Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 59-68, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1337 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Lu Feng, Fariba Noedoost (2021) Genetic diversity and relationships among Glaucium (Papaveraceae) spe- cies by ISSR Markers: A high value medicinal plant. Caryologia 74(4): 59-68. doi: 10.36253/caryologia-1337 Received: June 20, 2021 Accepted: November 18, 2021 Published: March 08, 2022 Copyright: © 2021 Lu Feng, Fariba Noe- doost. 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, distribution, and reproduction in any medium, 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 and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant Lu Feng1,*, Fariba Noedoost2 1 College of Medicine, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK 2 Department of Biology, Faculty of Science, Behbahan Khatam Alanbia University of Technology, Khuzestan, Iran *Corresponding author. E-mail: fatemeh.fat1990@gmail.com; fl337551127@outlook.com; Abstract. Glaucium Mill. (horned poppy), belonging to the family Papaveraceae, is represented by a total of 25 species worldwide, and especially distributed through- out Western, Northern and Eastern Asia, Europe, Northern Africa, and Australia. As a country, Iran harbors relatively more species of the genus Glaucium (11-13 spe- cies) and hence, this country is considered as the hot spot of the genus. As a result, we conducted a molecular analysis of the data for this genus due to the relevance of these species of plants. We employed 75 plants from seven species and seven prov- inces that were randomly picked for this investigation. Five primers were used to amplify genomic DNA, yielding 78 bands, 73 of which were polymorphic (97.78%). ISSR primers have a great capability to recognise polymorphic loci among Glaucium species, as evidenced by the high average PIC and MI values obtained. The genetic similarity of seven samples was calculated to be between 0.77 and 0.92. Glaucium corniculatum var. corniculatum and Glaucium elegans var. elegans showed the low- est similarity, while Glaucium oxylobum and Glaucium grandiflorum had the highest similarity, according to Inter-Simple sequence repeats (ISSR) markers analysis. The following are the study’s goals: 1) Is it possible to identify Glaucium species using ISSR markers? 2) In Iran, how are these taxa genetically structured? 3) what is the inter-species relationship? According to this study, ISSR markers can be utilized to distinguish species. Keywords: Iran, species identification, population structure, Glaucium species, ISSR markers. INTRODUCTION Having a better understanding of any biological investigations requires determining the exact boundaries of a species. As a result, in the context of biology, species delimitation is a topic that receives a lot of attention (Collard & Mackill 2009, Wu et al. 2013; Esfandani-Bozchaloyi et al. 2018a, 2018b, 2018c, 2018d; Pandey et al. 2008). Additionally, the research of intra-specific 60 Lu Feng, Fariba Noedoost levels of genetic diversity and the examination of genetic sequence of wild populations are essential for the devel- opment of effective conservation methods (Fujita et al., 2012; Hendrixson et al., 2013; Mckay et al., 2013). Che- lidoniodeae Ernest, Eschscholzioideae Ernest, Papa- veroideae Ernest, and Platystemonoideae Ernest were the four subfamilies of the Papaveraceae s. str (Ernest 1962- Kadereit 1993). Later on, Kadereit et al. (1994) included the sub- family of Platystemonoideae in Papaveroideae as well. Glaucium is a genus belonging to Papaveraceae sub- fam. Chelidonoideae Ernest that contains about 23 spe- cies (Kadereit 1993). Fedde (1909) listed 20 species, ten varieties, and one subvariety, but Boissier (1867) only approved 12 species. Mory (1979) divided the genus into two segments based on fruit dehiscence, morpho- logical and structural characteristics of leaves, stems, seeds, and pollen grains: G. sect. Acropetal Mory, with four species having acropetal dehiscence, and G. sect. Glaucium, with 18 species having basipetal dehiscence. The genus can be discovered in both dry and wet envi- ronments throughout Europe’s Atlantic coasts and the Canary Islands, as well as Mongolia’s Altai (Mory 1979). (Kadereit 1993). In Iran, it was represented by 11 (Cullen 1966) to 13 (Mobayen 1985; Gran and Sharifnia 2008) species, of these, five are endemics: G. calycinum Boiss., G. con- tortuplicatum Boiss., G. elegantissimum Mobayen, G. mathiolifolium Mobayen and G. golestanicum Gran & Sharifnia. Several taxonomic investigations have demonstrated that seed and trichome micromorphology can be used to classify and delimitate taxa at all taxonomic levels and even across plant families (Barthlott 1981, Krak and Mraz 2008, Salmaki et al. 2009, Satil et al. 2011, Salimi Moghadam et al. 2015, Tavakkoli and Assadi 2016, Arabi et al. 2017). Gran and Sharifnia also researched the seed ornaments of 14 Glaucium taxa in Iran (2008). Light microscopy (LM) and scanning electron microscopy (SEM) were used to examine the seeds and trichomes of 15 Glaucium taxa found in Iran (Tavakkoli and Assadi, 2019). The seeds are semicircular to reni- form in shape, however reniform and extended