ARTÍCULO

Comparative assessment of pollen micromorphology and meiotic observations in some species from the genus Salvia L. (Lamiaceae) in Iran

MASOUMEH SAFAEISHAKIB¹, BEHNAZ ALIJANPOOR² & MASOUD SHEIDAI³

^{1, 3} Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
² Research Center of Agriculture and Natural Resource of Tehran Province, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

ORCID iD. M. SAFAEISHAKIB: https://orcid.org/0000-0002-3557-0287, B. ALIJANPOOR: https://orcid.org/0000-0002-9698-2592,
M. SHEIDAI: https://orcid.org/0000-0003-3983-6852

Author for correspondence: M. Safaeishakib (safaeishakib@gmail.com)

Editor: S. Garcia

ABSTRACT
Comparative assessment of pollen micromorphology and meiotic observations in some species from the genus Salvia l. (Lamiaceae) in Iran.— This study presents some detailed observations on the meiotic behavior and a comparative palynological study of some selected species of Salvia L. sect. Aethiopis Benth. ANOVA test was used to compare chiasma frequency, distribution and chromosomal associations, revealing a significant difference in all meiotic characteristics among the S. hypoleuca, S. limbata, S. reuteriana, S. spinosa, and S. xanthocheila species. Moreover, some meiotic abnormalities such as chromosome stickiness, laggard chromosomes, as well as frequent tripolar, multipolar cell formation and cytomixis occurred in these species. Light Microscopy (LM) and Scanning Electron Microscopy (SEM) were used for analyzing pollen. Some micromorphological characteristics such as pollen shape, size, polar axis length, equatorial axis length, aperture numbers and exine ornamentation, exhibited remarkable differences amongst the studied species.
KEY WORDS: Light Microscopy (LM); meiotic behavior; pollen micromorphology; Salvia; Scanning Electron Microscopy (SEM).

RESUMEN
Evaluación comparativa de la micromorfología del polen y las observaciones meióticas en algunas especies del género Salvia l. (Lamiaceae) en Irán.— Este estudio presenta algunas observaciones detalladas sobre el comportamiento meiótico y un estudio palinológico comparativo de algunas especies seleccionadas de Salvia L. sect. Aethiopis. Se usa la prueba ANOVA para comparar la frecuencia, la distribución y las asociaciones cromosómicas de los quiasmas, lo que revela una diferencia significativa en todas las características meióticas entre las especies S. hypoleuca, S. limbata, S. reuteriana, S. spinosa y S. xanthocheila. Además, en estas especies se producen algunas anomalías meióticas, como la adherencia de los cromosomas, los cromosomas rezagados, así como la formación frecuente de células tripolares, multipolares y citomixis. Se usa microscopía de luz (LM) y microscopía electrónica de barrido (SEM) para analizar el polen. Algunas características micromorfológicas como la forma del polen, el tamaño, la longitud del eje polar, la longitud del eje ecuatorial, el número de aperturas y la ornamentación de la exina, exhiben diferencias notables entre las especies estudiadas.
PALABRAS CLAVE: comportamiento meiótico; micromorfología del polen; microscopía de luz (LM); microscopía electrónica de barrido (SEM); Salvia.

