Journal Pre-proof Pollen diversity in the genus Carlina L. (subtribe Carlininae, Compositae) and its systematic significance Mohamed O. Badry, Ahmed K. Osman, Ahmed Elkordy PII: S0034-6667(20)30059-2 DOI: https://doi.org/10.1016/j.revpalbo.2020.104243 Reference: PALBO 104243 To appear in: Review of Palaeobotany and Palynology Received date: 12 March 2020 Revised date: 17 April 2020 Accepted date: 20 April 2020 Please cite this article as: M.O. Badry, A.K. Osman and A. Elkordy, Pollen diversity in the genus Carlina L. (subtribe Carlininae, Compositae) and its systematic significance, Review of Palaeobotany and Palynology (2020), https://doi.org/10.1016/j.revpalbo.2020.104243 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier. Journal Pre-proof Pollen diversity in the genus Carlina L. (subtribe Carlininae, Compositae) and its systematic significance Mohamed O. Badry1*, Ahmed K. Osman1 & Ahmed Elkordy2 1 Department of Botany & Microbiology, Faculty of Science, South Valley University, Qena 83523, Egypt. 2 Department of Botany & Microbiology, Faculty of Science, Sohag University, Sohag oo f 82524, Egypt. Pr ABSTRACT e- E-mail address: mohamedowis@svu.edu.eg pr *Correspondence Author al Pollen morphology of 19 species, four subspecies, and one variety of Carlina was studied in detail by light and scanning electron microscopy. Qualitative and quantitative pollen Jo ur n morphological characters which vary between taxa are found in the length of the ectocolpi, presence/nature of the echinae, pollen size and shape, lumina area, P/E ratio, polar axis, equatorial diameter, pollen shape, ectocolpi length, sculpture of ectoaperture membranes, area of lumina, and exine ornamentation. Three main pollen types are discerned: the C. atlantica type characterized by P/E ≤ 1.00, the C. curetum type characterized by P/E = 1.03–1.11, and the C. acanthifolia type with ratio P/E= 1.24–1.50. HCA and PCA analyses were carried out to detect the potential palynological characters which could be used to identify taxa. The data suggest that several pollen morphological characters can be used to delimit the taxa of Carlina. Dichotomous artificial keys based on palynological data are also given. Journal Pre-proof 2 Keywords: Ectocolpi; Cardueae; pollen size; taxonomic significance; ectoaperture membrane 1. Introduction Carlina L is a well-defined genus belongs to the tribe Cardueae, subtribe Carlininae of Compositae. The Cardueae are known commonly as “thistles” and plants of Carlina are oo f known as “carline thistles”. The genus is medium sized, comprising a number of species and infrageneric taxa with estimates of by 28 species, 12 subspecies and fourteen 14 pr according to Kovanda (2002) and Susanna & Garcia-Jacas (2007), and 34 accepted species, e- 12 subspecies, and seven varieties, according to The Plant List (2013). The genus is Pr distributed from the Canary Islands and the Mediterranean (across Europe and northern Africa) to temperate Asia (central Siberia and northwestern China) (Kovanda, 2002; Peng al et al., 2012). Meusel & Kästner (1994) divided the genus Carlina into five subgenera, Jo ur n Carlina, Carlowizia, Heracantha, Lyrolepis, and Mitina, with an underlying hierarchy of sections and subsections. In their monograph of the genus, Meusel & Kastner (1990, 1994) provided a review of Carlina that covered their leaf morphology, inflorescence, flower and fruit, chorology, ecology, karyology, embryology, palynology, chemistry of secondary compounds, and taxonomy. A molecular phylogeny of the genus has been established (Susanna et al., 2006; Wahrmund et al., 2010). Palynological studies have been helpful tools in taxonomical revisions of numerous genera of Compositae and the Cardueae in particular (Bolick, 1978; Bordbar and Mirtadzadini, 2015; De Leonardis et al., 1984; Garnatje and Martín, 2007; López, 1981; Osman, 2009; Ozler et al., 2009; Pehlivan, 1995; Petit et al., 2001; Punt and Hoen, 2009; Journal Pre-proof 3 Shabestari et al., 2013; Skvarla et al., 1977; Skvarla and Turner, 1966; Stix, 1960; Velari, 1982; Wagenitz, 1976, 1955). To date details of pollen morphology are limited to only a few species of Carlina as part of studies at the family and tribal level and in the description of certain species (Beug, 2004; Dimon, 1971; Dittrich, 1977; La-Serna Ramos et al., 1999; Meusel et al., 1984; Petit, 1997; Punt and Hoen, 2009; Stix, 1960; Terrab et al., 2001; Tormo et al., 1984; Valdés et f al., 1987). oo In this study, we detail pollen morphological characters of 24 taxa of Carlina (19 pr species, four subspecies, and one variety). From this data set we assess pollen e- morphological support for the current infrageneric classification of Carlina. Provision of an identification key to species using pollen is aimed at further applications in other taxonomic Jo ur n 2.1. Pollen materials al 2. Materials and Methods Pr studies as well as expanding the palynological knowledge base of the Compositae. Pollen grains of the studied species of Carlina were extracted from fertile anthers of flowers in anthesis and/or from buds in pre-anthesis, derived from 26 herbarium specimens of 24 taxa deposited in MSB Herbarium at the Ludwig-Maximilians-Universität München, Germany (herbarium acronyms follow Thiers 2017) (Table 1). Nomenclature of the studied species was updated according to APG III (2009) and the Kew Garden plantlist website (The Plant List, 2013). 