Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 23-31, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1515 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Wei Cao, Xiao Chen, Zhi- wei Cao (2022) Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified poly- morphism. Caryologia 75(2): 23-31. doi: 10.36253/caryologia-1515 Received: December 01, 2021 Accepted: July 06, 2022 Published: September 21, 2022 Copyright: © 2022 Wei Cao, Xiao Chen, Zhiwei Cao. 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 Com- mons Attribution License, which per- mits 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. Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism Wei Cao1, Xiao Chen2,*, Zhiwei Cao3 1 College of Information Science and Engineering, Tongji University, Shanghai City, China 2 Koal Software Co., Ltd, Shanghai City, China 3 The Third Research Institute of the Ministry of Public Security, Shanghai City,China *Corresponding author. E-mail: ruirui5349@163.com Abstract. There are about 484 species of Hypericum in the Guttiferae family, which includes Hypericoideae.  In Iran, species of this genus are mainly found in the north, northwest, and center of the country, and they are key contributors to the floral ele- ments of the Hyrcanian mountains, Irano-Turanian, and Mediterranean regions (such as the Zagros).  Medicinal, commercial, and horticultural values are associated with these plants.The genetic diversity was assessed through Sequence-related amplified polymorphism. To uncover genetic diversity and species characteristics in Hypericum species, were studied through a combination of morphological and molecular data. Eighty-five individuals related to 7 Hypericum were collected in 6 provinces. A total of 76 (Number of total loci) (NTL) DNA bands were produced through polymerase chain reaction amplifications (PCR) amplification of seven Hypericum species. These bands were produced with the combinations of 5 selective primers. The total number of amplified fragments ranged from 10 to 20. According to the SRAP (Sequence-relat- ed amplified polymorphism) markers analysis, H. perforatum and H. asperulum had the lowest similarity. This study also detected a significant signature of isolation by dis- tance (Mantel test results). Present results showed that sequence-related amplified pol- ymorphism have the potential to identify and decipher genetic affinity in Hypericum species. Current results have implications in biodiversity and conservation programs. Besides this, present results could pave the way for selecting suitable ecotypes for for- age and pasture purposes in Iran. Keywords: sequence-related amplified polymorphism, gene flow, genetic diversity, morphometric analysis, Hypericum. INTRODUCTION: Genetic diversity is a basic component of biodiversity and its conserva- tion is essential for survival of any species in the changing environments (Si et al. 2020; Liu et al. 2021). Most authors agree that genetic diversity is nec- essary to preserve the long-term evolutionary potential of a species (Peng et al. 2021; Ma et al. 2021). This is very important in fragmented populations, 24 Wei Cao, Xiao Chen, Zhiwei Cao because they are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreased heterozygosity and eventual fixation of alleles) and inbreeding depression (increased homozygosity within populations; Chen et al. 2021; Bi et al. 2021). Therefore, understanding the genetic variabil- ity and diversity within and among different populations is crucial for their conservation and management (e.g., Esfandani-Bozchaloyi et al., 2018a, 2018b, 2018c). Sequence-related amplified polymorphism (SRAP) is PCR –based marker system. It is one of the efficient and simple marker systems to study gene mapping and gene tagging in plant species (Li and Quiros 2001), and SRAP are potential markers to assess plant systematics and genetic diversity studies (Wang et al. 2021; Yin et al. 2021; Zhao et al. 2021). Previously, Wu et al. (2010) assessed genetic diversity and population structure in Pogostemon cablin  with the aid of SRAP markers. SRAP markers were successfully implemented in Lamiaceae family to study natural populations and variations with- in the family. These past studies showed that molecular markers, including SRAP markers, are efficient to inves- tigate genetic diversity analyses and phylogenetic rela- tionship among Hypericum species in Guttiferae, Hyper- icoideae family. There are about 484 species of Hypericum in the Guttiferae family, which includes Hypericoideae.  