Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 31-43, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1109 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Jun Wang, Qiang Ye, Chu Wang, Tong Zhang, Xusheng Shi, Majid Khayatnezhad, Abdul Shakoor (2021) Palynological analysis of genus Gera- nium (Geraniaceae) and its systematic implications using scanning electron microscopy. Caryologia 74(3): 31-43. doi: 10.36253/caryologia-1109 Received: October 06, 2020 Accepted: July 21, 2021 Published: December 21, 2021 Copyright: © 2021 Jun Wang, Qiang Ye, Chu Wang, Tong Zhang, Xusheng Shi, Majid Khayatnezhad, Abdul Shakoor. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Palynological analysis of genus Geranium (Geraniaceae) and its systematic implications using scanning electron microscopy Jun Wang1,2,*, Qiang Ye1, Chu Wang2, Tong Zhang2, Xusheng Shi2, Majid Khayatnezhad3, Abdul Shakoor4,5 1School of Management, Harbin Institute of Technology; Heilongjiang Provice, China 2Beijing Jinghang Computation and Communication Research Institute; BeiJing, China 3Department of Environmental Sciences and Engineering, Ardabil Branch, Islamic Azad University, Ardabil, Iran 4College of Environment and Planning, Henan University, Kaifeng, 475004, Henan, China 5Key Laboratory of Geospatial Technology for the Middle and Lower Yellow River Regions, Ministry of Education, Kaifeng 475004, Henan, China *Corresponding authors. E-mail: jxwxuwei@163.com; abdul_shakoor954@yahoo.com Abstract. Pollen morphology of 23 species belonging to Geranium have been studied in details, which represent eight sections of two subgenera i.e., G. sect. Dissecta, Gera- nium, and Tuberosa of subgen. Geranium, Divaricata, Lucida, Ruberta and Trilopha of subgen. Robertium. These plant species were collected from different phytogeographi- cal regions of Iran. The palynological investigation was done using scanning electron microscopy (SEM) techniques. Different palyno-morphological features have been observed, and the closely related species were distinguished. We used different multi- variate statistical methods to reveal the species relationships. Ward clustering analyses have been done to check out the relationship among the species. The shapes of pol- len grains were monad, radially symmetric, isopolar, apertures were tricolporate, and of spheroid, prolate-spheroid or sub-prolate classes. Three pollen types were recog- nized on the basis of differences in exine sculpturing pattern: reticulate-clavate, striate- rugulate, reticulum cristatum with clavae. Observed differences were not of diagnostic importance in subgenera and sections level. The main objective of this study is to find distinguish pollen characters in the species of the genus Geranium and to elucidate their systematics importance. Keywords: Geranium, pollen morphology, systematics, phytogeographical regions. RESEARCH HIGHLIGHTS – Pollen grains of the studied species were investigated by light and scan- ning electron microscope – Using scanning electron microscopy techniques for the easy and quick identification of plant species – Using micro-morphological (SEM) characters for the identification and species delimitations. 