ACTA BOT. CROAT. 80 (2), 2021 125 Acta Bot. Croat. 80 (2), 125–130, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-012 ISSN 0365-0588 eISSN 1847-8476 Morphological and genetic diversity of Senecio vulgaris L. (Asteraceae) in Iran Mostafa Ebadi1*, Rosa Eftekharian2 1 Azarbaijan Shahid Madani University, Faculty of Science, Department of Biology, 53714-161, Tabriz, Iran 2 Shahid Beheshti University, Faculty of Life Sciences and Biotechnology, Tehran, Iran Abstract – Senecio vulgaris L., an annual herb belonging to the Asteraceae, is widely distributed in different regions of the world. There is no information on the intraspecific variations of the morphological and molecular features of this species. In the present investigation, we studied the morphological and genetic diversity of 81 accessions of S. vulgaris collected from 10 geographical populations. Eleven inter simple sequence repeat (ISSR) primers were used for the ex- amination of genetic variations among the populations. Analysis of molecular variance (AMOVA) and GST analyses revealed significant differences among the investigated populations. A significant correlation between genetic distance and geographical distance was revealed by the Mantel test. However, reticulation analysis indicated the occurrence of gene flow among most of the populations studied. Principal component analysis (PCA) plot showed that the number of capitula, length of the cauline leaf and plant height were the most variable morphological characters. Principal co- ordinates analysis (PCoA) plot revealed two groups of populations, according to molecular and morphological data. The results suggested the existence of possible intraspecific taxonomic ranks within this species. Keywords: gene flow, groundsel, morphology, Principal Component Analysis Introduction The genus Senecio L. (Asteraceae) includes ca. 1250 spe- cies and is one of the largest genera of flowering plants (Bremer 1994, Calvo et al. 2015). In Iran, some species of Senecio have a widespread distribution in the different geo- graphical regions, such as S. vulgaris L., S. glaucus L., and S. leucanthemifolius subsp. vernalis (Waldst. et Kit.) Greuter. Senecio vulgaris, commonly called groundsel or old- man-in-the-Spring, is an annual herb that grows in a variety of habitats from open grassland to woodland. It is usually regarded as a temperate weed and although it is almost cos- mopolitan it appears to be more localized in temperate re- gions of America and Asia, Mediterranean areas and South Africa (Chater and Walters 1976). Senecio vulgaris has a long history of herbal use and is widely used as an anthelmintic, antiscorbutic, diaphoretic, diuretic, emmenagogue and purgative (Hatfield 1977, Launert 1981). A homeopathic remedy is made from aerial parts of the plant. The therapeutic activity of groundsel is related to a variety of biologically active substances such as phenols and flavonoids, essential oils, polysaccharides, tri- terpenes, amines, saponins, tannins and mucilage (Uzun et al. 2004, Conforti et al. 2006). A population genetics survey plays the main role in the planning of genetic and breeding programs. It provides data about genetic diversity, gene migration, allelic drift, genetic fragmentation, genetic bottleneck and any other evolutionary forces acting on population divergence (Sheidai et al. 2013). Intraspecific taxonomic entities can be identified after a detailed population-based investigation within plant spe- cies. In general, extensive morphological and genetic diver- gences among populations result in speciation events. In- traspecific variation occurs within both wild and cultivated crop plants (Maxted and Hunter 2011). In Iran, S. vulgaris grows in different areas and forms numerous local geographical populations. There is no in- formation about its genetic diversity and adaptation against population divergence. Population genetic studies not only can provide information on these aspects but can also un- ravel the speciation process of Senecio in general. One meth- od for the study of genetic diversity employs morphological * Corresponding author e-mail: ebadi2023@yahoo.com EBADI M., EFTEKHARIAN R. 126 ACTA BOT. CROAT. 80 (2), 2021 Material and methods Plant specimens Eighty-one plant specimens were collected from ten geo- graphical regions (On-line Suppl. Fig. 1). Details of localities and voucher specimens are showed in Tab. 1. Morphologi- cal characteristics of S. vulgaris accessions were measured by a binocular microscope (Olympus BH2 ×400). DNA extraction and PCR amplification For the molecular study, a sub-sample of 0.5 g leaves from each population was taken and genomic DNA was ex- tracted by the CTAB method (Sheidai et al. 2013). Eleven ISSR primers were tested for PCR amplification; (AGC) 5GG, (AGC) 5GT, (CA) 7GT, (CA) 7AT, (GA) 9C, UBC807, UBC810, UBC811, UBC823, UBC834, and UBC847. PCR reactions were done in a total volume of 25 μL contain- ing 1 μL primer (12.5 pmol), 0.5 μL dNTPs mix (10mM), 