations. AMOVA produced significant genetic difference among populations. The Mantel test showed a positive significant correla- tion between the genetic and geographic distance of the studied populations. STRUC- TURE analysis showed that there are different genetic groups in the studied popula- tions. Morphometric analysis showed that one population differed in seed color and mean stem diameter. The same population contained specific allele combinations and differed genetically from the rest of the studied populations. Therefore, we considered it as a new variety within Plantago afra. Keywords: Plantago afra, ISSR, PCoA, STRUCTURE analysis. INTRODUCTION Genus Plantago L. is the largest genus of the Plantaginaceae family which contains more than 200 annual and perennial herbs and subshrubs with a worldwide distribution (Rahn 1996; Rønsted et al. 2002). Most species of the genus Plantago are small, with rosette leaves, ovoid and cylindrical spikes that contain tiny flowers. Plantago species have been used in both conventional and traditional systems of medicine throughout Asia, Europe, and North America (Sarihan et al. 2005; Goncalves and Roma- no 2016). Moreover, few species like P. afra L. and P. ovata Forssk. are high- ly valued in the nutraceutical, pharmaceutical and cosmetic industries The polysaccharides obtained from husks in these species can improve intestinal performance, obesity, high cholesterol, colon cancer, constipation and diabe- tes (Goncalves and Romano 2016). 74 Saeed Mohsenzadeh, Masoud Sheidai, Fahimeh Koohdar P. afra (syn. P. psyllium L.) is native to the western Mediterranean region, West Asia, and North Africa. It is an Annual herb with well-developed stems (grow up to 40 cm long), leaves narrow-linear and opposite or whorl covered sparsely with short, hard and glandular hairs (Kazmi 1974). This medicinal plant species grows in different regions of the country and forms several local populations. We have no information on genetic diversity and available gene pools in this species. Popu- lation genetic studies is a proper approach to investigate geographical populations within a single species and to identify divergent plant populations, both in genetic content as well as morphological differentiation (Sheidai et al. 2016a,b, 2018). Population genetic analyses provide valuable data on the rate of genetic divergence, genetic variability within/ between populations, self-pollination versus outcrossing, gene flow and inbreeding. Also, data regarding morpho- logical differentiation among populations, together with data on genetic diversity, are vital to support population management and conservation strategies (Zanella et al. 2011; Sheidai et al. 2016a). Different molecular markers have been used in population genetic studies such as neutral multi-locus markers (RAPD, RFLP, ISSR, SSR, SRAP, AFLP, etc.) and gene sequence data (cp-DNA, nuclear ITS, ETS, etc.) (Ferreira et al. 2013; Mosaferi et al. 2015; Sheidai et al. 2013, 2016a,b, 2018). In the present work, we carried out population genetic analyses of P. afra by using inter-sim- ple sequence repeats (ISSR) markers as they are repro- ducible, and cheap in cost (Sheidai et al. 2013; 2016 a,b; Safaei et al. 2016). Morphological analyses of these populations were also performed to study if genetic divergence in popula- tions is accompanied by morphological differentiation. MATERIAL & METHODS Plant materials For the present study, 88 plant accessions were col- lected from 10 geographical populations in two provinc- es of Fars and Bushehr, that are located in South of Iran. Details of localities are provided in Table 1. The voucher specimens are deposited in the Herbarium of Shahid Beheshti University (HSBU). Identification of P. afra was done by using different references (Patzak & Rechinger 1965; Kazmi 1974; Sell et al. 2010). DNA extraction and PCR details Total DNA was extracted from 40 mg of leaf tissue by using CTAB-activated charcoal protocol (Križman et al. 2006). Quality of extracted DNA was examined by running on 0.8% agarose gel. Each ISSR amplification was performed in a 25μL volume containing 20 ng of genomic DNA, 10 mM Tris- HCl buffer at pH 8, 50 mM KCl, 1.5 mM MgCl2, 0.2 μM of a single primer, 0.2 