Micromorphological Characteristics for the Differentiation of 16 Centaurea Species

Micromorphological Characteristics for the Differentiation of 16 Centaurea Species | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 27 January 2025 V1 Latest version Share on Micromorphological Characteristics for the Differentiation of 16 Centaurea Species Authors : morteza tavassol 0009-0004-3041-1529 [email protected] and Farideh Attar Authors Info & Affiliations https://doi.org/10.22541/au.173798677.70923240/v1 304 views 166 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Centaurea L. is one of the largest genera within the Asteraceae family globally, encompassing approximately 400 to 700 species. This genus presents significant taxonomic challenges. The Iranian flora region comprises 28 sections and 89 species, of which 74 species are native to Iran. Recent studies have introduced several new species. The primary objective of this research was to investigate micro-morphological traits for the identification and differentiation of 16 Centaurea species. Micromorphological studies on plants allow for micro-meter level analysis of leaves, tissues, seeds, etc., using scanning electron microscopy (SEM). In this study, 18 micromorphological traits of the leaf abaxial epidermis, pappus, and achene were examined. The results indicated that among all measured traits, the leaf abaxial epidermis type (ETL), the number of pappus secondary bristles (NFP), abaxial wax density (WDL), pappus width (WOP), and seed surface texture (SST) had the greatest impact on species differentiation. The presence of glandular trichomes (SGL), glandular trichomes diameter (AGL), glandular trichomes density (DGL), achene hairs (ACH), and pappus twist (PAT) were also influential in species discrimination. A principal component analysis (PCA) graph confirmed the effectiveness of these micro-morphological traits in species differentiation. Introduction Iran is recognized as one of the world’s richest regions in terms of plant genetic diversity. Botanists estimate that there are between 10,000 and 12,000 plant species in Iran, positioning it as one of the most diverse centers of plant genetic resources globally. Understanding the genetic diversity of plant species is crucial for conservation efforts, agricultural improvement, and basic biological research. Genetic diversity is assessed and determined based on morphological, biochemical, cytological, and molecular traits. Before the development of molecular markers, morphological traits, which served as diversity metrics, were more commonly used (Iwamoto & Bull-Hereñu, 2018)\RL.In the past, scientists relied heavily on morphological descriptions to investigate population structure and genetic diversity. Mendel utilized morphological traits to develop his understanding of genetics and heredity. Similarly, Darwin employed various morphological characteristics in plants and animals to formulate his theory of natural selection and evolution(Atefe et al., 2015)\RL. Morphological traits have been the most widely used characteristics in plant taxonomy and identification. These traits have been employed for a longer period compared to anatomical and molecular evidence and have been the primary source of taxonomic data since the inception of plant systematics. Morphological assessment is a direct, cost-effective, and easily accessible method that does not require specialized technology. These traits are readily observable and are based on visible and accessible characteristics such as flower color, seed shape, growth habits, and pigmentation. In this approach, genetic diversity is evaluated in the presence of environmental variations, thus making it impossible to overlook genotypic changes (Chourasia & Mishra, 2018)\RL. These characteristics have significant practical applications in plant description and the development of botanical keys. Considering the evolutionary lineages of flowering plants, each of which may encompass thousands of species, it is not expected that all morphological features will appear identically in all species. In some cases, the absence of a combined or descriptive trait in a group that usually possesses it, or the rare occurrence of a feature in a group that is typically lacking it, is expected. Among reproductive characters, floral characters provide the most valuable features and are considered fundamental in defining natural groups. In a species, there is a clear relationship between changes in environmental conditions and morphological diversity, and species with wide geographic ranges tend to be more morphologically diverse than those with restricted ranges (Richards et al., 2005)\RL. Moreover, species with higher