reniform seeds have been seen in G. oxylobum and G. elegans, respectively. The sculpturing of the testa surface are ver- rucate–rugulate (most frequent type), verrucate–granu- late, verrucate–perforate, verrucate–lineolate, rugulate– granulate, rugulate and ocellate. Their findings reveal that seed and ovary trichome micromorphological traits give helpful and critical information for separating spe- cies and taxa within species, as well as a diagnostic key for the taxa. Glaucium taxa were researched in terms of their morphological, palynological, and phylogenetical char- acteristics, according to Fatma Mungan Kiliç et al (2019). Several of these characteristics differ between taxa, particularly in micromorphology and the establish- ment of clades in phylogenetic trees based on matK and ITS3-6 DNA sequence data, according to their findings. The genus Glaucium of Turkey was separated into subsections Glabrousae and Pubescentae based on the results of DNA investigations and morphological data (stem trichomes). For researching genetic diversity, molecular markers are a useful tool. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers are among the most common- ly utilized advanced genetic markers for diversifica- tion assessments (Pharmawati et al. 2004). The RAPD method is quick and easy to use, and it doesn’t need any clear insight of sequences. Using a single primer of any nucleotide sequence, the approach detects nucleotide sequence polymorphism (Moreno et al., 1998). A single 16-18 bp. long primer consists of a repeating sequence attached at the 3’ or 5’ end of 2-4 arbitrary nucleotides is used to amplify DNA for ISSR markers. The method is faster, easier, less expensive, and more repeatable than RAPD (Esfandani-Bozchaloyi et al. 2017a, 2017b, 2017c, 2017d; Collard & Mackill 2009, Wu et al. 2013). The cur- rent study used new gene-targeted molecular markers, namely ISSR markers, to assess the genetic diversity and relationships among different Glaucium species. Because this is the first research of ISSR markers in the Glau- cium genus, we conducted a molecular analysis on 75 collected specimens from seven Glaucium species. We attempt to respond to the following questions: 1) Does the researched species have infraspecific and interspe- cific genetic diversity? 2) Is there a link between genetic distance and geographical distance among these spe- cies? 3) How do populations and taxa differ genetically? 4) Does the Glaucium genus exchange genes with other Glaucium species in Iran? MATERIALS AND METHODS Plant materials During the months of July to August 2016, 75 individuals representing seven geographical popula- tions of Glaucium species were sampled in the Iranian provinces of Lorestan, Guilan, Mazandaran, Esfahan, Golestan, Hamadan, and Kohgiluyeh, as well as Boyer- Ahmad (Table 1). 75 plant accessions (eight to thirteen samples from each population) were collected from seven distinct pop- 61Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers ulations of different eco-geographic features and stored in -20 until used for ISSR analysis. Table 1 and Fig. 1 provide more information on the geographical distribu- tion of accessions. Morphological studies Morphometry was performed on eight to thirteen samples from each species. A total of 36 morphologi- cal features (13 qualitative, 23 quantitative) were inves- tigated. The data was normalized (Mean=0, variance=1) and used to calculate Euclidean distance for clustering and ordination analysis (Podani 2000). Corolla form, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx margins, bract length, corolla length, corolla width, corolla apex, leaf length and width, leaf apex, leaf margins, leaf shape, leaf gland, and bract margins are among the morphological features analyzed. DNA extraction and ISSR assay Young leaves were utilized at random from one to twelve plants in each of the populations studied. Silica gel powder was used to dry them. To extract genom- ic DNA, the CTAB activated charcoal procedure was applied (Esfandani-Bozchaloyi et al. 2019). The purity of the extracted DNA was tested using an 8% agarose gel. 22 primers from the UBC (University of British Colum- bia) series were evaluated for DNA amplification for the ISSR study. Based on band reproducibility, ten primers were chosen for ISSR study of genetic diversity (Table 2). PCR reactions were carried in a 25μl volume con- taining 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany). The reactions and amplifications were carried out in a Techne thermocycler (Germany) using the follow- ing program: Initial denaturation at 94°C for 5 minutes, then 40 cycles of 1 minute at 94°C, 1 minute at 52-57°C, and 2 minutes at 72°C. A last extension step of 7-10 min- utes at 72°C brought the reaction to a close. Running the amplification results through a 1% agarose gel and stain- ing with ethidium bromide revealed the amplification products. Using a 100-bp molecular size ladder, the frag- ment size was determined (Fermentas, Germany). Data analyses Morphological investigations First, morphologica l characters were norma l- ized (Mean = 0, Variance = 1) and utilized to calculate Euclidean distance between taxonomic pairs (Podani 2000). The UPGMA (Unweighted paired