Genetic and micromorphological differentiation within Sambacus ebulus (Adoxaceae) suggests divergence of populations from Iran

doi:10.21203/rs.3.rs-4998002/v1

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Genetic and micromorphological differentiation within Sambacus ebulus (Adoxaceae) suggests divergence of populations from Iran

https://doi.org/10.21203/rs.3.rs-4998002/v1

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Sambucus ebulus is mainly distributed in the northern, northwestern and northeastern regions of Iran and is characterized by its perennial growth habit and distinctive long, creeping and branched rhizomes. In this study, genetic diversity of seven populations S. ebulus based on molecular ISSR data and the micromorphology of seeds and leaves of this species in Iran were investigated. Micromorphological examination revealed the anomocytic type of stomata and indicated that the epidermal cells of various S. ebulus populations exhibited irregular cell shapes with anticlinal walls ranging from straight to curved. The seed shape was described as predominantly almond-shaped, and the surface of the seed coat of the studied taxa showed a reticulate pattern. The AMOVA (analysis of molecular variance) test showed that 87% of the total genetic variance was due to differences inter-populations genetic differences, while 13% was due to within-populations genetic variability, indicating a high degree of genetic variation among S. ebulus populations. The discriminating power of ISSR (inter simple sequence repeat) loci as determined by Gst against Nm (the number of migrants) analysis, showed that almost all ISSR loci have an excellent discriminating power. Thus, ISSR markers are efficient in differentiating of the studied S. ebulus populations. The Mantel test presented a significant correlation between genetic and geographic distance. These results suggest that the selected markers for genetic analysis represent different regions of the genome and effectively capture the genetic variation within the studied populations.

AMOVA

Genetic

Iran

Populations

SEM

The genus Sambucus, belonging to the Adoxaceae family, comprises approximately 30 species of deciduous shrubs, small trees, and a few perennial herbs (Mabberley, 2008) predominantly found in the northern hemisphere. In Iran, the genus is represented by two species: Sambucus ebulus L. and Sambucus nigra L. (Jamzad, 1993). Sambucus ebulus is mainly distributed in the northern, northwestern, and northeastern regions of Iran and is characterized by its perennial growth habit and distinctive long, creeping, and branched rhizomes (Judd et al., 2007). In recent years, research into the pharmacology of S. ebulus has increased, revealing both traditional and modern medicinal uses of the plant. Various sources have documented its diuretic, antiseptic, tonic, and laxative properties, highlighting its medicinal potential (Novelli, 2003; Uncinimangianelli et al., 2005).

Genetic diversity plays a crucial role in the survival of populations, as highlighted in studies such as Pourmeidani et al., 2024. To understand the genetic information of plants, researchers often use markers and PCR techniques. Among these markers, ISSR (inter simple sequence repeat) molecular markers stand out for their stability, high reproducibility, and ease of use. This has made them a popular choice in various biological investigations, as noted in studies by Sheidai et al. (2012, 2013, 2014) and Atazadeh et al. (2020).

The molecular study of Sambucus species has been a focus in a limited number of studies thus far. For example, Waswa et al. (2022) conducted research on the taxonomy and classification of Sambucus species. Their analysis revealed that Sambucus species form a monophyletic group with two main clades: one containing S. nigra, S. peruviana, S. maderensis, and S. canadensis, and the other containing the remaining species. Amini et al. (2019a) carried out a molecular study in Iran using the ITS marker to evaluate 18 populations of two Sambucus species, S. ebulus and S. nigra, which led to the differentiation of these two species. Additionally, Shen et al. (2019) investigated genetic diversity in mackerel germplasm using ISSR markers. Their study did not find a clear correlation between clustering results and fruit characteristics, as the mackerel germplasms are related to native superior members.

Micromorphological studies, particularly using scanning electron microscopy (SEM), play a significant role in botany by providing valuable diagnostic information for the identification of plant taxa, especially at the species level (Amini et al., 2018a). This technique allows for the examination of stable traits that are not influenced by environmental conditions, making it useful for accurate plant identification. Epidermal traits are particularly effective for this purpose, as they are present on most green organs and fruits and can even be observed in herbarium samples.

