Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications

Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications | 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 Search Search ADVANCED SEARCH SCROLL Microscopy Research and Technique This is a preprint and has not been peer reviewed. Data may be preliminary. 17 January 2025 V1 Latest version Share on Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications Authors : Alireza Dolatyari and Mehdi Dehghani 0000-0002-7616-6715 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173712970.07182095/v1 Published Microscopy Research and Technique Version of record Peer review timeline 371 views 186 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Pollen from 20 populations representing 13 Iranian species of the genus Crocus was analyzed using light microscopy (LM) and scanning electron microscopy (SEM) to investigate the palyno-morphological features and their taxonomic significance. Polliniferous materials were sourced from either fresh plants or herbarium samples. For LM analysis, pollen grains were acetolyzed, while intact pollen grains were used for SEM micrographs. The study examined various pollen traits, including polar and equatorial diameters, aperture and meso-aperture width, exine thickness, and P/E ratios in LM analyses. Additionally, SEM observations focused on spinule width and length and the density of spinules and perforations on the exine. The investigation revealed that the pollen grains of Crocus are monads, oblate-spheroidal in shape, and measure approximately 61 to 106.48 micrometers along their equatorial axis. The smallest pollen grains were observed in C. reinhardii , while the largest were found in C. archibaldiorum . The pollen grains were intectate, with the exine irregularly perforated and covered with microechinate ornamentation. Two distinct types of pollen apertures were identified in the studied species: polyaperturoidate apertures in the two populations of Crocus haussknechtii from section Crocus and spiraaperturate pollen in the remaining taxa belonging to section Nudiscapus . Our data suggest that pollen characteristics offer valuable synapomorphies for delimiting sections within the genus Crocus , and also provide significant support for understanding and clarifying relationships within taxonomic aggregates and complexes. Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications Alireza Dolatyari 1 and Mehdi Dehghani 2* 1 Plant Bank, Iranian Biological Resource Centre (IBRC), Iranian Academic Centre for Education, Culture & Research (ACECR), Tehran, Iran. Email: [email protected] . 2* Department of Biology, Faculty of Science, University of Zabol, Zabol, Iran. https://orcid.org/0000-0002-7616-6715 Corresponding author E-mail: [email protected] Acknowledgments We extend our heartfelt thanks to Mr. Hossein Salimi for his invaluable assistance with fieldwork activities. Our sincere appreciation also goes to Mr. Javad Shaghaghi for his dedicated efforts in maintaining our living collections. Additionally, we are deeply grateful to Mrs. Mahnaz Amirabadi from the Central Laboratory of the University of Sistan and Baluchestan, Zahedan, Iran, for capturing the micrographs of pollen grains. Funding interests: This work received financial support from The University of Zabol under grant number UOZ-GR-0331. Also, the Iranian Biological Resource Center is gratefully acknowledged for funding project No: P-1401-01. Conflict of Interest Statement The authors declare that they have no financial or non-financial competing interests regarding this paper. Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications Abstract Pollen from 20 populations representing 13 Iranian species of the genus Crocus was analyzed using light microscopy (LM) and scanning electron microscopy (SEM) to investigate the palyno-morphological features and their taxonomic significance. Polliniferous materials were sourced from either fresh plants or herbarium samples. For LM analysis, pollen grains were acetolyzed, while intact pollen grains were used for SEM micrographs. The study examined various pollen traits, including polar and equatorial diameters, aperture and meso-aperture width, exine thickness, and P/E ratios in LM analyses. Additionally, SEM observations focused on spinule width and length and the density of spinules and perforations on the exine. The investigation revealed that the pollen grains of Crocus are monads, oblate-spheroidal in shape, and measure approximately 61 to 106.48 micrometers along their equatorial axis. The smallest pollen grains were observed in C. reinhardii , while the largest were found in C. archibaldiorum . The pollen grains were intectate, with the exine irregularly perforated and covered with microechinate ornamentation. Two distinct types of pollen apertures were identified in the studied