Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 149-161, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1235 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Jing Ma, Wenyan Fan, Shu- jun Jiang, Xiling Yang, Wenshuai Li, Di Zhou, Amir Abbas Minaeifar (2021) Molecular techniques in the assess- ment of genetic relationships between populations of Consolida (Ranuncu- laceae). Caryologia 74(2): 149-161. doi: 10.36253/caryologia-1235 Received: March 05, 2021 Accepted: July 20, 2021 Published: October 08, 2021 Copyright: © 2021 Jing Ma, Wenyan Fan, Shujun Jiang, Xiling Yang, Wensh- uai Li, Di Zhou, Amir Abbas Minaeifar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) 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. Molecular techniques in the assessment of genetic relationships between populations of Consolida (Ranunculaceae) Jing Ma1, Wenyan Fan1,*, Shujun Jiang1, Xiling Yang1, Wenshuai Li1, Di Zhou1, Amir Abbas Minaeifar2,* 1.Agronomy College, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China 2 Department of Biology, Payame Noor University, P.O. Box19395-3697 Tehran, Iran Corresponding author. E-mail: fwyjsj2021@126.com; aaminaeifar@pnu.ac.ir Abstract. Genetic diversity studies are essential to understand the conservation and management of plant resources in any environment. The genus Consolida (DC.) Gray (Ranuculaceae) belongs to tribe Delphinieae. It comprises approximately 52 species, including the members of the genus Aconitella Spach. No detailed Random Amplified Polymorphic DNA (RAPD) studies were conducted to study Consolida genetic diversi- ty. Therefore, we collected and analyzed 19 species from 12 provinces of regions. Over- all, one hundred and twenty-seven plant specimens were collected. We showed signifi- cant differences in quantitative morphological characters in plant species. Unweighted pair group method with arithmetic mean and principal component analysis (PCA) divided Consolida species into two groups. All primers produced polymorphic ampli- cons though the extent of polymorphism varied with each primer. The primer OPA- 06 was found to be most powerful and efficient as it generated a total of 24 bands of which 24 were polymorphic. The Mantel test showed correlation (r = 0.34, p=0.0002) between genetic and geographical distances. We reported high genetic diversity, which clearly shows the Consolida species can adapt to changing environments since high genetic diversity is linked to species adaptability. Present results highlighted the utility of RAPD markers and morphometry methods to investigate genetic diversity in Con- solida species. Our aims were 1) to assess genetic diversity among Consolida species 2) is there a correlation between species genetic and geographical distance? 3) Genetic structure of populations and taxa. Keywords: Consolida, population structure, gene flow, network, genetic admixture. INTRODUCTION Genetic diversity is a vital feature that helps plant species survive in an ever-changing environment, and it sheds light on understanding the phylo- genetic affinity among the species (Erbano et al. 2015; Ellegren and Galtier 2016; Turchetto et al. 2016 ). Quite a significant number of genetic resources and materials programs of plant species have been carried out to preserve 150 Jing Ma et al. the plant species worldwide. Scientific data indicate that genetic diversity plays a pivotal role in conservation pro- grams (Gomez et al. 2005; Frankham 2005; Cires et al. 2013). The genus Consolida (DC.) Gray (Ranuculaceae) belongs to tribe Delphinieae. It comprises approximately 52 species, including the members of the genus Aconi- tella Spach. Iran is one of the richest countries for the genus in South-West Asia, since it has 24 species (Iran- shahr et al., 1992). The genus Consolida S.F. Gray was considered as a separate genus based on one species (C. regalis) by Gray (1821), who worked on British flora. But some research- ers considered Consolida as a section of Delphinium (De Candole 1824; Boissier 1867; Huth 1895; Nevskii 1937). Unlike the others based on annual life form, sin- gle spured petal, single