In Iran, species of this genus are mainly found in the north, northwest, and center of the country, and they are key contributors to the floral elements of the Hyrcanian mountains, Irano-Turanian, and Mediterranean regions (such as the Zagros).  Medicinal, commercial, and hor- ticultural values are associated with these plants. They prefer steep-sloped rocky and calcareous cliffs, as well as the edges of highland woods (Robson 1968; Azadi 1999). Robson (1968) expanded the Flora Iranica region by 21 species. H. fursei N. Robson and H. dogonbadani- cum were described by Robson (1977) and Assadi (1980), respectively (1984). Assadi can only be found in Iran’s north and southwestern regions. Azadi (1999) identi- fied 19 species in the Flora of Egypt, four subspecies divided into five sections (Campylosporus (Spach) R. Keller, Hypericum, Hirtella Stef., Taeniocarpum Jaub. & Spach., and Drosanthe (Spach) Endl.) and two doubtful species (H. heterophyllum Vent. and H. Olivieri) and two doubtful species (H (Spach) The term “Hofariqun” was used by Bo Ebn Sina (or Bo Ali Sina) to denote Hyperi- cum species in Iran (Rechinger, 1986). St. John’s wort (Hypericum perforatum L.) is the most important medic- inal species of the genus and its main uses in medicine includes treatment of mild and moderate depression, skin wounds and burns (Barnes et al. 2001). The plant contains a vast array of secondary metabolites, among which naphthodianthrones (hypericin and pseudohy- pericin), acylphloroglucinols (hyperforin and adhyper- forin) and essential oil can be mentioned (Jia et al. 2020; Shi et al. 2021; Zheng et al. 2021; Zhu et al. 2021). The present study investigated the molecular variation of seven species in Iran. Objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using NJ approaches. Current results have implications in breeding and conservation programs. MATERIALS AND METHODS Plants collection Eighty-five (85) individuals were sampled. Seven Hypericum species in East Azerbaijan, Esfahan, Hamed- an, Tehran, Mazandaran, Kermanshah and Kohgilouye- Boirahmad Provinces of Iran were selected and sampled during may-August 2014-2020 (Table 1). We employed 85 plant accessions (five to twelve samples from each community) from 7 distinct populations with various eco-geographic features for SRAP analysis, which were sampled and kept in -20 till further use. Table 1 provide further information on the geographical distribution of accessions. Morphological studies Each species was subjected to morphometric anal- ysis and twelve samples per species were processed. Qualitative (10) and quantitative (11) morphological characters were studied. Data were transformed before calculation. Different morphological characters of flow- ers, leaves, and seeds were studied. Ordination analyses were conducted while using Euclidean distance (Podani 2000). Sequence-related amplified polymorphism method In each of the tested populations, fresh leaves were taken at random from one to twelve plants. Silica gel powder was used to dry them. To extract genomic DNA, the CTAB activated charcoal procedure was applied (Esfandani-Bozchaloyi et al., 2019). SRAP assay was per- formed as described previously (Li and Quiros 2001). Five SRAP in different primer combinations were used (Table 2). A 25μl volume containing 10 mM of Tris- HCl buffer at pH 8; 50 mM of KCl; 1.5 mM of MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of 25Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism single primer; 20 ng of genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany) were subjected to PCR reactions. The overall reaction volume consisted of 25 μl. This PCR reaction was carried out in Techne ther- mocycler (Germany). The following cycles and programs were observed. The initial denaturation step was per- formed for 5 minutes at 94°C. The initial denaturation step was followed by 40 cycles for 1 minute at 94°C; 1 minute at 52-57°C, and 2 minutes at 72°C. The reaction was completed by a