2.2. Light Microscopy (LM) For light microscopy, pollen material was acetolyzed according to Erdtman (1960). The acetolyzed pollens were mounted in glycerin jelly on glass slides and sealed with paraffin wax (melting point 60–62 °C) according to Salgado-Labouriau & Rinaldi (1990) and Journal Pre-proof 4 (Osman, 2003). Prepared slides were then examined under 720× and 1800× magnifications with a Labomed light microscope, powered by an ocular micrometer and stage (Labomed, Inc., USA), and average measurements obtained from 15–20 readings per slide from each specimen when possible. Pollen shape classes were identified, described and subdivided according to the product of the ratio between the polar axis and the equatorial diameter (P/E) in equatorial view following Erdtman’s (1969) system. Quantitative measurements of pollen characters from the LM and SEM images were compiled using the program ImageJ oo f v1.45 (Schneider et al., 2012). pr 2.3. Scanning Electron Microscopy (SEM) e- Acetolyzed and non-acetolyzed pollen samples were detached from the 26 herbarium specimens with fine clean needles and forceps and were mounted onto clean aluminum Pr stubs using double-sided adhesive tape and coated with gold using a JEOL JFC 1100 E ion sputtering device. The pollen samples were then examined and photographed using a JEOL al JSM 5400LV Scanning Electron Microscope (at an accelerating voltage of 15 kV) at the Jo ur n Electron Microscopy Unit (EMU) at Assiut University, Egypt. Pollen terminology and descriptions were based on the classification proposed by Punt et al. (2007) and Halbritter et al. (2018). 2.4. Data Analyses For all measured qualitative and quantitative characters, data analysis was implemented using STATISTICA (STATISTICA software, version 5.0). Pollen characters of the 24 taxa of Carlina studied were organized in a data matrix with 60 pollen characters that exhibited variation between taxa. The described qualitative features were coded in the data matrix as present (1) or absent (0), while the average of quantitative characters was used. Hierarchical Cluster Analysis (HCA) based on Euclidian distance measure and using the Unweighted Journal Pre-proof 5 Pair Group Method using the Arithmetic averages (UPGMA) clustering method (Seberg et al., 1991; Sokal, 1958) was generated with the aim of classifying the studied taxa into clusters based on overall pollen variables similarity. A Principal Component Analysis (PCA) was implemented to detect whether the analyzed pollen features could cluster species and to identify the most distinguishing morphological character(s) for the studied taxa as well, following exemplary studies (e.g. f Coutinho et al., 2011; Lopes et al., 2013). Eigenvalues were plotted in a two-dimensional oo scatter plot along the first two principal component axes (PCA1, PCA2), accounting for the Jo ur n al Pr e- pr highest pollen variation. Journal Pre-proof 6 3. Results 3.1. General pollen morphology Pollen grains of the studied taxa of Carlina are quite homogeneous in their characters. Main features of the analyzed pollen grains of 19 species, four subspecies, and one variety of Carlina are summarized in detail in Tables 2–5 and Plates I–V. Pollen grains are monads and 3-zonocolporate. Most of the studied taxa of Carlina have a sub-spheroidal shape. Among other taxa, prolate (C. acanthifolia, C. biebersteinii subsp. oo f brevibracteata, C. corymbosa var. graeciformis, and C. sitiensis, Plate I, 1; Plate II, 6, 8; Plate V, 23), sub-prolate (C. sicula, Plate V, 22), and prolate-spheroidal (C. curetum, C. pr frigida subsp. fiumensis, and C. graeca, Plate II, 9; Plate III, 12, 13) in shape. e- The polar perimeter is triangular, ovate to slightly circular, and the equatorial Pr perimeter ovate to circular. The ectoapertures are of the colpus type (ectocolpus), usually long, narrow to slightly wide at the equator, acute or acuminate to tapered towards the ends, al and with a granulate, scabrate, and striate membrane. However, the ectocolpus in C. frigida Jo ur n and C. oligocephala is short (Plate III, 11; Plate IV, 19) a and the colpus membrane is granulate-rugulate in C. involucrata (Plate IV, 16) and microrugulate in C. frigida (Plate III, 11). Mesoapertures are generally covered by ectoapertures, blurring their observation (Tormo Molina and Ubera Jiménez, 1995, 1990). The tectum ornamentation pattern is quite variable amongst Carlina species. All 24 taxa of Carlina studied have echinate ornamentation with lengths of 1.49–2.94 µm. The tectum in the space between echinae is nanoreticulate, microreticulate, microrugulate, rugulate, nanoverrucate, perforate or striate. No significant difference between the ornamentation pattern at the Apocolpium and Mesocolpium is observed. Exine sculpture Journal Pre-proof 7 elements have the same size or are slightly decreased towards the pollen apertures to form a perforate pattern or towards the aperture's edges to form a margo. The palynological results of the studied taxa of Carlina reveal the presence of three pollen types: the C. atlantica type, C. curetum type, and C. acanthifolia type. They can be distinguished through the following key. 3.2. Artificial key to the pollen types of the studied taxa of Carlina based on the f palynological results of this study oo 1. a. P/E of pollen grains ≤ 1.00 …………………………………… Carlina atlantica type pr 1. b. P/E of pollen grains > 1.00 ………….…………………………..……………………. 2 e- 2. a. Pollen prolate-spheroidal in shape, P/E = 1.03–1.11……..…...… Carlina curetum type Pr 2. b. Pollen sub-prolate to prolate in shape, P/E = 1.24–1.50.....… Carlina acanthifolia type 3.3. Descriptions of pollen types (Tables 2–5) al 3.3.1. Carlina atlantica type Jo ur n Pollen grains are 3-colporate, sub-spheroidal (P/E = 0.87 (0.72–1.11)), triangular to circular outline in polar view, polar axis 29.70 (22.67–35.76) µm, with total area 931.83 (528.10–1260.88) µm, ovate to circular outline in equatorial view, with 34.29 (25.51– 41.45) µm diameter and 795.38 (498.33–1031.76) µm total area. Polar distance is 14.85 (11.34–17.88) µm. Apocolpium field is 29.11 (13.78–48.52) µm, with 11.84 (5.41–18.62) μm diameter and 13.76 (6.41–21.42) index. Ectoaperture colpus type, 22.37 (10.68–40.00) µm length, 11.11 (2.69–18.60) µm width, and 43.23 (10.32–102.47) µm total area, long, wide at the equator, tapered to acute towards the ends, with scabrate, striate, granulate to rugulate membrane. Mesocolpium is 20.84 (7.45–28.93) µm in width. Tectum sculpture is echinate, perforate, microrugulate-rugulate, nano-microreticulate, nanoverrucate or striate, spines with ± rounded tops, 2.52 (1.49–2.94) µm length, and 13.80 (8.55–21.67) µm base Journal Pre-proof 8 area. Sculpture elements are the same on all the pollen surface. Lumina with 0.98 (0.11– 2.52) µm area and 0.32 (0.11–0.67) µm diameter, and 0.35 (0.14–0.98) µm thickness, enclosed by the muri. The following species belong to this type: 1- Carlina acaulis L. s.l. (Plate I, 2). 