morphological diversity are better adapted to various environmental conditions compared to those with lower morphological diversity (Chen et al., 2010)\RL. The Asteraceae family, comprising more than 25,000 to 30,000 species worldwide, is recognized as the largest plant family and is unequivocally monophyletic. Excluding Antarctica, this family encompasses 1600 to 1700 genera distributed globally. Given an estimated 250,000 to 350,000 species of flowering plants worldwide, approximately 10% belong to this family (Funk et al., 2009)\RL. The genus Centaurea L. Is one of the largest genera within the Asteraceae family. Due to significant taxonomic challenges, the number of species within this genus is estimated to range from 400 to 700, based on accepted classifications (Aboul-Soud et al., 2022; Garcia-Jacas et al., 2006)\RL. The Flora Iranica recognizes 28 sections and 90 species within this genus, with only 74 species recorded in Iran. Subsequent studies have added 7 new species, 2 new subspecies, and 2 new records to the Iranian flora (Attar & Ghahreman, 2003; RAHIMINEJAD et al., 2010; Ranjbar & Negaresh, 2013)\RL. The primary objective of this study is to investigate micro-morphological characters for the identification and delimitation of 16 Centaurea species. The necessity of this research stems from the fact that the genus Centaurea L. Is one of the largest genera within the Asteraceae family, and it has long been plagued by taxonomic complexities (Aboul-Soud et al., 2022; Garcia-Jacas et al., 2006). \RL These taxonomic challenges in Centaurea have been particularly evident in research conducted in the Middle East (Asadipour et al., 2005; Ranjbar & Negaresh, 2013)\RL. Moreover, several species under study are endemic to Iran, making them valuable for multifaceted research. The genus Centaurea is one of the largest genera within the Asteraceae family globally and the second largest in Iran. Due to its extensive distribution and high species diversity, it is the most complex genus in the Asteraceae family and is constantly under revision. Numerous taxonomic problems exist within this genus, particularly at the sectional level. These issues include name changes, transfers between sections, transfers from other genera, reductions to subspecies, and the merging of species into a single taxon. In other words, a thorough and comprehensive investigation of the species within this genus is essential to resolve ambiguities and uncertainties regarding the precise boundaries of taxa, especially among closely related sections. Extensive field studies and the collection of numerous samples from all regions of Iran, as well as the examination of ecological factors influencing the distribution of these species, are crucial for taxonomic studies (Garcia-Jacas et al., 2001; Häffner & Hellwig, 2015; Wagenitz, 1980)\RL. Moreover, morphological characteristics are considered the most commonly used features in plant taxonomy and identification. These characteristics have been employed for a longer duration compared to anatomical and molecular evidence and have been the primary source of taxonomic evidence since the inception of plant systematics. In this method, genetic diversity is evaluated in the presence of environmental changes; therefore, genotypic changes cannot be ignored in this approach (Chourasia & Mishra, 2018)\RL. In other words, plant morphology is a branch of science that deals with the internal and external structures of plants and is intertwined with plant anatomy, which studies the cells and tissue structures of plant organs. Micromorphological studies on plants allow for micro-level analysis of pollen, leaves, tissues, seeds, and other plant parts through scanning electron microscopy (SEM). In general, high-capacity light or electron microscopes are used to analyze plant anatomical features. Scanning electron microscopy provides high-magnification imaging through high-resolution imaging techniques. Therefore, morphological, structural, and elemental information about plants can be obtained at high magnifications of surfaces (Yigit, 2016)\RL. Materials and Methods Preparation of plant samples In this study, 16 species and subspecies were examined for micromorphological characteristics. Plant specimens were obtained from the University of Tehran Herbarium (TUH) and the Research Institute of Forests and Rangelands (TARI). Herbarium specimens belonging to the genus Centaurea L. Were specifically studied at the Central Herbarium of the University of Tehran (TUH). Including duplicates, a total of 39 species were examined. These species were identified using standard botanical methods and relevant references, and the necessary traits for subsequent studies were