mM of each dNTP and 3 U of Taq DNA polymerase (Bioron Germany). ISSR analyses The ISSR primers employed were (GA)9A, (GA)9T, UBC 807, UBC 811, UBC 810, UBC 834, CAG(GA)7, (CA)7AC, (CA)7AT and (CA)7GT commercialized by the University of British Columbia. Amplification reactions were done in a Techne thermocycler (Germany) with the following program: 5 min for initial denaturation step at 94 °C, 1 min at 94 °C, 45s at 55 °C, 2 min at 72 °C and a final run of 10 min at 72°C. The amplification products were visualized by running on 1% agarose gel, followed Table 1. The studied populations, their localities and voucher numbers. Pop. Locality Longitude Latitude Altitude (m) Voucher no. 1 Fars, Kazeroun, Taleghanei mountain 51°40’13” 29°38’29” 970 HSBU-2018410 2 Fars, Kazeroun, Aboali village 51°42’2” 29°31’32” 838 HSBU-2018411 3 Fars, Kazeroun, mountains around Baladeh village 51°56’48” 29°17’22” 781 HSBU-2018412 4 Fars, Farashband, Nougin village 52°0’46” 29°10’16” 740 HSBU-2018413 5 Fars, Bishapour 51°35’21” 29°44’43” 840 HSBU-2018414 6 Fars, mountains around Ghaemieh 51°25’21” 29°50’26” 928 HSBU-2018415 7 Fars, mountains around Noorabad 51°35’1” 29°58’51” 1080 HSBU-2018416 8 Fars, Konartakhteh 51°24’40” 29°31’45” 512 HSBU-2018417 9 Bushehr, Dalaki 51°17’10” 29°25’13” 83 HSBU-2018418 10 Bushehr, Chahkhani village 51°6’27” 29°11’42” 20 HSBU-2018419 75Populations genetic study of Plantago afra L. by the Ethidium Bromide staining. The fragment size was estimated by employing a 100 bp molecular size lad- der (Fermentas, Germany). DATA ANALYSES Morphological analysis Morphological characters studied are stem diameter, peduncle length, internode length, leaf length and width, spike length, seed color. We used Ward clustering (mini- mum spherical cluster method) based on Euclidean dis- tance after 100 times bootstrapping for grouping of the accessions. Data analysis performed by using PAST ver. 2.17 software (Hammer et al. 2012). Molecular analysis We used the ISSR bands as binary characters and coded them accordingly (absence = 0, presence = 1,). The number of common bands versus private bands was determined. Genetic diversity parameters such as the percentage of allelic polymorphism, diversity (He), allele diversity, Nei’s gene and Shannon information index (I) were determined (Weising et al. 2005). We used GenAl- ex 6.4 for these analyses (Peakall and Smouse 2006). Nei’s genetic distance (Weising et al. 2005) was determined among the studied populations followed by Neighbor Joining (NJ). AMOVA test with 1000 permuta- tions performed for examining the genetic difference of the studied populations (Peakall and Smouse 2006). DCA (detrended correspondence analysis) was performed for estimating the distribution of loci in the genome. PCoA (Principal Coordinate analysis) analysis was performed to group the plant specimens according to ISSR data. The Mantel test (Podani 2000) was implemented to study the association between genetic distance and geographical distance of the studied populations. Data analyses were performed by using GenAlex 6.4 (Peakall and Smouse 2006) and PAST ver. 2.17 (Hammer et al. 2012). Bayesia n model-based STRUCTUR E a na lysis (Pritchard et al. 2000) was utilized to examine the genetic structure of the studied populations. For this analysis data were scored as dominant markers and analysis developed the method advised by Falush et al. (2007). The STRUCTURE Harvester website (Dent and von Holdt 2012) to perform the Evanno method (Evan- no et al. 2005) was used For the optimal value of K in the studied populations. The selection of the most likely number of clusters (K) was performed by calculating an ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values, as defined by Evanno et al. (2005). RESULTS Morphometry The WARD tree (Fig. 1), of the selected studied populations based on morphological features, separated plants of population 1, from the others. This population differed in stem diameter (Fig. 2a,b) and seed color (Fig. 2c,d) from other populations. The mean stem diameter was 4mm in population 1, while it ranged from 1mm up to 3mm in other populations. Similarly, the seed color was dark brown in population 1, while it was light brown in the other studied