final extension step of 7-10 min at 72°C. Staining was performed with the aid of ethidium bromide. DNA bands/fragments were compared against a 100 bp molecular size ladder (Fermentas, Germany). Data Analyses Morphological characteristics were first normalized (Mean = 0, Variance = 1) before being utilized to calcu- late Euclidean distance between taxonomic pairs (Poda- ni 2000). The UPGMA (Unweighted paired group using average) ordination techniques were utilized to group the plant specimens (Podani 2000). Molecular analyses Sequence-related amplified polymorphism (SRAP) bands were coded as binary characters (presence = 1, absence = 0). Total loci (NTL) and the number of poly- morphism loci (NPL) for each primer were calculated. Furthermore, the polymorphic ratio was assessed based on NPL/NTL values. Polymorphism information content was calculated as previously suggested by Roldan-Ruiz et al. (2000). Resolving power for individual marker sys- tem was calculated as: Rp = ΣIb. Ib (band informative- ness) was estimated while following equation: proposed as: Ib= 1 - [2 x (0.5-p)]. In the equation, p indicates the presence of bands (Prevost and Wilkinson, 1999). To quantify the capability of each primer to identify poly- morphic loci among the genotypes, two measures, poly- morphism information content (PIC) and marker index (MI) were utilized to assess its discriminatory abil- ity (Powell et al. 1996). The Mantel test was used to see whether there was a link between the analyzed popula- tions’ geographical and genetic distances (Podani 2000). PAST ver. 2.17 (Hammer et al. 2012) and DARwin ver. 5 (2012) software were used to conduct these studies. Table 1. Voucher details of Hypericum species in this study from Iran. No Section Sp. Locality Sp1 Hypericum H. perforatum L. Esfahan:, Ghameshlou, Sanjab Sp2 Hirtella Stef. H. lysimachioides Boiss. & Noe in Boiss. Kermanshah, Islamabad Sp3 H. asperulum Jaub. & Spach. Hamedan, Nahavand Sp4 H. helianthemoides (Spach) Boiss. Tehran, Damavand Sp5 H. vermiculare Boiss. & Hausskn Hamedan, Alvand Sp6 Taeniocarpium H. hirsutum L. Mazandaran, Nowshahr Sp7 H. linarioides Bosse. Azarbaiejan, West of Tabriz Table 2. SRAP primer information and results. Primer name NTLa NPLb Pc PICd RPe Em1-Me1 25 20 88.22% 0.29 34.71 Em2-Me2 14 14 100.00% 0.46 32.16 Em1-Me4 16 13 83.4% 0.35 40.16 Em2-Me4 13 13 100.00% 0.22 31.30 Em2-Me5 10 10 100.00% 0.40 49.94 Mean 17 16 93.50% 0.33 39.14 Total 76 70 198.33 a: Number of total loci (NTL). b: Number of polymorphic loci (NPL). c: Polymorphic ratio(P %). d: Polymorphic information content (PIC). e: Resolving power (Rp). 26 Wei Cao, Xiao Chen, Zhiwei Cao To reveal genetic differences across the populations, the AMOVA (Analysis of molecular variance) test (with 1000 permutations) was utilized, which was imple- mented in GenAlex 6.4 (Peakall & Smouse 2006). 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 popula- tions. Gene flow was conducted in POPGENE software, version 1.32 (Yeh et al. 1999). RESULTS Mophometery The ANOVA findings showed substantial differences (p<0.01) between the species in terms of quantitative mor- phological characteristics. Principal component analysis results explained 77% cumulative variation. The first PCA axis explained 43% of the total variation. The highest cor- relation (> 0.7) was shown by morphological characters such as calyx length, calyx width, corolla length, corolla color. The morphological characters of Hypericum spe- cies are shown in UPGMA tree (Figure 1). Each species formed separate groups based on morphological charac- ters. The morphometric analysis showed clear difference among Hypericum species and separated each groups. Species identification and genetic diversity Five (5) suitable primer combinations (PCs), out of 10 PCs were screened in this research. Figure 2 illus- trates the banding pattern of Em2-Me4, Em1-Me1 and Em2-Me2 primer by the SRAP marker profile. Seventy (70) amplified polymorphic bands (number of polymor- phic loci) were produced. These bands (fragments) had different range i.e. 150bp to 3000 bp. Maximum and minimum numbers of polymorphic bands were 20 for Em1-Me1 and 10 Em2-Me5, respectively. Each primer produced 16 polymorphic bands on average. The PIC ranged from 0.22 (Em2-Me4) to 