investigated. Morphological studies Initially, to accurately identify and verify the herbarium codes of plant species under study, reliable botanical resources such as species descriptions and identification keys were utilized. Additionally, various morphological characteristics of each species, including vegetative (e.g., stem, leaf, root) and reproductive (e.g., flower, fruit) organs, were empirically examined and measured using tools like millimeter rulers and stereomicroscopes equipped with graduated lenses. Micromorphological studies Given the unique characteristics of cornflower leaves and seeds, scanning electron microscopy (SEM) was employed to conduct a detailed examination of features such as the adaxial epidermal tissue, seed surface texture, pappus type, and guard cells. These micrographs were essential for taxonomic classification and species identification. A total of 18 traits were analyzed as outlined in Table 1. To prepare samples for SEM imaging, plant parts were initially observed under a stereomicroscope to ensure the absence of contaminants. Subsequently, approximately 2 mm² sections of the adaxial epidermis were excised. For seed analysis, individual seeds and pappi were isolated. To enhance statistical power and account for ecological variability, two to three populations of each species from different ecological regions were sampled. The excised samples were mounted on SEM stubs using double-sided carbon tape without any pretreatment. To improve image quality, the samples were sputter-coated with a 24 nm layer of gold using a Hummer 11 sputter coater. Images were captured using a KYKY 3200 EM scanning electron microscope operating at an accelerating voltage of 24 kV. Based on the SEM micrographs, qualitative traits of the adaxial epidermis, seed surface, and pappus were recorded. Additionally, stomatal density was determined by counting the number of stomata per 100 μm². Stomatal length and width were measured using ImageJ software at a scale of 10 μm. The obtained data were tabulated and analyzed in Excel. Table 1- Micromorphological traits studied in species 1 Width of the pappus WOP 2 Number of secondary filaments of the pappus NFP 3 Lateral branches of the pappus LBP 4 Pappus torsion PAT 5 Achenes hair \RL ACH 6 Seed surface texture SST 7 Seed surface hair SSH 8 Epidermal hairs on the back of the leaf EHL 9 The density of epidermal hairs on the back of the leaf DHL 10 Width of the back of leaf stomata (µm) WLS 11 Length of the back of the leaf stomata (µm) LLS 12 Epidermal tissue on the back of the leaf ETL 13 Shape of wax on the back of the leaf SWL 14 Wax density on the back of the leaf WDL 15 Shape of the glandular trichomes on the back of the leaf SGL 16 Density of the glandular trichomes behind the leaf DGL 17 Average diameter of the glandular trichomes behind the leaf (µm) AGL 18 Number of leaf stomata on a fixed surface (145924 µm) NLS Morphological and micromorphological data analysis Before conducting any analysis, all quantitative data obtained from the morphometric and micro morphometric studies were standardized by converting them to micrometers. Both quantitative and qualitative attributes were recorded in Excel 2016. \RL Data normality was assessed using the Shapiro-Wilk test and the Kolmogorov-Smirnov test in SPSS version 26. Levene’s test was employed to evaluate the homogeneity of variances. For normally distributed variables, ANOVA was used for group comparisons, while the Kruskal-Wallis test was applied to compare non-normally distributed variables. \RL Attributes that did not show significant differences between groups were excluded. Pearson correlation was used to assess the relationship between normally distributed variables, while Spearman correlation was employed for categorical variables and those that did not follow a normal distribution. \RL Attributes exhibiting a correlation coefficient greater than 95% confidence level were considered highly correlated. To address multicollinearity, one of the highly correlated attributes was either removed or a ratio was calculated between the attributes. All analyses were conducted using SPSS version 26 and GraphPad Prism 10. dendrogram and clustering, variables were standardized using the Scale command to ensure comparability and facilitate the interpretation of results. This method, also known as PCA, is a variable reduction technique that shares many similarities with exploratory factor analysis. \RL The primary goal of Principal Component Analysis (PCA) is to reduce a large set of variables into a smaller set of ’artificial’ variables, called principal components, which account for most of the variance in the original variables. Results Examining the