32 Jun Wang et al. INTRODUCTION The Geranium L., is the largest genus of Gerani- aceae and contains 325 species. Geranium species are distributed worldwide except in lowland tropical areas (Aedo 2017). Most of the species belong to subg. Gera- nium (Aedo 2001). Traditionally the genus Geranium was classified into three subgenera: 1- subg. Geranium, 2- subg. Erodioidea (Picard) Yeo, and 3- subg. Rober- tium (Picard) Rouy. According to Yeo (1984), the subg. Robertium primary diagnostic characteristics are carpel projection of fruit discharge. This subgenus has thirty species, and these are distributed in eight sections. The subgenus. Geranium, is the largest subgenus containing 300 species. These 300 species were in at least ten sec- tions. The genus is characterized by a seed-ejection fruit type in which the explosive recurvature of the awn car- ries the mericarp upwards in an arc, throwing the seed out (Marcussen and Meseguer 2017). The family Gerani- aceae Juss., is a worldwide family and comprising addi- tionally five genera: Hypeocharis J. Remy (subfam. Hyp- seocharitoideae), Erodium L’Herit ex Aiton., Geranium L., Monsonia L., Pelargonium L’Herit. ex Aiton and Cali- fornia being successive sisters to Geranium L. (subfam. Geranioideae) (Fiz et al. 2008; Weng et al. 2014). These genera have a moderate number of species than Gerani- um and had a more narrow distribution. T he cha rac ter ist ic f ive-c a r pel led sch i zoc a r p Geranioideae fruit with a prominent central rostrum has a great taxonomic value in this genus. The Geranium shows four different fruit discharge methods: carpel- projection type, Erodium-type, seed-ejection type, and the inoperative type (Marcussen and Meseguer 2017). These methods have been considered in the delimitation of major groups within the Geranium. Marcussen and Meseguer (2017) pointed that phylogenetic relationships within Geranium and the evolution of the different seed discharge methods are still unknown. The new classifi- cation has shifted some of the studied taxa from subg. Robertium to subg. Geranium, while a third subgenus (Erodioidea) has been reduced to synonymy under subg. Geranium (Marcussen and Meseguer 2017). The Geraniaceae is a eurypalynous family (Baser et al. 2016). The pollen morphological features of the fam- ily have a great importance in the discrimination at the generic level (Perveen and Gaiser 1999). The pollen grains are usually isopolar with radial symmetry; gen- erally have oblate-spheroidal shape, rarely sub-oblate; three-lobed, tri-colporate, rarely colpate and the colpi are short; the sexine is thicker than nexine, and the tec- tum is reticulate with densely baculate or gemmate muri or striate (Perveen and Gaiser 1999). Various workers described the pollen morphology of different species of Geraniaceae, species delimita- tion and species relationships in the genus Geranium L. Since Bortenschlager’s (1967) preliminary study on the pollen morphology of the Geraniaceae, the pollen morphology of Monsonia (Verhoeven and Venter 1986), Sarcocaulon (Verhoeven and Venter 1988), and Erodi- um (Verhoeven andVenter 1987) of the southern Afri- can Geraniaceae has been investigated. Erodium pollen grains of the Middle East (EI-Oqlah 1983) and north- western Europe have been studied (Stafford and Black- more 1991). Stafford andBlackmore (1991) reviewed the pollen grains of Geranium species in north-western Europe. Pollen morphology of 35 taxa of the genus Geranium in Asia has been observed through light and scanning electron microscopy (SEM) by Park and Kim (1997). They found high variation in pollen grains size and exine sculpture (muri, lumina, excrescences distri- bution) among the examined taxa. Also, palynological characters were useful to establish the phylogeny with- in the genus. However, they are not