group using aver- age) ordination methods were utilized to group the plant specimens (Podani 2000). ANOVA (analysis of variance) was used to show morphological differences between Table 1. Voucher details of Glaucium species in this study from Iran. No Sp. Locality Latitude Longitude Altitude (m) Sp1 G. corniculatum var. corniculatum (L.) Curtis Kohgiluyeh and Boyer-Ahmad 38°52’37” 47°23’92” 1144 Sp2 G. elegans var. elegans Fisch. & C.A.Mey. Mazandaran, Haraz road, Emam Zad-e-Hashem 32°50’03” 51°24’28” 1990 Sp3 G. oxylobum var. oxylobum Boiss. & Buhse Guilan, Sangar, Road sid 29°20’07” 51°52’08” 1610 Sp4 G. flavum var. serpieri (Heldr.) Halácsy Esfahan:, Ghameshlou, Sanjab 38°52’373 47°23’92” 1144 Sp5 G. fimbrilligerum Boiss. Lorestan, Oshtorankuh, above Tihun village 33°57’12” 47°57’32” 2500 Sp6 G. contortuplicatum var. cantortuplicatum Boiss. Golestan, gorgan 34°52’373 48°23’92” 2200 Sp7 G. grandiflorum Boiss. & A.Huet Hamedan, Nahavand 38°52’373 47°23’92” 1144 Figure. 1. Map of Iran shows the collection sites and provinces where Glaucium species were obtained for this study. 62 Lu Feng, Fariba Noedoost groups, while a biplot of PCA (principal components analysis) was employed to determine the most variable morphological features among the populations investi- gated (Podani 2000). For multivariate statistical analysis of morphological data, Hammer et al. (2012) employed PAST version 2.17 (Hammer et al. 2012). Molecular analyses The ISSR bands were coded as binary charac- ters (presence = 1, absence = 0) and utilized to analyze genetic diversity. To quantify the capacity of each primer to distinguish polymorphic loci among the genotypes, two measures, polymorphism information content (PIC) and marker index (MI), were utilized to assess its dis- criminatory ability (Powell et al. 1996). MI = PIC× EMR is the formula for calculating MI for each primer, where EMR is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic frag- ments (β) (Heikrujam et al. 2015). For each primer, the number of polymorphic bands (NPB) and effective mul- tiplex ratio (EMR) were measured. The number of effec- tive alleles, Nei’s gene diversity (H), Shannon informa- tion index (I), and percentage of polymorphism (P per- cent = number of polymorphic loci/number of total loci) were all calculated (Weising et al, 2005, Freeland et al. 2011). Shannon’s index was calculated by the formula: H’ = -Σpiln pi. Rp is defined per primer as: Rp = ∑ Ib, were “Ib” is the band informativeness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. GenAlEx 6.4 software was used to calculate the percentage of polymorphic loci, the mean loci by accession and population, UHe, H’, and PCA (Peakall & Smouse 2006). Neighbor Joining (NJ) clustering and Neighbor- Net networking were based on Nei’s genetic distance between populations (Freeland et al. 2011, Huson & Bry- ant 2006). The Mantel test was used to see if there was a link between the analyzed populations’ geographical and genetic distances (Podani 2000). PAST ver. 2.17 (Ham- mer et al. 2012) and DARwin ver. 5 (2012) software were used to conduct these searches. For demonstrating genetic differences between the populations, the AMOVA (Analysis of molecular vari- ance) test (with 1000 permutations) was utilized, which was performed in GenAlex 6.4 (Peakall & Smouse 2006). The genetic organization of populations was investigated using the Bayesian-based model STRUCTURE analysis (Pritchard et al. 2000) and GenoDive ver. 2’s maximum likelihood-based K-Means clustering approach (2013). Data were evaluated as dominating markers for STRUC- TURE analysis (Falush et al. 2007). By using ΔK value, the Evanno test was run on the STRUCTURE output to identify the right number of K. (Evanno et al., 2005). Two summary statistics, pseudo-F and Bayesian Infor- mation Criterion (BIC), give the best fit for k in K-Means clustering (Meirmans, 2012). Gene flow was calculated by (i) using PopGene ver. 1.32 (1997) to calculate Nm, an estimate of gene flow from Gst, as follows: Nm = 0.5(1 - Gst)/Gst. This method takes into account the same amount of gene flow in all populations (Yeh et al. 1999). RESULTS Species identification and inter-relationship. Morphometry In quantitative morphological features, ANOVA revealed significant differences (P<0.01) among the sam- ples analyzed. PCA analysis was used to discover the most changeable characteristics among the taxa inves- tigated. The first three factors accounted for more than 75% of the overall variation. Characters like corolla form, calyx shape, calyx length, bract length, and leaf shape had the largest correlation (>0.7) in the first PCA axis, with 33 percent of total variation, whereas leaf apex, corolla length, leaf length, and leaf width influ- enced PCA axis 2 and 3 accordingly. Because the find- ings of several clustering and ordination approaches were similar, a PCA plot of morphological features is shown here (Fig. 2). Plant samples from different spe- cies were put together and generated various groups in general. This finding indicates that the examined species were divided into various groups based on both quanti- tative and qualitative morphological characteristics. We found no transitional forms in the specimens that we looked at. Species identification and genetic diversity To examine genetic links among Glaucium species, five ISSR primers were tested; all of the primers yielded replicable polymorphic bands in all seven Glaucium spe- cies. Figure 3 depicts the ISSR amplification produced by the ISSR-2, ISSR-4 primer. Seven Glaucium species yielded a total of 73 amplified polymorphism bands. The amplified fragments had different size from 100 to 3000 bp. ISSR-3 had the most polymorphic bands (22), where- as ISSR-2 had the fewest (only 7), with an average of 14 polymorphic bands per primer. The average PIC of the 5 ISSR primers was 0.22, ranging from 0.14 (ISSR-3) to 0.29 (ISSR-5). The primers’ MI ranged from 2.85 (ISSR- 2) to 5.47 (ISSR-5), with an average of 3.7 per primer. 63Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers ISSR primers had an EMR ranging from 2.56 (ISSR- 4) to 6.23 (ISSR-5), with an average of 4.6 per primer (Table 2). The primers with the highest EMR values were thought to be more useful in separating the genotypes. For all 7 Glaucium species amplified with ISSR primers, the genetic parameters were computed (Table 3). Unbi- ased predicted heterozygosity (H) ranged from 0.10 to 0.30 (Glaucium corniculatum var. corniculatum), with a mean of 0.21. With a mean of 0.26, Shannon’s informa- tion index (I) showed a similar pattern, with the maxi- mum value of 0.38 in Glaucium corniculatum var. cor- niculatum and the lowest value of 0.15 in Glaucium con- tortuplicatum var. cantortuplicatum. Glaucium oxylobum var. oxylobum has a number of alleles (Na) ranging from 0.261 to 0.667. The effective number of alleles (Ne) ranged from 1.011 (Glaucium contortuplicatum var. cantortupli- catum) to 1.495 (Glaucium elegans var. elegans). The AMOVA test revealed a substantial genetic difference (P = 0.001) between the species investigated. It was dis- covered that 55 percent of overall variance occurred between species and 45 percent occurred within spe- cies (Table 4). Furthermore, significant Nei’s GST (0.88, P = 0.001) and D est (0.389, P = 0.001) values revealed genetic difference between these species. In comparison to within-species genetic diversity, these findings dem- onstrated a larger distribution of genetic variety among Glaucium species. Because the findings of other clus- tering and ordination approaches were similar, NJ clus- tering is reported here (Figure 4). In general, two main clusters appeared in the NJ tree (Figure 4). Populations of Glaucium fimbrilligerum, G. contortuplicatum, and G. oxylobum were put in the first major cluster, sepa- rated from the other species by a great distance. Two sub-clusters made up the second major cluster. The first sub-cluster consisted of Glaucium corniculatum var. corniculatum and G. grandiflorum plants, whereas Figure 2. PCA plots of morphological characters revealing species delimitation in the Glaucium species. Figure 3. Electrophoresis gel of studied ecotypes from DNA frag- ments produced by ISSR-2, ISSR-4; 1, 8= G. corniculatum var. cor- niculatum; 2, 9= G. elegans var. elegans; 3, 10= G. oxylobum; 4, 11= G. flavum var. serpieri; 5, 12=; G. fimbrilligerum; 6, 13= G. contortu- plicatum; 7, 14= G. grandiflorum 64 Lu Feng, Fariba Noedoost the second sub-cluster consisted of G. flavum var. ser- pieri and G. elegans var. elegans plants. In general, ISSR data aligns well with morphological data in terms of species relationships. This is in line with the AMOVA and genetic diversity factors discussed previously. The species are genetically distinct. These findings show that ISSR molecular markers can be utilized to clas- sif y Glaucium species. The Nm analysis by Popgene software also produced mean Nm= 0.768, that is con- sidered very low value of gene flow among the studied species. Isolation by distance (IBD) occurred among the Glaucium species tested, as the Mantel test with 5000 permutations revealed a substantial correlation (r = 0.87, p=0.0002) between genetic distance and geo- graphical distance. The genetic identity of Nei and the genetic distance between the species examined (Table not included). Glaucium corniculatum var. cornicula- tum and G. elegans var. elegans had the highest degree of genetic similarity (0.92), according to the findings. Between G. oxylobum and G. grandiflorum, there was the least genetic resemblance (0.77). The low Nm val- ue (0.768) indicates minimal gene flow or ancestrally shared alleles between the species investigated, as well as considerable genetic divergence between and within Glaucium species. The ΔK =6 was obtained by STRUC- TURE analysis and the Evanno test. The Organization plot (Figure 5) revealed further details regarding the genetic structure of the species investigated, as well as common ancestral alleles and/or gene flow between Glaucium species. Due to shared common alleles, this plot demonstrated genetic affinity between G. cornicu- latum var. corniculatum and G. grandiflorum (similarly colored, No. 1, 7) and G. fimbrilligerum and G. contort- Table 2. ISSR primers used for this study and the extent of polymorphism. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI ISSR-1 DBDACACACACACACACA 14 14 100.00% 0.22 2.66 3.55 4.45 ISSR-2 GGATGGATGGATGGAT 8 7 84.99% 0.25 4.91 4.43 2.85 ISSR-3 GACAGACAGACAGACA 22 22 100.00% 0.14 5.34 5.55 5.44 ISSR-4 AGAGAGAGAGAGAGAGYT 13 13 100.00% 0.27 2.88 2.56 3.85 ISSR-5 ACACACACACACACACC 12 12 100.00% 0.29 1.23 6.23 5.47 Mean 16 14 97.78% 0.22 3.5 4.6 3.7 Total 78 73 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 CAAT box- derived polymorphism (CBDP) primers. Table 3. Genetic diversity parameters in the studied Glaucium species. SP N Na Ne I He UHe %P G. corniculatum var. corniculatum (L.) Curtis 13.000 0.358 1.380 0.384 0.30 0.31 66.50% G. elegans var elegans Fisch. & C.A.Mey. 8.000 0.299 1.495 0.231 0.18 0.23 44.38% G. oxylobum var. oxylobum Boiss. & Buhse 13.000 0.667 1.062 0.24 0.224 0.213 44.73% G. flavum var. serpieri (Heldr.) Halácsy 8.000 0.499 1.067 0.19 0.181 0.14 49.26% G. fimbrilligerum Boiss. 9.000 0.261 1.034 0.172 0.13 0.13 33.15% G. contortuplicatum var. cantortuplicatum Boiss. 11.000 0.545 1.011 0.15 0.10 0.10 23.53% G. grandiflorum Boiss. & A.Huet 13.000 0.352 1.083 0.23 0.22 0.14 45.05% 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 48 1201.364 22.789 17.154 55% 55% Within Pops 50 104.443 1.805 1.888 45% Total 98 1355.807 19.060 100% 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). 65Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers uplicatum (sp No. 5,6). This aligns with the NJ dendro- gram that was previously displayed. The allele composi- tions of the other species are different. DISCUSSION In the biology of long-term evolution of a taxon or population, genetic diversity plays a significant role. The foundation for a taxon’s existence, expansion, and evo- lution. To recognize the taxonomy, origin, and evolution of a taxon, it is necessary to investigate its genetic diver- sity. Furthermore, such research will provide a theoreti- cal foundation for the conservation, development, use, and breeding of germplasm resources (Lubbers et al., 1991; Ma, et al., 2021; Peng, et al 2021; Jia, et al., 2020; Karasakal, et al., 2020a; 2020b). The recent study dis- covered fascinating information about genetic diver- gence, genetic differentiation, and physical differences in Iran’s north and west. The degree of genetic diver- sity inside a species is intimately linked to its breeding technique; the higher the percentage of open pollina- tion/cross breeding, the higher the level of genetic diver- gence in the clade under investigation (Meusel et al., 1965). A primer’s PIC and MI features aid in establish- ing its efficacy in genetic diversity analysis. According to Sivaprakash et al. (2004), the level of polymorphism may be more directly related to an indicator technique’s ability to address level of genetic diversity. PIC values of zero to 0.25 indicate very low genetic variation among genotypes, 0.25 to 0.50 indicate a mid-level of genetic diversity, and 0.50 indicate a high level of genetic diver- sity (Tams et al., 2005; Si et al., 2020; Sun et al., 2021). The PIC values of the ISSR primers in this study ranged from 0.14 to 0.29, with a mean value of 0.22, indicating that ISSR primers had a mid-level ability to determine genetic diversity among Glaucium species. In the Glau- cium taxon, all five primer pairs demonstrated good pol- ymorphism. For the species under investigation, a total of 78 alleles were discovered. The total number of poly- morphic bands per primer ranged from 8 to 22, and the average allele number in loci was 16. In most studies, population size is limited to several vegetative accession (Meusel et al., 1965; Uotila, 1996). This population may have experienced genetic drift, as evidenced by the high degree of FIS and minimal genetic diversity. The isolation of the population and absence the gene flow led to fragmentation of the Glaucium populations. Between genetic diversity parameters and population size were showing positive correlations that confirmed various studies (Leimu et al. 2006). The positive asso- ciation across genetic diversity and size of population Figure 4. UPGMA tree of ISSR data revealing species delimitation in the Glaucium species. Figure 5. STRUCTURE plot of Glaucium species based on ISSR data. 1= G. corniculatum var. corniculatum; 2= G. elegans var. elegans; 3= G. oxylobum; 4= G. flavum var. serpieri; 5=; G. fim- brilligerum; 6= G. contortuplicatum; 7= G. grandiflorum. 66 Lu Feng, Fariba Noedoost can be explained in two ways (Leimu et al., 2006). 1- A positive connection may indicate the existence of an extinction vortex, in which a decrease in population size reduces genetic variety, resulting in inbreeding depres- sion. Plant fitness separates populations depending on habitat quality changes, which is the second cause (Ver- geer et al., 2003). Low genetic variety, according to Booy et al. (2000), can impair plant fitness and limit a population’s capa- bilities to react to changes in environmental conditions by selection and adaptation. Within populations, 45 per- cent of genetic variety was achieved, while 55 percent of genetic variance was gained among the assessed groups. The reproductive system in plant species is important member of the primary elements controlling the dis- tribution of genetic variation (Duminil, 2007). Couvet (Booy et al., 2000) found that one migrant per genera- tion is insufficient to maintain