In the genus Sambucus, micromorphological studies have contributed to a better understanding of the morphological and anatomical characteristics of various species. For instance, Sedláčková et al. (2018) focused on flower morphology and antioxidant activities in the inflorescence of Sambucus. Alerico et al. (2016) studied the chemical compounds present in stem bark, while Amini et al. (2021) investigated the morphology and anatomy of Sambucus species, including S. ebulus and S. nigra. Their studies highlighted the micromorphological characteristics (such as pollen and seeds) that supported the differentiation of S. nigra and S. ebulus. Elkiran et al. (2021) examined the anatomy of S. nigra and S. ebulus in northern Turkey, focusing on differences in features such as drusen crystals, vascular bundle cells, margin shape, and collenchyma cells on the stems.

The present study aims to 1) assess the genetic diversity and investigate the relationships among populations of S. ebulus using molecular ISSR data in seven populations of S. ebulus, and 2) examine the micromorphology of seeds and leaves in populations of this species. These comprehensive analyses, combining the genetic structure of the populations and micromorphological data, may provide valuable insights into the evolutionary relationships and characteristics of S. ebulus.

Plant material

Genetic and morphological data from seven populations of Sambucus ebulus in North Iran were examined in this study (Figs. 1, 2). Plant samples were collected from various locations and stored at the Gonbad Kavous University Herbarium (GKUH). Geographic coordinates and elevation of each genotype's habitat were determined using GPS technology.

Table 1

Investigated *Sambucus ebulus* populations
Pop.	Province	Locality	Voucher number	Latitude	Longitude	Altitude (m)
1	Mazandaran	Babol	804509	E 52.6625°	N 36.6001°	-11
2	Golestan	Golestan Forest	804502	E 55.7828°	N 37.5793°	457
3	Mazandaran	Noor	804500	E 53.6402°	N 36.7013°	45
4	Gilan	Amlash	804508	E 50.1844°	N 37.0961°	-7
5	Gilan	Lahijan	804506	E 50.0028°	N 37.2060°	-3
6	Golestan	Kordkoy	804504	E 54.1480°	N 36.6686°	2302
7	Golestan	Bandar Gaz	804503	E 54.5838°	N 36.7813°	445

DNA extraction

Genomic DNA from the plant samples was extracted using the CTAB-activated charcoal protocol as described in various studies (Doyle & Doyle 1987; Doyle & Dickson 1987; Cullings 1992; Amini et al., 2018b; Nasrollahi et al., 2019). The quality and quantity of the extracted DNA were assessed by running it on a 0.8% agarose gel.

ISSR-PCR

The PCR experiment was conducted using 12 samples and four ISSR primers (refer to Table 2) in a 25 µL reaction volume. The reaction mixture included 2.5 mM MgCl2 (Cinna Gen Co, Iran), 0.2 µM of primer (Cinna Gen Co, Iran), 10 mM Tris-HCl pH 8.3, 1 mM dNTP mix (Cinna Gen Co, Iran), 1 U of Taq DNA polymerase-500 (Cinna Gen Co, Iran), and approximately 40 ng of template DNA. The ISSR-PCR was carried out using a thermocycler (Biorad, USA) with the program outlined in Table 3. The amplified products were visualized by running them on a 1% agarose gel electrophoresis.

Table 2

The used ISSR primers
Code	Sequences
ISSR13	(ACG) ₆Ga
ISSR826	CCCGGATCC (CA)₈
ISSR810	(GA) ₈ T
ISSR7	(AC) ₈ AG

Table 3

PCR program for ISSR primers
Step	Temperature	Time	Cycling
Initial denaturation	94 ºC	3 min	-
Denaturation	94 ºC	50 s	-
Annealing	58 ºC	42 s	38
Extension	72 ºC	50 sec	-
Final extension	72 ºC	7 min	1

ISSR analysis

The ISSR bands obtained were treated as binary characters, with the presence of a band coded as 1 and absence as 0. Genetic diversity parameters, including Nei's gene diversity (H), Shannon information index (I), number of effective alleles, and percentage of polymorphism, were calculated for each population following the methods described by Freeland et al. (2011). Nei's genetic distance was used for clustering analysis using Neighbor-Joining (NJ), WARD, and Unweighted Paired Group using Average (UPGMA) algorithms. Principal Coordinate Analysis (PCoA) and Multidimensional Scaling (MDS) methods were also employed for grouping the populations after 100 permutations, as outlined in Freeland et al. (2011). To assess the correlation between genetic and geographical distances among the populations, Mantel's test was conducted using PAST ver. 2.17 (Hammer et al., 2012) and DARwin ver. 5 (Perrier and Jacquemoud-Collet, 2006) software.