20 ng template DNA, 1.5 μL reaction buffer (10×), 1.2 μL MgCl2, 1.5 U Taq DNA polymerase and 18.5 μL deionized water. The PCR reaction was performed with a thermal cycler (Techne, Germany) with the following program: 94 °C for 5 min, 30 cycles at 94 °C for 30 sec, 55 °C for 1 min, 72 °C for 2 min and a final extension at 72 °C for 10 min. The PCR products were electrophoresed at 85 V on 1.5% agarose gels and the resulting bands were observed under a UV transil- luminator. The DNA fragment sizes were estimated after comparison with a 1-kb DNA ladder. Polymorphic ISSR markers were manually scored as binary data with presence as “1” and absence as “0”. Multivariate analysis The one-way analysis of variance (ANOVA) was used to indicate a significant difference of morphological characters among the studied populations. Principal component anal- ysis (PCA) was used to recognize important variable mor- phological characters in the studied populations. Principal coordinate analysis (PCoA) was carried out based on mor- phological and molecular data with Euclidean distance as a characteristics, but molecular markers are the potential tools to study genetic relations, phylogeny, population dy- namics or gene- and genome mapping. . The advent of molecular markers resulted in an im- proved ability to track evolution through a good under- standing of genetic diversity among populations and new phylogenetic viewpoints (Müller-Schärer and Fischer 2001, Stuessy et al. 2014). Among molecular markers, inter simple sequence repeat (ISSR) deserves special attention as a tool for analyzing diversity. ISSR is easy to use, quick, simple and highly reproducible (Azizi et al. 2014, Eftekharian et al. 2016). ISSR markers usually show high polymorphism and have the very important advantage that no prior informa- tion about the genomic sequence is required (Kojima et al. 1998, Bornet and Branchard 2001). This study aimed to investigate the genetic variation be- tween S. vulgaris populations collected from different geo- graphic regions by using ISSR and morphological characters. Also, gene flow and correlation of genetic and geographical distances were estimated. Fig. 1. Principal component analysis (PCA) among studied popu- lation based on quantitative morphological characters. Compo- nent 1 and component 2 refer to the first and second principal components, respectively. Arrows represent the correlations be- tween the independent variables and the two principal compo- nents represented. Each point represents an individual sample. Tab. 1. Geographical information concerning the studied populations of Senecio vulgaris. Population Locality Habitat type Altitude (m a.s.l.) Longitude (E) Latitude (N) Voucher No. Pop1 Fars, Kazerun Waste ground 904 29.64 51.64 95109 Pop2 Khuzestan, Andimeshk Roadsides 185 32.44 48.34 95105 Pop3 Khuzestan, Masjed soleiman Roadsides 150 31.13 49.20 95104 Pop4 Fars, Nurabad Roadsides 985 30.11 51.54 95110 Pop5 Tehran, Velenjak Mountain meadows 1758 35.48 51.23 95101 Pop6 Lorestan, Khorramabad Roadsides 1215 33.29 48.22 95103 Pop7 Alborz, Karaj Mountain meadows 1327 35.38 50.92 95107 Pop8 Ilam, Ivan Waste ground 1144 33.82 46.30 95106 Pop9 Tehran, Lavasan Mountain meadows 1720 35.75 51.60 95102 Pop10 Khuzestan, Ahwaz Roadsides 15 31.31 48.67 95108 INTRASPECIFIC DIVERSITY OF Senecio vulgaris L. ACTA BOT. CROAT. 80 (2), 2021 127 similarity index. Statistical analysis was performed using the program PAST v. 2.17c. (Hammer et al. 2001). Genetic diversity and population variation parameters were performed by PopGene software v. 1.32 (Yeh et al. 1999). Mantel permutation test was used to check the cor- relation between genetic distances and corresponding geo- graphical distances of the studied populations by GenAlex software v. 6.5. (Peakall and Smouse 2006). Analysis of molecular variance (AMOVA) test was used to determine significant genetic differences among the stud- ied populations with the use of GenAlex v. 6.5. Genetic dif- ferentiation was estimated by D,ST = Jost measure of differ- entiation (Jost 2008) and G,ST = standardized measure of genetic differentiation (Hedrick 2005). The occurrence of allele flow among populations was estimated by reticulation analysis (Legendre and Makaren- kov 2002). Results Morphological variability Twenty-three qualitative and quantitative characters were selected for morphological evaluation of S. vulgaris populations (Tab. 2). ANOVA test showed that there are sig- nificant differences (P ≤ 0.01) among the quantitative mor- phological characters of studied populations. The most important morphological characters among the studied populations were identified by PCA-biplot. The bi- plot of the PCA based on quantitative morphological charac- ters showed that the number of capitula, length of the cauline leaf and plant height are important markers in the distribu- tion of the populations studied (Fig. 1). It showed two factors (PC1 and PC2) which together explained 82% of the total variance. The