the long-term existence of small populations, but also that the numbers of immi- grants is governed by phenotypic traits and population genetics (Vergeer et al., 2003). Despite the fact that the genetic variations across the three groups were identical, they were statistically mean- ingful. For the lack of distinctions across isolated groups, there are two explanations. The initial hypothesis pro- posed that genetic variety within and between popula- tions demonstrates gene flow patterns, resulting in popu- lation fragmentation (Dostálek et al., 2010). According to the second hypothesis, populations that are geographi- cally close are more clearly related through gene transfer than species that are divided by a great distance. The morphological, palynological, and phyloge- netic parameters of ten Glaucium taxa were investigated (Fatma Mungan Kiliç et al., 2019). Although several of the morphological attributes of the taxa surveyed were matched with those listed in Cullen’s Flora of Turkey (Cullen, 1965), certain properties were revealed to be dif- ferent. In particular, the results of Mory’s (1979) study were compared to those acquired by our methods. In this assessment, the morphological and palynological characteristics were determined to be the most equiva- lent. Gran and Sharifnia (2008) identified G. haussknech- tii as homologous with G. grandiflorum depending on 28 qualitative and 37 quantitative features in a micromacro- morphological examination of 18 Glaucium taxa. According to Fatma Mungan Kiliç et al (2019) the Glaucium taxa were divided into two groups with respect to stem hairs. Taxa with pubescence stems were G. corniculatum subsp. corniculatum and G. cornicula- tum subsp. refractum, G. grandiflorum var. grandiflorum, G. grandiflorum var. torquatum, G. grandiflorum var. haussknechtii and G. secmenii, while the taxa with hair- less stems were G. flavum, G. leiocarpum, G. acutiden- tatum and G. cappadocicum. The results of phylogenetic analyses showed that the Glaucium taxa were grouped into two main clades in the ML trees based on the matK and ITS3-6 DNA sequences, which is in compatible with the hairness of their stems, petal color and testa outline of the seeds. The taxa included in these two sub-clades were also compatible with ovary tubercle. Finally, the findings of this study revealed that prim- ers obtained from ISSR were more successful than other molecular markers in determining the genetic diversity of the Glaucium genus. In addition, the dendrogram and PCA clearly distinguished Glaucium species, demon- strating that the ISSR approach is more effective in iden- tifying Glaucium species. ACKNOWLEDGMENT The authors express their gratitude to anonymous reviewers who provided helpful feedback on an earlier edition. REFERENCES Arabi, Z. et al. 2017. Seed micromorphology and its sys- tematic significance in tribe Alsineae (Caryophyllace- ae). – Flora 234: 41–59. Barthlott, W. 1981. Epidermal and seed surface characters of plants: systematic applicability and some evolu- tionary aspects. – Nord. J. Bot. 1: 345–355 Booy G, Hendriks RJJ, Smulders MJM, Van Groenendael JM, Vosman B. 2000. Genetic diversity and the sur- vival of populations. Plant Biol. 2: 379–395. Collard BCY. Mackill DJ. 2009. Start codon targeted (SCoT) polymorphism: a simple novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93. Cullen, J., 1966: Glaucium. In: Rechinger, K. H. (ed.), Flora Iranica 34, 2–7. Akad. Druck- und Verlagsan- stalt. Dostálek T, Münzbergová Z, Plačková I. 2010. Genetic diversity and its effect on fitness in an endangered plant species, Dracocephalum austriacum L. Conserv Genet. 11:773–783. Duminil J, Fineschi S, Hampe A, Jordano P, Salvini D, Vendramin GG. 2007. Can population genetic struc- ture be predicted from life-history traits? Amer Nat. 169: 662–672. Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2017a. Genetic Diversity and Mor- 67Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers phological Variability In Geranium Purpureum Vill. (Geraniaceae) Of Iran. Genetika 49: 543-557. https:// doi.org/10.2298/GENSR1702543B Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2017b. Species Delimitation In Geranium Sect. Batrachioidea: Morphological and Molecular. Act Bot Hung 59(3–4):319–334. doi: 10.1556/034.59.2017.3-4.3 Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2017c. Genetic and morphological diversity in Geranium dissectum (Sec. Dissecta, Gera- niaceae) populations. Biologia 72(10): 1121- 1130. DOI: 10.1515/biolog-2017-0124 Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2017d. Analysis of genetic diversity in Geranium robertianum by ISSR markers. Phytolo- gia Balcanica 23(2):157–166. Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2018a. Species Relationship and Pop- ulation Structure Analysis In Geranium Subg. Rober- tium (Picard) Rouy with The Use of ISSR Molecular Markers. Act Bot Hung, 60(1–2), pp. 47–65. Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2018b. Species Identification and Population Structure Analysis In Geranium Subg. Geranium (Geraniaceae) . Hacquetia, 17/2 , 235–246 DOI: 10.1515/hacq-2018-0007 Esfandani Bozchaloyi S, Sheidai M, Keshavarzi M. Noor- mohammadi Z. 2018c. Morphometric and ISSR-anal- ysis of local populations of Geranium molle L. from the southern coast of the Caspian Sea. Cytology and genetics, 52, No. 4, pp. 309–321. Esfandani –Bozchaloyi S, Sheidai M. 2018d. Molecu- lar diversity and genetic relationships among Gera- nium pusillum and G. pyrenaicum with inter simple sequence repeat (ISSR) regions, Caryologia, vol 71, No. 4, 1-14.https://doi.org/10.1080/00087114.2018.15 03500 Esfandani-Bozchaloyi S, Sheidai M. 2019. Comparison of Dna Extraction Methods from Geranium (Geraniace- ae), Acta Botanica Hungarica 61(3–4): 251–266 Ernest, W. R., 1962: A comparative morphology of Papa- veraceae, PhD Dissertation. Standford University, Standford, California. Fedde, F. 1909. Glaucium Mill. – In: Engler, A. (ed.), Das Pflanzenreich, Vol. 4. Leipzig, pp. 221–238. Fujita,M.K.,Leache,A.D.,Burbrink,F.T.,Mcguire,J.A.,and Moritz,C.(2012). Coalescent-based species delimita- tion in an integrative taxonomy. TrendsEcol. Evol. 27,480–488.doi:10.1016/j.tree.2012.04.012 Freeland JR, Kirk H. Peterson SD. 2011. Molecular Ecol- ogy (2nded). Wiley-Blackwell, UK, 449 pp. Gran, A., Sharifnia, F., 2008: Micro–macrophological studies of the genus Glaucium (Papaveraceae) in Iran. The Iranian Journal of Botany 14, 22–38. Huson DH. Bryant D. 2006. Application of Phylogenetic Networks in Evolutionary Studies. Molecular Biology and Evolution 23: 254−267. Hammer O, Harper DA. Ryan PD. 2012. PAST: Paleonto- logical Statistics software package for education and data analysis. Palaeonto Electro 4: 9. Heikrujam M, KumarJ. Agrawal V. 2015. Genetic diversi- ty analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism andCAAT box-derived polymorphism (CBDP) markers. Meta Gene 5, 90–97. Hendrixson BE, Derussy BM, Hamilton CA, Bond JE. (2013). Anexploration of species boundaries in tur- ret-building tarantulas of the Mojave Desert (Ara- neae, Mygalomorphae, Theraphosidae, Aphonopel- ma). Mol. Phylogenet. Evol. 66,327–340.doi:10.1016/j. ympev.2012. 10.004 Jia, Y., M. Khayatnezhad and S. Mehri 2020. Population differentiation and gene flow in Rrodium cicutarium: A potential medicinal plant. Genetika 52: 1127-1144. Kadereit, J. W., 1993: Glaucium. In: Kubitzki, K. Rohwer, J. C., Bittrichotteidedelberg (eds.), The families and Gen- era of Vascular Plants, 1–663. Springer Verlag, Berlin. Kadereit, J. W., Blattner, F. R., Jork, K. B., Schwarzbach, A. E., 1994: Phylogenetic analysis of the Papaver- ceae s. 1. (including Fumariaceae, Hypecoaceae and Pteridophyllum) based on morphological characters. Botanische Jahrbücher für Systematik und Pflanz- engeographie 116, 361–390. Karasakal, A., M. Khayatnezhad and R. Gholamin 2020a. The Durum Wheat Gene Sequence Response Assess- ment of Triticum durum for Dehydration Situations Utilizing Different Indicators of Water Deficiency. Bioscience Biotechnology Research Communications 13: 2050-2057. Karasakal, A., M. Khayatnezhad and R. Gholamin 2020b. The Effect of Saline, Drought, and Presowing Salt Stress on Nitrate Reductase Activity in Varieties of Eleusine coracana (Gaertn). Bioscience Biotechnol- ogy Research Communications 13: 2087-2091. Krak, K. and Mraz, P. 2008. Trichomes in the tribe Lac- tuceae (Asteraceae)–taxonomic implications. – Bio- logia 63/5: 1–15. Lubbers EL, Gill KS, Cox TS, Gill BS. 1991. Variation of molecular markers among geographically diverse accessions of Triticum tauschii. Genome 34:354–361 Leimu R, Mutikainen P, Koricheva J, Fischer M. 2006. How general are positive relationships between plant 68 Lu Feng, Fariba Noedoost population size, fitness and genetic variation? J Ecol. 94: 942–952. Meusel H, Jäger EJ, Weinert E. 1965. Vergleichende Chorologie der zentraleuropäischen Flora. Text u. Karten. Bd. 1. VEB Fischer, Jena. Mckay,B.D.,Mays,H.L.,Wu,Y.,Li,H.,Yao,C.T.,Nishiumi ,I., etal.(2013).An empirical comparison of charac- ter-based and coalescent-based approachesto spe- cies delimitation in a youngavian complex. Mol.Ecol. 22,4943–4957.doi: 10.1111/mec.12446 Mobayen, S., 1985: Glaucium. In: Flora of Iran, vascular plants 3, 154 –170. Tehran University, Iran. Mory, B., 1979: Beitragezur Kenntnis der Sippenstruktur der Gattung Glaucium Miller (Papaveraceae). Feddes Repertorium 39, 499–595. Ma, S., M. Khayatnezhad, A. A. Minaeifar 2021b. Genetic diversity and relationships among Hypericum L. spe- cies by ISSR Markers: A high value medicinal plant from Northern of Iran. Caryologia 74: 97-107. Peng, X., M. Khayatnezhad, L. Ghezeljehmeidan 2021. Rapd profiling in detecting genetic variation in stel- laria l. (caryophyllaceae). Genetika-Belgrade 53: 349- 362. Pandey A, Tomer AK, Bhandari D, Pareek S. 2008. Towards collection of wild relatives of crop plants in India. Genet Resour Crop Evol 55(2):187–202 Peakall R. Smouse PE. 2006. GENALEX 6: genetic analy- sis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288–295. Podani J. 2000. Introduction to the Exploration of Multi- variate Data English translation. Backhuyes publisher, Leide,407 pp. Powell W, Morgante M, Doyle JJ, McNicol JW, Tingey SV. Rafalski A J. 1996. Gene pool variation in genus Gly- cine subgenus Soja revealed by polymorphic nuclear and chloroplast microsatellites. Genetics 144, 793–803. Sivaprakash KR, Prasanth S R, Mohanty BP. Parida A. 2004. Genetic diversity of