helpful characters for separation at subgenera and sections. Perveen and Gaiser (1999) studied the pollen morphology of Paki- stani Geraniaceae taxa and based on exine ornamenta- tions and apertural type. They divided the pollen grains into three groups i.e., Erodium cicutariumGeranium himalayense, and Monsonia senegalensis-types. They observed that the pollen grains were mostly oblate- spheroidal, rarely prolate-spheroidal or spheroidal, and often sub-oblate in the examined taxa. Shehata (2008) analyzed pollen grains of the Geraniaceae family from Eg ypt. The results showed that pollen morphologi- cal characters, i.e., pollen size, pollen shape, and aper- ture, were taxonomically significant at the generic level and up to some extent at the specific level. Ilcim et al. (2008) analyzed the morphological and palynological properties of G. tuberosum. Palynological investigation of 13 species of genus Geranium in Turkey by Deniz et al. (2013), did not reflect significant differences between the sections, except some differences in pollen grains of the subgenus Geranium. According to Deniz et al. (2013), all examined species’ pollen grains were large, and exine ornamentation was reticulate-clavate; and the apertures were tri-colporate. In Iran, the only study on pollen morphology of five Geranium species was assessed by Keshavarzi et al. (2016). There is no comprehensive palynological inves- tigation of Geranium species in Iran. The present study aimed to use the pollen grains morphological features as a source of diagnostic characters to distinguish different Iranian Geranium species. 33Palynological analysis of genus Geranium and its systematic implications using scanning electron microscopy MATERIAL AND METHODS Totally 60 populations were collected and studied from 23 taxa of Geranium from different habitats in Iran to explore the pollen features (Table 1). Ten individuals of each location were studied and examined for three qualitative and eight quantitative features (Table 2). For scanning electron microscopy (SEM) examinations, pol- len grains were not acetolyzed according to the method of Erdtman (1960). The pollens were suspended in a drop of water for a while and then directly transferred to a metallic stubby a fine pipette, and double-sided cello tape was used. And then the pollens were sputtered in a chamber coated with gold (Sputter Coater BALTEC, SCDOOS). Coating with gold by the physical vapor dep- osition method (PVD) was restricted to 100 Å. The SEM examination was carried out on a TESCAN microscope. Pollen sculptures were described according to previous terminology (Hesse et al. 2009). The evaluated and measured characters of the spe- cies studied of Geranium are summarized in Tables II. To detect significant differences in the studied characters among the various studied species, an analysis of vari- ance (ANOVA) was performed. For multivariate analy- sis, the mean of the quantitative characters was used. Principal Components Analysis (PCA) was performed among the species to determine palynological charac- ters useful for separating the species. In order to group the species, cluster analysis using WARD (Minimum spherical variance) methods and PCA ordination plot were performed by PAST software (Hammer et al. 2001), using Euclidean and taxonomic distance among the spe- cies was calculated (Podani 2000). The qualitative traits were coded as binary or multi- state. Variables were systematized for multivariate statis- tical analysis. Average