follicle compared to 3 or 5 sessile follicles of Delphinium recognized Consolida as a sepa- rate genus (Tutin et al. 1964; Davis 1965; Munz 1967; Hayek 1970; Iranshahr 1992; Ertugrul et al. 2016; Khalaj 2013). Kemularia-Nathades (1939) recognized a new genus Aconitopsis from species of Consolida based on peculiar formation of the petal, upper sepal, and spur. The name Aconitopsis was later rejected by Sojak (1969) and being replaced by Aconitella because of nomencla- ture priority. Some researchers have studied these genera taxonomically (Soo 1922; Munz 1967 ; Davis 1965; Iran- shahr et al., 1992; Constantinidis et al., 2001). Consolida has about 40 species, of which 19 have been recorded from Iran. Aconitella with ca. 10 species (3 species in Iran) and 31 species of Delphinium (species in Iran) are centred in Irano-Turanian and Mediterranean phyto- geographic regions (Trifonova, 1990; Hasanzadeh et al. 2017). Consolida has been separated from Delphinium by De Candolle based on single spurred petals, one follicle and annual life cycle and has occurred in separate sec- tion. Finally, it introduced as a separate genus by Gray in 1821 (Triffonova, 1990). Based on phylogenetic studies of Jabbour and Renner (2011), Aconitella is part of Consol- ida, both being embedded in Delphinium. The Jabbour & Renner (2011) results showed that Consolida diverged from Delphinium relatives in the Early to Middle Mio- cene, a period of increasing aridity, caused primarily by decrease in sea level in the Mediterranean (Hayek 1970; Iranshahr 1992; Ertugrul et al. 2016) and desertification in Asia (Triffonova 1990). Some biosystematic studies have carried out in vari- ous field such as chromosomal studies (Trifonova 1990; Koeva 1992; Hong, 1986) chemical studies (Aitzetmul- ler et al.1999), palynological studies (Munz, 1967) and phylogenetic investigations by using DNA sequence data (Johansson 1995; RO et al.1997; Jabbour and Ren- ner 2011; 2012; Yosefzadeh et al. , 2012). In the recent molecular studies (Jabbour and Renner 2001; 2012) it was showed that Consolida and Aconitella form a clade embeded in Delphinium and also Aconitella is embedded within Consolida. The Jabbour and Renner (2011) results showed that Consolida diverged from Delphinium rela- tives at least in the early of middle Miocene. Genetic diversity studies are usually tapped due to molecular markers. Molecular markers are an excellent method to disentangle phylogenetic association between species and population. Among molecular methods or markers, RAPD (Random Amplified Polymorphic DNA) are sensitive to detect variability among indi- viduals of species. RAPD method is cost-effective and can work with limited sample quantities. In addition to this, RAPD can amplify and target genomic regions with potential and several markers (Esfandani-Bozch- aloyi et al. 2017a). Taxonomical Systematics studies were conducted in the past to identify the Consolida species. According to the best of our knowledge, there is no existing RAPD data on genetic diversity investigations in Iran. We studied one hundred and twenty-seven sam- ples. Our aims were 1) to assess genetic diversity among Consolida species 2) is there a correlation between spe- cies and geographical distance? 3) Genetic structure of populations and taxa 4) Are the Consolida species able to exchange genes? MATERIALS AND METHODS Plant materials 19 Consolida species were collected from different regions of Iran (Table 1). These species were studied via morphological and molecular methods. 127 plant sam- ples (10-25 per plant species) were examined for mor- phometry purposes (Figure 1). The random amplified polymorphic DNA analysis method was limited to 110 samples. According to previous references, all the species were identified (Iranshahr, 1992; Ertugrul et al., 2016; Khalaj, 2013). Voucher specimens were deposited in Her- barium of Azad Islamic University (HAIU). Morphometry We studied 18 qualitative and 7 quantitative mor- phological characters (Table 2). Data were transformed (Mean= 0, variance = 1) prior to ordination . Euclidean distance was implemented to cluster and ordinate plant species (Podani 2000). 