Molecular techniques in the assessment of genetic relationships between populations of Consolida (Ranunculaceae)

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 diversity. Therefore, we collected and analyzed 19 species from 12 provinces of regions. Overall, one hundred and twenty-seven plant specimens were collected. We showed significant 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 amplicons though the extent of polymorphism varied with each primer. The primer OPA06 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 Consolida 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.


INTRODUCTION
Genetic diversity is a vital feature that helps plant species survive in an ever-changing environment, and it sheds light on understanding the phylogenetic 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 the plant species worldwide. Scientific data indicate that genetic diversity plays a pivotal role in conservation programs (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 Aconitella Spach. Iran is one of the richest countries for the genus in South-West Asia, since it has 24 species (Iranshahr et al., 1992).
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 section. 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 Consolida, both being embedded in Delphinium. The Jabbour & Renner (2011) results showed that Consolida diverged from Delphinium relatives in the Early to Middle Miocene, 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 various field such as chromosomal studies (Trifonova 1990;Koeva 1992;Hong, 1986) chemical studies (Aitzetmuller et al.1999), palynological studies (Munz, 1967) and phylogenetic investigations by using DNA sequence data (Johansson 1995;RO et al.1997;Jabbour and Renner 2011;Yosefzadeh et al. , 2012). In the recent molecular studies (Jabbour and Renner 2001; 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 relatives 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 individuals 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-Bozchaloyi 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 samples. Our aims were 1) to assess genetic diversity among Consolida species 2) is there a correlation between species and geographical distance? 3) Genetic structure of populations and taxa 4) Are the Consolida species able to exchange genes?

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 samples (10-25 per plant species) were examined for morphometry 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 Herbarium of Azad Islamic University (HAIU).

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 technology, 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 contained 25μl volume. This 25 volume had ten mM Tris-HCl buffer, 500 mM KCl; 1.5 mM MgCl 2 ; 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, Germany). 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 scaling and principal coordinate analysis were also performed (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 population morphological traits. Multivariate and all the necessary 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- 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 (Heikrujam 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), Shannon 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 heterozygosity were assessed in GenAlEx 6.4 software (Peakall and Smouse 2006). Neighbor-joining (NJ) and networking were studied to fathom genetic distance plant populations (Huson and Bryant 2006;Freeland et al. 2011). The comparison of genetic divergence or genetic distances, estimated by pairwise F ST and related statistics, with geographical distances by Mantel test is one of the most popular approaches to evaluate spatial processes driving population structure. The Mantel test was performe as implemented in PAST. For this, Nei genetic distance was determined for RAPD data, while Geographic distance 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 interested in knowing the genetic structure and diversity, we also investigated the genetic difference between populations 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 Evanno test was conducted (Evanno et al. 2005).

Morphometry
Significant ANOVA results (P <0.01) showed differences in quantitative morphological characters in plant species. Principal component results explained 80% variation. Firs component of PCA demonstrated 57% of the total variation. Traits such as presence of petiole in caulin leaves, overtopping the bract from fruit, proportion of petal middle lobes to lateral lobes, presence of hair on the filament positively correlated with firs component (>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 species were separated from each other due to differences in morphology. Our PCA results also confirmed the application 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 (Consolida ) DNA ( Figure 3). 133 polymorphic bands were generated and amplified. Amplified products ranged from 100 to 3000 bp. We recorded the highest polymorphic bands for OPA-06. OPD-08 had the lowest polymorphic bands. The average polymorphic bands ranged to 13.3 for each primer.
tive multiplex ratio (EMR) values are useful to distinguish 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 features calculated of 19 Consolida species are shown (Table  4). C. linorioides depicted unbiased expected heterozygosity (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 highlighted 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 differentiation ( Figure 4). Genetic similarity and dissimilarity assessed through Genetic statistics (GST) showed significant 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 repeated the same pattern as indicated in figures 2. In current work, molecular findings also coincided with the traditional taxonomical (morphology) approaches for Consolida 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 consisted of two sub-groups. C. stocksiana; C. ambigua ; C. oliveriana; C. flava formed the first sub-group. C. trigonelloides; C. oligantha; C. linorioides; C. leptocarpa and C. persica formed the second sub-group. These groups and sub-groups were formed due to molecular differences among the individuals of Consolida. Gene flow (Nm) was relatively low (0.54) in Consolida species. Genetic identity and phylogenetic distance in the Consolida members are mentioned (Table 5). C. camptocarpa and C. anthoroidea were genetically closely related (0.907) to each other. C. persica and C. rugulosa were dissimilar due to low (0.702) genetic similarity.
Mantel test after 5000 permutations produced significant correlation between genetic distance and geographical 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 isolation by distance (IBD) in Consolida species. The most popular approaches for estimating divergence include calculation of genetic distances and variance partition- Evanno test performed on STRUCTURE analysis produced the best number of k = 10 ( Figure.6). The STRUCTURE plot has revealed the allele combination difference among the studied populations and the occurrence of genetic admixture among them.
Inspite of genetic stratification and isolation by distance 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. Similarly, 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 morphological findings (results not shown). The Consolida populations showed divergence due to genetic and morphological characters.

DISCUSSION
The Consolida is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify Consolida species (Ertugrul 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 (reduction 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 Aconitum. Most Consolida species are adapted to the Mediterranean 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 biogeography 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 Aconitum. Some authors propose a direct evolution line of Consolida from Delphinium (Tamura 1966).
We examined genetic diversity in Consolida by morphological and molecular methods. We mainly used RAPD markers to investigate genetic diversity and genetic affinity in Consolida. Our clustering and ordination techniques showed similar patterns. Morphometry results clearly showed the utilization or significance of morphological characters in Consolida species. PCA results also confirmed the application of morphological characters to separate Consolida species. The present study also high-   lighted that morphological characters such as bract exerting 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 dissimilarity was due to differences in quantitative and qualitative traits. Present findings on morphological differences are in line with the previous studies (Iranshahr, 1992;Ertugrul 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 sufficient 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 previous scientific data (Kurata et al. 2019) supports our current 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 populations from 6 arable plant species throughout Central Germany. Random amplified polymorphic DNA analyses (RAPD) were applied to calculate measures of genetic diversity at the population level and genetic differentiation. 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 differentiation 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 detrimental genetic structure.

Genetic diversity and population size
Our data suggest that the 19 study species differed highly in their genetic diversity. Populations of C. rugulosa; 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 influences gene diversity dramatically ( Mable and Adam 2007). For example Nybom and Bartish (2004) extracted 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. camptocarpa; 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. oliveriana; 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 correlated to genetic variation (Leimu et al. 2006). Molecular markers (RAPD) and morphometry analysis were useful to study genetic diversity and population structure in Consolida species identification. All the species had distinct genetic differentiation. Present results highlighted isolation and limited gene flow are the main deterministic 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 adaptability. Table 6. The matrix of Nei genetic similarity (Gs) estimates using SCoT molecular markers among 19 Consolida species.sp1= C. tehranica; sp2= C. camptocarpa; 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. orientalis; sp13= C. regalis; sp14= C. oliveriana; sp15= C. flava; sp16= C. trigonelloides; sp17= C. oligantha; sp18= C. linorioides; sp19= C. rugulosa f. paradoxa. sp1 sp2 sp3 sp4 sp5 sp6 sp7 sp8 sp9 sp10 sp11 sp12 sp13 sp14 sp15 sp16 sp17 sp18 sp19