taxonomic distances and squared Euclidean distances were applied as dissimilarity coef- ficients in the pollen data cluster analysis. Image Tool Version 3.0 (http://ddsdx.uthscsa.edu/dig/itdesc.html) was used to carry out the required measurements. RESULTS Infrageneric variation We showed that G. purpureum, G. robertianum and G. sylvaticum possess type I pollen grains. Sub-genus Geranium had type II pollens. Species, i.e., G. biuncina- tum ,G. mascatense and G. trilophum belonging to Trilo- pha section .showed the type III of pollen grains. Factor analysis shows three factors explained more than 77% of the total observed variation in studied pol- len grains. The first factor revealed equatorial length, pollen shape importance. Aperture condition, colpus length, and exine thickness illustrated more than 44% of the observed variation in the second factor. Principal component analysis (PCA) based on pollen grain quali- tative and quantitative traits confirms the cluster analy- sis results by Ward’s method. General pollen grain features The majority of Geranium species depicted prolate- spheroidal pollen types. For instance, G. columbinum, G. collinum, G. sylvaticum, G. pratense, G. dissectum, G. lin- earilobum, G. tuberosum, G. kotschyi, G. platypetalum, G. gracile, G. ibericum, G. purpureum, G. pyrenaicum, G. robertianum, G. divaricatum, G. lucidum, and G. molle had prolate-spheroidal shape (Figures. 2 A1, B1, C1, D1; 3 E1, F1, G1; 4 M1, O1; 5 P1, Q1; 6T1, W1, Y1, Z1; 7 X1, U1). Spheroidal pollen types were observed in G. rotundi- folium, G. pusillum, G. albanum (Figs. 4 L1, 3 H1, 4 N1). G. biuncinatum, G. mascatense, G. trilophum species had sub-prolate pollen morphology (Figures. 5 R1, S1; Figures. 7, U4-U6). We found that G. lucidum and G. robertianum had spheroidal-prolate pollen grains. While G. rotundi- folium had spheroidal pollen shape (Table II, Figures 4 L1-L2). Our statistical and microscopy analyses depicted that G. platypetalum had the largest (Table II, Figure. 6 Z1, Z2) and G. pusillum possessed smallest pollen grains. (Table II, Figures 3 H1, H2).Our observations revealed that pollen grains were generally prolate-spheroidal except G. pusillum, G. rotundifolium, and G. albanum (Fig- ures 3H1-H2, Figures 4 L1-L2; N1-N2).Mean polar axis length varied from 38.55 μm (G. pusillum) to 104.88 μm (G. platypetalum), while the mean of the equatorial axis length varied from 37.55 μm (G. pusillum) to 105μm (G. platypetalum). The main colpus length varied from 12 μm (G. molle) to 58 μm (G. trilophum). P/E ratio differed from 0.89 μm (G. albanum) to 1.5 μm (G. trilophum). The main features of the investigated pollen grains are summarized in Table II. The basic ornamentation of the exine surface in the Geranium was reticulate-clavate, striate-rugulate, and reticulum cristatum with clavae. On the basis of dif- ferences in exine sculpturing pattern, the following 3 types are recognized: reticulate-clavate, striate-rugulate, reticulum cristatum with clavae. Most of the specimens belong to reticulum cristatum with clavae pattern. Type I: Geranium robertianum- type (“reticulate- clavate”) Species: Geranium robertianum (Figure 5, P3; Figure 4, O3; Figure 3, E2) Pollen class: Tricolporate 34 Jun Wang et al. Table 1. List of Geranium species in Iran their localities and voucher numbers. No Section Sp. Locality 1 Dissecta Yeo G. dissectum L. Guilan, Lahijan East Azerbaijan, Kaleybar, Cheshme Ali Akbar East Azerbaijan, Kaleybar, Shoj-abad Tehran, Damavand