151Molecular techniques in the assessment of genetic relationships between populations of Consolida Random Amplified Polymorphic DNA We extracted DNA from fresh leaves. Leaves were dried. DNA extraction was carried out according to the previous protocol (Esfandani-Bozchaloyi et al. 2019; Niu et al., 2021; Sun et al., 2021). DNA quality was checked on an agarose gel to confirm the purity. We amplified the DNA with the aid of RAPD primers (Operon tech- nology, Alameda, Canada). These primers belonged to OPA, OPB, OPC, OPD sets. We selected those primers (10) which could show clear bands and polymorphism (Table 3). Overall, the polymerase chain reaction con- tained 25μl volume. This 25 volume had ten mM Tris- HCl buffer, 500 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP; 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Ger- many). We observed the following cycles and conditions for the amplification. Five minutes initial denaturation step was carried out at 94°C after this forty cycles of 1 minute at 94°C were observed. Then 1-minute cycle was at 52-57°C followed by two minutes at 72°C. In the end, the final extension step was performed for seven to ten minutes at 72°C. We confirmed the amplification steps while observing amplified products on a gel. Each band size was confirmed according to 100 base pair molecular ladder/standard (Fermentas, Germany). Data analyses Ordination methods such as multidimensional scal- ing and principal coordinate analysis were also per- formed (Podani 2000). The morphological difference among species and population was assessed through analysis of variance (ANOVA). PCA analysis (Podani 2000) was done to find the variation in plant popula- tion morphological traits. Multivariate and all the neces- sary calculations were done in the PAST software, 2.17 (Hammer et al. 2001). To assess genetic diversity, we encoded RAPD bands as present and absent. Numbers 1 and 0 were used to show the presence and absence of bands. It is essential to know the polymorphism infor- Table 1. Location and herbarium accession numbers of the studied populations of Consolida species collected by Mehri in Iran. No Sp. Locality Latitude Longitude Altitude (m) Sp1 C. tehranica (Boiss.) Rech.f. Tehran: Damavand Tehran: Rodehen Golestan, Ramian 38°52’37” 32°50’03” 35°50’03” 47°23’92” 51°52’08” 48°52’08” 1144 1066 1234 Sp2 C. camptocarpa (Fisch. &C.A.Mey.) Nevski Khorassan: Sarakhs, 14 km to Mozduran 32°50’03” 51°24’28” 1990 Sp3 C. lorestanica lRANSHAHR, Lorestan: 110 km Khorram abad Markazi:Arak 29°20’07” 36°14’14” 51°52’08” 51°18’07” 1610 1807 Sp4 C. leptocarpa Nevski Golestan: Golestan national park, Mirzabailoo 38°52’37” 47°23’92” 1144 Sp5 C. persica (Boiss.) Grossh. Fars: Bamo national park Fars: Shiraz Keramn: Jiroft Zanjan: Abhar 33°57’12” 47°57’32” 2500 Sp6 C. aucheri (Boiss.) Iranshahr Khorassan: Neyshabur 34°52’373 48°23’92” 2200 Sp7 C. anthoroidea (Boiss.) Schrödinger East Azerbaijan: kaleybar, Cheshme Ali Akbar 38°52’373 47°23’92” 1144 Sp8 C. hohenackeri (Boiss.) Grossh. Arak: Komayjan, Pass of Chehregan village, the margin road 35°50’03” 51°24’28” 1700 Sp9 C. stocksiana Nevski Golestan: Golestan national park, Mirzabailoo 36°14’14” 51°18’07” 1807 Sp10 C. rugulosa Schrödinger Esfahan: Semirom to Keikha 32°36’93” 51°27’90” 2500 Sp11 C. ambigua (L.) Ball & Heywood Tehran: Between Karaj and Eshtehard 37°07’02” 49°44’32” 48 Sp12 C. orientalis (Gray) Schrödinger Azarbaijan: 20 km from Jolfa to Marand 28°57’22” 51°28’31” 430 Sp13 C. regalis S.F. Gray Azarbaijan: Tabriz 30°07’24” 53°59’06” 2178 Sp14 C. oliveriana (DC.)Schrod. Kermanshah: 31 km to Ghasre-shirin 28°57’22” 51°28’31” 288 Sp15 C. flava (DC.)Schrod Khuzestan: Do-gonbadan 34°46’10” 48°30’00” 1870 Sp16 C. trigonelloides (Boiss.) Munz Fars: Bamo national park 35° 37’77” 46°20’25” 1888 Sp17 C. oligantha (Boiss.)Schrod Ardabil 33°47’60” 46°07’58” 1250 Sp18 C. linorioides (BOIss.) MUNZ, Esfahan: Ghamishloo protected area 37°07’02” 49°44’32” 48 Sp19 C. rugulosa f. paradoxa (Bunge) Iranshahr Hamedan: Khan Abad 28°57’22” 51°28’31” 288 152 Jing Ma