Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 69-76, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1380 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani (2021) Morpho- metric analysis and genetic diversity in Rindera (Boraginaceae-Cynoglosseae) using sequence related amplified poly- morphism. Caryologia 74(4): 69-76. doi: 10.36253/caryologia-1380 Received: August 24, 2021 Accepted: December 17, 2021 Published: March 08, 2022 Copyright: © 2021 Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani. 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. Morphometric analysis and genetic diversity in Rindera (Boraginaceae-Cynoglosseae) using sequence related amplified polymorphism Xixi Yao1, Haodong Liu2,*, Maede Shahiri Tabarestani3 1 College of Agriculture and Animal Husbandry, Qinghai University, Xining,Qinghai, 810016, China 2 Gansu Polytechnic College of Animal Husbandry & Engineering, Wuwei, Gansu, 733006, China 3 Assistant Professor, Department of Agriculture, Payame Noor University, Tehran, Iran *Corresponding author. E-mail: mindkeeper@126.com; Chunou41@gmail.com Abstract. The genus Rindera comprises about 20–25 species distributed in central east- ern Europe to central Asia. Ninety-five individuals related to six Rindera were collected in 9 provinces. A total of 147 (Number of total loci) (NTL) DNA bands were produced through polymerase chain reaction amplifications (PCR) amplification of six Rindera species. These bands were produced with the combinations of 10 selective primers. The total number of amplified fragments ranged from 8 to 22. ). The predicted unbi- ased heterozygosity (H) varied between 0.15 (Rindera media) and 0.30 (Rindera regia). High Shannon’s information index was detected in Rindera regia. The genetic similari- ties between six species are estimated from 0.73 to 0.95. Clustering results showed two major clusters. According to the SRAP (Sequence-related amplified polymorphism) markers analysis, Rindera regia and Rindera media had the lowest similarity. This study also detected a significant signature of isolation by distance (Mantel test results). Pre- sent results showed that sequence-related amplified polymorphism have the potential to identify and decipher genetic affinity in Rindera species. Current results have impli- cations in biodiversity and conservation programs. Keywords: sequence-related amplified polymorphism, population structure, gene flow, network, genetic admixture, Rindera. INTRODUCTION: Sequence-related amplified polymorphism (SRAP) is PCR –based mark- er 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; Guo, et al. 2021; Cheng, et al. 2021), and SRAP are potential markers to assess plant sys- tematics and genetic diversity studies (Robarts and Wolfe 2014). These past studies showed that molecular markers, including SRAP markers, are effi- cient to investigate genetic diversity analyses and phylogenetic relationship among Paracaryum species in Boraginaceae family. The family Boraginaceae 70 Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani s.str consists of approximately 131 genera and 2,500 spe- cies, mainly distributed in dry, cliffy and sunny habi- tats of Eurasia, the Mediterranean region and the west- ern North America (Binzet and Akcin 2009). They are mainly annual, bi-annual or perennial herbs and shrubs, some trees and a few lianes, distributed throughout the temperate and subtropical regions of the world (Retief and Vanwyk 1997), with a high distribution in Iran (Willis 1973). Given the negative impact of biodiversity threats and over exploitation of Rindera plant species in Iran, it is necessary to conduct genetic diversity studies on Rindera species. Genetic diversity based studies pave our understanding to develop conservation strategies (Esfandani-Bozchaloyi et al. 2017). Subfamily Cynoglossoideae Weigend., is the larg- est subfamily having about 900 species and 50 genera. Recent molecular studies have shown that a wide range of the previously recognized tribes places into this sub- family (Chacón et al. 2016). The subtribe Cynogolossi- nae Dumort. (tribe Cynoglosseae W.D.J.Koch) is entirely restricted to the Old