populations. ISSR analyses We obtained 31 ISSR bands (Loci) in total (Table 2). The highest mean number of bands occurred in popu- lations 1 and 3 (31 and 28 bands, respectively). Some of the populations had private bands while, few common bands occurred in the studied population. These are shared alleles among these populations. The studied P. afar populations varied in genetic variability (Table 3), for example, population 1 had the highest degree of genetic polymorphism (58.97%), while the population 10 showed the lowest degree (5.13%). The highest value of New gene diversity was observed in population 1 (0.15) followed by populations 3-5 (>0.10). However, the studied populations had almost a similar value for the mean effective alleles. Figure 1. Ward tree of morphological data of the selected popula- tions of P. afra. 76 Saeed Mohsenzadeh, Masoud Sheidai, Fahimeh Koohdar DCA detrended correspondence analysis plot of ISSR alleles (Fig. 3) revealed that the loci studied are dis- tributed in the genome and are not closely linked as they are scattered in this plot. Therefore, ISSR loci studied are suitable molecular markers for genetic variability inves- tigation in P. afar. The discriminating power of the ISSR loci is present- ed in Table 4. We presented only the loci with at least 0.70 Gst value/ or above 1 Nm indicating their migration and shared value. The mean Gst value 0.62 for all ISSR loci, indicates that these molecular markers have a good discriminating power and can be used in date Plantago Figure 2. a: stem of population 1; b: stem of other populations; c: dark brown seed in population 1; d: light brown seed in other popula- tions. Figure 3. DCA plot of ISSR alleles of P. afra. Figure 4. PCoA plot of the studied populations based on ISSR data. Table 2. ISSR bands in P. afar populations studied. Population Pop1 Pop2 Pop3 Pop4 Pop5 Pop6 Pop7 Pop8 Pop9 Pop10 No. Bands 31 26 28 24 23 17 14 17 16 14 No. Bands Freq. >= 5% 31 26 28 24 23 17 14 17 16 14 No. Private Bands 3 0 1 1 1 0 0 0 0 0 No. LComm Bands (<=25%) 4 2 2 1 1 0 0 0 1 1 No. LComm Bands (<=50%) 13 9 10 5 6 2 1 1 2 2 77Populations genetic study of Plantago afra L. cultivar genetic fingerprinting. The studied P. afar populations almost showed a high degree of genetic similarity (Mean = 0.84) (Table 4). The highest degree of genetic distance (0.32) occurred between populations 3 and 9, while the lowest degree (0.03) between populations 9 and 10. PCoA grouping of the P. afar populations based on ISSR data (Fig. 4), placed the studied specimens in 4 major groups. Individuals of populations 9 and 10 were intermixed and formed the first major group at the left top part of the PCoA plot. Individuals from three pop- ulations 1-3, are also close to each other and comprised the second major group, at the right top corner of this plot. Similarly, plants in populations 6-8 and 4 and 5 comprised the next two major groups, which are placed in lower parts of the PCoA plot. These results indicate that a limited degree of genetic admixture has occurred in some of the studied populations. NJ tree obtained (Fig. 5), revealed the genetic affin- ity between P. afar populations. The four genetic groups are well separated in distinct clusters. As also indicated before, the populations 6-8 formed a distinct cluster, in which populations 6 and 7 showed closer genetic similar- ity. The populations 9 and 10 formed the second genetic Figure 5. NJ tree of the studied populations based on ISSR data. Figure 6. STRUCTURE plot of the studied P. afra populations. Table 3. Genetic diversity parameters in the studied populations (N = number of samples, Na= number different alleles, Ne = number of effective alleles, I= Shannon’s information index, He gene diver- sity, UHe = unbiased gene diversity, P%= percentage of polymor- phism). Pop Na Ne I He uHe %P Pop1 1.385 1.236 0.241 0.151 0.160 58.97% Pop2 1.000 1.120 0.124 0.077 0.081 33.33% Pop3 1.154 1.216 0.202 0.131 0.139 43.59% Pop4 0.974 1.179 0.168 0.109 0.114 35.90% Pop5 0.897 1.180 0.160 0.106 0.116 30.77% Pop6 0.692 1.176 0.143 0.097 0.105 25.64% Pop7 0.538 1.089 0.084 0.054 0.058 17.95% Pop8 0.641 1.128 0.111 0.075 0.079 20.51% Pop9 0.513 1.031 0.035 0.021 0.022 10.26% Pop10 0.410 1.035 0.029 0.020 