black gram landraces as evaluated by AFLP markers. Curr. Sci. 86, 1411–1415. Salimi Moghadam, N., Saeidi Mehrvarz, S. , Ahamadian, A. & Shahi Shavvon, R . (2015) Data from: Micro- morphological studies on fruits and seeds of the genus Geranium (Geraniaceae) from Iran and their systematic significance. Nordic Journal of Botany 000: 001–011. doi: 10.1111/njb.00859 Si, X., L. Gao, Y. Song, M. Khayatnezhad and A. A. Minaeifar 2020. Understanding population differen- tiation using geographical, morphological and genet- ic characterization in Erodium cicunium. Indian J. Genet 80(4): 459-467. Sun, Q., D. Lin, M. Khayatnezhad and M. Taghavi 2021. Investigation of phosphoric acid fuel cell, linear Fresnel solar reflector and Organic Rankine Cycle polygeneration energy system in different climatic conditions. Process Safety and Environmental Protec- tion 147: 993-1008. Salmaki, Y. et al. 2009. Trichome micromorphology of Iranian Stachys (Lamiaceae) with emphasis on its sys- tematic implication. – Flora 204: 371–381. Satil, F. et al. 2011. The taxonomic value of leaf anatomy and trichome morphology of the genus Cyclotrichium (Lamiaceae) in Turkey. – Nord. J. Bot. 29: 38–48. Tavakkoli, Z. and Assadi, M. 2016. Evaluation of seed and leaf epidermis characters in the taxonomy of some annual species of the genus Papaver (Papaveraceae). – Nord. J. Bot. 34: 302–321. Tavakkoli, Z. and Assadi, M. 2019. A taxonomic revision of the genus Glaucium (Papaveraceae) in Iran. – Acta Bot. Croat. 78: 57–65. Tams SH, Melchinger AE. Bauer E. 2005. Genetic similar- ity among European winter triticale elite germplasms assessed with AFLP and comparisons with SSR and pedigree data. Plant Breed. 124, 154–160. Wu JM, Li YR, Yang LT, Fang FX, Song HZ, Tang HQ, Wang M. Weng ML. 2013. cDNA-SCoT: a novel rap- id method for analysis of gene differential expression in sugarcane and other plants. AJCS 7:659–664 Weising K, Nybom H, Wolff K. Kahl G. 2005. DNA Fin- gerprinting in Plants. Principles, Methods, and Appli- cations. 2nd ed. CRC Press, Boca Rayton, 472 pp. Uotila P. 1996. Decline of Anemone patens (Ranuncu- laceae) in Finland. Symb. Bot. Ups. 1996; 31: 205– 210. Vergeer P, Rengelink R, Copal A, Ouborg NJ. 2003.The Interacting Effects of Genetic Variation, Habitat Quality and Population Size on Performance of Suc- cisa pratensis. J Ecol. 91:18–26. Yeh FC, Yang R, Boyle T 1999. POPGENE. Microsoft Windows-based freeware for population genetic anal- ysis. Release 1.31. University of Alberta 1-31. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 4 - 2021 Firenze University Press Cytogenetic analyses in three species of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina) Kevin I. Sánchez1,*, Fabio H. Takagui2, Alberto S. Fenocchio3 Genetic variations and interspesific relationships in Lonicera L. (Caprifoliaceae), using SCoT molecular markers Fengzhen Chen1, Dongmei Li2,* , Mohsen Farshadfar3 The new chromosomal data and karyotypic variations in genus Salvia L. (Lamiaceae): dysploidy, polyploidy and symmetrical karyotypes Halil Erhan Eroğlu1,*, Esra Martin2, Ahmet Kahraman3, Elif Gezer Aslan4 Cytogenetic survey of eight ant species from the Amazon rainforest Luísa Antônia Campos Barros1, Gisele Amaro Teixeira2, Paulo Castro Ferreira1, Rodrigo Batista Lod1, Linda Inês Silveira3, Frédéric Petitclerc4, Jérôme Orivel4, Hilton Jeferson Alves Cardoso de Aguiar1,5,* Molecular phylogeny and morphometric analyses in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f. Neda Atazadeh1,*, Masoud Sheidai1, Farideh Attar2, Fahimeh Koohdar1 A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) Melika Tabasi1, Masoud Sheidai1,*, Fahimeh Koohdar1, Darab Hassani2 Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant Lu Feng1,*, Fariba Noedoost2 Morphometric analysis and genetic diversity in Rindera (Boraginaceae-Cynoglosseae) using sequence related amplified polymorphism Xixi Yao1, Haodong Liu2,*, Maede Shahiri Tabarestani3 Biosystematics, fingerprinting and DNA barcoding study of the genus Lallemantia based on SCoT and REMAP markers Fahimeh Koohdar*, Neda Aram, Masoud Sheidai Karyotype analysis in 21 plant families from the Qinghai–Tibetan Plateau and its evolutionary implications Ning Zhou1,2, Ai-Gen Fu3, Guang-Yan Wang1,2,*, Yong-Ping Yang1,2,* Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis (Scopoli, 1786) and Tachybaptus ruficollis (Pallas, 1764) in Thailand Isara Patawang1,*, Sarawut Kaewsri2, Sitthisak Jantarat3, Praween Supanuam4, Sarun Jumrusthanasan2, Alongklod Tanomtong5 Centromeric enrichment of LINE-1 retrotransposon in two species of South American monkeys Alouatta belzebul and Ateles nancymaae (Platyrrhini, Primates) Simona Ceraulo, Vanessa Milioto, Francesca Dumas* Repetitive DNA mapping on Oligosarcus acutirostris (Teleostei, Characidae) from the Paraíba do Sul River Basin in southeastern Brazil Marina Souza Cunha1,2,*,#, Silvana Melo1,3,#, Filipe Schitini Salgado1,2, Cidimar Estevam Assis1, Jorge Abdala Dergam1,* Karyomorphology of some Crocus L. taxa from Uşak province in Turkey Aykut Yilmaz*, Yudum Yeltekin Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis L. Egizia Falistocco1,*,+, Gianpiero Marconi1,+, Lorenzo Raggi1, Daniele Rosellini1, Marilena Ceccarelli2, Emidio Albertini1