For determining the genetic differentiation of the species, an AMOVA (Analysis of Molecular Variance) test was performed using GenAlex 6.4 (Peakall and Smouse, 2006). Gene flow was estimated using the Nm value, calculated from GST using Pop Gene ver.1.32 (Nm = 0.5(1 - GST) / GST) and by the least square method as implemented in T-REX (Boc et al., 2012).

Scanning electron microscopy

In the analysis process, three samples of each species were utilized. The samples were boiled for 20 minutes, and the openings were observed. Subsequently, they were preserved in 70% alcohol post-placement in Carnoy's solution for 24 hours. To prepare them for observation under a light microscope, the samples were initially immersed in distilled water until they became colorless and then rinsed with water. Light microscopy was employed to create slides, and an 8-megapixel Canon digital camera (model a63) was utilized for examination.

The use of a scanning electron microscope enables the acquisition of high zoom images through advanced imaging techniques, allowing for the investigation of morphological, structural, and elemental details at intricate levels (URL-2, 2016). The leaf epidermis of each species was scrutinized under a stereomicroscope to verify normal size and development. The specimens were affixed directly onto aluminum stubs using double-sided adhesive, coated with a thin layer of gold (approximately 25 nm), and examined using a VEGA II TESCAN-LMU Electron Microscope at an accelerating voltage of 15–22 kV at the Research Institute of Razi in Tehran, Iran. Subsequent to this procedure, analysis and imaging were carried out utilizing the scanning electron microscope. Microphotographs of the samples were captured using suitable applications, micrometric measurements were taken, and data were digitized.

ISSR assay and genetic diversity

The DNA extraction process yielded high-quality molecules suitable for the genetic diversity study. Gel electrophoresis results indicated clear nucleic acid molecules without smearing, confirming their suitability for polymerase chain reaction (PCR) amplification. All examined primers generated bands within the desired size range of 200 to 900 bp. To ensure accuracy, only bands with optimal resolution were considered. ISSR primers revealed 19 bands per locus, with predominantly polymorphic bands and only two monomorphic bands. Population 4 and 5 exhibited the greatest band diversity (12 bands), while populations 1 and 3 displayed the lowest band count (9 bands). Notably, populations 5 and 6 uniquely featured two specific bands amid more general bands across all populations (Table 4). Tables 5 and 6 show the parameters of genetic diversity and Nei^’s genetic distance for S. ebulus in detail.

The discriminating power of ISSR loci as determined by Gst against Nm (the number of migrants) analysis, showed that almost all ISSR loci have an excellent discriminating power. Thus, ISSR markers are efficient in differentiating of the studied S. ebulus populations.

The AMOVA test allows the calculation of the molecular variance components, which illustrate the contributions of the variances intra- and inter-groups to the total genetic variation. The test results revealed significant genetic differentiation among the studied populations (P = 0.001), indicating distinct genetic clustering (Table 7). The AMOVA test showed that 87% of the total genetic variance was due to differences inter-populations genetic differences, while 13% was due to within-populations genetic variability (Fig. 3). This substantial inter-population genetic diversity highlights the valuable genetic resources present in these populations for future breeding programs. The results emphasize the marked genetic diversity observed in samples of the same species from different regions.

Moreover, the Mantel's test conducted to assess the relationship between geographic and genetic distances demonstrated a significant correlation among populations (P = 0.01). This implies that as populations become geographically more distant from each other, they also exhibit increasing genetic divergence.