CONTENIDOS

INTRODUCTIONTop

Salvia L. (family Lamiaceae) consists of nearly 1000 species with a notable diversity and cosmopolitan distribution. Salvia species are used in traditional medicine throughout the world, possessing antioxidant, antiplasmodial and anti-inflammatory features (Ulubelen, 2003Ulubelen, A. 2003. Cardioactive and antibacterial terpenoids from some Salvia species. Phytochemistry 64(2): 395–399. https://doi.org/10.1016/S0031-9422(03)00225-5; Kamatou et al., 2008Kamatou, G. P. P., Makunga, N. P., Ramogola, W. P. N. & Viljoen, A. M. 2008. South African Salvia species: a review of biological activities and phytochemistry. Journal of Ethnopharmacology 119(3): 667–672. https://doi.org/10.1016/j.jep.2008.06.030; Safaeishakib & Ghaffarzadegan, 2022Safaeishakib, M. & Ghaffarzadegan, R. 2022. Herbal drug technology 1 [translation]. Institute of Medicinal Plants (IMP) Press, Iran. ). Four regions such as central and South America, western and eastern Asia are the major distribution centers of this large genus (Hedge, 1982aHedge, I. C. 1982a. Salvia L. In: Davis, P. H. (Ed.), Flora of Turkey and East Aegean Islands 7. University of Edinburgh Press, Edinburgh: 400–461.; Wu & Li, 1982Wu C. & Li H. 1982. On the evolution and distribution in Labiateae. Acta Botanica Yunnanica 4: 97–118 [in Chinese].; Walker & Sytsma, 2007Walker, J. B. & Sytsma, K. J. 2007. Staminal evolution in the genus Salvia (Lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. Annals of Botany 100(2): 375–391. https://doi.org/10.1093/aob/mcl176). Based on Flora Iranica (Hedge, 1982aHedge, I. C. 1982a. Salvia L. In: Davis, P. H. (Ed.), Flora of Turkey and East Aegean Islands 7. University of Edinburgh Press, Edinburgh: 400–461., bHedge, I. C. 1982b. Labiatae. In: Rechinger, C. H. (Ed.), Flora Iranica 150. Akademische Druck-u, Verlagsanstalt, Graz: 403–476.), the genus Salvia consists of 58 species in Iran, among which 17 are endemic (Hedge, 1982aHedge, I. C. 1982a. Salvia L. In: Davis, P. H. (Ed.), Flora of Turkey and East Aegean Islands 7. University of Edinburgh Press, Edinburgh: 400–461., bHedge, I. C. 1982b. Labiatae. In: Rechinger, C. H. (Ed.), Flora Iranica 150. Akademische Druck-u, Verlagsanstalt, Graz: 403–476.). Cytological studies in Salvia species have been conducted by many researchers from Europe, America and Asia; however, most of this research has been carried out only based on chromosome counts (Patudin et al., 1975Patudin, A. V., Yurtsev, V. N. & Pakaln, D. A. 1975. Chromosome number in some species of Salvia L. (Lamiaceae). Botanicheskii Zhurnal (Moscow & Leningrad) 60: 529–534 [in Russian].; Afzal-Rafii, 1976Afzal-Rafii, Z. 1976. Etude cytotaxonomique et phylogénétique de quelques Salvia de la région méditerranéenne: Groupe du Salvia officinalis L. Bulletin de la Société Botanique de France 123(9): 515–531. https://doi.org/10.1080/00378941.1976.10835708; Bhattacharya, 1978Bhattacharya, S. 1978. Study of some Indian members of the genus Salvia with references to the cytological behavior. Cytologia 43: 317–324. https://doi.org/10.1508/cytologia.43.317; Haque & Ghoshal, 1980Haque, M. D. & Ghoshal, K. K. 1980. Karyotypes and chromosome morphology in the genus Salvia Linn. Cytologia 45: 627–640. https://doi.org/10.1508/cytologia.45.627; Mercado et al., 1989Mercado, P., Ramamoorthy, T. P. & Palomino, G. 1989. Karyotypes of five Mexican species of Salvia subgenus Calosphace (Lamiaceae). Cytologia 54(3): 605–608. https://doi.org/10.1508/cytologia.54.605; Harley & Heywood, 1992Harley, R. M. & Heywood, C. A. 1992. Chromosome numbers in tropical American Labiatae. In: Harley R. M. & Reynolds, T. (Eds.), Advances in Labiatae Science. Royal Botanical Gardens, Kew: 211–246.; Yang et al., 2004Yang, Z., Gong, X. & Pan, Y. 2004. Cytological study of six Salvia species (Lamiaceae) from the Hengduanshan Mountains region of China. Caryologia 57(4): 360–366. https://doi.org/10.1080/00087114.2004.10589417; Song & Li, 2009Song, Z-Q. & Li, X-F. 2009. Cytomixis in pollen mother cells of Salvia miltiorrhiza. Caryologia 62(3): 213–219. https://doi.org/10.1080/00087114.2004.10589687; Wang et al., 2009Wang, M., Zhang, L., Ding, C-B., Yang, R-W., Zhou, Y-H., Yang, Z-J. & Yin, Z-Q. 2009. Meiotic observations of eight taxa in the genus Salvia. Caryologia 62(4): 334–340. https://doi.org/10.1080/00087114.2004.10589699).