2- Carlina acaulis subsp. caulescens (Lam.) Schübl. & G.Martens (Plate I, 1). 3- Carlina atlantica Pomel (Plate I, 4). oo f 4- Carlina biebersteinii Bernh. ex Hornem. (Plate I, 5). 5- Carlina corymbosa L. (Plate I, 7). pr 6- Carlina diae (Rech.f.) Meusel & Kästner (Plate II, 10). e- 7- Carlina frigida Boiss. & Heldr. (Plate III, 11). Pr 8- Carlina hispanica Lam. (Plate III, 14). 9- Carlina hispanica subsp. major (Lange) Meusel & Kästner (Plate III, 15). al 10- Carlina involucrata Poir. (Plate IV, 16). Jo ur n 11- Carlina lanata L. (Plate IV, 17). 12- Carlina macrocephala Moris (Plate IV, 18). 13- Carlina oligocephala Boiss. & Kotschy (Plate IV, 19). 14- Carlina racemosa L. (Plate IV, 20). 15- Carlina salicifolia (L.f.) Cav. (Plate V, 21). 16- Carlina vulgaris L. (Plate V, 24). 3.3.2. Artificial key to Taxa of Carlina atlantica pollen type based on the palynological results of this study 1.a. Mesocolpium diameter range = 8.97–15.30 µm …………………...……...………… 2 1.b. Mesocolpium diameter range = 18.97–26.51 µm …………………………….………4 2.a. Apocolpium diameter medium = 12.79 (11.98–14.00) µm …………...……..C. acaulis Journal Pre-proof 9 2.b. Apocolpium diameter range = 7.38–8.98 µm ……………………………………….. 3 3.a. Apocolpium index medium = 11.01 (10.16–12.60) µm …………….……....C. atlantica 3.b. Apocolpium index medium = 8.64 (7.70–9.82) µm ………. C. hispanica subsp. major 4.a. Apocolpium field range = 15.49–27.91 µm ………………….……………………….5 4.b. Apocolpium field range = 30.42–41.12 µm …………………………..………………8 f 5.a. Apocolpium field range = 15.49–19.84 µm …………………….…………………….6 oo 5.b. Apocolpium field range = 26.46–27.91 µm …………………………………………..7 pr 6.a. Apocolpium index medium = 10.19 (9.17–11.59) µm …………………….… C. lanata e- 6.b. Apocolpium index medium = 7.56 (6.41–8.63) µm …...…………..… C. oligocephala Pr 7.a. Polar axis medium = 29.20 (28.56–30.24) µm ……………………..…. C. biebersteinii 7.b. Polar axis medium = 32.10 (31.54–33.08) µm ………………………………… C. diae al 8.a. Apocolpium field range = 40.13–41.12 µm …………………………………………..9 Jo ur n 8.b. Apocolpium filed range = 30.42–35.14 µm ……………………………...………….10 9.a. Apocolpium index medium = 17.68 (15.18–19.63) µm….. C. acaulis subsp. caulescens 9.b. Apocolpium index medium = 20.61 (19.59–21.42) µm ……...……….…. C. hispanica 10.a. Colpus length range = 16.64–21.33 µm …………...……………………………… 11 10.b. Colpus length range = 25.13–33.38 µm ……………………………………………13 11.a. Apocolpium index medium = 14.27 (13.64–14.84) µm…..…………..……. C. frigida 11.b. Apocolpium index range = 16.15–17.11 µm …...……………………….…………12 12.a. Polar axis medium = 26.40 (24.82–27.48) µm…….……………….…… C. corymbosa 12.b. Polar axis medium = 30.79 (29.54–32.56) µm ……...………………..….. C. vulgaris Journal Pre-proof 10 13.a. Apocolpium index medium = 11.73 (10.81–12.73) µm ……….…...……. C. racemosa 13.b. Apocolpium index range = 14.98–15.73 µm ………………………………………14 14.a. Equatorial diameter medium = 39.57 (36.69–41.45) µm….…............….C. involucrata 14.b. Equatorial diameter range = 31.14–34.05 µm…….……………………………….. 15 15.a. Equatorial diameter medium = 31.14 (29.48–32.26) µm….………….… C. salicifolia f 15.b. Equatorial diameter medium 34.05 (33.15–35.16) µm……….……. C. macrocephala oo 3.3.3. Carlina curetum type pr Pollen grains are 3-colporate, prolate-spheroidal (P/E = 1.06 (1.03–1.11)), triangular e- to lobate outline in polar view, polar axis 39.20 (31.84–44.66) µm, with total area 1161.59 (977.76–1368.85) µm, ovate outline in equatorial view, with 36.79 (30.19–40.27) diameter Pr µm and 1148.60 (726.49 –1446.65) µm total area. Polar distance is 19.60 (15.92–22.33) al µm. Apocolpium field is 30.68 (26.33–33.17) µm, with 13.12 (12.24–14.46) μm diameter and 15.18 (14.14–16.25) index. Ectoaperture colpus type, 27.63 (21.24–32.54) µm length, Jo ur n 12.21 (10.26–13.31) µm width, and 52.73 (29.42–97.66)µm total area, long, wide at the equator, acute towards the ends, with scabrate to granulate membrane. Mesocolpium is 20.33 (11.44–27.85) µm width. Tectum sculpture echinate, rugulate-microrugulate to perforate, spines with ± rounded tops, 2.33 (2.02–2.60) µm length and 15.76 (10.99–19.34) µm base area. Sculpture elements are slightly decreased towards the pollen apertures to form echinate, perforate, microrugulate to nanoreticulate sculpture. Lumina with 1.16 (0.87–1.67) µm area and 0.43 (0.39–0.52) µm diameter, and 0.37 (0.33–0.43) µm thickness, enclosed by the muri. The following species belong to this type: 1- Carlina curetum Heldr. ex Halácsy (Plate II, 9). 2- Carlina frigida subsp. fiumensis (Simonk.) Meusel & Kästner (Plate III, 12). Journal Pre-proof 11 3- Carlina graeca Heldr. & Sart (Plate III, 13). 3.3.4. Artificial key to Taxa of Carlina curetum pollen type based on the palynological results of this study 1.a. Polar axis medium = 31.84 (30.82-32.81) µm …………………………...……C. graeca 1.b. Polar axis range = 41.10 - 44.66 µm ……………………………………………..…… 2 2.a. Mesocolpium diameter medium = 21.69 (19.75-32.53) µm …...