species: polyaperturoidate apertures in the two populations of Crocus haussknechtii from section Crocus and spiraaperturate pollen in the remaining taxa belonging to section Nudiscapus . Our data suggest that pollen characteristics offer valuable synapomorphies for delimiting sections within the genus Crocus , and also provide significant support for understanding and clarifying relationships within taxonomic aggregates and complexes. Keywords: Crocus , Nudiscapus, pollen morphology, spiraperturate, taxonomy Research highlights Palynological analysis of 13 Iranian Crocus species revealed significant taxonomic value of pollen traits. Two distinct aperture types were identified: spiraperturate (sect. Nudiscapus ) and polyaperturoidate (sect. Crocus ). Principal component analysis confirmed sectional distinctions and provided insights into species and aggregate relationships Novel palynological data were supplied for nine Iranian endemic species. Graphical abstract Introduction Belonging to the family Iridaceae, the genus Crocus currently consists of 262 species (Rukšāns, 2023, Advay and Rukšāns, 2024). The genus exhibits a wide distribution range, approximately between 10°W to 80°E and 30°N to 50°N (Saxena, 2010). The Balkan Peninsula and Asia Minor are its main diversity centers, harboring over half of the recognized species (Rukšāns, 2017b). Crocuses have shown remarkable adaptability to a wide range of ecological conditions, from shaded forests to alpine and desert environments (Dolatyari et al., 2024). The genus Crocus is characterized as perennial, cormous plants with three stamens and actinomorphic (radially symmetrical) flowers. Phylogenetic studies strongly supported the monophyly of the genus, but have questioned the monophyly of several already accepted intra-generic and completely all infra-specific groupings (Harpke et al., 2013, Nemati et al., 2018, Petersen et al., 2008, Harpke et al., 2015). Additionally, crocuses are systematically problematic due to the shortage of diagnostic traits and character heterogeneity so that significant morphological variability is displayed even within single populations (Kerndorff et al., 2015). Among Crocus species, C. sativus L. is of high pharmacological, nutritional, and economic importance and C. damascenus Herb. provides edible corms while many other species and their artificial hybrids are good candidates for ornamental uses (Dolatyari et al., 2024). Except for C. sativus L. which is a cultivated crop, nine wild species have been reported within the Flora Iranica territory, of which eight occurred in Iran (Wendelbo and Mathew, 1975). Over the past 15 years, the number of Iranian species has tripled (Advay and Rukšāns, 2024, Dolatyari and Rukšāns, 2022, Rukšāns, 2022, Rukšāns, 2014a, Rukšāns, 2014b, Rukšāns, 2017a, Rukšāns, 2017b). Presently, 25 Crocus species are growing within the political borders of Iran, categorized into two sections: Crocus (4 autumn-flowering species) and Nudiscapus Mathew (21 species, mostly spring-blooming species), of them, on the whole, 19 species (about three quarters) are endemic elements. The conservation status of most species were assigned as critically endangered according to IUCN criteria (Dolatyari et al., 2024). Palynological studies contribute significantly to taxonomic differentiation especially in higher taxonomic ranks, however, in particular cases, pollen characters are useful in species delimitation as well (Perveen and Qaiser, 2012, Dehghani et al., 2021). Pollen morphological studies within the family Iridaceae are limited, with only one previous study focusing on the pollen morphology of Iranian crocuses (Tabasi and Mehrabian, 2019) where species identification was mostly based on herbarium specimens and outdated identification literature. There is a pressing need for a comprehensive taxonomic understanding of the whole genus using all sources of taxonomic evidence. Further, many of the species already described for Iran were delineated solely based on morphological and gross anatomical characters (Advay and Rukšāns, 2024, Dolatyari and Rukšāns, 2022, Rukšāns, 2014a, Rukšāns, 2014b, Rukšāns, 2017b, Rukšāns, 2017a). To address part of this uncertainty and to clarify taxonomic relations, this study focuses on the pollen morphology of Iranian crocuses, following the species concept presented in the last taxonomic revision on the genus in Iran (Dolatyari et al., 2024). Specifically speaking, by conducting a detailed pollen morphological analysis on the 20 accessions of the genus Crocus , we aimed to enhance our understanding of its palynology and taxonomic significance of pollen characters in 13 Iranian species. Material and Methods Material collection and identification The studied plants were collected, mostly in flowers, from the natural habitats of 11 Iranian provinces. Immediately after returning home, the