Detailed chromosomal data and meiotic behavior with palynological research reports on the genus grown in Iran are limited to these studies (Jafari & Nikian, 2008Jafari, A. & Nikian, M. 2008. Micromorphological, anatomical and pollen ornamentation study on four desert species of Salvia in center of Iran. Asian Journal of Plant Sciences 7: 736–741. https://doi.org/10.3923/ajps.2008.736.741; Sheidai et al., 2010Sheidai, M., Alijanpoor, B. & Khayyami, M. 2010. Contribution to cytology of genus Salvia L. (Lamiaceae) in Iran. Caryologia 63(4): 405–410. https://doi.org/10.1080/00087114.2010.10589753; Sheidai & Alijanpoor, 2011Sheidai, M. & Alijanpoor, B. 2011. Karyotype analysis in some Salvia species (Lamiaceae) of Iran. Cytologia 76(4): 425–429. https://doi.org/10.1508/cytologia.76.425; Kharazian, 2011Kharazian, N. 2011. Karyotypic study of some Salvia Lamiaceae species from Iran. Journal of Applied Biological Sciences 5(3): 21–25.; Ranjbar et al., 2015Ranjbar, M., Pakatchi, A. & Radjabian, T. 2015. Pollen morphology study of Salvia sect. Plethiosphace and sect. Hemisphace (Lamiaceae) in Iran. Nova Biologica Reperta 1(2): 16–28. https://doi.org/10.29252/nbr.1.2.16; Alijanpoor & Safaeishakib, 2022Alijanpoor, B. & Safaeishakib, M. 2022. Cytomixis and meiotic abnormal behavior related in some selected species of the genus Salvia L. (Lamiaceae) from Iran. Acta Scientifica Malaysia 6(2): 34–37. https://doi.org/10.26480/asm.02.2022.34.37). Besides, studies on pollen morphology in Salvia have been conducted by many authors worldwide (see e.g. Henderson et al., 1968Henderson, D. M., Prentice, H. & Hedge, I. C. 1968. Pollen morphology of Salvia and some related genera. Grana Palynologica 8: 70–85. https://doi.org/10.1080/00173136809427461 ; Trudel & Morton, 1992Trudel, M. C. G. & Morton, J. K. 1992. Pollen morphology and taxonomy in North American Labiatae. Canadian Journal of Botany 70: 975–995. https://doi.org/10.1139/b92-122; Hassan et al., 2009Hassan, N., Osman, A. K. & El Garf, I. A. 2009. Pollen types of the Egyptian species of the genus Salvia (Lamiaceae). Feddes Repertorium 120: 394–404. https//doi.org/10.1002/fedr.200911118; Kahraman et al., 2009Kahraman, A., Celep, F. & Dogan, M. 2009. Morphology, anatomy and palynology of Salvia indica L. (Labiatae). World Applied Sciences Journal 6(2): 289–296., 2010Kahraman, A., Celep, F. & Dogan, M. 2010. Anatomy, trichome morphology and palynology of Salvia chrysophylla Stapf (Lamiaceae). South African Journal of Botany 76(2): 