World, with a center of diversity in western Asia and the Mediterranean (Chacón et al. 2016). The genus Rindera Pallas (1771: 486), compris- es about 20–25 species distributed in central eastern Europe to central Asia (Bigazzi et al. 2006). This taxon is closely related to Paracaryum Boissier (1849: 128) and Mattiastrum Brand (1915: 150), nested in Cynoglossum Linnaeus (1753: 134) s.str. (Weigend et al. 2013, Weigend et al. 2016). All species of Rindera are perennial and linked to the dry and continental climate of the steppe and semidesertic belts (Bigazzi et al. 2006). Rindera is represented by 6 species in Iran, 4 of which Rindera albida (Wettst.) Kusn.; Rindera bungei (Boiss.) Gürke; Rindera regia Kusn., rindera media (Turrill) Riedl. are endemic (Khatamsaz 2001). Rindera is characterized by tubular corollas, stamens usually inserted at the throat of the corolla, with a style mostly exserted from the corolla, and usually eglochidiate large mericarpids with a broad, membranous wing (Bigazzi et al. 2006). Rindera species are widely known as “Yünlü gelin” and used as an anti-inflammatory agent in Anatolian folk medicine (Altundag and Ozturk 2001). R. lanata is used to alleviate joint pains in Iranian folk medicine (Mosaddegh et al. 2012). In order to develop conservation strategies and proper utilization of plant genetic resources, it is impor- tant to characterize plant species based on genetic stud- ies (Kharazian et al. 2015), particularly this approach will serve better to understand genotypes of the geo- graphically differentiated genus, such as Echium L. and Onosma (Boraginaceae) (Maria et al. 2007; Dana et al. 2007). The present study investigated the molecular varia- tion of six species in Iran. Objectives of the study were; a) to estimate genetic diversity; b) to evaluate population relationships using WARD approaches. Current results have implications in breeding and conservation pro- grams. MATERIALS AND METHODS: Plants collection Ninety-five (95) individuals were sampled. Six Rindera species in west Azerbaijan, Mazandaran, Hama- dan, Kurdistan, Esfahan, Semnan, Khorasan and Razavi Khorasan Provinces of Iran were selected and sampled during July-August 2018-2020 (Table 1). Morphomet- ric and SRAP analyses on 95 plant accessions were car- ried out. Five to twelve samples from each population belonging to six different species were selected based on other eco-geographic characteristics. Samples were stored at -20 °C till further use. Detailed information about locations of samples and geographical distribution Table 1. List of the investigated taxa including origin of voucher specimens. All material is collected by Majid Khayatneshad. Taxa Locality Latitude Longitude Altitude(m) Rindera albida (Wettst.) Kusn. Kurdestan, Sanandaj Hamedan, 20km s of Nahavand 37°07’48” 49°54’04” 165 Rindera bungei (Boiss.) Gürke Razavi Khorasan, Kashmar, Kuhsorkh District 37°07’08” 49°54’11” 159 Rindera lanata (Lam.) Bunge Kurdestan, Sanandaj Esfahan, Ardestan on road to Taleghan 38°52’93” 47°25’92” 1133 Rindera cyclodonta Bunge Bojnord, Ghorkhod protected area Semnan, 20km NW of Shahrud 38°52’93” 47°25’92” 1139 Rindera regia Kusn v Mazandaran, 40 km Tonekabon to Janat abad Mazandaran, Nowshahr 35°50’36” 51°24’28” 2383 Rindera media (Turrill) Riedl n West-Azarbaijan, Urumieh, Silvana 35°42’29” 52°20’51” 2421 71Morphometric analysis and genetic diversity in Rindera using sequence related amplified polymorphism of species are mentioned (Table 1 and Fig 1). Morphological studies Each species was subjected to morphometric analy- sis and twelve samples per species were processed. Qual- itative (3) and quantitative (4) morphological characters were studied. Data were transformed before calculation. Different morphological characters of flowers, leaves, and seeds were studied. Ordination analyses were con- ducted while using Euclidean distance (Podani 2000). Sequence-related amplified polymorphism method: Fresh leaves were used randomly from one to twelve plants. These were dried with silica gel powder. Genomic DNA was extracted while following previous protocol (Esfandani-Bozchaloyi et al. 2019). SRAP assay was per- formed as