corms were transplanted in trial fields of the Iranian Biological Resource Center (IBRC), Karaj, Alborz province. Moreover, herbarium vouchers were deposited in the herbarium of this Center (IBRC). We used the identification key presented by Dolatyari et al. (2024) for scientific name determinations. Finally, a total of 20 accessions, representing 13 Crocus species—including nine endemics to Iran—were sampled for pollen analysis. The stamens were obtained from flowering plants in the living collections or herbarium sheets. Detailed information about localities, herbarium numbers, and distribution ranges of the studied populations are presented in Table 1. In addition, Figure 1 illustrates the detailed morphological traits and habitat characteristics of all studied accessions. Light microscopic analysis Pollen samples from 20 populations of 13 species of the genus Crocus from Iran were examined using both light (LM) and scanning electron microscopes (SEM). The acetolysis method outlined by Erdtman (2023) was followed to prepare pollen grains for LM observations. This involved treating the pollen grains with a mixture of acetic anhydride and sulfuric acid (9:1 v/v) at 90°C for four minutes, followed by centrifugation and repeated decantation using distilled water. The acetolysed pollen grains were then mounted between microscopic slides using glycerol jelly as a mounting medium (for details see (Dehghani and Akhani, 2009). An Olympus BX51 light microscope equipped with a DP25 digital camera was utilized to photograph a minimum of 25 mature pollen grains at a magnification of 600x. Motic software was used to measure pollen characteristics including polar and equatorial axes, exine thickness, and colpi and intercolpi widths. The average and standard deviation of each pollen character was calculated for all studied samples using Microsoft Excel (Table 2). Scanning electron microscopic analysis Untreated pollen grains were dissected out of the mature anthers and mounted directly on double-sided sellotape on SEM stubs. The pollen grains were coated with gold, and examined, by a KYKY-EM8000F scanning electron microscopy at the Central Laboratory of the University of Sistan and Baluchestan, Zahedan, Iran. At least three micrographs were captured at 1500 x and 3000 x magnifications for each sample. Fine pollen morphological characters including colpus type, spinule length and width, spinule, and peroration densities on exine were measured using Image J software (Table 2). Statistical analysis Measurements were recorded for a minimum of 25 pollen grains per specimen, and the means and standard deviations of quantified pollen grain characteristics were calculated using Microsoft Excel (Table 2). Subsequently, a matrix comprising 20 accessions (samples) and seven pollen traits (variables) was constructed for light microscopy (LM) data analyses. Principal component analysis (PCA) was then performed to explore variations in pollen characteristics, employing the PAST v. 3.14 software (Hammer and Harper, 2001). Correlation matrices were computed, from which eigenvalue and eigenvector matrices were derived, leading to the extraction of a PCA biplot. An identical analytical approach was applied to the combined LM and scanning electron microscopy (SEM) data. The new dataset, containing 13 samples and 12 variables was subjected to another PCA, utilizing the same software. Results and Discussion The list of all studied accessions and the measured data are presented in Table 2. The LM and SEM micrographs of 13 species of the examined specimens are also arranged in Figures 2-4. The pollen grains under analysis are monad, oblate-spheroidal, and measure about 61-106.48 micrometers in their longest axes which fall into the large pollen grain category based on six classes of pollen sizes described by (Erdtman, 2023). The smallest pollen grain was found in C. reinhardi and the largest in C. archibaldiorum (P1015056 accession) (Table 2 and Figs. 2-4). Notably, the total haploid length of chromosomes in C. archibaldiorum was found to be the largest among the five autumn-flowering species studied from Iran (48.19 µm, Dolatyari 2025). The pollen grains are intectate, and the exine is covered with microechinate ornamentations. Perforations are irregular and visible at a magnification of 3,000 x in all examined samples (Figs 3-4). Two types of apertures exist in the studied species: polyaperturoidate in the two populations of C. haussknechtii from section Crocus (Fig. 3 H) and spiraloaperturate pollen in the rest of the taxa belonging to sect. Nudiscapus Mathew (Figs 2-3). Erdtman (2023) described pollen in Crocuses as inaperturate, spiraperturate, and polyrugoidate. However, there has been considerable disagreement among researchers regarding aperture types in the genus Crocus , described as inaperturate, pantoporate, polyaperturoidate, and