187–195. https://doi.org/10.1016/j.sajb.2009.10.003; Özler et al., 2011Özler, H., Pehlivan, S., Kahraman, A., Doğan, M., Celep, F., Başer, B., Yavru, A. & Bagherpour, S. 2011. Pollen morphology of the genus Salvia L. (Lamiaceae) in Turkey. Flora 206: 316–327. https://doi.org/10.1016/j.flora.2010.07.005, 2020Özler, H., Kahraman, A., Pehlivan, S., Doğan, M., Başer, B., Fişne, A. Y. & Bagherpour, S. 2020. Contribution to the knowledge of the pollen morphology of the genus Salvia (Lamiaceae). Phytotaxa 428(3): 228–240. https://doi.org/10.11646/phytotaxa.428.3.4). Yet, research on this genus and related taxa had been mainly based on observations with light microscopy (LM) in the Aethiopis Benth. section (Afzal-Rafii, 1980Afzal-Rafii, Z. 1980. In: Löve, A. (Ed.), Chromosome number reports LXVII. Taxon 29: 365–366., 1981Afzal-Rafii, Z. 1981. In: Löve, A. (Ed.), Chromosome number reports LXX. Taxon 30: 73–74.), the focus of the present work. The main features of this section are false upper crown lips, long-attached stamens, reduced lower theca to usually double-shaped plate, and articular stamens (Hedge, 1982aHedge, I. C. 1982a. Salvia L. In: Davis, P. H. (Ed.), Flora of Turkey and East Aegean Islands 7. University of Edinburgh Press, Edinburgh: 400–461.; Walker & Sytsma, 2007Walker, J. B. & Sytsma, K. J. 2007. Staminal evolution in the genus Salvia (Lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. Annals of Botany 100(2): 375–391. https://doi.org/10.1093/aob/mcl176). There are 34 species from sect. Aethiopis in Iran, 11 being endemic to the country (Boissier, 1879Boissier, E. 1879. Salvia L. In: Flora orientalis: sive, Enumeratio plantarum in Oriente a Graecia et Aegypto ad Indiae fines hucusque observatarum 4. Basileae, H. Georg: 591–636. https://doi.org/10.5962/bhl.title.20323; Hedge, 1982bHedge, I. C. 1982b. Labiatae. In: Rechinger, C. H. (Ed.), Flora Iranica 150. Akademische Druck-u, Verlagsanstalt, Graz: 403–476.). From this section, species S. hypoleuca Benth. (one of the endemisms growing in the north and central parts of Iran; Hedge, 1982bHedge, I. C. 1982b. Labiatae. In: Rechinger, C. H. (Ed.), Flora Iranica 150. Akademische Druck-u, Verlagsanstalt, Graz: 403–476.), S. limbata C. A. Mey., S. reuteriana Boiss., S. spinosa L. and S. xanthocheila Boiss. have been investigated in this study. The present survey describes the behavior of the chromosomes in meiosis and the occurrence of unreduced pollen grain formation, in addition to the micromorphology of their pollen grain.