described previously (Li and Quiros 2001). Ten 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 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: UPGMA (Unweighted paired group using average) ordination method was implemnented to assess morpho- logical characters. ANOVA (Analysis of variance) was conducted to assess morphological differences among species. Principal component analysis (PCA) was imple- mented to identify variable morphological characters in Rindera species. Multivariate statistical analyses i.e., PC analysis, were performed in PAST software version 2.17 (Hammer et al. 2001). Molecular analyses Sequence-related amplified polymorphism (SRAP) bands were recorded. Presence and absence of bands were scored present (1) and absent (0), respectively. Total loci (NTL) and the number of polymorphism loci (NPL) for each primer were calculated. Furthermore, the polymor- phic ratio was assessed based on NPL/NTL values. Poly- morphism information content was calculated as previ- ously suggested by Roldan-Ruiz et al. (2000). Resolving power for individual marker system was calculated as: Rp = ΣIb. Ib (band informativeness) was estimated while Figure 1. Provinces and collection sites of Rindera species. Table 2. SRAP primer information and results. Primer name NTL NPL P PIC RP Em1-Me1 13 12 92.31% 0.44 43.77 Em2-Me2 12 12 100.00% 0.66 36.77 Em1-Me4 18 17 94.4% 0.43 40.46 Em2-Me4 15 15 100.00% 0.49 33.76 Em2-Me5 8 8 100.00% 0.44 50.99 Em3-Me4 10 10 100.00% 0.41 32.24 Em3-Me1 24 19 79.00% 0.30 26.55 Em4-Me1 11 11 100.00% 0.44 44.23 Em5-Me1 16 16 100.00% 0.47 38.55 Em5-Me2 22 22 100.00% 0.35 29.65 Mean 16 15 94.00% 0.48 37.55 Total 147 133 359.85 Abbreviations: NTL = Number of total loci; NPL = Number of pol- ymorphic loci; P = Polymorphic ratio; PIC = Polymorphic informa- tion content; RP = Resolving power. 72 Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani following equation: proposed as: Ib= 1 - [2 x (0.5-p)]. In the equation, p indicates the presence of bands (Prevost and Wilkinson, 1999). Pairwise genetic similarity between species was evaluated to reveal genetic affinity between species (Jaccard, 1908). Unbiased expected heterozygo- sity and Shannon information index were calculated in GenAlEx 6.4 software (Peakall and Smouse, 2006). Gene flow was conducted in POPGENE software, version 1.32 (Yeh et al. 1999). Analysis of molecular variance test was conducted in GenAlEx (Peakall and Smouse 2006). Man- tet test was performed with 5000 permutations in PAST, version 2.17 (Hammer et al. 2001). The comparison of genetic divergence or genetic distances, estimated by pair- wise FST and related statistics, with geographical distanc- es by Mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. The Mantel test, as originally formulated in 1967, Zm = gij × dij where gij and dij are, respectively, the genetic and geo- graphic distances between populations i and j, consid- ering populations. Because Zmis given by the sum of products distances its value depends on how many pop- ulations are studied, as well as the magnitude of their distances. The Zm-value can be compared with a null distribution, and Mantel originally proposed to test it by the standard normal deviate (SND), given by SND =Zm/ var(Zm)1/2 (Mantel 1967). These analyses were done by PAST ver. 2.17 (Hammer et al. 2012), DARwin ver. 5 (2012) software. RESULTS Mophometery The ANOVA findings showed substantial differ- ences (p<0.01) between the species in terms of quantita- tive morphological characteristics. Principal component analysis results explained 55% cumulative variation. The first PCA axis explained 40% of the total variation. The highest correlation (> 0.7) was shown by morphologi- cal characters such as calyx length, calyx width, corolla length, corolla color. The morphological characters of Rindera species are shown in WARD tree (Fig. 2). Each species formed separate groups based on morphologi- cal characters. The morphometric analysis showed clear difference among Rindera species and separated each groups. In Rindera albida and R. bungei nutlets are 8–14 mm, two-winged; outer wing 3 mm broad, margin undulate, inner 2 mm broad, incurved, margin