0.021 5.13% Table 4. Discriminating power of ISSR loci studied in P. afar popu- lations. Locus Sample Size Ht Hs Gst Nm* Locus2 88 0.0333 0.0306 0.0823 5.5745 Locus4 88 0.4011 0.0982 0.7949 0.1290 Locus7 88 0.4790 0.0982 0.7949 0.1290 Locus11 88 0.1378 0.1267 0.0808 5.6895 Locus12 88 0.0215 0.0205 0.0440 10.8533 Locus13 88 0.0333 0.0306 0.0823 5.5745 Locus15 88 0.0233 0.0208 0.1075 4.1492 Locus17 88 0.0233 0.0208 0.1075 4.1492 Locus24 88 0.5000 0.1022 0.7956 0.1284 Locus25 88 0.0360 0.0300 0.1682 2.4719 Locus26 88 0.4434 0.0536 0.8790 0.0688 Locus28 88 0.3200 0.0000 1.0000 0.0000 Locus30 88 0.4592 0.0929 0.7977 0.1268 Locus32 88 0.4899 0.0531 0.8915 0.0608 Locus34 88 0.1238 0.1071 0.1343 3.2217 Locus35 88 0.2314 0.1685 0.2716 1.3407 Locus36 88 0.0604 0.0487 0.1933 2.0870 Locus36 88 0.0730 0.0664 0.0899 5.0614 Mean 88 0.2266 0.0840 0.6293 0.2945 St. Dev 0.0335 0.0041 * Nm = estimate of gene flow from Gst or Gcs. E.g., Nm = 0.5(1 - Gst)/Gst; See McDermott and McDonald, Ann. Rev. Phytopathol. 31: 353-373 (1993). Ht = Totale diversity, Hs = Diversity due to population. 78 Saeed Mohsenzadeh, Masoud Sheidai, Fahimeh Koohdar group and were positioned in a separate cluster. The same holds true for the populations 4 and 5. Similarly, popula- tions 1-3 comprised the last genetic group, with popula- tions 2 and 3 showing closer genetic similarity. AMOVA produced significant genetic difference among the studied populations (P = 0.001). It reveals that 61% of total genetic difference occurred due to among populations difference, while 39% was due to within populations genetic variability. Similarly, pair- wise AMOVA among populations (Table 6), revealed that most of the populations differed significantly in their genetic content. STRUCTURE analysis revealed more detailed infor- mation on the genetic structure of the studied P. afra populations (Fig. 6). The populations 2-3, and the popu- lations 9-10 are genetically similar due to a high degree of shared common/ancestral alleles (similarly colored segments). Population 1 and 4, contained distinct allele combinations and differed in color segments. The Mantel test performed between the genetic and geographical distance of the studied populations pro- duced a significant positive correlation (p = 0.0002). Therefore, with an increase in the population geographi- cal distance, they become genetically more diverged. This is called isolation by distance (IBD). This also indi- cates that gene flow may occur among the neighboring populations only, which is in agreement with low degree of Nm and genetic admixture obtained. IDENTIFICATION OF NEW VARIETY IN PLANTAGO AFAR Based on both morphological and genetic results, we consider population 1 as a new variety for Plantago afra. Seed color and diameter of the stem are among impor- tant morphological characters that can be used in the taxonomy of the genus at the bellow species rank. Discussion P. afra is medicinally important plant and producing data on its genetic affinity, genetic structure and vari- ability can be used in conservation and probably future breeding programs. In the process of populations divergence, they may form new taxonomic entity bellow the species rank. This may be, ecotype, variety or subspecies (Sheidai et al. 2013, 2014). The species delimitation in complex groups and in cases where the species have morphological over- lap is a tedious and difficult task. Therefore, using mul- tiple approaches is suggested to determine the species boundaries (Carstens et al. 2013). In the recent years, combined approaches of morphological and molecular studies have been used to delimit the species and sub- Table 5. Nei genetic distance versus genetic identity of P. afar popu- lations. Nei’s genetic identity (above diagonal) and genetic distance (below diagonal). Pop ID 1 2 3 4 5 6 7 8 9 10 1 **** 0.87 0.81 0.77 0.76 0.82 0.80 0.76 0.78 0.76 2 0.13 **** 0.92 0.83 0.84 0.78 0.73 0.83 0.76 0.75 3 0.20 0.08 **** 0.84 0.84 0.78 0.74 0.83 0.72 0.74 4 0.25 0.17 0.16 **** 0.89 0.90 0.80 0.85 0.77 0.78 5 0.26 0.17 0.16 0.11 **** 0.89 0.81 0.86 0.75 0.78 6 0.19 0.23 0.24 0.10 0.11 **** 0.95 0.91 0.85 0.85 7 0.21 0.31 0.30 0.21 0.20 0.04 **** 0.92 0.842 0.84 8 0.26 0.17 0.18 0.16 0.15 0.08 0.08 **** 0.87 