0.46 (Em2-Me2) for the 5 SRAP primers, with an average of 0.33 per primer. RP of the primers ranged from 31.30 (Em2-Me4) to 49.94 (Em2-Me5) with an average of 39.14 per primer (Figure 2, Table 2). The calculated genetic parameters of Hyperi- cum species are shown (Table 3). The unbiased heterozy- gosity (H) varied between 0.17 (H. helianthemoides) and 0.32 (H. hirsutum) with a mean of 0.32. Shannon’s infor- mation index (I) was maximum in H. hirsutum (0.49), where as we recorded minimum Shannon’s information index in H. helianthemoides (0.18). The observed num- ber of alleles (Na) ranged from 0.113 in H. perforatum to 1.222 in H. lysimachioides. The significant number of alleles (Ne) ranged from 1.011 (H. helianthemoides) to 1.190 (H. lysimachioides). Analysis of Molecular Variance results in significant genetic difference (p = 0.01) among Hypericum species. The majority of genetic variation occurred among spe- cies. AMOVA findings revealed that 75% of the total variation was between species and comparatively less genetic variation was recorded at the species level (Table 4). Genetic difference between Hypericum species was highlighted by genetic statistics (Nei’s GST), as evident by significant p values i.e. Nei’s GST (0.476, p = 0.01) and D_est values (0.843, p = 0.01) . Different clustering and ordination methods pro- duced similar results therefore, WARD clustering are presented here (Figure 3). In general, plant samples of each species belong to a distinct section, were grouped Figure 1. Morphological characters analysis of Hypericum species by UPGMA tree. Figure 2. Electrophoresis gel of studied ecotypes from DNA frag- ments produced by SRAP profile; 1,8: H. perforatum; 2, 9: H. lysi- machioides; 3,10: H. asperulum ; 4, 11: H. helianthemoides; 5,12: H. vermiculare; 6,13: H. hirsutum; 7,14: H. linarioides. 27Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism together and formed separate cluster. This result show that molecular characters studied can delimit Hyperi- cum species in two different major clusters or groups. In the studied specimens we did not encounter intermedi- ate forms. In general, two major clusters were formed in WARD tree (Figure 3), Populations of H. perforatum were placed in the first major cluster and were placed with great distance from the other species. The second major cluster included two sub-clusters. Plants of H. hir- sutum and H. linarioides comprised the first sub-cluster, while plants of H. lysimachioides, H. asperulum, H. heli- anthemoides and H. vermiculare formed the second sub- cluster. We detected strong correlation between geographi- cal and genetic distances (r = 0.88, p=0.0002) and gene flow (Nm) score of 0.265 was reported among species. Detailed information about genetic distances and genetic identity (Nei’s) are described (Supplementary Table). The findings suggested that there was the highest degree of genetic similarity (0.89) between H. hirsutum and H. lin- arioides. On the contrary to this, H. perforatum and H. asperulum (0.68) had lowest genetic resemblance. We performed STRUCTURE analysis followed by the Evanno test to identify the optimal number of genet- ic groups. We used the admixture model to illustrate interspecific gene flow or / and ancestrally shared alleles in the species studied. K-Means clustering showed k = 7 according to pseudo-F and k = 5 according to BIC. K = 7 is in agreement with WARD grouping and AMOVA. K = 7 reveal the presence of 7 genetic group. Similar result was obtained by Evanno test performed on STRUC- TURE analysis which produced a major peak at k = 7. The STRUCTURE plot (Figure not included) produced more detailed information about the genetic structure of the species studied as well as shared ancestral alleles and / or gene flow among Hypericum species. This plot revealed that genetic difference of species 1 and 2 (dif- ferently colored), as well as 3 and 4. This is in agreement with Neighbor joining dendrogram presented before. The other species are distinct in their allele composition and differed genetically from each other. Table 3. Genetic diversity parameters in the studied Hypericum species. SP N Na Ne I He UHe %P H. perforatum L. 20.000 0.113 1.099 0.262 0.27 0.22 38.23% H. lysimachioides Boiss. & Noe in Boiss. 