et al. mation content and marker index (MI) of primers because these parameters serve to observe polymorphic loci in genotypes (Ismail et al. 2019). Marker index was calculated according to the previous protocol (Heikru- jam et al. 2015). Other parameters such as the number of polymorphic bands (NPB) and effective multiplex ratio (EMR) were assessed. Gene diversity associated characteristics of plant samples were calculated. These characteristics include Nei’s gene diversity (H), Shan- non information index (I), number of effective alleles (Ne), and percentage of polymorphism (P% = number of polymorphic loci/number of total loci) (Shen et al. 2017). Unbiased expected heterozygosity (UHe), and heterozy- gosity were assessed in GenAlEx 6.4 software (Peakall and Smouse 2006). Neighbor-joining (NJ) and network- ing were studied to fathom genetic distance plant popu- lations (Huson and Bryant 2006; Freeland et al. 2011). The comparison of genetic divergence or genetic distanc- es, estimated by pairwise FST and related statistics, with geographical distances by Mantel test is one of the most popular approaches to evaluate spatial processes driv- ing population structure. The Mantel test was performe as implemented in PAST. For this, Nei genetic distance was determined for RAPD data, while Geographic dis- tance of PAST was determined for geographical data. It is calculated based on the sum of the paired differences among both longitude as well as latitude coordinates of the studied populations (Podani 2000). As we were inter- ested in knowing the genetic structure and diversity, we also investigated the genetic difference between popula- tions through AMOVA (Analysis of molecular variance) in GenAlEx 6.4 (Peakall and Smouse 2006). Gene flow (Nm) which were calculated using POPGENE (version 1.31) program [Yeh et al. 1999]. Gene flow was estimated indirectly using the formula: Nm = 0_25(1 _ FST)/FST. We also did STRUCTURE analysis to detect an optimum number of groups. For this purpose, the Evan- no test was conducted (Evanno et al. 2005). RESULTS Morphometry Significant ANOVA results (P <0.01) showed differ- ences in quantitative morphological characters in plant species. Principal component results explained 80% vari- ation. Firs component of PCA demonstrated 57% of the total variation. Traits such as presence of petiole in cau- lin leaves, overtopping the bract from fruit, proportion of petal middle lobes to lateral lobes, presence of hair on the filament positively correlated with firs compo- nent (>0.7). The second and third components explained characters such as number of petal lobes, position of hair on filament, colour of anther, shape of follicle beak, shape of follicle. Unweighted pair group method with arithmetic mean (UPGMA) and principal component analysis (PCA) plots showed symmetrical results (Figure 2). Generally, plant specimens belonging to different spe- cies were separated from each other due to differences in morphology. Our PCA results also confirmed the appli- cation of morphological characters in separating and clustering the species in separate groups (Figure 2). Species identification and genetic diversity The primers, i.e., OPD-05, could amplify plant (Con- solida ) DNA (Figure 3). 133 polymorphic bands were generated and amplified. Amplified products ranged from 100 to 3000 bp. We recorded the highest polymor- phic bands for OPA-06. OPD-08 had the lowest poly- morphic bands. The average polymorphic bands ranged to 13.3 for each primer. The polymorphic information content (PIC) had values in the range of 0.38 (OPC-04) to 0.57 (OPB-02). Primers had 0.52 average polymorphic information content values. Marker index (MI) values were 4.18 (OPD-05) to 8.87 (OPA-06), with an average of 6.87 per primer. Effec- Figure 1. Map of distribtion of populations Consolida species in Iran; sp1= C. tehranica; sp2= C. camptocarpa; sp3= C. lorestani- ca; sp4= C. leptocarpa; sp5= C. persica; sp 6= C. aucheri; sp7= C. anthoroidea; sp8= C. hohenackeri; sp9= C. stocksiana; sp10: C. rugu- losa; sp11: C. ambigua ; sp12= C. orientalis; sp13= C. regalis; sp14= C. oliveriana; sp15= C. flava; sp16= C. trigonelloides; sp17= C. oli- gantha; sp18= C. linorioides; sp19= C. rugulosa f. paradoxa. 