0.87 9 0.23 0.26 0.32 0.25 0.27 0.15 0.17 0.13 **** 0.96 10 0.26 0.28 0.30 0.24 0.23 0.15 0.16 0.13 0.03 **** Table 6. Pair-wise AMOVA among P. afar populations. PhiPT Values below diagonal. Probability, P(rand >= data) based on 999 permuta- tions is shown above diagonal. Pop1 Pop2 Pop3 Pop4 Pop5 Pop6 Pop7 Pop8 Pop9 Pop10 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Pop1 0.431 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Pop2 0.459 0.263 0.000 0.001 0.001 0.001 0.001 0.002 0.001 0.001 Pop3 0.567 0.547 0.475 0.000 0.001 0.001 0.001 0.001 0.001 0.001 Pop4 0.555 0.559 0.452 0.459 0.000 0.005 0.002 0.001 0.001 0.001 Pop5 0.506 0.615 0.542 0.392 0.477 0.000 0.004 0.001 0.001 0.001 Pop6 0.590 0.732 0.653 0.623 .658 0.245 0.000 0.001 0.001 0.001 Pop7 0.596 0.589 0.526 0.534 0.558 0.360 0.463 0.000 0.001 0.001 Pop8 0.679 0.756 0.714 0.724 0.783 0.660 0.750 0.692 0.000 0.001 Pop9 0.698 0.775 0.715 0.731 0.764 0.682 0.750 0.701 0.575 0.000 Pop10 79Populations genetic study of Plantago afra L. species (Seif et al. 2014; Mosaferi et al. 2015; Sheidai et al. 2016a). This multiple approaches seems to be an effi- cient method in P. afra as Wolff & Morgan-Richards (1988) concluded that PCR-generated polymorphic markers like RAPD and inter-SSR are useful tools to study populations of the two subspecies of P. major and to group the plants into the two subspecies. Population genetic study produces important infor- mation on the genetic structure of plants, the stratifica- tion versus gene flow among the species populations, genetic divergence of the populations, etc. (Sheidai et al. 2014). The ISSR-PCR marker technique is also efficient for genetic characterization even at the varietal level of a species. For example, Charters et al. (1996) distin- guished 20 cultivars of Brassica napus using ISSR mark- ers, Similarly, Seif et al. (2012) used combined analysis of morphological and ISSR-RAPD molecular markers in 13 populations of Cirsium arvense to recognize new varie- ties within this species. Meyers and Liston (2008) recog- nized 4 varieties of P. ovata in the New and Old World by using sequence data of ITS and morphological char- acters included corolla lobe length/width ratio, Trichome length to length of bracts, color midrib on corolla lobes and bracts. The occurrence of IBD in the studied populations indicates that the neighboring populations are genetical- ly more alike than distantly placed populations. There- fore, the reason for the genetic similarity of population 2 with 3, and Populations 9 and 10 together, and popu- lation 6 with 7 and 8 revealed by STRUCTURE plot is probably their geographical vicinity, followed by their pollination system and the distribution of their seeds by the wind, which can bring about a frequent gene flow among these populations. Genetic study revealed that there are different genetic groups in the studied populations. Morphologi- cal study of the selected studied population showed that we have two different groups based on morphological features. Therefore, we suggest the existence of new taxa within this species. Taxonomy Plantago afra L. var. kazerunensis Sheidai var. nov. Iran. Fars Prov.: Kazerun, 970 m, Saeed Mohsenzadeh, 10 April 2018, 2018410 (HSBU). Description: Plants annual, ca. 20 cm tall; all parts covered with short, hard and glandular hairs, stems, erect, highly branched usually of basal, diameter ca. 4 mm; internodes 3–3.5 cm; Leaves opposite 3–3.5 long up to 1mm broad, linear-lanceolate or linear, margins entire; Spikes ovate, 8–10 mm; peduncle 3–3.5 cm; fertile bracts 3-5 mm long, covered with glandular hairs, nar- row-ovate to ovate, lower bracts in the upper part pro- duced into a long, narrow acuminate part; Sepals 3-3.5 mm long narrow-ovate covered with similar hairs as on the bracts; Corolla tube up to 4 mm long, rugulose, lobes 2 mm long, narrow-ovate, acute; Seeds 2, dark brown, narrow-elliptic, shining, 3 mm long. Distribution and habitat: Plantago afra var. kazerun- ensis was found at only one locality in Kazerun, in the west Fars Province, Iran. Dozens of individuals were found at the type locality in the hillside of Talegha- nei mountain to 970 m above sea level. 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