………….... C. curetum f 2.b. Mesocolpium diameter medium = 27.85 (26.82-28.54) µm…C. frigida subsp. fiumensis oo 3.3.5. Carlina acanthifolia type pr Pollen grains are 3-colporate, sub-prolate to prolate (P/E = 1.34 (1.17–1.62)), lobate, e- circulate to triangular outline in polar view, polar axis 43.90 (38.71–49.85) µm, with total Pr area 902.72 (661.53–1189.64) µm, ovate outline in equatorial view, with 32.91 (26.70– 36.77) µm diameter and 1156.43 (922.31–1363.84) µm total area. Polar distance is 22.46 al (19.54–24.93) µm. Apocolpium field is 31.17 (22.49–45.35) µm, with 12.18 (7.96–16.36) Jo ur n μm diameter and 14.67 (10.23–19.59) index. Ectoaperture colpus type, 27.99 (17.58–56.50) µm length, 5.67 (1.29–9.04) µm width, with total area 29.57 (17.98–48.64) µm, long, wide to narrow at the equator, acuminate towards the ends, with scabrate to granulate membrane. Mesocolpium is 22.54 (14.50–26.98) µm in width. Tectum sculpture echinate, perforate, reticulate-microreticulate or rugulate, spines with ± rounded tops, 2.37 (1.96–2.67) µm length, and 13.27 (8.66–17.30) µm base area. Sculpture elements are decreased towards the apertures to form echinate, microreticulate, rugulate or perforate patterns. Lumina with 1.73 (0.83–3.58) µm area and 0.58 (0.18–1.30) µm diameter, and 0.36 (0.20–0.63) µm thickness, enclosed by the muri. The following species belong to this type: 1- Carlina acanthifolia All. (Plate I, 1). 2- Carlina biebersteinii subsp. brevibracteata (Andrae) K.Werner (Plate II, 6). Journal Pre-proof 12 3- Carlina corymbosa var. graeciformis Meusel & Kästner (Plate II, 8). 4- Carlina sicula Ten. SEM (Plate V, 22). 5- Carlina sitiensis Rech.f. SEM (Plate V, 23). 3.3.6. Artificial key to Taxa of Carlina acanthifolia pollen type based on the palynological results of this study 1.a. Mesocolpium diameter medium = 15.88 (14.50-16.85) µm……………..C. biebersteinii f subsp. brevibracteata oo 1.b. Mesocolpium diameter range = 22.52 - 25.57 µm ………………..……………..…… 2 pr 2.a. Apocolpium index medium = 11.98 (10.37–13.40) µm ……….………. C. acanthifolia 2.b. Apocolpium index range = 15.55 – 18.90 µm ………………………………….……....3 Pr var. graeciformis e- 3.a. Apocolpium diameter medium = 15.54 (14.59–16.36) µm………………..C. corymbosa 3.b. Apocolpium diameter range = 12.06 – 13.61 µm …………………………….………. 4 al 4.a. Mesocolpium diameter medium = 25.29 (22.86 – 26.98) µm ………….…….. C. sicula Jo ur n 4.b. Mesocolpium diameter medium = 22.52 (19.54-24.53) µm ……………..…. C. sitiensis 3.3.7. Statistical analyses The phenetic relationships of the taxa of Carlina studied reflected in the pollen morphological diversity is presented through two statistical analyses. The scatter plot (Figure 1) for PCA 1 and PCA 2 revealed that the palynological data can clearly separate almost all taxa studied. Mesocolpium diameter, murus thickness, echinus length, polar axis and the polar distance were the most variable characters among taxa studied. Most taxa belonging to Carlina formed a close cluster/group in an intermediate position. Journal Pre-proof 13 The results of the clustering analysis (Fig. 2) are consistent with the PCA analysis and show a clear separation among taxa. Clustering divides the species in two main groups (A, B). The first one comprising all the taxa of pollen type I except for C. gracea (of pollen type II). The second group includes the remaining taxa of pollen type II along with the taxa which belong to pollen type III. 4. Discussion f Pollen of the taxa of Carlina studied are consistently 3-zonocolporate monads with oo elongate ectoapertures and echinate ornamentation. The taxa are diverse in shape (sub- pr spheroidal, sub-prolate, prolate or prolate-spheroidal), polar view area (small, medium or e- large), length of ectocolpus, tectum ornamentation pattern, and P/E ratio. These results are Pr in agreement with the findings of Tormo et al. (1984) and Punt & Hoen (2009). Although morphologically pollen grains of the studied taxa of Carlina are very similar, al the analyses performed here highlight the potential importance of both qualitative and Jo ur n quantitative characters in distinguishing the studied taxa. Based on our observations, three main pollen types were distinguished: C. atlantica type characterized by P/E ≤ 1.00 C. curetum type characterized by P/E = 1.03–1.11, and C. acanthifolia type with ratio P/E= 1.24–1.50. Among the morphological characters analyzed, the quantitative characters such as mesocolpium diameter, murus thickness, echinus length, polar axis, and the polar distance are useful for the discrimination among the taxa studied, in agreement with the results of the statistical analyses. These characters together with the P/E ratio make it feasible to distinguish between the taxa of the genus Carlina. Pollen morphology clearly separates C. graeca and C. acaulis from other Carlina taxa (Figs. 1, 2). Carlina graeca has a prolate-spheroidal shape, and lumina with 0.94 (0.69– 1.15) µm area and C. acaulis possesses sub-spheroidal pollen shape and lumina with 0.32 Journal Pre-proof 14 (0.24–0.39) µm area. Furthermore, the sculpture of ectoaperture membranes classifies the studied taxa into five groups. The first group has colpi with scabrate ectoaperture membranes and includes C.acaulis, C. atlantica, C. biebersteinii subsp. brevibracteata, C corymbosa, C corymbosa var. graeciformis, C. curetum, C. frigida subsp. fiumensis, C. macrocephala, C. racemosa, C. sicula, C. sitiensis and C. vulgaris. The second group has colpi with striate ectoaperture membranes and includes C. acaulis subsp. caulescens, C. diae, C. hispanica, C. lanata, and C. salicifolia. The third group possesses colpi with oo f granulate ectoaperture membranes, and comprises C. acanthifolia, C. biebersteinii, C. graeca, C. hispanica subsp. major, and C. oligocephala. The fourth group included only C. pr involucrate, having colpi with granulate-rugulate ectoaperture membrane. Likewise, the e- fifth group represented by C. frigida, having colpi with microrugulate ectoaperture Pr membrane (Table 5). Pollen size of the studied taxa has a taxonomic significance, it ranged from 23.87 to al 46.71 µm mean value in polar axis and 26.98–40.27 µm mean value in equatorial diameter. Jo ur n According to measurements of the polar axis, the smallest pollen grains are those of C. atlantica 23.87 (23.18–24.69) µm and the largest ones are those of C. corymbosa var. graeciformis 