Khorassan, Kashmar-Darvaneh 2 Geranium G. columbinum L. Guilan, Siahkal, Ezbaram; Guilan, Langerud, chaff; Guilan, Bandar-e Anzali; Tehran, Damavand; Khorassan, Kashmar-darvaneh 3 G. rotundifolium L. Tehran, Tuchal Mazandaran, Kandovan-Siahbisheh East Azerbaijan, Kaleybar, Shoj-abad 4 G. collinum Stephan ex Willdenow Mazandaran, Tonekabon-Jannat Rudbar Hamedan, 20km s of Nahavand 5 G. sylvaticum L. East Azerbaijan, Kaleybar, Cheshme Ali Akbar 6 G. pratense L. East Azerbaijan, Kaleybar, Shoj-abad Mazandaran, 40 km Tonekabon to Janat Abad 7 Tuberosa (Boiss.) Reiche G. platypetalum Fisch. & C. A. Mey. East Azerbaijan, Kaleybar Razavi Khorasan, Kashmar Tehran, Damavand 8 G. ibericum Cav. Tehran, Damavand Kordestan, Sanandaj Khorassan, Kashmar-Darvaneh 9 G. gracile Ledeb. ex Nordm. Mazandaran, Noshahr, Kheyrud Kenar Forest Kerman, Lalehzar, Baghabad 10 G. linearilobum DC. Tehran, Firuz kuh Mazandaran, 40 km Tonekabon to janat abad 11 G. kotschyi Boiss. Alborz, Karaj- Qazvin Tehran, Desin 12 G. tuberosum L. East Azerbaijan, Kaleybar Cheshme Ali Akbar, Tehran, Tuchal 13 Batrachioidea W.D.J. Koch G. molle L. East Azerbaijan, Kaleybar, Shojabad East Azerbaijan, Kaleybar, Cheshme Ali Akbar Hamedan, 20km s of Nahavand 14 G. pyrenaicum Burm. f. East Azerbaijan, Kaleybar, roadside East Azerbaijan, Kaleybar, Cheshme Ali akbar East Azerbaijan, Kaleybar, Shojabad East Azerbaijan, Babak fort Tehran, Damavand 15 G. pusillum L. East Azerbaijan, Kaleybar, roadside East Azerbaijan, Kaleybar Cheshme Ali Akbar East Azerbaijan, Kaleybar, Shojabad Hamedan, 20km s of Nahavand Tehran, Damavand 16 Ruberta Dumort. G. purpureum Vill. East Azerbaijan, Kaleybar, Cheshme Ali Akbar Guilan, Gole rodbar Guilan, Gole rodbar, roadside Guilan, Jirandeh Khorassan, Kashmar-darvaneh Kerman, Lalehzar, Baghabad 17 G. robertianum L. Guilan,Gole rodbar Esfahan, 50 km Delijan Tehran, Damavand 18 Divaricata Rouy G. albanum M. Bieb. Guilan, Sangar, roadside Guilan, Lahijan Guilan, Jirandeh Mazandaran, Siah bisheh to Chalus Golestan, Ramian 35Palynological analysis of genus Geranium and its systematic implications using scanning electron microscopy No Section Sp. Locality Esfahan, 50 km delijan Khorasan, Birjand Tehran, Darakeh Hamedan, 20km s of Nahavand 19 G. divaricatum Ehrh. East Azerbaijan, Kaleybar Tehran, Darband Khorasan, Birjand Tehran, Darakeh Kerman, Lalehzar, Baghabad 20 Lucida R. Knuth G. lucidum L. East Azerbaijan, Kaleybar Cheshme Ali Akbar 21 Trilopha Yeo G. trilophum Boiss. Hormozgan, Amani village, Kushk-e Nar Rural Hamedan, 20km s of Nahavand 22 G. mascatense Boiss. Hormozgan, Qeshm, Bakho Mountain 23 G. biuncinatum Kokwaro Khuzestan, Shushtar- Masjed soleyman Table. 2. Evaluated characters of pollen grains in Geranium species studied (values M ± SD μm). M- Mean value; SD- Standard deviation. Aperture: At the same level =2, protruding = 1; Pollen shape: prolate-spheroidal =1, spheroidal= 2, subprolate=3; Exine ornamentation type: reticulate-clavate=1, striate-rugulose =2, reticulum cristatum with clavae= 3 Taxon Po la r ax is le ng th (µ m ) Ex in e th ic kn es s (µ m ) Eq ua to ri al a xi s le ng th ( µm ) P/ E ra tio A po co lp iu m le ng th ( µm ) M es oc ol pi um le ng th ( µm ) C ol pu s le ng th (µ m ) Po re d ia m et er (µ m ) A pe rt ur e Po lle n sh ap e Ex in e or na m en ta tio n ty pe G. dissectum 55.67±2.1 4.5 55.87±3.3 1.05±0.05 ±0.03 23.67 35.66 36.44 6.87 1 1 3 G. columbinum 62.76±3.4 5.88 62.66±2.9 1.00±0.03 35.87 52.54 22 15 1 1 3 G. rotundifolium 66.44±3.8 5.87 65.54±2.3 1.01±0.02 