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, 2003; Kamatou et al., 2008; Safaeishakib & Ghaffarzadegan, 2022). Four regions such as central and South America, western and eastern Asia are the major distribution centers of this large genus (Hedge, 1982a ; Wu & Li, 1982; Walker & Sytsma, 2007). Based on Flora Iranica (Hedge, 1982a , b ), the genus Salvia consists of 58 species in Iran, among which 17 are endemic (Hedge, 1982a , b ). 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., 1975; Afzal-Rafii, 1976; Bhattacharya, 1978; Haque & Ghoshal, 1980; Mercado et al., 1989; Harley & Heywood, 1992; Yang et al., 2004; Song & Li, 2009; Wang et al., 2009).

Detailed chromosomal data and meiotic behavior with palynological research reports on the genus grown in Iran are limited to these studies (Jafari & Nikian, 2008; Sheidai et al., 2010; Sheidai & Alijanpoor, 2011; Kharazian, 2011; Ranjbar et al., 2015; Alijanpoor & Safaeishakib, 2022). Besides, studies on pollen morphology in Salvia have been conducted by many authors worldwide (see e.g. Henderson et al., 1968; Trudel & Morton, 1992; Hassan et al., 2009; Kahraman et al., 2009, 2010; Özler et al., 2011, 2020). 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, 1980, 1981), 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, 1982a ; Walker & Sytsma, 2007). There are 34 species from sect. Aethiopis in Iran, 11 being endemic to the country (Boissier, 1879; Hedge, 1982b ). From this section, species S. hypoleuca Benth. (one of the endemisms growing in the north and central parts of Iran; Hedge, 1982b ), 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.

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., 2010). 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).

A detailed cytological study of Salvia species showed the occurrence of abnormalities (Sheidai et al., 2010; Alijanpoor & Safaeishakib, 2022), responsible for the production of unreduced pollen grains (Bretagnolle & Thomson, 1995). The present work demonstrates that the investigated whole pollen grains are hexacolpate and not octacolpate as Erdtman (1945) 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. (1968). 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, 1979). 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., 2011, 2020). 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.

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.