glandular trichomes by species and section The mean diameter of glandular trichomes, in micrometers, classified by species and section. The results indicate that species belonging to the section Cyanus lack glandular trichomes on the leaf back, while species in sections Phaeopappus and Cynaroides predominantly possess these trichomes. \RL Figure (1) shows the epidermis texture and the shape of the leaf glandular trichomes in the species and subspecies studied, which were imaged and measured with an electron microscope\RL. Examining the characteristics of the pappus by species and section As shown in Table 1, this study investigated the average width, curvature, lateral veins, and subsidiary vein count of the pappus. The characteristics of the pappus are presented in Table 2, and qualitative traits have been quantified for analysis. Results showed that species from different sections were highly similar in terms of pappus characteristics, except for a few exceptions. \RL Within the section Cynaroides, except for the species C. Gigantea, the measured traits were similar and homogeneous. Figure 2 shows the various shapes of the pappus in the studied species and subspecies, as imaged and measured using a scanning electron microscope. Table 2: Pappus characteristics by species and section NO Section Species Average width of pappus\RL) µ m \RL ( Pappus torsion Lateral branches of pappus Number of substrings 1 Phaeopappus C. albonitens 110 Smooth Yes 15 2 Phaeopappus C. aucheri subsp aucheri 130 Smooth Yes 18 3 Phaeopappus C. aucheri subsp szowitsii 90 Smooth Yes 12 4 Phaeopappus C. aucheri subsp elburzensis 160 Smooth Yes 21 5 Phaeopappus C. aucheri subsp indistincta 120 Smooth Yes 20 6 Phaeopappus C. aucheri subsp farsistanica 120 Smooth Yes 20 7 Cyanus C. cheiranthifolia var. cheiranthifolia 70 Smooth Yes 10 8 Cyanus C. cheiranthifolia var. purpurascens 70 Smooth Yes 10 9 Cyanus C. triumfettii 90 Smooth No 10 10 Cyanus C. depressa 50 Smooth Yes 8 11 Cyanus C. cyanus 70 Smooth Yes 11 12 Cynaroides C. regia 90 Twisted No 9 13 Cynaroides C. imperialis 90 Twisted No 9 14 Cynaroides C. gigantea 100 Smooth Yes 12 15 Cynaroides C. phlomoides 80 Twisted No 7 16 Cynaroides C. geluinsis 80 Twisted No 6 Figure 1- Electron microscope images of the epidermis and leaf glandular trichomes on the back of the leaf in different Centaurea species\RL. A\RL) C. regia \RL، (B \RL) C. gigantea \RL، (C\RL) C. imperialis \RL ، ( D \RL) C. geluinsis ) \RL، E \RL) C. phlomoides \RL، (F \RL) C. albonitens \RL، ( G \RL) C. aucheri subsp szowitsii \RL، ( H \RL) C. aucheri subsp elburzensis )\RL، I\RL) C. aucheri subsp indistincta ). Figure 2- Electron microscope images of the stamens in different Centaurea species. (A) Pappus torsion in C. Regia , (B) Pappus torsion in C. imperialis , (C) Lateral tentacles of \RL Pappus in C. gigantea , (D) Pappus torsion in C. geluinsis , (E) Pappus tendrils in C. phlomoides , (F) Lateral tentacles of \RL Pappus in C. albonitens , (G) Lateral tentacles of \RL Pappus in C. aucheri subsp szowitsii , (H) Lateral tentacles of \RL Pappus in C. aucheri subsp elburzensis , (I) Lateral tentacles of \RL Pappus in C. cyanus . Multivariate analysis based on micromorphological traits The results of the analysis of 18 micromorphological traits indicate that among all the measured traits, the traits of epidermal tissue of the back of the leaf (ETL), number of secondary filaments of the pappus (NFP), wax density of the back of the leaf (WDL), Width of the pappus (WOP) and seed surface texture (SST) show the greatest effect in the separation of the studied species in the first dimension (Figure 3). In the second dimension, the presence of the glandular trichomes on the back of the leaf (SGL), Average diameter of the glandular trichomes behind the leaf ( \RL µm) (AGL), density of the glandular trichomes behind the leaf (DGL), Achenes hair (ACH) and Pappus torsion (PAT) show the greatest effect in the separation of the studied species (Figure 4). In Figure (5), the graph of the intensity of the effect of quantitative traits in the separation of the species confirms. In the final graph of the principal component analysis (PCA), the species of this genus are well separated from each other (Figure 6)\RL. Figure 3 - Diagram of the influence of micromorphological traits in the first dimension Figure 4 - Diagram of the influence of micromorphological traits in the second dimension Figure 5 - Graph of the intensity of the effect of micromorphological traits in species differentiation Figure 6- Final graph of principal component analysis (PCA) based on micromorphological traits Discussion The results showed that species in the Cyanus section lacked glandular trichomes on the back