cristate- dentate, glochids entirely absent, while in R. lanata, R. cyclodonta nutlets are 15.8–23 mm, smooth, wing with smooth or undulate often blue margin, without glochids. Species identification and genetic diversity Ten (10) suitable primer combinations (PCs), out of 25 PCs were screened in this research. Figure 3 illustrates the banding pattern of Em3-Me4, Em1-Me4, Em5-Me2 and Em1-Me1 primer by the SRAP marker profile. One hundered and thirty three (133) amplified polymorphic bands (number of polymorphic loci) were produced. These bands (fragments) had different range i.e. 150bp to 3000 bp. Maximum and minimum numbers of poly- morphic bands were 22 and 8 for Em5-Me2 and 8 Em2- Me5, respectively. Each primer produced 15 polymorphic bands on average. The PIC ranged from 0.30 (Em3-Me1) to 0.66 (Em2-Me2) for the 10 SRAP primers, with an average of 0.48 per primer. RP of the primers ranged Figure 2. Morphological characters analysis of Rindera species by WARD. Figure 3. Electrophoresis gel of studied ecotypes from DNA frag- ments produced by SRAP profile; 1,7,14,20: Rindera albida ; 2, 8,15,21: Rindera bungei ; 3,9, 16, 22: Rindera lanata; 4, 10, 17, 23: R. cyclodonta;; 5, 11, 18, 24: Rindera regia and 6, 12-13, 19, 25-26: Rindera media; L = Ladder 100 bp. 73Morphometric analysis and genetic diversity in Rindera using sequence related amplified polymorphism from 26.55 (Em3-Me1) to 50.99 (Em2-Me5) with an aver- age of 37.55 per primer (Fig. 3, Table 3). The calculated genetic parameters of Rindera species are shown (Table 3). The unbiased heterozygosity (H) varied between 0.15 (Rindera media) and 0.30 (Rindera regia) with a mean of 0.23. Shannon’s information index (I) was maximum in Rindera regia (0.37), where as we recorded minimum Shannon’s information index in Rindera media (0.18). The observed number of alleles (Na) ranged from 0.299 in Rindera albida to 1.155 in Rindera cyclodonta. The sig- nificant number of alleles (Ne) ranged from 1.016 (Rinde- ra lanata) to 1.440 (Rindera regia). Analysis of Molecular Variance results in significant genetic difference (p = 0.01) among Rindera species. The majority of genetic variation occurred among species. AMOVA findings revealed that 82% of the total variation was between species and comparatively less genetic vari- ation was recorded at the species level (Table 4). Genetic difference between Rindera species was highlighted by genetic statistics (Nei’s GST), as evident by significant p values i.e. Nei’s GST (0.66, p = 0.01) and D_est values (0.122, p = 0.01) .Mantel test after 5000 permutations produced significant correlation between genetic distance and geographical distance in these populations (r = 0.77, P = 0.001). Therefore, the populations that are geographi- Table 3. Genetic diversity parameters. SP N Na Ne I He UHe P Rindera lanata 8.000 0.333 1.016 0.192 0.17 0.22 48.23% R. cyclodonta 12.000 1.155 1.190 0.271 0.184 0.192 55.91% R. regia 5.000 0.358 1.440 0.374 0.30 0.29 66.50% R. albida 6.000 0.299 1.029 0.231 0.18 0.23 44.38% R. bungei 5.000 0.462 1.095 0.288 0.25 0.22 62.05% R. media 5.000 0.358 1.117 0.18 0.15 0.12 34.30% Abbreviations: N = number of samples, Na= number of different alleles; Ne = number of effective alleles, I= Shannon’s information index, He = genetic diversity, UHe = unbiased gene diversity, P = percentage of polymorphism, populations. Table 4. Molecular variance analysis. Source df SS MS Est. Var. % ΦPT Among Pops 30 1501.364 92.789 16.154 82% 82% Within Pops 100 334.443 3.88 2.888 18% Total 130 1955.807 20.060 100% 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). Figure 4. Dendrograms of Rindera species. 