46.71 (41.91–49.85) µm. Pollen sizes of other species range between 24.45– 46.00 µm. While the measurements of the equatorial diameter showed that the smallest pollen grains are those of C. hispanica subsp. major 26.98 (25.51-28.32) µm and the largest one is those of C. curetum 40.27 (38.65-41.55) µm. Also, the equatorial diameter of other species ranges between 27.35–39.91 µm (Table 2). Moreover, the length of ectocolpi can be used to differentiate between the studied taxa. It ranged between 12.96–37.66 µm. The longest colpus is 37.66 (29.90–56.50) µm in C. acanthifolia and the shortest one 12.96 (10.97–15.29) µm is in C. corymbosa var. Journal Pre-proof 15 graeciformis. Colpus length of the remaining species ranges between 16.01–33.38 µm (Table 4). The results presented herein show that pollen grains of the studied taxa of Carlina have echinate tectum sculpture, the spines are with ± rounded tops, with 1.49–2.94 length µm, and large bases range between 8.55–21.67 µm broad. C. diae possesses the shortest spines, with 1.49 (0.65-2.41) µm length, and 8.55 (6.63-10.54) µm base. While C. racemose has f the longest spines 2.94 (1.58-3.94) µm length. Both belong to our C. atlantica type. oo In an in-depth palynological study on Compositae, Punt & Hoen (2009) identified and pr described different pollen types along with identification keys. Although the genus Carlina e- was represented by only two taxa, viz C. acaulis, and C. vulgaris. Their description of the Pr C. vulgaris-type is, however, conform our C. atlantica type. It is evident from the cluster analysis of the palynological characters that the clustering al of Carlina taxa studied did not correlate well with the sectional classification of Meusel & Jo ur n Kästner (1994). while, C. lanata and C. racemosa, belonging to Sect. Mitina grouped together. Moreover, C. diae was a sister to C. oligocephala (C. subgenus Lyrolepis) this relationship was supported by both morphological and molecular data (Wahrmund et al., 2010). Likewise, the palynological data was not recovered Carlina Sect. Heracantha as a monophyletic group, these results are in line with the molecular data, supporting its paraphyly in relation to the rest of the genus (Wahrmund et al., 2010). Our palynological results show significant differences between the Carlina taxa studied and suggest that some palynological characters such as length of the ectocolpi, echinae, pollen size, lumina area, P/E ratio and sculpture of ectoaperture membranes bring new taxonomic data to the genus Carlina. These results are in line with other palynological Journal Pre-proof 16 studies on different genera within the tribe Cardueae and Compositae (Coutinho et al., 2011; Ferrauto et al., 2017). 5. Conclusion This study contributes to the understanding of pollen diversity in Carlina with substantial variation across a range of characters. The palynological characters of the studied taxa serve for the identification and classification of taxa at the specific level. The qualitative and f quantitative pollen characters provide a helpful taxonomic key for the isolation of closely oo related species. Further work will be required to determine if the significant differences pr between single samples of the species and infraspecific taxa studied stand up with Pr Declaration of Competing Interest e- additional sampling. article. Jo ur n Acknowledgments al The authors declare that there is no conflict of interest regarding the publication of this We would like to express our deep gratitude to the director of MSB Herbarium for allowing the study of their specimens. Sincere thanks to Dr. Murray Fagg, Australian National Herbarium & Australian National Botanic Gardens, Canberra, Australia, Dr. W.R. (Bill) Barker and Dr. Robyn Barker, the State Herbarium of South Australia for their diligent proofreading and comments which improved this manuscript. Sincere thanks and deepest appreciation to Dr. Jennifer A. Tate, Senior Lecturer in Plant Systematics & Evolution, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand, for her guidance and support that always inspired us to learn something new. Journal Pre-proof 17 References APG III, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants. Bot. J. Linn. Soc. 161, 105–121. Beug, H.J., 2004. Leitfaden der Pollenbestimmung für Mitteleuropa und angrenzende Gebiete. Verlag Dr. Friedrich Pfeil, München. Bolick, M.R., 1978. Taxonomic, evolutionary, and functional considerations of Compositae oo f pollen ultrastructure and sculpture. Plant Syst. Evol. 130, 209–218. Bordbar, F., Mirtadzadini, M., 2015. Palynological study of Jurinea (Asteraceae) and pr related genera from flora of Iran. Plant Syst. Evol. 301, 151–162. e- Coutinho, A.P., Aguiar, C.F., da Bandeira, D.S., Dinis, A.M., 2011. 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Zeitung 142, 213–279. oo f Wahrmund, U., Heklau, H., Röser, M., Kästner, A., Vitek, E., Ehrendorfer, F., Bernhard von Hagen, K., 2010. A molecular phylogeny reveals frequent changes of growth form Jo ur n al Pr e- pr in Carlina (Ateraceae). Taxon 59, 367–378. Journal Pre-proof Plates & Figures legends Plate I. SEM micrographs of pollen grains of Carlina. (a) polar view, (b) equatorial view, (c) surface ornamentation of exine. (1) C. acanthifolia; (2) C. acaulis; (3) C. acaulis ssp. caulescens; (4) C. atlantica; (5) C. biebersteinii. Plate II. SEM micrographs of pollen grains of Carlina. (a) polar view, (b) equatorial view, (c) surface ornamentation of exine. (6) C. biebersteinii ssp. brevibracteata; (7) C. corymbose; (8) C. corymbose var. graeciformis; (9) C. curetum; (10) C. diae. Plate III. SEM micrographs of pollen grains of Carlina. (a) polar view, (b) equatorial view, oo f (c) surface ornamentation of exine. (11) C frigida; (12) C frigida ssp. fiumensis; (13) C. graeca; (14) C. hispanica; (15) C. hispanica ssp. major. pr Plate IV. SEM micrographs of pollen grains of