spiraperturate to net-like apertures (Muradyan et al., 2023). In addition, it is noteworthy that a high percentage of anomalous grains, characterized by smaller size, collapsed, and with broken walls, was seen in the studied samples (see Fig 2 as two instances in C. archibaldiorum (P1015056) and C. dolatyarii ). A high percentage of anomalous grains (47%) was previously reported in C. sativus, while the lowest (1%) was observed in C. hadriaticus Herb. (Grilli Caiola et al., 2000), however, neither species was included in the present study. Multivariate analysis Principal Component Analysis of LM Pollen Features in 20 Accessions Figure 5 shows the PCA scatter plot for pollen features of 20 investigated populations of the genus Crocus measured under LM based on the first two principal components. PC1 captures 57.63% of the variance, and the first two components together account for 77.12% of the total variability among the features. The horizontal axis correlates with pollen size and the vertical axis is mainly associated with aperture type and aperture width (Fig. 5). In the PCA biplot, the two samples of C. haussknechtii group together in the positive part of PCA2 and the negative side of PCA1. The isolated position of this species (sect. Crocus ) distinct from the other 12 species (sect. Nudiscapus ) indicates section-specific pollen characteristics. The studied accessions of C. damacenus are roughly grouped together, but two distinct subgroups can be identified: the two populations from Esfahan (P1015064 and P1015065 accessions) exhibit greater similarity to each other, while populations from Kermanshah (P1015138), Kurdistan (P1015128) and Markazi (P1015062) demonstrate closer similarities among themselves. The same heterogeneity in karyotypic features, both chromosome number and morphology, has already been reported for different investigated materials of this widely distributed species from Iran (Sanei et al., 2007, Brighton et al., 1973, Ghaffari and Djavadi, 2007, Ebrahirnzadeh et al., 1998, Dolatyari, 2025). It is worth mentioning that palynological data verifies the previous assumption based on morphological, ecological, and karyological characteristics that this species includes still unrecognized species in Iran (Dolatyari et al., 2024, Dolatyari, 2025). Also, the two examined populations of C. caspius (P1015046, P1015052) are located close to one another but pollen features of C. archibaldiorum populations (P1015056, P1015060) manifest considerable dissimilarities in terms of pollen size and meso-aperture and aperture widths (Figure 1). C. speciosus aggregate is one of the most complicated yet unresolved taxonomic groups within the genus Crocus (Brighton et al., 1983, Rukšāns, 2023). Until now, C. archibaldiorum and C. zubovii Rukšāns have been split from the Iranian C. speciosus aggregate solely based on morphological characters (Rukšāns, 2014a, Rukšāns, 2017b). Our data here confirm morphological variation, though strongly against uniform karyotypes we already saw in the same examined accessions (Dolatyari 2025 in press). These two populations are somewhat geographically close to each other but drastically differ in habitat conditions (Figs. 1b and 1d). We have recognized another new species of this aggregate from north Iran (unpublished data), adding more ambiguity to the existing condition. Certainly, before any concrete conclusion, much data should be provided on this complex. The rest of the investigated species are represented by one population in our study (Table 1, Fig 5). However, the PCA diagram reveals indications of grouping among morphologically, ecologically, or phylogenetically related species. C. marandicus , C. azerbaijanicus , and C. chionophilus which are morphologically similar and belong to the so-called “ Crocus biflorus Miller aggregate” are arranged together in the PCA plot. Moreover, the two morphologically similar species C. iranicus and C. peudoiranicus are positioned close to each other. All five latter species are distributed in NW Iran, hardly distinguishable by amateurs based on morphological features. There are no previous chromosome and molecular data for this group and it could be highly invaluable that for the first time, except morphology, pollen data corroborates their position as separate entities. Principal component analysis of combined LM and SEM datasets in 13 species The PCA scatter plot of combined LM and SEM data has been shown in Fig 6 where PCA1 accounts for 41.24% and PCA2 explains 18.47% of the variance in the dataset. In the PCA plot, PCA axis 1 is primarily associated with variations in polar and equatorial axes as well as exine thickness while PCA axis 2 is mainly reflects differences in aperture type and aperture width. In the combined plot, the distinct positioning of C. haussknechtii (belonging to section Crocus ) relative to other taxa (from section Nudiscapus ) supports again the presence of section-specific pollen traits among the examined species of the genus in Iran. C. michelsonii , along with the other two Central Asian species, C. alatavicus Regel & Semenow and C. korolkowii Maw & Regel, constitutes the series Orientales Mathew. Interestingly, molecular analyses revealed that a very close relative of this trio is the autumn-blooming C. caspius from northern Iran and southern Azerbaijan (Harpke et al., 2013, Petersen et al., 2008). However, our analysis indicates the distinct position of C. michelsonii , separate from C. caspius , confirming the morphological, phenological, and habitat differences between these two species. Three of the 13 investigated species are autumn-blooming ( C. archibaldiorum , C. caspius , and C. damascenus ). Their positioning in the PCA plot, both relative to each other and to other species, suggests that phenological data has limited significance in the taxonomy of the genus. This aligns with molecular studies, which have ruled out the monophyly of some previously proposed groupings based on a common flowering season (Harpke et al., 2013, Petersen et al., 2008, Harpke et al., 2015, Mathew, 1983) . Among the studied species within the so-called Crocus biflorus aggregates, the distinct positions of C. dolatyarii and C. chiaicus compared to other species ( C. azerbaijanicus , C. chionophilus , C. iranicus , C. marandicus , C. pseudoiranicus , and C. reinhardii ) are noteworthy. C. dolatyarii is differentiated from the other species by the color of its flower throat (white versus yellow), while C. chiaicus exhibits the highest number of ribs in the lateral channels of its leaves among the Iranian species—six well-developed ribs, with occasionally up to two additional, less conspicuous ribs. Comparison of our results with previous findings Comparison of our results with the only palynological study on Iranian crocuses (Tabasi and Mehrabian, 2019) reveals some congruencies, despite differences in the literature used for identifications. Both studies demonstrate that pollen characters offer valuable synapomorphies for delimiting sections within Crocus . While the pollen of C. sativus was not analyzed in this study, the micrograph provided by Tabasi and Mehrabian (2019) indicated its polyaperturoidate condition as a synapomorphy for members of section Crocus . However, their study reported a wider range of pollen sizes (33.95 to 130 μm) (Tabasi and Mehrabian, 2019) compared to the present study (61-106.48 μm). Pollen size variability has been observed both within and between species of Crocus (Isik and Donmez, 2006, Muradyan et al., 2023). Katya Spasova and Popova Todorova (2012) analyzed the pollen of 10 wild Crocus species from Bulgaria and identified two distinct pollen types based on SEM observations: Inaperturate C. vernus (L.) Hill pollen type with a warty exine, which was found only in C. pallasii Goldb. (sect. Crocus ), and Syncolpate C. biflorus Mill. pollen type featuring one spiral groove and an echinate exine, found in eight other species, mostly from section Nudiscapus . Additionally, C. reticulatus Steven ex Adams exhibited smooth exine and indistinct apertures (KATYA SPASOVA and POPOVA TODOROVA, 2012). In contrast, Işık and Oybak (2006) studied 29 Crocus taxa from Turkey and identified three types of apertures: a spiral furrow, more or less extensive furrows, and short furrows. They described the pollen of C. pallasii as having short furrows (not inaperturate), and the pollen of C. reticulatus subsp. reticulatus with more or less extensive furrows and echinate exine, rather than having a non-sculptured surface and indistinct apertures. It is important to note that none of the Bulgarian or Turkish species examined in these studies were included in our research, though results of Işik and Oybak (2006) in C. pallasii are congruent with ours in C. haussknechtii , confirming section-specific pollen features (Isik and Donmez, 2006). Furthermore, similar to ours, all species of the sect Nudiscapus examined by Katya Spasova and Popova Todorova (2012) possessed spiraperturate pollen, even though they described spiraperturate pollen of C. reticulatus as having indistinct aperture. However, inaperturate pollen was not observed in any of the 20 taxa examined in our study from Iran. Furthermore, in agreement with the previous studies (Isik and Donmez, 2006, Tabasi and Mehrabian, 2019), spinule size was found to have no significant taxonomic value in crocuses. However, spinule density varied significantly among the species examined by us, providing a more notable distinguishing feature for taxonomic analysis (Table 2). Pollen Evidence and Evolutionary Trajectories in Crocus A developmental investigation into the pollen wall and tapetum across several Crocus species revealed