MATERIAL AND METHODSTop

Plant material

The materials for cytological and pollen micromorphology analysis are presented in Table 1. Voucher specimens were deposited at the herbarium of Shahid Beheshti University (HSBU). Samples were collected from five different regions of Iran.

Cytological and pollen grain preparation

To check for stages of meiosis, the presence of abnormalities and the possible failure of meiosis, preparation and counting of pollen were performed according to previous works (Sheidai et al., 2002Sheidai, M., Koobaz, P., Termeh, F. & Zehzad, B. 2002. Phenetic studies in Avena species and populations of Iran. Journal of Sciences, Islamic Republic of Iran 13(1): 19–28.; Safaei et al., 2016Safaei, M., Sheidai, M., Alijanpoor, B. & Noormohammadi, Z. 2016. Species delimitation and genetic diversity analysis in Salvia with the use of ISSR molecular markers. Acta Botanica Croatica 75(1): 45–52. https://doi.org/10.1515/botcro-2016-0005 ; Alijanpoor & Safaeishakib, 2022Alijanpoor, B. & Safaeishakib, M. 2022. Cytomixis and meiotic abnormal behavior related in some selected species of the genus Salvia L. (Lamiaceae) from Iran. Acta Scientifica Malaysia 6(2): 34–37. https://doi.org/10.26480/asm.02.2022.34.37). For the assessment of the behavior of chromosomes in meiosis, young flower buds were selected and fixed in an acetic alcohol mixture (1:2) for 24 hours. For further storage, 70% ethanol at 4°C was adopted. Pollen fertility size frequencies and stain ability tests were done with 2% acetocarmine:50% glycerin (1:1) for about 30 min. Approximately 1000 pollen grains were evaluated. The squashing technique and 2% acetic-orcein (as the stain) were applied for cytological preparations. Round complete pollen grains with stained nuclei were taken as apparently fertile, while shriveled and unstained pollen grains were considered sterile. One hundred pollen mother cells (PMCs) were analysed for chiasma frequency, distribution at diakinesis/metaphase stage and 500 PMCs were analysed for chromosome segregation during the anaphase and telophase stages. All stages of meiosis and morphology of the chromosomes, presence of abnormalities and possible meiosis failure were all considered (Fig. 1). The present study of pollen morphology was evaluated by LM (Light Microscopy) and SEM (Scanning Electron Microscopy). Freshly prepared slides were observed by Nikon 80i Eclipse digital imaging system. For SEM the acetolyzed pollen grains were attached and fixed to aluminum stubs with double-sided cellophane tape, air-dried at room temperature and coated with gold (JEOL 6060, JSM 6400). The specimens were tested with a Philips XL 20 SEM at 20kV. The UTHSCSA Image Tool v5 software was applied for pollen measurements. Quantitative and qualitative characters such as diameter of the mesocolpial area, diameter of lumina and polar axis length, equatorial axis length, thickness and number of colpus, and pollen type were considered. The terminology and pollen shape classification used is in accordance with Punt et al. (2007Punt, W., Hoen, P. P., Blackmore, S., Nilsson. S. & Le Thomas, A. 2007. Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology 143(1–2): 1–81. https://doi.org/10.1016/j.revpalbo.2006.06.008) and Özler et al. (2011Özler, H., Pehlivan, S., Kahraman, A., Doğan, M., Celep, F., Başer, B., Yavru, A. & Bagherpour, S. 2011. Pollen morphology of the genus Salvia L. (Lamiaceae) in Turkey. Flora 206: 316–327. https://doi.org/10.1016/j.flora.2010.07.005, 2013Özler, H., Pehlivan, S., Celep, F., Doğan, M., Kahraman, A., Fişne, A. Y., Başer, B. & Bagherpour, S. 2013. Pollen morphology of Hymenosphace and Aethiopis sections of the genus Salvia (Lamiaceae) in Turkey. Turkish Journal of Botany 37: 1070–1084. https://doi.org/10.3906/bot-1209-50). Correspondence Analysis (CA) is a multivariate graphical technique based on Hill & Gauch (1980Hill, M. O. & Gauch, H. G. 1980. Detrended correspondence analysis: an improved ordination technique. In: van der Maarel, E. (Eds.), Classification and Ordination (Advances in Vegetation Science, 2). Springer, Dordrecht: 47–58. https://doi.org/10.1007/978-94-009-9197-2_7) that was performed with PAST software v4.06b (Hammer et al., 2001Hammer, Ø., Harper, D. A. T. & Ryan, P. D. 2001. PAST: PAleontological STatistics software package for education and data analysis. Palaeontologia Electronica 4: 9.).

Figure 1. Representative miotic cells in Salvia species studied. Scale bar = 10 μm. (A–C), S. xanthocheila: (A), micronucleus (arrow) telophase I; (B), metaphase II; (C), laggard (arrow). (D–F), S. hypoleuca: (D), triad; (E), diakinesis; (F), tetrad. (G–I), S. reuteriana: (G), diakinesis; (H), metaphase I stickiness; (I), laggard (arrow). (J–L), S. spinosa: (J), cytomixis; (K), diakinesis; (L), metaphase I stickiness. (M–O), S. limbata: (M), pachyten; (N), telophase II; (O), anaphase II stickiness. [View full size] [Descargar tamaño completo]

Statistical analyses

One-way Analysis of Variance (ANOVA) and t-test were applied (p < 0.05) to decide the contrasts in chiasma frequency, chromosomes association and size of unreduced and reduced pollen grains findings. The Pearson coefficient of correlation was applied to address the relationship between pollen fertility, anaphase, metaphase and telophase stickiness.