Carlina. (a) polar view, (b) equatorial view, (c) surface ornamentation of exine. (16) C. involucrate; (17) C. lanata; (18) C. e- macrocephala; (19) C. oligocephala; (20) C. racemose. Pr Plate V. SEM micrographs of pollen grains of Carlina. (a) polar view, (b) equatorial view, (c) surface ornamentation of exine. (21) C. salicifolia; (22) C. sicula; (23) C. sitiensis; (24) al C. vulgaris. Fig. 1. Scatterplot of first two axes from the principal component analysis (PCA) of the Jo ur n pollen morphology of the 24 Carlina taxa studied. The percentage of variability explained by each component is indicated on the axis. Fig. 2. Cluster analysis performed with the measured variables of pollen from the 24 Carlina taxa studied using the Unweighted Pair Group Method with arithmetic Averages (UPGMA method). o. Journal Pre-proof 24 Table 1. Taxa of Carlina investigated for the palynological study. Studied taxa Collector/field number Date of collection Locality Carlina acanthifolia All. H. Merxmüller & J. Graw, Nr. 21968 26-Aug-1966 Spain Carlina acaulis L. F. Schuhwerk, N. 99/350 11-Sep-1999 Austria Carlina acaulis subsp. caulescens (Lam.) Schübl. & G.Martens J. Ewald & F. Schuhwerk, N. 94/921 30-Aug-1994 Bavaria, Germany 23-Sep-1999 Slovenia D. Podlech, Nr. 55433 19-Aug-1999 Morocco Carlina biebersteinii Bernh. ex Hornem. J. Koch 28-Jul-1950 Swabia, Germany Carlina biebersteinii subsp. brevibracteata (Andrae) K.Werner F. Schuhwerk, 84/621 16-Oct-1984 Bavaria, Germany Carlina corymbosa L. M. Erben, Nr-55875 16-Jun-1996 Sardinia, Italy Carlina corymbosa var. graeciformis Meusel & Kästner Ernst Vitek, 99/396 7-Aug-1999 Spain Carlina curetum Heldr. ex Halácsy H. Merxmüller & D. Podlech, Nr. 30798 20-Sep-1975 Crete, Greece 0 Carlina diae (Rech.f.) Meusel & Kästner H. Merxmüller & D. Podlech, Nr-30921 29-Sep-1975 Crete, Greece 1 Carlina frigida Boiss. & Heldr. E. et M. Mayer det. E. Mayer, N. 10405 11-Oct-1979 Yugoslavia 2 Carlina frigida subsp. fiumensis (Simonk.) Meusel & Kästner O. Angerer 6-Oct-1986 Yugoslavia 3 Carlina graeca Heldr. & Sart. 4 Carlina hispanica Lam. F. Schuhwerk, N. 84/465 R. Morales & E. Vitex, 97-228 28-Aug-1984 16-Jul-1997 Croatia Spain 5 Carlina hispanica subsp. major (Lange) Meusel & Kästner E. Vitek, 96.1044 26-Jun-1996 South Portugal 6 Carlina involucrata Poir. D. Podlech N-47795 8-Jul-1989 Morocco 7 Carlina lanata L. J. Bormüller, N-883 (34586) 14-Jul-1897 Lebanon 8 Carlina macrocephala Moris H. Merxmüller & Horis, J. Graue Nr-20409 4-Aug-1965 Palermo-Sicily, Italy 9 Carlina oligocephala Boiss. & Kotschy F. Schuwerk, Nr-99/312 30-Aug-1999 Turkey 0 Carlina racemosa L. R. Morales, N. 18518 25-Jul-1997 Italy 1 Carlina salicifolia (L.f.) Cav. I. & H. Hertel, 33.523 5-Sep-1986 Madeira, Portugal 2 Carlina sicula Ten. H. Merxmüller & J. Graw, Nr. 20355 2-Aug-1965 Palermo, Sicily, Italy 3 Carlina sitiensis Rech.f. H. Merxmüller & D. Podlech, Nr-30927 29-Sep-1975 Lasithi, Crete 4 Carlina vulgaris L. Doppelbaur, 5822 10-Aug-1962 Reisensburg, Germany Jo ur n al Pr e- pr oo f Ernst Vitek, N. 99/424 Carlina atlantica Pomel Journal Pre-proof 25 Table 2. Quantitative pollen morphological characteristics through light microscopy and scanning electron microscopy of the studied taxa of Carlina. Equatorial Polar Distance Diameter View area 46.00 (43.11-48.34) 23.00 (21.56-24.17) 958.20 (838.56-1189.64) 35.17 (33.61-36.39) 1284.49 (1238.15-1313.54) 1.31 Carlina acaulis L. 30.24 (26.84-31.85) 15.12 (13.42-15.93) 977.53 (818.22-1017.76) 30.24 (27.76-32.81) 720.43 (657.88-782.15) 1.00 Carlina acaulis subsp. caulescens (Lam.) chübl. & G.Martens Carlina atlantica Pomel 34.18 (32.48-35.12) 17.09 (16.24-17.57) 1219.84 (1153.78-1260.88) 38.56 (37.61-39.56) 1004.04 (997.17-1007.79) 0.88 23.87 (23.18-24.69) 11.93 (11.59-12.34) 592.94 (562.54-617.10) 27.35 (25.59-28.43) 499.52 (498.33-500.34) 0.88 Carlina biebersteinii Bernh. ex Hornem. 29.20 (28.56-30.24) 14.60 (14.28-15.12) 980.61 (976.90-983.86) 33.45 (31.74-35.62) 759.52 (758.07-761.80) 0.88 Carlina biebersteinii subsp. brevibracteata Andrae) K.Werner Carlina corymbosa L. 43.71 (38.71-49.84) 24.39 (24.02-24.92) 665.04 (661.53-668.57) 29.01 (26.70-31.32) 1068.12 (922.31-1202.52) 1.50 26.40 (24.82-27.48) 13.20 (12.41-13.74) 835.55 (770.36-852.53) 34.56 (32.97-36.15) 746.50 (731.72-755.88) 0.77 Carlina corymbosa var. graeciformis Meusel & Kästner Carlina curetum Heldr. ex Halácsy 46.71 (41.91-49.85) 23.36 (20.96-24.93) 965.57 (943.51-974.07) 35.00 (33.33-36.77) 1290.17 (1180.28-1363.84) 1.34 44.66 (40.87-47.52) 22.33 (20.44-23.76) 1368.85 (1296.70-1422.15) 40.27 (38.65-41.55) 1446.65 (1358.20-1517.07) 1.11 Carlina diae (Rech.f.) Meusel & Kästner 32.10 (31.54-33.08) 16.05 (15.77-16.54) 864.84 (727.28-998.87) 36.85 (36.69-36.98) 864.65 (821.31-895.93) 0.87 Carlina frigida Boiss. & Heldr. 28.88 (27.38-29.88) 14.44 (13.69-14.94) 791.94 (750.82-826.96) 32.86 (31.56-33.96) 677.03 (672.93-687.41) 0.88 Carlina frigida subsp. fiumensis (Simonk.) Meusel & Kästner Carlina graeca Heldr. & Sart. 41.10 (36.64-45.00) 20.55 (18.32-22.50) 1138.16 (1137.40-1139.35) 39.91 (39.03-41.15) 1272.65 (1266.45-1281.34) 1.03 31.84 (30.82-32.81) 15.92 (15.41-16.41) pr View area 977.76 (774.69-1051.19) 30.19 (28.97-31.21) 726.49 (725.35-728.68) 1.05 Carlina hispanica Lam. 33.62 (32.87-34.99) 16.81 (16.43-17.50) 1208.90 (1190.35-1218.02) 39.33 (38.78-40.00) 1026.83 (1021.90-1031.76) 0.85 Carlina hispanica subsp. major (Lange) Meusel & Kästner Carlina involucrata Poir. 24.80 (23.89-25.69) 12.40 (11.94-12.85) 576.36 (574.48-578.57) 26.98 (25.51-28.32) 515.58 (513.88-518.09) 0.92 31.98 (31.72-32.41) 15.99 (15.86-16.20) 1114.19 (1112.41-1116.96) 39.57 (36.69-41.45) 1015.32 (1013.98-1017.21) 0.79 Carlina lanata L. 35.21 (34.90-35.76) 17.60 (17.45-17.88) 1030.37 (988.64-1070.90) 37.86 (36.36-39.27) 981.81 (923.45-1017.74) 0.93 Carlina macrocephala Moris 28.66 (27.22-29.75) 14.33 (13.61-14.87) 1184.12 (1183.71-1184.45) 34.05 (33.15-35.16) 