of the leaf, and species in the Phaeopappus and Cynaroides sections mostly had glandular trichomes. In the Phaeopappus section, only the species C. Aucheri subsp aucheri lacked of \RL glandular trichomes, which requires further investigation. The results showed that species of different sections are very similar in terms of leaf characteristics, except for exceptions. In the Cynaroides section, except for the species C. Gigantea , the measured traits are close and homogeneous, which is why in Figure (6) the species C. Gigantea (code 14) is farther away from other species. As can be seen in Figure (6), species of the Phaeopappus and Cyanus sections are well separated based on quantitative traits. However, in the Cynaroides section, further investigation is needed, which could be due to the complexity of the relationships between species in this section. Not many studies have been conducted on the micromorphology of this genus, but in relation to its morphology, Garcia et al. (2001) stated that the systematic classification of the genus Centaurea has its own complexities and is directly related to the morphology of the bract and achene appendages (Garcia-Jacas et al., 2001)\RL. The present study also showed the relationships of some bract appendage traits such as the length of the terminal spine of the bract (LSB), and the achene, such as the achene length (mm) (ACL) with the differentiation of plant species\RL. References Aboul-Soud, M. A. M., Ennaji, H., Kumar, A., Alfhili, M. A., Bari, A., Ahamed, M., Chebaibi, M., Bourhia, M., Khallouki, F., Alghamdi, K. M., & Giesy, J. P. (2022). Antioxidant, Anti-Proliferative Activity and Chemical Fingerprinting of Centaurea calcitrapa against Breast Cancer Cells and Molecular Docking of Caspase-3. Antioxidants, 11(8). Https://doi.org/10.3390/antiox11081514Asadipour, A., Mehrabani, M., & Najafi, M. L. (2005). Volatile oil composition of Centaurea aucheri (DC.) Wagenitz. Daru, 13(4), 160–164.Atefe, K., Abkenar, K., & Torkman, J. (2015). Variations in Leaf and Fruit Morphological Traits of Sweet Chestnut (Castanea Sativa) in Hyrcanian Forests, Iran. International Journal of Plant Research, 1, 155–161.Attar, F., & Ghahreman, A. (2003). Two new species of the genus Cousinia (Asteraceae) from Iran. Nordic Journal of Botany, 23(5), 589–592. Https://doi.org/10.1111/j.1756-1051.2003.tb00438.xChen, X., Gao, Y., Zhao, T., ZHU, M.-J., Ci, H., & Xiao-yan, S. (2010). Morphological variations of Caragana microphylla populations in the Xilingol steppe and their relationship with environmental factors. Acta Ecologica Sinica, 30, 50–55. Https://doi.org/10.1016/j.chnaes.2010.03.001Chourasia, H. K., & Mishra, D. P. (2018). Plant Systematics and Biotechnology: Challenges and Opportunities. Today and Tomorrow’s Printers and Publishers.Funk, V. A., Susanna, A., Stuessy, T. F., & Bayer, R. J. (2009). Systematics, Evolution, and Biogeography of Compositae. In Madroño (Vol. 56, Issue 3).Garcia-Jacas, N., Susanna, A., Garnatje, T., & Vilatersana, R. (2001). Generic delimitation and phylogeny of the subtribe Centaureinae (Asteraceae): A combined nuclear and chloroplast DNA analysis. Annals of Botany, 87(4), 503–515. Https://doi.org/10.1006/anbo.2000.1364Garcia-Jacas, N., Uysal, T., Romashchenko, K., Suárez-Santiago, V. N., Ertuǧrul, K., & Susanna, A. (2006). Centaurea revisited: A molecular survey of the Jacea group. Annals of Botany, 98(4), 741–753. Https://doi.org/10.1093/aob/mcl157Häffner, E., & Hellwig, F. H. (2015). Phylogeny of the tribe Cardueae (Compositae) with emphasis on the subtribe Carduinae: an analysis based on ITS sequence data. Willdenowia, 29(1/2), 27. Https://doi.org/10.3372/wi.29.2902Iwamoto, A., & Bull-Hereñu, K. (2018). Floral development: re-evaluation of its importance. Journal of Plant Research, 131(3), 365–366. Https://doi.org/10.1007/s10265-018-1034-9RAHIMINEJAD, M. R., MOZAFFARIAN, V., & MONTAZEROLGHAEM, S. (2010). A taxonomic revision of Centaurea section Acrocentron (Asteraceae) in Iran. Botanical Journal of the Linnean Society, 163(1), 99–106. Https://doi.org/10.1111/j.1095-8339.2010.00772.xRanjbar, M., & Negaresh, K. (2013). A revision of Centaurea sect. Phaeopappus (Asteraceae, Cardueae). Phytotaxa, 123(1), 1–40. Https://doi.org/10.11646/phytotaxa.123.1.1Richards, C. L., Pennings, S. C., & Donovan, L. A. (2005). Habitat range and phenotypic variation in salt marsh plants. Plant Ecology, 176(2), 263–273. Https://doi.org/10.1007/s11258-004-0841-3Wagenitz, G. (1980). Centaurea L. In: Flora Iranica (Ed. Rechinger, K. H.). Akademische Druck-Und, Verlagsanstalt, Graz., 139b, 356–362.Yigit, N. (2016). Micromorphological Studies On Plants And Their Importance. 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