Table 4

Items of ISSR bands in *Sambacus ebolus* populations (Populations 1–7 matching Fig. 1)
	Pop1	Pop2	Pop3	Pop4	Pop5	Pop6	Pop7
No. Bands	9	10	9	12	12	10	10
No. Bands Freq. (≥ 5%)	9	10	9	12	12	10	10
No. Private Bands	0	0	0	0	1	1	0
No. Common Bands (≤ 25%)	0	0	0	0	0	0	0
No. Common Bands (≤ 50%)	0	0	0	2	2	0	0

Table 5

Genetic diversity details in *Sambacus ebolus* populations (Pop. 1–7 are according to Fig. 1)
Pop	N	Na	Ne	I	He	uHe	%P
Pop1	3.000	0.474	1.000	0.000	0.000	0.000	0.00%
Pop2	3.000	0.579	1.050	0.036	0.026	0.031	5.26%
Pop3	3.000	0.474	1.000	0.000	0.000	0.000	0.00%
Pop4	3.000	0.684	1.050	0.036	0.026	0.031	5.26%
Pop5	3.000	0.684	1.050	0.036	0.026	0.031	5.26%
Pop6	3.000	0.526	1.000	0.000	0.000	0.000	0.00%
Pop7	3.000	0.526	1.000	0.000	0.000	0.000	0.00%

Note: N = Number of studied plants, Na = Number of Different alleles, Ne = Number of Effective alleles, I = Shannon's Information Index, He = Expected Heterozygosity, uHe = Unbiased gene diversity, and P% = Percentage of Polymorphic Loci.

Table 6

**Nei**^,**s genetic identity (above diagonal) and genetic distance (below diagonal) (Populations numbers matching to** Fig. 1)
Pop ID	1	2	3	4	5	6	7
1	***	0.991	1.000	0.884	0.777	0.947	0.947
2	0.009	***	0.991	0.918	0.810	0.937	0.982
3	0.000	0.009	***	0.884	0.777	0.947	0.947
4	0.123	0.085	0.123	***	0.856	0.831	0.937
5	0.252	0.210	0.252	0.156	***	0.724	0.831
6	0.054	0.065	0.054	0.186	0.323	***	0.895
7	0.054	0.018	0.054	0.065	0.176	0.111	***

Table 7

**Pair-wise AMOVA between** **Sambacus ebolus** **populations (PhiPT Values below diagonal. Probability values based on 99 permutations are shown above diagonal)**
AMOVA	Pop 1	Pop 2	Pop 3	Pop 4	Pop 5	Pop 6	Pop 7
Pop1	0.000	0.354	0.001	0.123	0.089	0.001	0.001
Pop2	0.500	0.000	0.421	0.135	0.080	0.090	1.00
Pop3	0.686	0.500	0.000	0.113	0.111	0.001	0.001
Pop4	0.875	0.667	0.875	0.000	0.117	0.113	0.124
Pop 5	0.929	0.833	0.929	0.750	0.000	0.095	0.111
Pop 6	1.00	0.800	1.00	0.909	0.941	0.000	0.001
Pop 7	1.00	0.000	1.00	0.800	0.909	1.00	0.000

The population clustering and grouping based on ISSR data by NJ (Figure not given), UPGMA tree, PCO and MDS plot (Figure not given) showed similar results. Upon evaluation of the ISSR data, the UPGMA analysis illustrated the presence of two primary clusters (Fig. 4). Within the first cluster, population 6 (Golestan, Kordkoy) appeared as a distinct sub-cluster from the other populations. Furthermore, populations 1 (Mazandaran, Babol) and 3 (Mazandaran, Noor) clustered together, separate from populations 2 (Golestan, Golestan Forest) and 7 (Golestan, Bandar Gaz) which formed another close cluster. The second cluster also exhibited two sub-clusters, with populations 5 (Gilan, Lahijan) and 4 (Gilan, Amlash) forming a separate cluster.

The results of the PCOA plot aligned with those of the UPGMA chart. Populations 2 (Golestan, Golestan Forest) and 7 (Golestan, Bandar Gaz) along with populations 1 (Mazandaran, Babol) and 3 (Mazandaran, Noor) appeared to constitute closely related genetic groups. Population 6 (Golestan, Kordkoy) exhibited genetic resemblance to populations 1 and 3. Populations 4 (Gilan, Amlash) and 5 (Gilan, Lahijan) displayed significant genetic differentiation from the other populations (Fig. 5).