74 Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani cally more distant have less amount of gene flow, and we have isolation by distance (IBD) in the Rindera. The constructed dendrogram highlighted two major clusters (Fig. 4). Group A consisted of 3 species Rinde- ra lanata; R. cyclodonta and Rindera regia .Two sub- clusters were in the B group: three species of Rindera bungei, Rindera albida and Rindera media. We detected strong correlation between geographi- cal and genetic distances (r = 0.22, p=0.0002) and gene flow (Nm) score of 0.356 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 (0.95) between Rindera lanata and R. cyclodonta. On the contrary to this, Rindera regia and Rindera media (0.73) had lowest genetic resemblance. DISCUSSION In the present study, we used morphological and molecular (SRAP) data to evaluate species relationships in Rindera species. Morphological analyses of Rindera species showed that quantitative indicators (ANOVA test results) and qualitative characteristics are well dif- ferentiated from each other. PCA analysis suggests that morphological characters such as corolla color, nutlet shape, nutlet length, stamens position, nutlet margin, nutlet disc have the potentials to identify and delimitate Rindera species. Principal component analysis results suggests the utilization of morphological characters to identify and delimitate Rindera species. Morphological characters including nutlet shape, nutlet length, stamens position, nutlet margin play key role in plant systemat- ics and taxonomy. Our work also highlighted the signifi- cance of morphological characters and molecular data to identify and study species genetic 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 differ- ence among species. These results indicate that SRAP have potentials to study plant systematics and taxonomy in Rindera members. Genetic diversity studies are conducted through appropriate selection of primers and indexes includ- ing Polymorphic information content (PIC) and marker index (MI) are important indexes to fathom genetic variation in species (Sivaprakash et al. 2004). Com- mon logic suggests 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 0.30 to 0.66, with a mean value of 0.48. PIC values indeed show low and high genetic diver- sity among genotypes. Values are ranging from zero to 0.25 show low genetic diversity; in contrast to this, 0.25 to 0.50 highlight mid-level of genetic diversity. In addi- tion 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 esti- mate genetic diversity in Rindera species. In our study, SRAP markers detected average percentage of polymor- phism (94%). Current research results also described average PIC values of SRAP makers (0.48) and average RP (resolving power) values i.e. 37.55 of SRAP mark- ers. These current reported values are higher than other reported markers on Rindera species (Maria et al. 2007; Dana et al. 2007). In the recent study, low gene flow (Nm) was detected among Rindera species. Despite the pres- ence of limited gene flow in Rindera species, two dis- tinct ecotypes were reported previously. These ecotypes were formed due to reproductive isolation caused by alti- tude gradient and different niches (Moein et al., 2019). The present study also depicted a significant correlation between genetic and geographical distances. Our find- ings revealed that isolation by distance (IBD) existed between Rindera species (Mantet test results). Several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentia- tion (Frichot et al. 2013; De Kort et al. 2014; Zhang et al. 2021; Zheng et al. 2021; Guo et al. 2021). The magnitude of variability among Na, Ne, H, and I indices demon- strated a high level of genetic diversity among Rindera species. Dendrogram and principal component analysis results showed clear difference among Rindera species . This shows the high utilization of the SRAP technique to identify Salvia species. Our results have implications for conservation and breeding programs. Furthermore, it may identify suitable ecotypes for forage and pasture. CONCLUSIONS The present study investigated the molecular vari- ation of six species. Molecular and morphometric analysis confirmed morphological and genetical dif- ference between Rindera 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. These two major groups were separated on the basis of genetic and morphological characters. The genetic simi- larities between six species was estimated from 0.73 to 75Morphometric analysis and genetic diversity in Rindera using sequence related amplified polymorphism 0.95. SRAP (Sequence-related amplified polymorphism) markers analysis, showed that Rindera regia and