830.92 (826.09-834.42) 0.84 Carlina oligocephala Boiss. & Kotschy 24.45 (22.67-26.8) 12.22 (11.34-13.43) 529.54 (528.10-530.76) 30.22 (29.66-30.91) 601.29 (578.46-614.75) 0.81 Carlina racemosa L. 33.79 (32.40-34.91) 16.90 (16.20-17.46) 1141.44 (1088.73-1180.61) 38.36 (36.06-39.93) 943.18 (921.58-951.74) 0.88 Carlina salicifolia (L.f.) Cav. 26.99 (25.27-27.87) 13.49 (12.63-13.94) 1008.14 (875.03-1099.04) 31.14 (29.48-32.26) 661.30 (650.53-667.55) 0.87 Carlina sicula Ten. 41.64 (40.80-42.85) 20.82 (20.40-21.43) 934.63 (933.00-935.94) 33.62 (32.67-35.04) 1116.37 (1098.06-1122.98) 1.24 Carlina sitiensis Rech.f. 41.42 (39.09-43.48) 20.71 (19.54-21.74) 990.15 (975.86-1003.42) 31.76 (30.33-33.30) 1023.01 (1019.48-1025.71) 1.31 Carlina vulgaris L. 30.79 (29.54-32.56) 15.39 (14.77-16.28) 852.96 (790.40-902.71) 37.30 (35.02-38.71) 878.13 (873.56-881.73) 0.83 Jo ur n Pr e- oo f Axis Carlina acanthifolia All. al Studied taxa Table 3. Quantitative pollen morphological characteristics through light microscopy and scanning electron microscopy of the studied taxa of Carlina. Apocolpium Studied taxa Mesocolpium Pore or Lum Diameter index field Diameter Diameter 9.67 (7.96-10.82) 11.98 (10.37-13.40) 25.39 (23.48-27.73) 23.44 (20.79-25.87) 0.88 (0.60-1.30) 1 a acaulis L. 12.79 (11.98-14.00) 12.78 (10.84-14.81) 30.61 (28.81-31.69) 8.97 (7.45-10.40) 0.23 (0.18-0.31) 0 a acaulis subsp. caulescens (Lam.) . & G.Martens a atlantica Pomel 15.39 (14.29-16.43) 17.68 (15.18-19.63) 40.13 (33.08-48.52) 18.74 (16.66- 21.73) 0.33 (0.21-0.46) 0 8.98 (8.67-9.51) 11.01 (10.16-12.60) 22.15 (21.08-23.42) 10.25 (7.55-11.48) 0.29 (0.20-0.47) 1 a biebersteinii Bernh. ex Hornem. 11.11 (10.24-11.63) 13.77 (13.04-14.39) 27.91 (26.20-29.65) 23.87 (22.63-25.04) 0.30 (0.13-0.58) 1 a biebersteinii subsp. brevibracteata e) K.Werner a corymbosa L. 10.01 (8.30-10.94) 11.33 (10.23-12.42) 23.39 (22.49-23.86) 15.88 (14.50-16.85) 0.44 (0.18-0.73) 1 14.15 (12.78-15.24) 17.11 (14.76-18.74) 35.14 (33.40-38.75) 24.42 (22.83-25.79) 0.30 (0.21-0.45) 0 15.54 (14.59-16.36) 18.90 (18.56-19.59) 41.39 (36.97-45.35) 25.57 (22.82-26.53) 0.60 (0.25-0.84) 1 12.67 (10.60-15.10) 14.14 (13.07-15.15) 26.33 (24.90-27.85) 21.69 (19.75-23.53) 0.39 (0.21-0.53) 0 a acanthifolia All. a corymbosa var. graeciformis l & Kästner a curetum Heldr. ex Halácsy Journal Pre-proof 26 Apocolpium Studied taxa Mesocolpium Pore or Lum index field Diameter Diameter a diae (Rech.f.) Meusel & Kästner 11.07 (9.28-12.28) 12.56 (9.73-13.86) 26.46 (24.02-30.50) 22.94 (21.31-24.44) 0.22 (0.11-0.33) 0 a frigida Boiss. & Heldr. 12.89 (11.25-14.21) 14.27 (13.64-14.84) 30.55 (29.59-32.79) 21.24 (18.59-23.59) 0.29 (0.16-0.47) 1 a frigida subsp. fiumensis (Simonk.) l & Kästner a graeca Heldr. & Sart. 14.46 (13.54-15.98) 16.25 (14.23-19.17) 32.53 (30.96-34.28) 27.85 (26.82-28.54) 0.52 (0.26-1.01) 1 12.24 (10.79-13.59) 15.16 (11.76-17.62) 33.17 (29.12-36.53) 11.44 (10.08-12.95) 0.39 (0.24-0.56) 0 a hispanica Lam. 17.32 (16.46-18.62) 20.61 (19.59-21.42) 41.12 (39.51-42.58) 26.76 (25.19-28.93) 0.43 (0.23-0.67) 1 7.38 (5.87-8.32) 8.64 (7.70-9.82) 17.11 (15.39-18.95) 15.30 (14.45-15.98) 0.38 (0.20-0.56) 1 11.87 (11.50-12.33) 14.98 (14.60-15.39) 31.00 (29.47-33.06) 24.49 (22.80-25.98) 0.49 (0.28-0.64) 1 8.52 (7.64-9.09) 10.19 (9.17-11.59) 19.84 (18.85-21.99) 26.51 (25.80-27.80) 0.31 (0.19-0.44) 0 12.63 (10.417-14.23) 15.57 (13.43-17.44) 30.42 (28.15-33.40) 21.92 (18.88-24.45) 0.27 (0.19-0.37) 0 7.11 (5.41-9.91) 7.56 (6.41-8.63) 15.49 (13.78-18.94) 18.97 (16.50-21.90) 0.28 (0.18-0.38) 1 a racemosa L. 10.18 (8.91-12.07) 11.73 (10.81-12.73) 31.14 (24.52-39.98) 23.73 (21.93-25.57) 0.36 (0.22-0.53) 1 a salicifolia (L.f.) Cav. 13.72 (12.88-15.00) 15.53 (14.81-16.36) 32.42 (31.81-33.40) 22.11 (21.03-23.85) 0.39 (0.18-0.63) 1 a sicula Ten. 13.61 (12.31-14.27) 15.55 (14.17-16.30) 34.62 (31.62-36.23) 25.29 (22.86-26.98) 0.41 (0.21-0.78) 1 a sitiensis Rech.f. 12.06 (10.83-13.59) 15.61 (12.94-17.01) 31.06 (27.06-35.19) 22.52 (19.54-24.53) 0.56 (0.20-1.03) 2 a vulgaris L. 14.39 (13.15-15.98) 16.15 (15.16-16.99) 23.15 (22.36-24.37) 0.22 (0.13-0.39) 0 a macrocephala Moris a oligocephala Boiss. & Kotschy oo a lanata L. pr a hispanica subsp. major (Lange) l & Kästner a involucrata Poir. f Diameter 34.31 (33.70-35.18) e- Table 4. Quantitative pollen morphological characteristics through light microscopy and scanning electron microscopy of the studied taxa of Carlina. Murus thickness Pr Spines Studied taxa length Base area Colpus Length Width Area Carlina acanthifolia All. 0.42 (0.29-0.52) 2.67 (2.35-3.23) 12.95 (9.86-15.26) 37.66 (29.90-56.50) 8.50 (7.46-9.04) 46.15 (43.67 Carlina acaulis L. 0.69 (0.42-0.90) 2.15 (1.63-2.71) Carlina acaulis subsp. caulescens (Lam.) Schübl. & G.Martens Carlina atlantica Pomel 0.68 (0.43-0.98) 12.25 (10.63-14.44) 49.97 (47.05 23.57 (20.82-25.64) 15.86 (12.01-18.37) 60.79 (41.52 2.25 (1.49-3.04) 10.25 (7.55-11.48) 20.18 (16.42-25.14) 8.35 (7.09-9.18) 29.47 (26.78 al 19.26 (16.17-21.23) 14.53 (13.30-15.69) Jo ur n Carlina biebersteinii Bernh. ex Hornem. 0.32 (0.26-0.37) 21.67 (20.50-22.49) 1.88 (1.04-3.33) 0.29 (0.23-0.34) 2.32 (1.36-3.32) 17.32 (15.80-18.88) 23.17 (19.93-25.85) 12.52 (12.19-13.12) 36.28 (34.42 0.29 (0.20-0.50) 1.96 (1.28-2.54) 11.57 (9.32-14.08) 28.18 (25.08-35.91) 2.41 (1.29-4.23) 21.36 (19.44 0.31 (0.21-0.39) 1.80 (1.10-2.61) 11.33 (7.54-14.25) 21.23 (15.92-25.48) 10.42 (9.89-11.01) 44.06 (36.81 0.43 (0.30-0.63) 2.20 (1.32-2.87) 8.66 (6.72-11.23) 12.96 (10.97-15.29) 3.23 (2.01-4.33) 13.06 (12.86 0.34 (0.23-0.43) 2.60 (1.46-3.55) 10.99 (8.23-12.95) 29.11 (22.46-41.22) 13.31 (10.13-16.14) 57.07 (29.42 0.27 (0.14-0.49) 1.49 (0.65-2.41) 8.55 (6.63-10.54) 26.56 (19.04-30.20) 11.59 (10.76-12.93) 34.34 (26.70 Carlina frigida Boiss. & Heldr. 