RESULTS AND DISCUSSIONTop

The highest mean number of ring bivalents occurred in S. spinosa (3.33) while the lowest value occurred in S. xanthocheila (1.1). The highest mean number of rod bivalents occurred in S. hypoleuca, S. limbata (0.64), while the lowest value occurred in S. reuteriana (0.52). Subsequently, the highest mean number of quadrivalents occurred in S. hypoleuca (2.44) and the lowest value gained in the S. spinosa (0.18) (Table 2). Chromosome stickiness was observed in all three phases (anaphase I, metaphase I and telophase). The highest value of stickiness during anaphase and telophase I occurred in S. xanthocheila. Significant correlation was observed between pollen fertility and the anaphase I laggard chromosomes. Therefore, this meiotic abnormality produced pollen sterility (Sheidai et al., 2010Sheidai, M., Alijanpoor, B. & Khayyami, M. 2010. Contribution to cytology of genus Salvia L. (Lamiaceae) in Iran. Caryologia 63(4): 405–410. https://doi.org/10.1080/00087114.2010.10589753). The occurrence of meiotic abnormalities such as chromosome stickiness, laggard chromosome, formation of micronuclei in tetrad cells and cytomixis are provided in Fig. 1 and Table 3. ANOVA test conducted on chiasma frequency, distribution and chromosomal association revealed a significant difference in all meiotic characteristics among the species studied, indicating genomic differences. Correspondence Analysis (CA) (Fig. 2) was done to elucidate the correspondence between profiles of selected species and meiotic data. The main features of the pollen are illustrated in Figs. 3 and 4 and summarized in Table 4. The t-test analysis disclosed a significant difference (p < 0.05) in the size of unreduced pollen grains as compared to that of reduced pollen grains. Pollen grains (n, 2n, and infertile) were observed in all studied species. The diameter of normal pollen grains ranged from 25.44 to 50.81 μm while the diameter of the 2n pollen grains ranged from 34.50 to 49.55 μm in the studied species. The frequency of 2n pollen grains varied from 8.5 to 19.5 μm and the highest occurrence of large pollen grains (2n pollen grains), with a frequency of 19.5%, was observed in S. xanthocheila. Pollen grain type ranged from sub-oblate to oblate and subspherodial; also, exine sculpturing (ornamentation) showed bireticulate perforation. Correspondence Analysis (CA) showed that species were mainly influenced by wall thickness and pore diameters than polar and equatorial axis, ratio of polar axis/equatorial diameter, lumen and mesocolpia diameter traits (Fig. 5). The smallest ratio of polar axis/equatorial diameter (P/E) was detected in S. limbata, whereas the largest was observed in S. hypoleuca. The thickness of exine varied between 372.3 μm in S. hypoleuca and 642.9 μm in S. xanthocheila. The diameter of the mesocolpial area varied from 3.92 μm in S. hypoleuca to 16.41 μm in S. limbata. Moreover, the observation of whole pollen grains displayed radial symmetry with isopolar character. Among the investigated species the highest lumen size was obtained in S. limbata. Types of pollen apertures were hexacolpate with the highest diameter gained in S. hypoleuca (Table 2).

Figure 2. Correspondence analysis (CA) biplots performed in the meiotic data: (TX), terminal chiasma; (IX), intercalary chiasma; (TOX), total chiasma; (IXN), intercalary chiasmata/bivalent; (TXN), terminal chiasmata/bivalent; (TOXN), total chiasmata/bivalent; (RODN), rod bivalents; (RB), ring bivalent; (U), univalent; (Q), quadrivalent. [View full size] [Descargar tamaño completo]

Figure 3. Representative LM photographs of pollen grains in Salvia species studied: (A–B), equatorial view of S. spinosa; (C–D), equatorial view of S. xanthocheila, 2n, n and abnormal pollen grain; (E–F), polar and equatorial views of different pollen grains in S. limbata, 2n and infertile; (G–H), equatorial and polar view of S. hypoleuca various pollen grains; (I), polar and equatorial views of infertile pollen grain in S. reuteriana. [View full size] [Descargar tamaño completo]

Figure 4a. SEM micrographs of pollen grains in Salvia species general appearance and exine ornamentation in detail. [View full size] [Descargar tamaño completo]

Figure 4b. SEM micrographs of pollen grains in Salvia species general appearance and exine ornamentation in detail. [View full size] [Descargar tamaño completo]

Figure 5. Correspondence analysis (CA) biplots carried out on pollen data: (P), polar axis; (E), equatorial axis; (P/E), ratio of polar axis/equatorial diameter. [View full size] [Descargar tamaño completo]