153Molecular techniques in the assessment of genetic relationships between populations of Consolida tive multiplex ratio (EMR) values are useful to distin- guish genotypes. In our study, we reported 9.34 (OPD- 08) to 16.55 (OPA-05) EMR values. EMR values averaged 13.57 per primer (Table 3). All the necessary genetic fea- tures calculated of 19 Consolida species are shown (Table 4). C. linorioides depicted unbiased expected heterozy- gosity (UHe) in the range of 0.15. C. orientalis showed a 0.34. UHe value heterozygosity had a mean value of 0.23 in overall Consolida species. Shannon information was high (0.32) in C. orientalis. C. linorioides showed the lowest value, 0.20. Mean values for Shannon information was 0.22. The observed number of alleles (Na) ranged from 0.201 to 0.555 in C. regalis and C. oligantha. The effective number of alleles (Ne) was in the range of 0.67- 1.876 for C. flava and C. leptocarpa. Analysis of Molecular Variance (AMOVA) test high- lighted genetic differences among Consolida species (P = 0.01). AMOVA revealed significant difference among the studied populations. It also revealed that, 46% of total genetic variability was due to within population diversi- ty and 54% was due to among population genetic differ- entiation (Figure 4). Genetic similarity and dissimilarity assessed through Genetic statistics (GST) showed signifi- cant differences i.e., (0.77, P = 0.001) and D_est values (0.256, p = 0.01). The neighbor-joining tree also revealed two major groups (Figure 5). The neighbor-joining tree also repeat- ed the same pattern as indicated in figures 2. In current work, molecular findings also coincided with the tradi- tional taxonomical (morphology) approaches for Con- solida species. The neighbor-joining tree divided Consolida species into two groups (Figure 4). Populations belonging to C. tehranica; C. camptocarpa; C. lorestanica; C. aucheri; C. rugulosa; C. orientalis and C. hohenackeri were in the first group. On the other hand, the second group con- sisted of two sub-groups. C. stocksiana; C. ambigua ; C. oliveriana; C. flava formed the first sub-group. C. trigo- nelloides; C. oligantha; C. linorioides; C. leptocarpa and C. persica formed the second sub-group. These groups Table 2. Characters used in this study from Iran. Character Character states Length of basal leaves 0: <55 mm 1: <55mm Number of bracts 0: 0 1: 1 2: 2 Broad of petal 0: 3-9 mm 1: 8-16 mm Number of bracteole 0: variable 1: constant Length of bracteole 0: ≤ 9mm 1: ≥12 mm Length of spure 0: ≤ 25 mm 1: ≥ 25 mm Shape of spure 0: curved 1: erect Hair on lateral sepal 0: scattered 1: on the middle vein Number of petal lobes 0: 5 1: 3 Proportion of petal middle lobes to lateral lobes 0: equal 1: shorter 2: longer Hair on the filament 0: absent 1: present Hair on filament 0: wing 1: total of filament Colour of anther 0: brown 2: yellow Shape of follicle beak 0: erect 1: curved Shape of follicle 0: falciform 1: erect Hair on the follicle surface 0: absent 1: present Shape of fruit stalk 0: antrorse 1: erect 2: decurved Proportion of pedicle to flower 0: shorter 1: longer Proportion of pedicle to fruit 0: shorter 1: longer Presence of petiole in caulin leaves 0: present 1: absent Presence of hair on the leaf surface 0: present 1: absent Overtopping the bract from flower 0:yes 1: no Overtopping the bract from fruit 0:yes 1: no Position of bract 0: near the flower 1: far from the flower Spure 0: present 1: absent 154 Jing Ma et al. and sub-groups were formed due to molecular differenc- es among the individuals of Consolida. Gene flow (Nm) was relatively low (0.54) in Consol- ida species. Genetic identity and phylogenetic distance in the Consolida members are mentioned (Table 5). C. camptocarpa and C. anthoroidea were genetically close- ly related (0.907) to each other. C. persica and C. rugu- losa were dissimilar due to low (0.702) genetic similarity. Mantel test after 5000 permutations produced significant correlation between genetic distance and geographi- cal distance in these populations (r = 0.34, P = 0.0002). Therefore, the populations that are geographically more distant have less amount of gene flow, and we have iso- lation by distance (IBD) in Consolida species. The most popular approaches for estimating divergence include calculation of genetic distances and variance partition- Table 3. RAPD primers and other parameters. Note: TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: polymorphism index, EMR, effective multiplex ratio; MI, marker index; PIC, polymorphism information content for each of CBDP primers. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI OPA-05 5’-AGGGGTCTTG-3’ 15 15 100.00% 0.46 5.34 16.55 6.44 OPA-06 5’-GGTCCCTGAC-3’ 24 24 100.00% 0.57 5.88 14.56 8.87 OPB-01 5’-GTTTCGCTCC-3’ 22 22 100.00% 0.55 6.23 12.23 6.47 OPB-02 5’-TGATCCCTGG-3’ 15 14 91.74% 0.57 5.66 14.56 5.67 OPC-04 5’-CCGCATCTAC-3’ 13 12 92.31% 0.38 3.21 15.60 5.55 OPD-02 5’-GGACCCAACC-3’ 14 13 97.74% 0.37 5.66 9.56 5.67 OPD-03 5’-GTCGCCGTCA-3’ 13 12 92.31% 0.54 8.21 10.23 5.55 OPD-05 5’ -TGAGCGGACA-3’ 12 12 100.00% 0.47 7.32 11.55 4.18 OPD-08 5’-GTGTGCCCCA-3’ 11 9 80.89% 0.43 6.56 9.34 7.18 OPD-11 5’-AGCGCCATTG-3’ 10 10 100.00% 0.49 4.25 14.11 7.87 Mean 14.5 13.3 96.22% 0.52 6.32 13.57 6.87 Total 145 133 Figure 2. PCA plot of morphological characters revealing species delimitation in the Consolida species; sp1= C. tehranica; sp2= C. campto- carpa; sp3= C. lorestanica; sp4= C. leptocarpa; sp5= C. persica; sp 6= C. aucheri; sp7= C. anthoroidea; sp8= C. hohenackeri; sp9= C. stocksi- ana; sp10: C. rugulosa; sp11: C. ambigua ; sp12= C. orientalis; sp13= C. regalis; sp14= C. oliveriana; sp15= C. flava; sp16= C. trigonelloides; sp17= C. oligantha; sp18= C. linorioides; sp19= C. rugulosa f. paradoxa. 155Molecular techniques in the assessment of genetic relationships between populations of Consolida ing among and within populations using Wright’s FST and other related statistics, such as GST, AST, RST, θST and ΦST. For instance, the FST gives an estimate of the balance of genetic variability among and within popu- lations, and is an unbiased estimator of divergence between pairs of populations under an island-model in which all populations diverged at the same time and are linked by approximately similar migration rates. How- ever, migration rates usually vary proportionally with geographical distances, so that pairwise FST estimates between pairs of populations vary. Evanno test performed on STRUCTURE analy- sis produced the best number of k = 10 (Figure.6). The STRUCTURE plot has revealed the allele combination difference among the studied populations and the occur- rence of genetic admixture among them. Inspite of genetic stratification and isolation by dis- tance observed in Consolida species STRUCTURE plot (Figure 7) showed high degree of gene flow among the studied populations, Although the studied populations contained some specific alleles. For example populations 8-14 and 2,19 (differently colored segments in Figure.7), they shared some similar alleles too. For example, it showed genetic similarity between populations 3 and 4 (similarly colored), as well as 5, 6 and 15,16. The plants of population 1 had some alleles of population 10. Simi- larly, population 5,6 had some alleles of population 14. Nonetheless, we were able to construct a consensus tree that agreed with our molecular (RAPD) and mor- phological findings (results not shown). The Consolida populations showed divergence due to genetic and mor- phological characters. Figure 3. Gel Electrophoresis image of DNA fragments produced by OPD-03 of Consolida species. sp1= C. tehranica; sp2= C. camp- tocarpa; sp3= C. lorestanica; sp4= C. leptocarpa; sp5= C. persica; sp 6= C. aucheri; sp7= C. anthoroidea; sp8= C. hohenackeri; sp9= C. stocksiana; sp10: C. rugulosa; sp11: C. ambigua ; sp12= C. ori- entalis; sp13= C. regalis; sp14= C. oliveriana; sp15= C. flava; sp16= C. trigonelloides; sp17= C. oligantha; sp18= C. linorioides; sp19= C. rugulosa f. paradoxa. L = Ladder 100 bp. Arrows show polymorphic bands. Table 4. Genetic diversity parameters in the studied