Further, The CCA plot of genetic data and environmental features revealed that geographic factors such as latitude and longitude are effective in separation of studied S. ebolus populations, particularly in populations 4 and 5 (Fig. 6).

Micromorphology

Leaf epidermal cell

In the research conducted, leaf epidermal traits were analyzed, focusing on characteristics such as the shape of epidermal cells, anticlinal wall, stomatal type, stomatal index, stomatal length, and width at the lower surface. Examination of the epidermis of the studied populations using electron microscopy revealed the presence of stomata on the lower surface across all populations, with all exhibiting the anomocytic type of stomata. The investigation indicated that the epidermal cells of various S. ebulus populations featured irregular cell shapes with anticlinal walls ranging from straight to curve. The electron microscope images in Figs. 7, 8, 9, 10, and 11 provide visual representations of the epidermis of different populations, showcasing the observed characteristics of the epidermal cells and stomata for comparative analysis and detailed examination. The measurements conducted revealed that the population from Golestan province had the highest average width and length of the aperture among the studied populations of this species, with an average width of 40.68 µm and an average length of 55.44 µm. In contrast, the population from Mazandaran province exhibited the lowest averages, with an average width of 41.28 µm and an average length of 52.24 µm. These comparative values are detailed in Table 8 of the study.

Additionally, the examination of trichome characteristics in the populations of the studied species highlighted various types of covering trichomes on the epidermis of S. ebulus. These included simple single-cell trichomes, multi-cell trichomes, glandular trichomes, and conical or triangular trichomes. Despite the presence of these different types of trichomes across populations, variations were observed in the density of trichomes among the studied populations. These findings add further insights into the morphological diversity within different populations of S. ebulus.

Table 8

Description of the observed variation in the epidermal features of *S. ebulus*
Population	Cell Shape	Anticlinal Wall	Mean Stomata size (width*Length)	Mean trichome length	Density of trichome
Golestan	irregular	straight to curved	62.44*47.97	3.62	dense
Gilan	irregular	straight to curved	62.18*37.55	3.44	sparse
Mazandaran	irregular	straight to curved	52.24*41.28	3.23	dense

Seed surfaces

The seed characteristics deemed to have potential taxonomic significance in S. ebulus have been summarized in Table 9. The seeds were found to range in length from 2.58 to 2.77 mm and in width from 1.19 to 1.35 mm. Upon examining the seeds from various populations of the species, it was observed that all seeds exhibited an almond shape. Furthermore, the seed shape was described as predominantly almond-shaped, and the seed-coat surface of the studied taxa displayed a reticulate pattern, as illustrated in Fig. 12. These findings provide valuable insights into the consistent seed morphology and surface features across different populations of S. ebulus, which can be important for taxonomic classification and identification purposes.

Table 9

Characteristic features of the investigated seeds in *S.ebulus*.
Population		Length (mm)	Width (mm)	Length/ Width	Seed surface
Golestan	Almond shape	2.58	1.30	1.98	Reticulate
Gilan	Almond shape	2.77	1.19	2.32	Reticulate
Mazandaran	Almond shape	2.60	1.35	1.92	Reticulate

Genetic relationship estimations among individuals are influenced by multiple factors, including the number and distribution of markers in the genome, as well as the underlying evolutionary mechanisms (Powell et al., 1996). The effectiveness of the ISSR method in providing genetic information is highly dependent on the choice of primers. Ellis et al. (1997) demonstrated that selecting six optimal primer combinations could account for 80% of the expected relationships. In this study, four primary primers were selected based on established protocols and previous successful outcomes (Talebi Kohyakhy et al., 2008; Ahkami et al., 2007; Antonio et al., 2004). The genetic diversity among populations was found to be 87% in this investigation. Melchinger (1997) emphasized that the accuracy of genetic similarity estimation relies on the number of markers, level of polymorphism, and genomic coverage of markers employed.