that the pollen is atectate and echinate. The apertures exhibit notable variability, encompassing furrows, colpi, pores, or heterogeneous forms, with their shapes and dimensions displaying intraspecific variation (Chichiriccò, 1999). Based on generally accepted evolutionary trends, furrows are considered a primitive character, pores represent a more specialized state, and colpi are viewed as intermediate between the two (Blackmore and Barnes, 1990). According to this framework of exine morphology, the pollen of section Crocus would appear to be more evolutionarily advanced than that of section Nudiscapus . However, this interpretation contradicts the recognized advanced phylogenetic status of the sect. Nudiscapus relative to sect. Crocus . Muradyan et al. (2023), however, proposed an alternative hypothesis, suggesting that in Crocus pollen, the spiraperturate condition represents the primitive state, while the polyaperturoidate condition (either pantocolpate or pantoporate) is a transitional state arising due to mutations. The most advanced form is characterized by net-like apertures, reflecting progressive evolutionary specialization (Muradyan et al., 2023). The spiraperturate pollen type has arisen independently multiple times in the course of evolution in both dicots and monocots (Hesse and Zetter, 2005, Furness and Rudall, 1999). Polyaperturoidate pollen is frequently regarded as an advanced evolutionary adaptation, as it enhances pollen germination efficiency and improves reproductive success (Hesse and Zetter, 2005, Oh, 2023). Despite their diversity among angiosperms, apertures are generally consistent within taxa, making them valuable taxonomic markers. Aperture variability or instability reflects active selective pressures and underlying genetic instability that drives adaptation and diversity. Genetic variation influencing aperture formation is further evidenced by polymorphism in karyotypes and variations in chromosomal behavior during meiosis, underscoring the intricate relationship between genetic mechanisms and evolutionary adaptation. The genus Crocus is an extreme example of karyological variation with an extensive range of previously reported chromosome numbers, 2n = 6 to 64, which is accompanied by the presence of 0-11 B chromosomes in some species; besides a great inter- and intra-specific variation in karyotypic features (Mather, 1932, Brighton et al., 1973, Brighton, 1977b, Brighton, 1977a, Feinbrun, 1958, Feinbrun, 1957, Brighton et al., 1983, Dolatyari, 2025). All these variations highlight the active role of karyological mechanisms e.g. dysploidy and polyploidy in the genus evolution (Raca et al., 2023, Harpke et al., 2013). Conclusion This study offers valuable insights into the palynomorphology and its taxonomic significance within the genus Crocus . For the first time, we present palynological data for nine Iranian endemic species of Crocus . 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New cytotype of crocus pallasii subsp. haussknechtii from west of Iran. Acta Horticulturae, 739 , 107-111.SAXENA, R. B. 2010. Botany, Taxonomy and Cytology of Crocus sativus series. Ayu, 31 , 374-81.TABASI, M. & MEHRABIAN, A. 2019. The palynological studies of Crocus L. (Iridaceae) with emphasis on taxonomy in Iran. Journal of Plant Research, 31 , 873-880.WENDELBO, P. & MATHEW, B. 1975. Iridaceae. In: RECHINGER, K. H. (ed.) Flora Iranica. Graz, Austria: Akademische Druck- und Verlagsanstalt. Table 1. Characterization of the studied taxa and accessions (in alphabetical order), Her. No. = number of herbarium voucher Crocus archibaldiorum (Rukšāns) Rukšāns P1015056 3489 Endemic to Iran Gilan: Asalem to Khalkhal, 2 km after Charasu village to Almas pass. N 37 55 53.8, E 48 48 2.2, 1566m P1015060 3499 Gilan: Rostamabad, hills NE Shamam village, among jungle trees. N 36 55 48.1, E 49 28 36.3, 879m. Crocus azerbaijanicus Dolatyari & Rukšāns P1015088 3527 Endemic to Iran W Azerbaijan: N Chaldoran. N 39 14 37.8, E 44 16 0.7, 2164m. Crocus caspius Fisch. & C.A.Mey. ex Hohen. P1015046 3496 Iran and Azerbaijan Mazandaran: 10 km S Amol to Tehran, 2 km in the road to Emamzadeh Abdollah, opposite of Holomsar jungle park. N 36 23 37.7, E 52 19 45.9, 227m. P1015052 3491 Mazandaran: Tonekabon, Sehezar to Alamut, 2 km after Pelet Haleh village. N 36 34 5.8, E 50 50 4.1, 954m. Crocus chiaicus Dolatyari & Rukšāns P1015098 3721 Endemic to Iran W Azerbaijan: Oshnaviyeh to Mahabad. N 36 44 46.1, E 45 31 30.3, 2060m Crocus chionophilus Dolatyari & Rukšāns P1015086 3525 Endemic to Iran E Azerbaijan: Varzaghan to Meshganbar, 9 km from T-Junction to Meshganbar. N 38 28 30.7, E 46 28 10.3, 2113m. Crocus damascenus Herb. P1015062 3639 Levantine region to Iran Markazi: Khomein to Golpayegan, ca. 30 km before Golpayegan. N 33 37 50.5, E 50 11 19.6, 1837m. P1015064 3640 Esfahan: Khonsar-Boein-Miandasht road, just 