37.22 35.77 25 8.5 1 2 3 G. collinum 86.5±2.8 5.34 77.32±4.8 1.11±0.04 48.55 34.33 55 12 1 1 3 G. platypetalum 103.32±4.1 4.45 101±2.7 1.01±0.05 68.87 54.77 33 24 1 1 3 G. sylvaticum 78.44±2.9 5.87 75.55±1.4 1.04±0.04 54.22 42.32 25 7 2 1 1 G. pretense 75.55±3.7 5.88 66.77±2.4 1.06±0.02 55.77 60.22 32 7 2 1 3 G. ibericum 81.44±3.9 4.34 78.33±3.6 1.03±0.05 47.22 54.55 28 13 2 1 3 G. gracile 76.44±2.4 5.45 74.56±2.9 1.02±0.03 37.55 53.77 27 16 1 1 3 G. linearilobum 71.88±2.8 5.65 61.55±4.2 1.13±0.06 38.43 38.34 39 14 1 1 3 G. kotschyi 63.66±4.1 5.8 59.55±4.5 1.06±0.03 42.66 35.77 39 11 1 1 3 G. tuberosum 81.55±2.8 5.3 75.66±2.2 1.08±0.02 48.77 46.44 33 12 1 1 3 G. molle 49.44±1.6 4.55 47.34±1.3 1.04±0.04 31 35 13 10 1 1 3 G. pyrenaicum 64.22±2.8 4.3 59.34±2.2 1.08±0.05 33 30 23 4 2 1 3 G. pusillum 39.65±1.4 3.7 38.22±5.3 1.02±0.03 26-27 24 15.66 7.87 1 2 3 G. purpureum 58.27±1.9 4.2 54.55±2.5 1.1±0.04 37.85 29.01 27.68 7.4 2 1 1 G. robertianum 61.76±4.1 4.7 51.54±1.8 1.02±0.02 30 22-28 21-22 7-8 2 1 1 G. albanum 63.44±2.5 5.45 66.34±2.6 0.95±0.03 38.76 39.44 26.33 14 1 2 3 G. divaricatum 59.57±3.7 4.55 51.66±3.9 1.13±0.01 33.66 35.33 32 9 1 1 3 G. lucidum 58.55±5.2 4.76 49.65±3.3 1.09±0.04 33.77 25.44 32 6 1 1 3 G. mascatense 64.66±6.1 5.88 60.33±2.9 1.1±0.05 23.77 47.55 50 14 1 3 2 G. trilophum 65.44±2.6 5.34 54.45±2.5 1.2±0.03 21.45 43.55 56 9 1 3 2 G. biuncinatum 63.25±5.1 4.35 63.76±7.9 1.1±0.03 22.65 45.98 59 12 1 3 2 36 Jun Wang et al. Shape: Prolate-spheroidal Apertures: Ectoaperture-colpi small Ornamentation: Tectum coarsely reticulate with clavae Measurements: polar length (P) (56-75 μm), equato- rial diameter (E) (55-78 μm), colpi (20-25μm) in diam- eter. Mesocolpium (20-40 μm). Apocolpium (33-59 μm). Exine 4.9-5.9 μm Species included: G. robertianum, G. purpureum, G. syl- vaticum Type II: Geranium molle -type (“reticulum cristatum with clavae”) Species: G. molle (Figures 2, A3, B3, C3, D3; Figures 3, F3, G3, H3; Figures 4, L3, M3, N3; Figure 5, Q3; Figures 6, T3, W3, Y3, Z3; Figures 7, X3, U3 ) Pollen class: Tricolporate Shape: Prolate- Spheroidal Apertures: Ectoaperture - colpus short (approx. 1/5 to 1/8 of polar axis) Ornamentation: Tectum reticulum cristatum with clavae Measurements: Polar length (P) (38-105 μm) , Equatorial diameter (E) (38-105μm), colpi (10-58μm) in diameter. Mesocolpium (22-67μm). Apocolpium (22-69μm) . Exine 3.7-5.8 μm Species included: G.gracile, G. ibericum, G. pyrenaicum, G. divaricatum, G. lucidum, and G. molle, G. pusillum ; G. columbinum, G. collinum, G. pratense, G. dissectum, G. linearilobum, G. tuberosum,G. kotschyi, G. platypeta- lum Pollen type III: Erodium-type (“striate-rugulose”) (Figures. 5, R3, S3; Figures. 7, U4-U6) Pollen class: Tricolporate Shape: Sub- Prolate Apertures: Ectoaperture-colpi large Ornamentation: Tectum striate-rugulose Measurements: Polar length (P) (68-69 μm) , Equatorial diameter (E) (50-64μm), colpi (53-59μm) in diameter. Mesocolpium (40-49μm). Apocolpium (20-25 μm). Exine 5.5-5.9 μm Species included: G. mascatense, G. trilophum, G. biunci- natum DISCUSSION Geranium species are relatively challenging to study due to the overlapping of morphological characters (Aedo and Pando 2017; Ji et al. 2020; Wang et al. 2020; Sun et al. 2021). Therefore, an attempt was carried out to investigate pollen grains of Geranium species by scan- ning electron microscopy. Our approach also included the usage of principal component analysis to verify our findings. The current study showed that Geranium