PC1 (51% of variance) had positive correlations with the number of capitula, and a negative significant cor- relation with length of the cauline leaf. PC2 (variance 31%) had positive correlations with plant height (Tab. 3). The PCoA plot of both quantitative and qualitative char- acters divided the studied populations into two groups (groups I and II) based on morphological characters (Fig. 2). Populations No. 5, 7 and 9 were grouped into I and the rest of the studied populations were placed in Group II. The capitula numbers of populations of the first group ranged between 6 to 9 and between 4 to 7 in populations of Group II. The means of the length of cauline leaf were 6 cm Tab. 2. Quantitative and qualitative morphological characters of Senecio vulgaris accessions (length measured values are in cm). SD – standard deviation; N – number of analyzed samples. Quantitative characters mean ± SD (N) Plant height 21.45 ± 8.71 (81) Length of basal leaf 3.96 ± 1.08 (176) Width of basal leaf 1.35 ± 0.53 (176) Thickness in base of stem 3.21 ± 0.91 (81) Length of cauline leaf 4.54 ± 1.16 (214) Width of cauline leaf 1.97 ± 0.62 (214) Number of capitula 6.13 ± 1.42 (81) Length of peduncle 0.21 ± 0.08 (295) Length of bract on a peduncle 0.37 ± 0.08 (205) Number of involucral bracts 21.19 ± 0.59 (81) Length of involucral bracts 0.64 ± 0.05 (232) Number of calyculus bracts 10.1 ± 2.49 (81) Length of corolla 0.67 ± 0.05 (217) Length of corolla tube 0.40 ± 0.05 (217) Length of corolla lamina 0.28 ± 0.04 (217) Length of anther in disc florets 0.18 ± 0.01 (110) Length of style in disc florets 0.09 ± 0.01 (192) Length of papus 0.6 ± 0.08 (305) Length of nutlet 0.31 ± 0.03 (273) Qualitative characters Type of stem Branched- unbranched Stem indumentum Yes - No Blackness of calyculus bracts Less than 1mm – More than 1mm Blackness of involucral bracts Less than 1mm – More than 1mm Tab. 3. Correlation of morphological characteristics of the studied Senecio vulgaris populations with two components of principal component analysis (PCA). Character Component PC1 PC2 Number of capitula 0.908 0.132 Length of the cauline leaf –0.895 0.115 Length of peduncle 0.531 0.043 Plant height 0.144 0.951 Fig. 2. Two dimensional plot of principal coordinate analysis (PCoA) of the studied Senecio vulgaris populations based on mor- phological characters. Different colors indicate the plant speci- mens from each geographical population. Group 1: the popula- tions in north Iran (Populations 5, 7 and 9); Group 2: the populations in west and south west Iran (Populations 1, 2, 3, 4, 6, 8 and 10). EBADI M., EFTEKHARIAN R. 128 ACTA BOT. CROAT. 80 (2), 2021 and 4 cm in Group I and II populations, respectively. Also, the mean of plant height of Group I populations was 25 cm whereas it was 18 cm in Group II populations. Moreover, the stem type of Group I populations was of- ten branched and the black tips of the bracts were more than 1 mm long whereas populations of another group mostly have an unbranched stem and the black tips of the bracts were less than 1 mm long (Fig. 3). ISSR marker In this study, 121 reproducible ISSR fragments (200-800 bp) were obtained utilizing the PCR amplification. ANOVA analysis revealed significant genetic differences among the studied populations (PhiPT = 0.83, P ≤ 0.01). It also showed that most of the genetic variation (60%) occurred within pop- ulations, while 40% was due to inter-population genetic dif- ferences. These results revealed the presence of a high level of genetic differentiation among S. vulgaris populations. This result was also supported by the Gst analysis and Hickory test. Moreover, population differentiation parameters deter- mined among the studied populations produced high values for Hedrick, standardized fixation index after 999 permu- tation (G’st = 0.886, P ≤ 0.001) and Jost, differentiation in- dex (D-est = 0.275, P ≤ 0.001). Various parameters of genetic diversity calculated in 10 geographical populations of S. vulgaris are shown in On- line Suppl. Tab. 1. The results obtained from the common genetic diversity indices are similar to the unbiased gene diversity parameter (which is free from the sampling size). The polymorphic loci percentage in each population varied from 10.54% to 55.61% (On-line suppl. Tab. 1). Kazerun population (Pop1) showed the highest percentage (55.61%) of polymorphic loci of all the populations while the Velen- jak population (Pop 5) exhibited the lowest amount of poly- morphism (10.54%). The values of Shannon’s information index (I) varied from 0.062 to 0.298 in all the populations. The gene diversity (He) for all loci in each of the population ranged from 0.039 to 0.189. PCoA analysis showed that there were genetic differ- ences among studied populations of S. vulgaris based on ISSR data. Populations 5, 7 and 9 were categorized into sep- arate groups while the rest of the studied populations were distanced from them (Fig. 4). The Mantel test showed that there is a significant corre- lation (r = 