A detailed cytological study of Salvia species showed the occurrence of abnormalities (Sheidai et al., 2010Sheidai, M., Alijanpoor, B. & Khayyami, M. 2010. Contribution to cytology of genus Salvia L. (Lamiaceae) in Iran. Caryologia 63(4): 405–410. https://doi.org/10.1080/00087114.2010.10589753; Alijanpoor & Safaeishakib, 2022Alijanpoor, B. & Safaeishakib, M. 2022. Cytomixis and meiotic abnormal behavior related in some selected species of the genus Salvia L. (Lamiaceae) from Iran. Acta Scientifica Malaysia 6(2): 34–37. https://doi.org/10.26480/asm.02.2022.34.37), responsible for the production of unreduced pollen grains (Bretagnolle & Thomson, 1995Bretagnolle, F. & Thomson, J. D. 1995. Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytologist 129: 1–22. https://doi.org/10.1111/j.1469-8137.1995.tb03005.x). The present work demonstrates that the investigated whole pollen grains are hexacolpate and not octacolpate as Erdtman (1945Erdtman, G. 1945. Pollen morphology and plant taxonomy. IV. Labiatae, Verbenaceae and Avicenniaceae. Svensk Botanisk Tidskrift 39: 279−285.) had reported in Nepetoideae subfamily. In accordance with LM observations, the pollen grains of these species of Salvia are oblate in equatorial and elliptic in polar view, with a narrowing at the poles. Lateral mesocolpia were longer and thicker than the four medial mesocolpia which supported the finding from Henderson et al. (1968Henderson, D. M., Prentice, H. & Hedge, I. C. 1968. Pollen morphology of Salvia and some related genera. Grana Palynologica 8: 70–85. https://doi.org/10.1080/00173136809427461 ). The existence of big grains is known as an indication of the production of 2n pollen. The most straightforward and the easiest method for screening of pollen grains involves the assessment of the range of pollen sizes generated by an individual; as the DNA content increases, the cell volume increases, which in turn affects the pollen diameter (Vorsa & Bingham, 1979Vorsa, N. & Bingham, E. T. 1979. Cytology of 2n pollen formation in diploid alfalfa, Medicago sativa. Canadian Journal of Genetics and Cytology 21(4): 525–530. https://doi.org/10.1139/g79-057). These observations were made on pollen morphological characters, and together with our previous work, they completely support the palynological findings of Salvia (Özler et al., 2011Özler, H., Pehlivan, S., Kahraman, A., Doğan, M., Celep, F., Başer, B., Yavru, A. & Bagherpour, S. 2011. Pollen morphology of the genus Salvia L. (Lamiaceae) in Turkey. Flora 206: 316–327. https://doi.org/10.1016/j.flora.2010.07.005, 2020Özler, H., Kahraman, A., Pehlivan, S., Doğan, M., Başer, B., Fişne, A. Y. & Bagherpour, S. 2020. Contribution to the knowledge of the pollen morphology of the genus Salvia (Lamiaceae). Phytotaxa 428(3): 228–240. https://doi.org/10.11646/phytotaxa.428.3.4). Micromorphological measurements such as pollen shape, size, polar axis length (P), equatorial axis length (E), aperture numbers, and exine ornamentation, in the pollen grains of these studied species of Salvia, can become useful at the systematics/taxonomic level, as they exhibit remarkable differences for the distinction of species.

CONCLUSIONSTop

This study examined five species of the genus Salvia from the sect. Aethiopis. The occurrence of meiotic abnormalities such as chromosome stickiness, laggard chromosomes, the formation of micronuclei in tetrad cells and cytomixis are reported in these species. Similarly, the pollen grain type ranged from sub-oblate to oblate and subspherodial; also, exine sculpturing displayed bireticulate perforation. Based on the present analysis, the pollen morphology of these species is more influenced by wall thickness and pore diameters than any other characteristics.

ACKNOWLEDGEMENTSTop

The authors would like to thank Shahid Beheshti University for providing the facilities necessary to carry out the research.

REFERENCESTop