spe- cies have eurypalynous pollens morphology. Present results corroborate with a previous study conducted on the Geraniaceae family (Baser et al. 2016). Most of the species showed prolate-spheroidal pollen types. This pat- tern of pollen type is previously reported in the Gera- niance family (Baser et al. 2016). We observed monad, isopolar, radial symmetry, tricolporate, and short linear colpi pollen grains in Geranium species. Similar pollen types have been reported in Iran while working on five Geranium species (Keshavarzi et al. 2016). Our result recorded spheroidal-prolate pollen grains in G. lucidum and G. robertianum species. However, previously oblate- spheroidal pollen grains were reported in G. lucidum, G. robertianum (Perveen and Gaisar 1999). We argue that such a difference in pollens is due to sampling site and habitat (Brodschneider et al. 2019). Annual or biennial species, i.e., G. lucidum, G. pusillum, G. molle, G. dissec- tum, G. rotundifolium showed the smaller pollen grains (from 39 to 66 μm). G. collinum, G. sylvaticum, G. prat- ense, G. platypetalum, G. gracile, and G. ibericum had larger (63 to 103 μm) pollen grains. Such variation in pollen grains has been described in the past (Aedo 2001; Aedo et al. 2007; Jurgens et al. 2012; Hao et al. 2020). Figure 1. Ward clustering of Geranium species based on observed pollen data. 37Palynological analysis of genus Geranium and its systematic implications using scanning electron microscopy Clustering results revealed pollen grain type III in sec- tion Trilopha. Henceforth, type III pollen is the diagnos- tic feature to identify Trilopha Section. Deniz et al. (2013) observed the exine ornamenta- tion to be reticulate-clavate in Geranium taxa. While in the present study, exine ornamentations were reticulate- clavate, striate-rugulose, reticulum cristatum with cla- vae. Most of the specimens belong to reticulum cristat- um with clavae pattern. The pollen of all species of Gera- nium sect. Trilopha is of the Erodium-type (Aedo et al. 2016), which is congruent with the present findings. The striate ornamentation is considered a diagnostic feature to differentiate Trilopha section. This distinguishing fea- ture may support the close relationship between G. sect. Figure 2. Pollen micrographs of Geranium species: A1-A3: G. collinum, B1-B3: G. columbinum, C1-C3: G. pratense, D1-D3: G. dissectum; A1, B1,C1, D1 – equatorial view; A2, B2, C2, D2 - polar view; A3, B3, C3, D3 -exine sculpture. 38 Jun Wang et al. Polyantha and G. sect. Trilopha. The scanning electron microscopy (SEM) tech- nique has a crucial role in observing the minute biologi- cal structure (Ullah et al. 2019). Several taxonomic and plant systematics questions are also addressed through the application of SEM. For instance, seed morphology in Caryophyllaceae was studied by scanning electron microscopy to identify Caryophyllaceae species correctly (Ullah et al. 2019; Zou et al. 2019). Besides this, detailed plant morphology, anatomy, and pollens in Spergula fal- lax and Spergula arvensis revealed the significance of SEM to resolve taxonomic complexity (Ullah et al. 2018). Authors developed identification key and distinguishing features of Spergula fallax and Spergula arvensis species Figure 3. Pollen micrographs of Geranium species: E1-E3: G. sylvaticum, F1-F3: G. molle, G1-G3: G. pyrenaicum, H1-H3: G. pusillum; E1, F1, G1, H1 – equatorial view; E2, F2, G2, H2 - polar view; E3, F3, G3, H3 -exine sculpture. 