0.5, P < 0.01) between genetic and geographic dis- tance (On-line Suppl. Fig. 2). This result demonstrated that gene exchange occurred between populations that were close to each other. A reticulogram revealed some degree of shared alleles among most of the populations. (Fig. 5) These shared alleles might be due to ancestral and or ongoing limited gene flow among the studied populations. Discussion Population genetic investigations are useful in under- standing genetic variability, allele flow, inbreeding against Fig. 3. Morphological characters of two different types of Senecio vulgaris. A – unbranched stem and bract black tip less than 1 mm long, B – branched stem and bract black tip more than 1 mm long (adapted and modified from Klinkenberg 2019). Fig. 5. Reticulogram of the studied Senecio vulgaris populations based on a neighbour joining tree of ISSR data. Populations are marked with numbers from 1-10 according to Tab. 1. Dashed lines indicate gene flow. Reticulation analysis revealed that limited amount of shared alleles or gene. Fig. 4. Two dimensional plot of principal coordinate analysis (PCoA) of the studied Senecio vulgaris populations based on ISSR data. Different colors indicate the plant specimens from each geo- graphical population. Group 1: the populations in north Iran (Populations 5, 7 and 9); Group 2: the populations in west and south west Iran (Popu;atopms 1, 2, 3, 4, 6, 8 and 10). INTRASPECIFIC DIVERSITY OF Senecio vulgaris L. ACTA BOT. CROAT. 80 (2), 2021 129 outbreeding and effective population size. This information is valuable in choosing effective management in conservative plans and also throws light on the presence of intraspecific taxonomic forms and suggests an appropriate hybridization strategy (Freeland et al. 2011, Sheidai et al. 2013, 2014). Genetic diversity of S. vulgaris was studied by Müller- Schärer and Fischer (2001). According to their investigation different geographical areas include different habitats and populations representing different habitats varied in their sizes. In our recent study, the genetic structure of S. glaucus showed that population fragmentation, restricted gene flow, genetic drift, and local adaptation have played a role in the genetic divergence of S. glaucus populations in Iran (Eft- ekharian et al. 2016). In the present investigation, S. vulgaris populations pre- sented a high degree of genetic variability. Species that are dispersed over a wide area face different environmental con- ditions and therefore can possess a wide genetic and mor- phological variability to manage ecological challenges (Freeland et al. 2011, El-Amier et al. 2014, Eftekharian et al. 2015, 2016). In this study, population fragmentation in S. vulgaris is revealed by PCoA. However, some degree of gene flow and genetic admixture occurred among populations as shown by a reticulogram. The probable loss of genetic variation within these populations can be prevented by this limited gene flow due to the action of genetic drift (Habibollahi et al. 2015). According to the Mantel test, which presented a pattern of isolation-by-distance in the studied S. vulgaris populations, adjacent populations have more chance for gene exchange than more distant populations, which is why more genetic similarities have been shown in closely situated populations. Comparison of the two datasets indicated that in some cases, the grouping of populations based on the morpho- logical method was consistent with molecular groupings. Therefore, the PCoA plot based on molecular and mor- phological data showed two groups of populations. Genetic and morphological differences between the two groups may be related to different ecological conditions. Eco- logical and environmental factors (e.g., temperature, humid- ity, and soil factors) can be significantly effective in shaping genetic diversity patterns (Huang et al. 2016). According to Müller-Schärer and Fischer (2001) different habitats can af- fect the genetic structure of S. vulgaris populations. Tang et al. (2015) showed that there was a strong relationship be- tween the soil factors (especially salinity and soil texture) and plant diversity. In our study, the habitat of studied pop- ulations varied from mountain meadows (Populations 5, 7 and 9) to roadsides (Populations 2, 3, 4, 6 and 10) and waste grounds (Populations 1 and 8). The mountain meadow hab- itats had gravelly soils while loamy soils were predominant in the waste ground and roadside habitats. However, the ef- fects of some factors in plant population genetic diversity remain unclear and more investigations have to be done. Conclusion Morphological characters revealed that two groups of the studied geographical populations differed from each other in two qualitative morphological features (black tips of calyculus bracts and type of stem), as evidenced in Fig. 3 and three quantitative characters (capitula number, length of the cauline leaf and plant height). 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