Rinde- ra media had the lowest similarity. Current study also reported correlation between genetic and geographi- cal distances. This clearly indicated isolation mecha- nism envloved in the ecology of Rindera species. Pre- sent results indicated the potential of sequence-related amplified polymorphism to assess genetic diversity and genetic affinitiy among Rindera species. Current results have implications in biodiversity and conservation pro- grams. Besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in Iran. ACKNOWLEDGEMENT The authors are grateful for the support by Grants from Research start up fund project of Qinghai Univer- sity (41510406) and Innovation Fund for Higher Educa- tion of Gansu Province (2021A-270). REFERENCES Altundag E, Ozturk M 2001. Ethnomedicinal studies on the plant resources of east Anatolia, Turkey. Procedia Soc Behav Sci. 19:756–77. Bigazzi M, Nardi E, Selvi F 2006. Palynological Contri- bution to the Systematics of Rindera and the Allied Genera Paracaryum and Solenanthus (Boraginaceae- Cynoglosseae). Willdenowia 36: 37–46. https://doi. org/10.3372/wi.36.36103 Binzet R, Akcin OE 2009.. Nutlet size, shape and surface ornamentation in 14 Onosma species (Boraginaceae). Acta Botanica Croatica 68: 117–126. Bi D., C. Dan, M. Khayatnezhad, Z. Sayyah Hashjin, Z. Li and Y. Ma 2021. Molecular Identification and Genetic Diversity In Hypericum L.: A High Value Medicinal Plant Using Rapd Markers Markers. Genetika 53(1): 393-405. Cheng, X., X. Hong, M. Khayatnezhad and F. Ullah 2021. Genetic diversity and comparative study of genomic DNA extraction protocols in Tamarix L. species. Car- yologia 74(2): 131-139. Chacón et al. 2016. The borage family (Boraginaceae s.str.): A revised infrafamilial classification based on new phylogenetic evidence, with emphasis on the placement of some enigmatic genera, Taxon 65: 523– 546. https://doi.org/10.12705/653.6 Cires E, De Smet Y, Cuesta C, Goetghebeur P, Shar- rock S, Gibbs D, Oldfield S, Kramer A, Samain M-S. 2013. Gap analyses to support ex situ conservation of genetic diversity in Magnolia, a flagship group. Biodi- vers Conserv. 22(3):567-590. De Kort H, Vandepitte K, Mergeay J, Honnay O 2014. Isolation, characterization and genotyping of single nucleotide polymorphisms in the non-model tree species Frangula alnus (Rhamnaceae). Conserva- tion Genetics Resources 6(2):267-269. https://doi. org/10.1007/s12686-013-0083-6 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. Cytol Genet. 52(4):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. 71(4):1-14. Esfandani-Bozchaloyi S, Sheidai M, Kalalegh M 2019. Comparison of DNA extraction methods from Gera- nium (Geraniaceae). Acta Bot. Hung. 61(3-4):251- 266. 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):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. Esfandani-Bozchaloyi S, Sheidai M, Keshavarzi M, Noor- mohammadi Z. 2017. Genetic and morphological diversity in Geranium dissectum (Sec. Dissecta, Gera- niaceae) populations. Biologia. 72(10):1121- 1130. Frankham R 2005. Stress and adaptation in conservation genetics. J Evol Biol. 18(4):750-755. Frichot E, Schoville SD, Bouchard G, François O 2013. Testing for Associations between Loci and Environ- mental Gradients Using Latent Factor Mixed Models. Molecular Biology and Evolution 30(7):1687-1699. https://doi.org/10.1093/molbev/mst063 Guo, L.-N., C. She, D.-B. Kong, S.-L. Yan, Y.-P. Xu, M. Khayatnezhad And F. Gholinia 2021. Prediction of the effects of climate change on hydroelectric gen- eration, electricity demand, and emissions of green- house gases under climatic scenarios and optimized ANN model. Energy Reports 7: 5431-5445. Hou, R., S. Li, M. Wu, G. Ren, W. Gao, M. Khayatnezhad And F. Gholinia 2021. Assessing of impact climate parameters on the gap between hydropower supply and electricity demand by RCPs scenarios and opti- 76 Xixi Yao, Haodong Liu, Maede Shahiri Tabarestani mized ANN by the improved Pathfinder (IPF) algo- rithm. Energy 237: 121621 Hammer O, Harper D, Ryan P 2001. PAST: Paleontological Statistics Soft- ware Package for Education and Data Analysis. Pal- aeontologia Electronica 4(1):1-9. Khatamsaz M 2001. Pollen morphology