39Palynological analysis of genus Geranium and its systematic implications using scanning electron microscopy (Ullah et al. 2018). This clearly shows the utilization of new techniques to solve complex questions in plant sys- tematics and biology (Pathan et al. 2010; Ullah et al. 2018; Ullah et al. 2019). Cluster revealed groupings of G. uncinatum, G. mas- catense, G. trilophum into the Trilopha section. Similar results at the section level was reported. Phylogenetic relationship based on chloroplast (rbcL, trnL-trnF) and nuclear (ITS) DNA sequences highlighted the grouping of G. uncinatum, G. mascatense into the Trilopha sec- tion (Marcussen and Meseguer 2017). The phylogenetic tree proved the Geraniaceae family as a monophyletic. Figure 4. Pollen micrographs of Geranium species: L1-L3: G. rotundifollium, M1-M3: G. lucidum, N1-N3: G. albanum, O1-O3: G. purpure- um ; L1, M1, N1, O1 – equatorial view; L2, M2, N2, O2 - polar view; L3, M3, N3, O3 -exine sculpture. 40 Jun Wang et al. The discrepancy in the phylogenetic results obtained by different molecular markers may be the reason for unre- solved species relationship in the genus Geranium. Evo- lutionary relationships between Geranium, Erodium, and are unclear because of the low support for the alternative topologies inferred from both markers (trnL–F and rbcL) Figure 5. Pollen micrographs of Geranium species: P1-P3: G. robertianum, Q1-Q3: G. divaricatum, R1-R3: G. trilophum, S1-S3: G. mascat- ense; P1,Q1, R1, S1 – equatorial view; P2,Q2, R2, S2 - polar view; P3,Q3, R3, S3 -exine sculpture. 41Palynological analysis of genus Geranium and its systematic implications using scanning electron microscopy (Fiz et al. 2008). Inter-simple sequence repeat (ISSR) markers separated Geranium species (Esfandani–Bozch- aloyi et al. 2018). The present study showed that pollen features are not capable of distinguishing different sec- tions in Geranium. Two subgenera elements were mixed. Nonetheless, we found and linked the diagnos- tic character (Pollen type III) of the Trilopha section. Future studies may incorporate phylogenetic data and chemotaxonomy methods to decipher genus and spe- cies level evolutionary relationship in Geranium. Linking the morphological and phylogenetic traits may further aid our apprehension in plant evolution and systematics (Cohen 2011). In this research, we studied different palynological characteristics of the genus Geranium. Various charac- ters were crucial for the taxonomic identification of the Figure 6. Pollen micrographs of Geranium species: T1-T3: G. kotschyi, W1-W3: G. linearilobum, Y1-Y3: G. tuberosum, Z1-Z3: G. platypeta- lum; T1,W1,Y1, Z1 – equatorial view; T2,W2,Y2, Z2 - polar view; T3,W3,Y3, Z3 -exine sculpture. 42 Jun Wang et al. species delimitations. This study could serve better to understand the pollen morphology of Geranium spe- cies in Iran. The pollen characters studied here were useful for the taxonomic identification of the species of the genus. ACKNOWLEDGEMENTS We would also thank Editor for his comments to improve the quality of the manuscript. REFERENCES Aedo C, Barbera P, Buira A. 2016. Taxonomic revision of Geranium sect. Trilopha (Geraniaceae). Syst Bot. 41:354–377. Aedo C, Garcia M, Alarcon M, Aldasoro J, Navarro C. 2007. Taxonomic Revision of Geranium Subsect. Mediterranea (Geraniaceae). Syst Bot. 32:93–128. Aedo C, Pando F. 2017. A distribution and taxonomic reference dataset of Geranium in the New World. Scientific Data. 4:170049. Aedo C. 2001. 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