1 km after the pass. N 33 13 10.2, E 50 15 29.7, 2801m. P1015065 3641 Esfahan: 25 km a Aligoudarz to Damaneh. N 33 15 36.5, E 49 56 10.9, 2403m. P1015128 3638 Kurdistan: Marivan to Tizh Tizh. N 35 31 29.6, E 46 23 1.3, 1561m. P1015138 3714 Kermanshah: between Songhor and Sahneh. N 34 38 39.8, E 47 35 21.7, 1983m Crocus dolatyarii Rukšāns P1015103 3528 Endemic to Iran W Azerbaijan: W Oshnaviyeh, Sheikh Rivas region. N 36 57 3.9, E 44 58 45.4, 2159. Crocus haussknechtii (Boiss. & Reut. ex Maw) Boiss. P1015067 3716 Iran, Iraq and Jordan Lorestan: Doroud to Khorramabad, 42 km before Khorramabad. N 33 31 18, E 48 45 37, 1818m. P1015106 3717 Kurdistan: Baneh to Marivan. N 35 57 33.9, E 46 05 3.7, 1943m. Crocus iranicus Rukšāns P1015114 3722 Endemic to Iran Kurdistan: Saghez to Marivan, before military base of Kani Kan. N 35 43 13, E 46 23 13.4, 2091m. Crocus marandicus Dolatyari & Rukšāns P1015087 3526 Endemic to Iran E Azerbaijan: Marand Payami ski resort. N 38 20 19.1, E 45 46 29.3, 1920m. Crocus michelsonii B.Fedtsch. P1015195 3595 Iran and Turkmenistan Razavi Khorassan: Ghochan to Dargaz, 80 km to Dargaz. N 37 26 18, E 58 35 34, 2250m. Crocus pseudoiranicus Dolatyari & Rukšāns P1015129 3642 Endemic to Iran Kurdistan: Marivan to Tizh Tizh. N 35 31 29.6, E 46 23 1.3, 1561m. Crocus reinhardii Rukšāns P1015074 3524 Endemic to Iran Zanjan: ca. 29 km to Zanjan from Gilvan. N 36 41 4.6, E 48 44 12.1, 2303m. Table 2. Measurements of the pollen grain characters in the studied taxa under Light and Scanning Electron Microscopy C. archibaldiorum P1015056 98.27±5.81 106.48±6.71 2.35±0.21 22.5±2.13 2.86±0.81 0.92±0.02 spiraaperturate oblate-spheroidal 0.54±0.04 0.69±0.07 10±0.71 24.25±3.77 C. archibaldiorum P1015060 65.56±14.98 68.13±15.77 1.91±0.41 11.24±3.09 1.71±0.49 0.96±0.02 spiraaperturate oblate-spheroidal 0.8±0.16 1.07±0.11 4.57±0.49 13.17±3.48 C. azerbaijanicus P1015088 83.61±5.43 85.21±5.77 2.13±0.16 18.95±2.09 1.51±0.28 0.98±0.04 spiraaperturate oblate-spheroidal 0.65±0.03 0.81±0.05 9.5±1.66 7±3.24 C. caspius P1015046 77.36±4.24 79.84±3.72 1.91±0.24 16.25±2.42 1.87±0.36 0.97±0.03 spiraaperturate oblate-spheroidal C. caspius P1015052 78.49±2.68 81.9±2.61 2.1±0.21 18.47±2.55 2.04±0.82 0.96±0.04 spiraaperturate oblate-spheroidal 0.82±0.08 0.87±0.03 6.33±0.47 11±0.82 C. chiaicus P1015098 80.86±2.56 86.26±2.89 2.13±0.27 15.6±2.84 2.34±0.42 0.94±0.04 spiraaperturate oblate-spheroidal 0.67±0.05 0.72±0.07 8±1.79 16.4±6.77 C. chionophilus P1015086 78.72±2.68 81.56±2.27 2.28±0.18 15.53±2 1.51±0.28 0.97±0.03 spiraaperturate oblate-spheroidal 0.72±0.05 0.74±0.05 11.2±1.47 15.25±4.44 C. damascenus P1015062 84.09±5.19 88.48±7.25 2.37±0.16 17.06±1.91 2.06±0.59 0.95±0.03 spiraaperturate oblate-spheroidal C. damascenus P1015064 80.04±17.86 80.72±18.67 2.18±0.54 14.28±5.16 2.22±0.68 0.99±0.04 spiraaperturate oblate-spheroidal C. damascenus P1015065 75.86±4.5 78.51±4.87 2.15±0.25 16.60±0.62 2.42±0.22 0.97±0.04 spiraaperturate oblate-spheroidal 0.67±0.03 0.73±0.02 6.75±0.83 24±3.74 C. damascenus P1015128 79.69±3.31 83.69±3.91 2.29±0.21 18.1±1.26 1.65±0.2 0.95±0.02 spiraaperturate oblate-spheroidal C. damascenus P1015138 78.46±2.61 80.97±2.66 2.67±0.18 13.94±1.98 1.76±0.27 0.97±0.04 spiraaperturate oblate-spheroidal 0.71±0.07 0.88±0.01 7±1 5.25±2.56 C. dolatyarii P1015103 84.17±6.17 87.86±3.99 2.41±0.28 19.66±2.1 2.13±0.95 0.96±0.03 spiraaperturate oblate-spheroidal 0.607±0.08 0.72±0.11 9.6±2.87 14±5.8 C. haussknechtii P1015067 67.68±2.91 69.77±2.45 1.71±0.14 15.42±2.1 1.89±0.61 0.97±0.02 polyaperturoidate oblate-spheroidal 0.78±0.11 0.83±0.11 7.67±0.94 18.67±4.64 C. haussknechtii P1015106 73.89±5.39 74.27±5.25 1.84±0.33 14.62±1.38 2.51±0.62 0.99±0.01 polyaperturoidate oblate-spheroidal C. iranicus P1015114 71.93±2.81 74.85±1.83 1.92±0.17 12.03±2.87 1.61±0.35 0.96±0.02 spiraaperturate oblate-spheroidal 0.69±0.01 0.7±0.02 7.75±0.75 5.75±0.75 C. marandicus P1015087 86.47±3.77 88.97±4.24 2.3±0.2 17.03±1.64 1.37±0.38 0.97±0.06 spiraaperturate oblate-spheroidal 0.52±0.06 0.77±0.1 10.33±0.94 3.33±1.7 C. michelsonii P1015195 66.23±4.8 66.95±5.14 1.97±0.24 10.11±0.81 1.60±0.19 0.97±0.05 spiraaperturate oblate-spheroidal 0.73±0.04 0.97±0.14 7.67±2.36 22.33±6.65 C. pseudoiranicus P1015129 75.44±2.71 76.74±3.66 2.13±0.25 13.28±1.74 1.85±0.46 0.98±0.02 spiraaperturate oblate-spheroidal 0.59±0.04 0.68±0.2 6.6±1.8 8.5±3.35 C. reinhardii P1015074 61.01±2.54 62.64±2.29 1.5±0.06 9±0.81 1.530.24 0.97±0.01 spiraaperturate oblate-spheroidal 0.64±0.12 0.74±0.05 8.25±1.1 18.25±2.05 Figure 1 . Photographs of the studied collections, showcasing their morphological traits and habitat characteristics. A) C. archibaldiorum (P1015056) in cultivation, B) its habitat, C) C. archibaldiorum (P1015060) in cultivation, D) its habitat, E) C . azerbaijanicus in locus classicus, F) C. caspius in cultivation, G) C. chiaicus in cultivation, H) C. Chionophilus