Consolida species. SP N Na Ne I He UHe %P C. tehranica 12.000 0.287 1.233 0.271 0.184 0.192 51.91% C. camptocarpa 5.000 0.358 1.430 0.28 0.20 0.29 43.50% C. lorestanica 6.000 0.299 1.029 0.231 0.18 0.23 44.38% C. leptocarpa 5.000 0.462 1.876 0.288 0.29 0.28 62.05% C. persica 8.000 0.399 1.167 0.24 0.21 0.113 52.88% C. aucheri 5.000 0.336 1.034 0.23 0.25 0.19 51.83% C. anthoroidea 4.000 0.344 1.042 0.28 0.23 0.27 57.53% C. hohenackeri 5.000 0.455 1.234 0.277 0.24 0.22 55.05% C. stocksiana 3.000 0.255 1.021 0.25 0.18 0.22 42.15% C. rugulosa 3.000 0.288 1.024 0.23 0.35 0.30 64.30% C. ambigua 5.000 0.462 1.095 0.288 0.25 0.27 62.05% C. orientalis 8.000 0.399 1.167 0.322 0.398 0.344 65.77% C. regalis 8.000 0.201 1.00 0.23 0.17 0.17 42.23% C. oliveriana 5.000 0.341 1.058 0.24 0.27 0.20 53.75% C. flava 5.000 0.455 0.67 0.277 0.24 0.22 55.05% C. trigonelloides 8.000 0.499 1.067 0.24 0.13 0.24 49.26% C. oligantha 6.000 0.555 1.020 0.22 0.25 0.28 43.53% C. linorioides 10.000 0.431 1.088 0.20 0.12 0.15 41.53% C. rugulosa f. paradoxa 3.000 0.255 1.021 0.25 0.18 0.22 47.15% 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. 156 Jing Ma et al. DISCUSSION The Consolida is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify Consolida species (Ert- ugrul et al., 2016). Given the complexity, it is necessary to explore other methods that could complement the traditional taxonomical approach (Erbano et al. 2015). Advent and developments in molecular techniques have enabled plant taxonomists to utilize molecular protocols to study plant groups (Erbano et al. 2015). Consolida is an evolved genus with precise synapomorphies (reduc- tion of carpels from three or more to one, complete loss of lateral petals, spur consisting of one petal) that are not found in any other species of Delphinium and Aco- nitum. Most Consolida species are adapted to the Medi- terranean type climate or more arid climate types of the Irano-Turanian zone (Ertugrul et al., 2016). Pronounced periods of drought in these areas have certainly favoured the exclusive annual life cycle of Consolida. The bioge- ography of the genus indicates that Turkey, in particular Anatolia (c. 29 taxa) should be considered as the center of diversity, with further radiation of species into the Irano-Turanian area (c. 23 taxa), Greece (c. 10 taxa) and countries around the Mediterranean. Consolida forms a coherent, monophyletic clade with Delphinium and Aco- nitum. Some authors propose a direct evolution line of Consolida from Delphinium (Tamura 1966). We examined genetic diversity in Consolida by mor- phological and molecular methods. We mainly used RAPD markers to investigate genetic diversity and genetic affinity in Consolida. Our clustering and ordination tech- niques showed similar patterns. Morphometry results clearly showed the utilization or significance of morpho- logical characters in Consolida species. PCA results also confirmed the application of morphological characters to separate Consolida species. The present study also high- Table 5. Analysis of molecular variance (AMOVA) of the studied species. Source df SS MS Est. Var. % ΦPT Among Pops 20 1701.364 55.799 12.189 54% 54% Within Pops 120 354.443 1.905 4.55 46% Total 150 2055.807 16.060 100% 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. AMOVA test of the studied populations. Figure 5. Neigbor-Joingin tree produced while using RAPD data. Branch support values are given as bootstrap (BP) value above branches. 157Molecular techniques in the assessment of genetic relationships between populations of Consolida lighted that morphological characters such as bract exert- ing from fruit, presence of spore, shape of spore apex, the number of petal, the number of petal lobes, could delimit the Consolida group. The Consolida species highlighted morphological differences. We argue that such a dissimi- larity was due to differences in quantitative and qualita- tive traits. Present findings on morphological differences are in line with the previous studies (Iranshahr, 1992; Ert- ugrul et al., 2016; Khalaj 2013). Genetic structure and gene flow Polymorphic information