0.32 (0.19-0.40) 2.72 (1.94-3.43) 13.45 (11.29-14.84) 16.64 (11.85-20.26) 8.13 (6.26-9.76) 24.30 (19.48 Carlina frigida subsp. fiumensis (Simonk.) Meusel & Kästner Carlina graeca Heldr. & Sart. 0.43 (0.36-0.55) 2.38 (1.83-3.82) 16.94 (15.41-18.77) 32.54 (29.64-34.77) 13.06 (11.40-14.38) 46.72 (46.42 0.33 (0.22-0.45) 2.02 (1.23-2.58) 19.34 (17.98-21.21) 21.24 (16.94-26.76) 10.26 (7.83-14.81) 54.39 (29.47 Carlina hispanica Lam. 0.32 (0.26-0.44 1.88 (1.23-2.65) 15.58 (12.71-18.73) 19.83 (12.67-25.76) 12.61 (11.53-15.01) 40.97 (25.59 Carlina hispanica subsp. major (Lange) Meusel & Kästner Carlina involucrata Poir. 0.25 (0.16-0.33) 2.36 (1.44-3.03) 11.07 (9.92-12.90) 17.75 (10.68-22.42) 4.13 (3.74-4.33) 21.71 (14.19 0.30 (0.19-0.39) 2.72 (2.02-3.59) 12.97 (11.61-13.80) 25.95 (19.24-30.95) 12.25 (9.44-15.06) 53.57 (30.50 Carlina lanata L. 0.34 (0.23-0.48) 1.60 (1.13-2.17) 14.38 (12.36-18.31) 22.86 (21.17-25.56) 14.39 (11.10-18.60) 50.65 (44.21 Carlina macrocephala Moris 0.32 (0.21-0.45) 2.33 (1.547-3.16) 16.02 (13.91-17.85) 27.93 (22.36-33.94) 13.11 (11.60-14.30) 63.02 (46.08 Carlina oligocephala Boiss. & Kotschy 0.26 (0.20-0.36) 2.19 (1.75-2.76) 11.25 (8.20-14.47) 16.01 (13.68-19.10) 3.33 (2069-4.40) 13.57 (10.32 Carlina racemosa L. 0.28 (0.17-0.48) 2.94 (1.58-3.94) 13.09 (10.19-15.46) 33.38 (24.01-40.00) 15.20 (11.74-17.43) 72.21 (51.79 Carlina salicifolia (L.f.) Cav. 0.34 (0.24-0.49) 2.60 (1.30-3.42) 15.44 (12.85-16.48) 25.13 (18.39-28.61) 13.75 (12.72-15.16) 56.87 (42.14 Carlina sicula Ten. 0.35 (0.27-0.48) 2.40 (1.48-3.30) 17.30 (16.43-18.31) 24.73 (17.58-28.79) 7.49 (5.96-8.33) 21.82 (17.98 Carlina biebersteinii subsp. brevibracteata Andrae) K.Werner Carlina corymbosa L. Carlina corymbosa var. graeciformis Meusel & Kästner Carlina curetum Heldr. ex Halácsy Carlina diae (Rech.f.) Meusel & Kästner Journal Pre-proof 27 Carlina sitiensis Rech.f. 0.33 (0.24-0.38) 2.63 (1.54-3.59) 15.87 (13.59-17.43) 21.48 (19.51-22.86) 6.71 (6.13-7.25) 32.40 (30.94 Carlina vulgaris L. 0.29 (0.21-0.38) 2.15 (1.36-2.67) 13.83 (12.14-15.52) 18.48 (12.02-22.70) 9.82 (7.42-11.62) 39.87 (25.81 Table 5. Qualitative pollen morphological characteristics through light microscopy and scanning electron microscopy of the studied taxa of Carlina. Shape Studied taxa Sculpture type Pollen Shape Polar Equatorial lobate ovate Surface Aperture Ecto echinate, rugulate, microreticulate echinate, rugulate, microreticulate granu echinate, nanoreticulate, perforate, rugulate scabr echinate, microrugulate echinate, nanoreticulate, perforate, rugulate echinate, rugulate, microreticulate, perforate echinate microreticulate, echinate gran microreticulate, echinate, perforate scabr scabr reticulate, echinate, perforate echinate, nanoreticulate, microrugulate echinate, perforate lina acanthifolia All. Prolate lina acaulis L. Sub-spheroidal lina acaulis subsp. caulescens (Lam.) übl. & G.Martens lina atlantica Pomel Sub-spheroidal lina biebersteinii Bernh. ex Hornem. Sub-spheroidal circular ovate microreticulate,echinate, rugulate lina biebersteinii subsp. vibracteata Andrae) K.Werner lina corymbosa L. Prolate lobate ovate microreticulate,echinate, perforate triangular ovate echinate, nanoreticulate, microrugulate lina corymbosa var. graeciformis usel & Kästner lina curetum Heldr. ex Halácsy Prolate circular ovate Prolate-spheroidal triangular ovate echinate, rugulate echinate, nanoreticulate scabr lina diae (Rech.f.) Meusel & Kästner Sub-spheroidal triangular ovate echinate, perforate echinate, perforate striat lina frigida Boiss. & Heldr. Sub-spheroidal circular ovate microrugulate, echinate, perforate microrugulate, echinate micro Prolate-spheroidal triangular ovate microrugulate, echinate, perforate microrugulate, echinate, perforate scabr Prolate-spheroidal lobate ovate echinate, rugulate echinate granu lina hispanica Lam. Sub-spheroidal circular rugulate, echinate, , perforate microrugulate, echinate striat lina hispanica subsp. major (Lange) usel & Kästner lina involucrata Poir. Sub-spheroidal circular circular microrugulate, echinate, perforate microrugulate, echinate granu Sub-spheroidal circular ovate microrugulate to nanoverrucate, echinate microrugulate, echinate granu lina lanata L. Sub-spheroidal triangular ovate microreticulate, echinate, perforate microreticulate, echinate, perforate striat lina macrocephala Moris Sub-spheroidal triangular ovate microreticulate, echinate, perforate microreticulate, echinate, perforate scabr lina oligocephala Boiss. & Kotschy Sub-spheroidal ovate echinate, nanoreticulate or perforate echinate, nanoreticulate or perforate gran lina racemosa L. Sub-spheroidal circular ovate microrugulate, echinate, perforate microrugulate, echinate, perforate scabr lina salicifolia (L.f.) Cav. Sub-spheroidal triangular ovate microreticulate, echinate, striate microreticulate, echinate, striate striat lina sicula Ten. lina sitiensis Rech.f. lina vulgaris L. Sub-prolate Prolate Sub-spheroidal triangular circular triangular ovate ovate ovate microreticulate, echinate, perforate reticulate-microreticulate, echinate, rugulate microreticulate, echinate, rugulate microreticulate, echinate microreticulate, echinate, rugulate microreticulate, echinate, rugulate scabr scabr scabr f oo circular pr circular echinate, rugulate, microreticulate, perforate e- circular Pr triangular ovate al Sub-spheroidal ovate Jo ur n lina frigida subsp. fiumensis monk.) Meusel & Kästner lina graeca Heldr. & Sart. Sub-spheroidal triangular triangular striat scabr scabr Journal Pre-proof 28 Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this Jo ur n al Pr e- pr oo f article. Journal Pre-proof 29 Highlights Pollen morphology of 24 taxa of Carlina was observed and studied by LM and SEM  HCA and PCA analyses applied to detect the potential characters separating the taxa  Dichotomous keys to pollen types of the studied taxa of Carlina were constructed  Qualitative and quantitative pollen characters can easily separate the Carlina taxa  ImageJ program is a useful tool in measuring the quantitative of pollen characters Jo ur n al Pr e- pr oo f  Figure 1 Figure 2