in locus classicus, I) C. damascenus (P1015062), J) C. damascenus (P1015064), K) C. damascenus (P1015065), L) C. damascenus (P1015128), M) C. damascenus (P1015138), N) C. dolatyarii in locus classicus, O) C. haussknechtii (P1015067), P) C. haussknechtii (P1015106), Q) C. iranicus , R) C. marandicus , S) C. michelsonii , T) C. pseudoiranicus in cultivation, U) C. reinhardii in locus classicus Figure 2. Light microscope (LM) images of selected pollen grains of studied Crocus species at two focal points to show both apertures and exine structures. A, B) C. chiaicus P1015098, C, D) C. chionophilus P1015086, E, F) C. damascenus P1015064, G, H) C. haussknechtii P1015106, I, K) C. iranicus P1015114, J, L) michelsonii P1015195, M, N) Anomalous pollen grains in C. dolatyarii P1015103 and C. archibaldiorum (P1015056) respectively. Scale bar for A-L= 5 µm and for M and N = 100µm. Figure 3. Scanning electron microscope (SEM) images of pollen grains of studied species of Crocus at a magnification of 3000 X. A) C. archibaldiorum P1015056, B) C. azerbaijanicus P1015088, C) . Crocus caspius P1015052, D) C. chiaicus P1015098, E) C.chionophilus P1015086, F) C. damascenus P1015064,G) C. dolatyarii P1015103, H ) C. haussknechtii P1015067, I) C. iranicus P1015114. scale bar = 20 µm. Figure 4. Scanning electron microscope (SEM) images of pollen grains of studied species at a magnification of 3000 X. A) C. marandicus P1015087, B) C. michelsonii P1015195, C) C. pseudoiranicus P1015129, D) C. reinhardii P1015074. scale bar = 20 µm. Figure 5. Principal component analysis of 20 Crocus taxa and 7 variables of pollen features projected onto axis 1 and axis 2. Figure 6. Principal component analysis of combined LM and SEM dataset including 13 species of Crocus and 12 pollen variables projected onto axis 1 and axis 2. Figure legends Figure 1 . Photographs of the studied collections, showcasing their morphological traits and habitat characteristics. A) C. archibaldiorum (P1015056) in cultivation, B) its habitat, C) C. archibaldiorum (P1015060) in cultivation, D) its habitat, E) C . azerbaijanicus in locus classicus, F) C. caspius in cultivation, G) C. chiaicus in cultivation, H) C. Chionophilus in locus classicus, I) C. damascenus (P1015062), J) C. damascenus (P1015064), K) C. damascenus (P1015065), L) C. damascenus (P1015128), M) C. damascenus (P1015138), N) C. dolatyarii in locus classicus, O) C. haussknechtii (P1015067), P) C. haussknechtii (P1015106), Q) C. iranicus , R) C. marandicus , S) C. michelsonii , T) C. pseudoiranicus in cultivation, U) C. reinhardii in locus classicus Figure 2. Light microscope (LM) images of selected pollen grains of studied Crocus species at two focal points to show both apertures and exine structures. A, B) C. chiaicus P1015098, C, D) C. chionophilus P1015086, E, F) C. damascenus P1015064, G, H) C. haussknechtii P1015106, I, K) C. iranicus P1015114, J, L) michelsonii P1015195, M, N) Anomalous pollen grains in C. dolatyarii P1015103 and C. archibaldiorum (P1015056) respectively. Scale bar for A-L= 5 µm and for M and N = 100µm. Figure 3. Scanning electron microscope (SEM) images of pollen grains of studied species of Crocus at a magnification of 3000 X. A) C. archibaldiorum P1015056, B) C. azerbaijanicus P1015088, C) . Crocus caspius P1015052, D) C. chiaicus P1015098, E) C.chionophilus P1015086, F) C. damascenus P1015064,G) C. dolatyarii P1015103, H ) C. haussknechtii P1015067, I) C. iranicus P1015114. scale bar = 20 µm. Figure 4. Scanning electron microscope (SEM) images of pollen grains of studied species at a magnification of 3000 X. A) C. marandicus P1015087, B) C. michelsonii P1015195, C) C. pseudoiranicus P1015129, D) C. reinhardii P1015074. scale bar = 20 µm. Figure 5. Principal component analysis of 20 Crocus taxa and 7 variables of pollen features projected onto axis 1 and axis 2. Figure 6. Principal component analysis of combined LM and SEM dataset including 13 species of Crocus and 12 pollen variables projected onto axis 1 and axis 2. Information & Authors Information Version history V1 Version 1 17 January 2025 Peer review timeline Published Microscopy Research and Technique Version of Record 30 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Microscopy Research and Technique Keywords crocus nudiscapus pollen morphology spiraperturate taxonomy Authors Affiliations Alireza Dolatyari Iranian Biological Resource Center View all articles by this author Mehdi Dehghani 0000-0002-7616-6715 [email protected] University of Zabol View all articles by this author Metrics & Citations Metrics Article Usage 371 views 186 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Alireza Dolatyari, Mehdi Dehghani. Palynomorphological analysis of the genus Crocus L. (Iridaceae) in Iran and its taxonomic implications. Authorea . 17 January 2025. 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