content (PIC) values are useful to detect genetic diversity. The current study recorded average PIC values of 0.52. This value is suffi- cient to study genetic diversity in the population (Kempf et al. 2016). High genetic diversity among the Consolida population was reported in the present study. The previ- ous scientific data (Kurata et al. 2019) supports our cur- rent high diversity results. Genetic analysis conducted via analysis of molecular variance and STRUCTURE showed genetic differences among the species. According to Bru¨tting et al (2012) sampled 53 pop- ulations from 6 arable plant species throughout Cen- tral Germany. Random amplified polymorphic DNA analyses (RAPD) were applied to calculate measures of genetic diversity at the population level and genetic dif- ferentiation. Their results showed that genetic diversity was found to be lowest in Bupleurum rotundifolium and Anagallis foemina, and highest in Consolida regalis and Nigella arvensis. The highest levels of genetic differentia- tion were observed among populations of An. foemina and B. rotundifolium but within populations in all other species. UST values differed strongly ranging between 0.116 for C. regalis and 0.679 for An. foemina. Patterns of genetic structure were related to the Red List status for all the species studied except An. foemina, for which it should consequently be raised. Them data confirm that even relatively recent threats are accompanied by detri- mental genetic structure. Figure 6. Delta k plot of Evanno’s test based on STRUCTURE anal- ysis. Figure 7. STRUCTURE plot of Consolida species based on k = 10 of RAPD data. 158 Jing Ma et al. Genetic diversity and population size Our data suggest that the 19 study species differed highly in their genetic diversity. Populations of C. rugu- losa; C. ambigua and C. orientalis showed the highest diversity, followed by C. leptocarpa and C. anthoroidea. Lowest values were found in C. regalis and C. linorioides. It is widely accepted that the breeding system influ- ences gene diversity dramatically ( Mable and Adam 2007). For example Nybom and Bartish (2004) extract- ed from literature that selfing taxa have a mean He of around 0.09. In contrast, plant species with a mixed or outcrossing breeding system show an He of around 0.22 to 0.26. For our study species, C. tehranica; C. camp- tocarpa; C. lorestanica; C. leptocarpa; C. anthoroidea; C. stocksiana; C. ambigua; C. orientalis and C. regalis tend to have a mixed breeding system and that C. oli- veriana; C. flava; C. trigonelloides are more outcrossing species. This assumption is certainly true for C. regalis because it is not self pollinating (Svensson and Wigren 1986). As inflorescences of outcrossing taxa are generally larger than inflorescences of selfing species (Hill et al. 1992), Lower genetic diversity could be an indication of higher fragmentation, as fragmentation leads to limited gene flow (Leimu et al. 2010). In fragmented populations pollinators struggle to reach the more distant popula- tions and may even also decline in abundance (Potts et al. 2010). However, the relationship is consistent with population genetic theory, predicting that genetic drift is particularly important in small populations (Ellstrand and Elam 1993) and population size is positively corre- lated to genetic variation (Leimu et al. 2006). Molecular markers (RAPD) and morphometry analysis were use- ful to study genetic diversity and population structure in Consolida species identification. All the species had dis- tinct genetic differentiation. Present results highlighted isolation and limited gene flow are the main determinis- tic factors that shape the Consolida population. We also reported high genetic diversity, which clearly shows the Consolida species can adapt to changing environments since high genetic diversity is linked to species adapt- ability. REFERENCES Aitzetmuller K,Tsevegsuren N, Werner F. 1999. Seed oil fatty acid patterns of Aconitum- Delphinium- Hel- leborous complex (Ranunculaceae). Pl Syst Evol 213: 37-47. Boissier E. 1841. 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