17.000 1.222 1.190 0.211 0.284 0.292 25.91% H. asperulum Jaub. & Spach. 12.000 0.228 1.180 0.414 0.22 0.25 46.50% H. helianthemoides (Spach) Boiss. 15.000 0.288 1.011 0.181 0.19 0.17 16.11% H. vermiculare Boiss. & Hausskn 9.000 0.352 1.083 0.27 0.29 0.24 45.05% H. hirsutum L. 8.000 0.333 1.016 0.492 0.33 0.32 48.23% Abbreviations: N = number of samples, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percent- age of polymorphism, populations. Table 4. Molecular variance analysis. Source df SS MS Est. Var. % ΦPT Among Pops 22 1116.114 77.111 24.100 75% 75% Within Pops 112 55.455 18.27 10.133 25% Total 134 1656.127 34.022 100% 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). Figure 3. WARD tree of SRAP data revealing species delimitation in the Hypericum species. 28 Wei Cao, Xiao Chen, Zhiwei Cao The low Nm value (0.265) indicates limited gene flow or ancestrally shared alleles between the species studied and supports genetic stratification as indicated by K-Means and STRUCTURE analyses. Population assignment test also agreed with Nm result and could not identify significant gene flow among members of the studied species. DISCUSSION In the present study, we used morphological and molecular (SRAP) data to evaluate species relation- ships in Hypericum species. Morphological analyses of Hypericum species showed that quantitative indicators (ANOVA test results) and qualitative characteristics are well differentiated from each other. PCA analysis sug- gests that morphological characters such as corolla color, pedicel hair, stem hair, leaf hair, petiole hair, width of petal have the potentials to identify and delimitate Hypericum species. Principal component analysis results suggests the utilization of morphological characters to identify and delimitate Hypericum species. Morpho- logical characters including corolla color, pedicel hair, stem hair, leaf hair, petiole hair, width of petal play key role in plant systematics and taxonomy. Our work also highlighted the significance of morphological characters and molecular data to identify and study species genet- ic diversity. In general, genetic relationships obtained from SRAP data coincides with morphometric results. This is in accordance with the parameters of AMOVA and genetic diversity results. SRAP molecular markers detected clear genetic difference among species. These results indicate that SRAP have potentials to study plant systematics and taxonomy in Hypericum members. Given the negative impact of biodiversity threats and overexploitation of Hypericum plant species in Iran, it is necessary to conduct genetic diversity studies on Hypericum species. Genetic diversity based studies pave our understanding to develop conservation strate- gies (Esfandani-Bozchaloyi et al. 2017a,b,c,d). Genetic diversity studies are conducted through appropriate selection of primers and indexes including Polymor- phic information content (PIC) and marker index (MI) are important indexes to fathom genetic variation in species (Sivaprakash et al. 2004). Common logic sug- gests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (Sivaprakash et al. 2004). In this research, we reported PIC values of SRAP primers from 0.22 to 0.46, with a mean value of 0.33. PIC values indeed show low and high genetic diversity among gen- otypes. Values are ranging from zero to 0.25 show low genetic diversity; in contrast to this, 0.25 to 0.50 high- light mid-level of genetic diversity. In addition to this, values higher than 0.5 are associated with high genetic diversity (Tams et al. 2005). Present results highlighted the efficiency of SRAP markers to estimate genetic diver- sity in Hypericum species. In our study, SRAP markers detected average percentage of polymorphism (93.50%). Current research results also described average PIC val- ues of SRAP makers (0.33) and average RP (resolving power) values i.e. 39.14 of SRAP markers. These current reported values are higher than other reported mark- ers on Hypericum species (Maria et al. 2007; Dana et al. 2007). In the recent study, low gene flow (Nm) was detected among Hypericum species. The present study also depicted a significant correlation between genetic and geographical distances. Our findings revealed that isolation by distance (IBD) existed between Hypericum species (Mantet test results). Several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentiation (Frichot et al. 2013; De Kort et al. 2014). The magnitude of variability among Na, Ne, H, and I indices demonstrated a high level of genetic diversity among Hypericum species. Dendro- gram and principal component analysis results showed clear difference among Hypericum species. This shows the high utilization of the SRAP technique to identify Hypericum species . Our results have implications for conservation and breeding programs. Furthermore, it may identify suitable ecotypes for forage and pasture. A high level of variation among H. perforatum popu- lations was also reported by Percifield et al. (2007) which confirms results of the present study. Similar results have been reported on this species using the RAPD markers by Hazler Pilepic et al. (2008). The high genetic diversity of H. perforatum populations is as a result of its mating systems. In fact, propagation method(s) of plant species is considered as one of the most important factors deter- mining their levels of genetic diversity (Hamrick 1982). Self-incompatibility is a wide spread phenomenon in the genus Hypericum (Robson 1981), resulting in the high levels of genetic variability (Borba et al. 2001). Further- more, this perennial plant produces a great number of seeds every year in favor of the high amounts of diversity in this species (Zhao et al. 2007). Bi et al (2021) were conducted to study Hyperi- cum genetic diversity by Random Amplified Polymor- phic DNA (RAPD) from seventy plant specimens. They showed significant differences in quantitative morpho- logical characters in plant species. H. dogonbadanicum depicted unbiased expected heterozygosity (UHe) in the range of 0.10. Shannon information was high (0.32) in H. 29Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism perforaturm. H. dogonbadanicum showed the lowest val- ue, 0.17. The observed number of alleles (Na) ranged from 0.22 to 0.53 in H. dogonbadanicum and H. elongaturn. Gene flow (Nm) was relatively low (0.87) in Hypericum. Ma et al. (2021) conducted a study in Iran on identi- fication of Hypericum population through morphological and ISSR Markers. They observed 10 primers produced 141 bands, of which 127 were polymorphic (95.78%). The obtained high average PIC and MI values revealed high capacity of ISSR primers to detect polymorphic loci among Hypericum species. The genetic similarities of 17 collections were estimated from 0.617 to 0.911. Accord- ing to Inter-Simple sequence repeats (ISSR) markers analysis, H. androsaemum and H. hirtellum had the low- est similarity and the species of H. perforatum and H. triquetrifolium had the highest similarity. Since widespread species may possess the higher lev- els of genetic diversity than narrowly distributed plants (Singh et al. 1998), the wide range of H. perforatum dis- tribution is an important factor in this respect. Consid- ering the low level of gene flow rate among studied wild populations of H. perforatum, therefore, genetic drift might be inevitable. In H. perforatum, the low rate of gene flow may be due to factors such as prevailing apomixes and short dis- tance of seed dispersal as stated by Hazler Pilepic et al. (2008). Molecular markers have been used to investigate the genetic diversity, population structure, and repro- ductive biology of H. perforatum. High among-popu- lation variation was previously reported in Hypericum species by Percifield et al. (2007), Pilepić et al. (2008), and Farooq et al. (2014). High differentiation among populations is mostly coupled with limited gene flow among them. The low gene flow and the high differen- tiation among populations has been explained mainly by founder events such as time since colonization (Jacque- myn et al., 2004). CONCLUSIONS The present study investigated the molecular vari- ation of seven species. Molecular and morphometric analysis confirmed morphological and genetical differ- ence between Hypericum species. This was first attempt to assess genetic diversity through Sequence-related amplified polymorphism and morphometrics analy- sis in Iran. Current study reported two major clusters. 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