The results of Canonical Correspondence Analysis (CCA) in our study indicate that environmental factors such as latitude, longitude, and altitude have a simultaneous impact on gene flow and genetic structure within the studied populations. These variations among populations may suggest distinct taxonomic groups within S. ebulus below the species level. Previous research on various plant species, including wheat (Triticum turgidum and Triticum durum), has also shown a correlation between genetic diversity and geographical distribution. Genotypes from different regions and latitudes often exhibit genetic differentiation (Omidbakhshfard et al., 2009; Ahkami et al., 2007). Many studies have reported the existence of different ecotypes resulting from genetic differences among populations, leading to morphological divergence (Sheidai et al., 2012; 2013; 2014; Minaeifar et al., 2015; Atazadeh et al. 2020). It is interesting to note that in Ferula gummosa, a lack of correlation between molecular diversity and geographic diversity has been reported (Khonani et al., 2009). This highlights the complexity and variability of genetic and environmental interactions across different plant species and populations.

In Principal Coordinate Analysis (PCO), when the principal components account for a lower percentage of variation, it suggests that they provide a more proper distribution at the genome level. This indicates that the selected markers are effectively capturing genetic diversity. By sampling markers from different parts of the genome that have less correlation with each other, the analysis can provide a broader representation of genetic variation. The study by Messmer et al. (1992) supports using principal component analysis as a complementary method to cluster analysis to optimize the utilization of molecular data and extract maximum information. The PCO plot (Fig. 5) and UPGMA clustering (Fig. 4) in our study show that the distribution of genotypes in the two-dimensional axis aligns well with the dendrogram, indicating consistency between the different analytical approaches. These results suggest that the selected ISSR markers for genetic analysis represent diverse regions of the genome and are effectively capture the genetic variation within the studied populations. This comprehensive approach can provide valuable insights into the genetic diversity and structure of the populations under investigation.

The results of our study suggest that there is a high level of diversity among the ecotypes of Sambucus from different geographical regions in the north of the country. The presence of morphological diversity indicates that the observed differences in the samples are not solely influenced by environmental factors but are also under genetic control. The study highlights the effectiveness of the Inter-Simple Sequence Repeat (ISSR) method as a rapid, powerful, and cost-effective tool for genetic analysis. This method requires minimal initial work and can identify numerous gene locations that may not be easily accessible with other techniques within the same time frame and budget constraints. The work of Powell et al. (1996) also supports the utility of the ISSR technique, emphasizing its high repeatability and ability to measure a large number of gene locations efficiently in a single test. The repeatability of the ISSR method is crucial in assessing the quality of genetic markers, as it ensures consistent results that can be used for accurate clustering and genetic analysis of diverse samples. The ISSR method provides researchers with a reliable and efficient approach to capture genetic variation and understanding the underlying factors influencing diversity within Sambucus ecotypes from different regions.

The use of scanning electron microscopy (SEM) in studying Sambucus ebulus has provided valuable insights into the plant's morphology, revealing significant variation and introducing novel morphological features that contribute to a better understanding of the species. The scanning electron microscopy (SEM) is a specialized tool that has undergone significant advancements in recent years, although it may not be readily available in all laboratories due to its complexity and cost. In our study, SEM was employed to characterize and describe the surface characteristics of leaves and seeds of Sambucus ebulus. The examination of epidermal structures in populations from Golestan, Gilan, and Mazandaran regions revealed differences in cellular and stomatal characteristics among the species, likely influenced by varying environmental conditions in those regions. Stomata, such as the anomocytic-type aperture found on the lower surface of the species, play a crucial role in species recognition across different taxonomic, ecological, and physiological levels. These apertures are important features that can help differentiate species and are indicative of the plant's evolutionary adaptations to its environment. By studying these structural features using SEM, researchers can gain a deeper understanding of the unique characteristics of Sambucus ebulus and its variations across different geographical regions. The stomatal density of plants, including Sambucus ebulus, is influenced by a combination of natural, environmental, and ecological factors. Studies have shown that factors such as water availability, leaf size, and stomatal size play a significant role in determining stomatal density (Blum et al., 1981). These characteristics are closely linked to the habitat of the species and can vary at different levels of taxonomic classification and across different environmental conditions.