of Iranian Bor- aginaceae family and its taxonomic significance. Iran. J. Bot. 9, 27–40. Jaccard P 1908. Nouvelles Recherches Sur la Distribution Florale. Bulletin de la Societe Vaudoise des Sciences Naturelles 44(163):223-270. https://doi.org/ 10.5169/ seals-268384 Kharazian N, Rahimi S, Shiran B 2015. Genetic diver- sity and morphological variability of fifteen Stachys (Lamiaceae) species from Iran using morphological and ISSR molecular markers. Biologia 70(4):438-452. https://doi.org/10.1515/biolog-2015-0051 Li G, Quiros CF2001. Sequence-related amplified polymor- phism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tag- ging in Brassica. Theoretical and Applied Genetics103(2): 455-461. https://doi.org/ 10.1007/s001220100570 Moein F, Jamzad Z, Rahiminejad M 2019. An integrat- ing study of genetic diversity and ecological niche modelling in Salvia aristata (Lamiaceae). Acta Botanica Hungarica 61(1-2):185-204. https://doi. org/10.1556/034.61.2019.1-2.10 Podani J 2000. Introduction to the exploration of multi- variate data. Backhuyes, Leide, Netherlands. Prevost A, Wilkinson MJ 1999. A new system of com- paring PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics 98(1):107-112. https://doi.org/10.1007/s001220051046 Peakall R, Smouse PE 2006. GENALEX 6: Genetic Analy- sis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6(1):288-295. https://doi.org/10.1111/j.1471-8286.2005.01155.x Retief E, Vanwyk AE 1997. Palynology of southern Afri- can Boraginaceae: the genera Lobostemon, Echios- tachys and Echium. Grana 36: 271–278. Robarts DWH, Wolfe AD 2014. Sequence-related ampli- fied polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology. Applications in Plant Sciences 2(7):apps.1400017. htt- ps://doi.org/10.3732/apps.1400017 Roldán-Ruiz I, Dendauw J, Van Bockstaele E, Depick- er A, De Loose M 2000. AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.). Molecular Breeding 6(2): 125-134. https://doi. org/10.1023/A:1009680614564 Saebnazar A, Rahmani F 2013. Genetic Variation Among Salvia Species Based on Sequence-Related Ampli- fied Polymorphism (SRAP) Marker. Journal of Plant Physiology and Breeding 3(1):71-78. Sivaprakash KR, Prashanth SR, Mohanty BP, Parida A 2004. Genetic diversity of black gram (Vigna mungo) landraces as evaluated by amplified fragment length polymorphism markers. Current Science 86(10): 1411-1416. Talebi M, Rahimmalek M, Norouzi M 2015. Genetic diversity of Thymus daenensis subsp. daenensis using SRAP markers. Biologia 70(4):453-459. https://doi. org/10.1515/biolog-2015-0059. 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 Breeding 124(2):154-160. https:// doi.org/10.1111/j.1439-0523.2004.01047.x Wu Y-G, Guo Q-S, He J-C, Lin Y-F, Luo L-J, Liu G-D 2010. Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markers. Biochemical Systematics and Ecology 38(1):63-72. https://doi.org/10.1016/j. bse.2009.12.006 Weigend M, Gottschling M, Selvi F., Hilger HH 2009. Marble seeds are gromwells –systematics and evolu- tion of Lithospermum and allies (Boraginaceae tribe Lithospermeae) based on molecular and morphologi- cal data. Molecular Phylogenetics and Evolution 52: 755–768. DOI: 10.1016/j.ympev.2009.05.013 Weigend M, Luebert F, Selvi F, Brokamp G. Hilger HH 2013. Multiple origins for Hound’s tongues (Cyno- glossum L.) and Navel seeds (Omphalodes Mill.). The phylogeny of the borage family (Boraginaceae s.str.). Molecular phylogenetics and evolution 68: 604–618. http://dx.doi.org/10.1016/j.ympev.2013.04.009 Willis JC 1973. A dictionary of the flowering plants and ferns. University Press, Cambridge 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. Zhang, H., M. Khayatnezhad and A. Davarpanah 2021. Experimental investigation on the application of car- bon dioxide adsorption for a shale reservoir. Energy Science & Engineering n/a(n/a). Zheng, R., S. Zhao, M. Khayyatnezhad and S. Afzal Shah 2021. Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers. Caryologia 74(2): 45-56. Zhu, K., L. Liu, S. Li, B. Li, M. Khayatnezhad and A. Sha- koor 2021. Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa. Caryologia 74(2): 121-130. 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