Genetic factors also have a notable influence on the development of stomatal traits in plants (Miller 1983). Research on S. ebulus has revealed variations in stomatal index among different populations, with the population in Golestan province exhibiting the highest stomatal index and the population in Mazandaran province having the lowest stomatal index. These differences are attributed to the specific humidity conditions and habitats in which the populations are found. In habitats with higher humidity and ample water resources, plants tend to have lower stomatal indexes compared to those in drier environments. This relationship highlights the adaptive responses of plants to their specific environmental conditions. The aperture index, which is influenced by stomatal density, is also lower in populations with sufficient humidity, such as those in Mazandaran province, compared to populations in drier regions like Golestan province. These findings are consistent with previous studies, such as those by Van de Roovaaort and Fuller (1935), which showed that plants subjected to drought stress tend to have higher stomatal density as a response to water scarcity. The data from this research provide valuable insights into the stomatal characteristics of S. ebulus and offer a foundation for further investigations into the species and its adaptation to varying environmental conditions.

The morphology of seeds, including characteristics such as shape, size, and surface ornamentation, can provide valuable insights for identifying and classifying plant species. Studies have indicated that these seed attributes are influenced by genetic and phylogenetic differences among taxa and are relatively stable across different environmental conditions (Barthlott 1984; Ozcan 2002).

In the case of the target species population, all seeds were observed to be almond-shaped with reticulate grain surfaces. This uniformity in seed morphology suggests that these features are genetically determined and can serve as distinguishing traits for identifying the species. The presence of such consistent seed characteristics supports the idea that these traits hold taxonomic significance and can help clarify relationships among species that share morphological similarities.

The importance of seed morphology in plant classification has been emphasized in various studies, validating its value as a systematic trait for species identification (Amini et al., 2019b; Amini et al., 2024). The results of the research highlight grain size and surface ornamentations as key distinguishing features that can aid in the identification of plant species in Iran. These seed attributes provide valuable systematic information that can enhance our understanding of plant diversity and assist in resolving taxonomic ambiguities.

In the present study, the genetic diversity of S. ebulus populations was investigated using ISSR molecular data. Our results showed a high degree of genetic variability among populations of S. ebulus, which is important for assessing the genetic diversity of this species. The UPGMA dendrogram based on the molecular data provided valuable information on the relationships among the studied populations. This comprehensive approach can provide valuable insights into the genetic diversity and genetic structure of the studied populations. The ISSR method provides researchers with a reliable and efficient approach to capture genetic variation and to understand the underlying factors that influence diversity within Sambucus ecotypes from different regions. Examination of the epidermal structures in populations from the Golestan, Gilan and Mazandaran regions revealed differences in the cellular and stomatal characteristics of the species, which are likely to be influenced by the different environmental conditions in these regions.

Clinical trial number

not applicable.

Ethics approval and consent to participate

Not applicable

Consent for publication

Informed consent was obtained from all individual participants included in the study.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research received no external funding.

Author Contribution

"Elham Amini wrote the main manuscript text and Ali Sattarian and Marzieh Bayrami and Mahmood Salehi prepared figures 1-12. Neda Atazadeh prepared the analyses. All authors reviewed the manuscript.

Acknowledgements

The authors would like to thank Miss Beyk Mohammadi for the advice lab and wish to express our gratitude to Mr. Hosseini for constative suggestion. The present study was financially supported by Gonbad Kavous University, Gonbad (Iran).

Availability of data and material

No data was used for the research described in the article.

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Genetic and micromorphological differentiation within Sambacus ebulus (Adoxaceae) suggests divergence of populations from Iran

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Abstract

Figures

Introduction

Materials and Methods

Plant material

DNA extraction

ISSR-PCR

ISSR analysis

Scanning electron microscopy

Results

ISSR assay and genetic diversity

Micromorphology

Leaf epidermal cell

Seed surfaces

Discussion

Conclusion

Declarations

Competing interests

Funding

Author Contribution

Acknowledgements

Availability of data and material

References

Additional Declarations

Supplementary Files

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