Contribution to cytology of genus Salvia L. (Lamiaceae) in Iran
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CARYOLOGIA Vol. 63, no. 4: 405-410, 2010 Contribution to cytology of genus Salvia L. (Lamiaceae) in Iran Sheidai Masoud1,*, Behnaz Alijanpoo2 and Masoud Khayyami2 1 Faculty of Biological Sciences, Shahid Beheshti University, GC, Tehran, Iran 2 Biology Department, Urmia University, Urmia, Iran Abstract — The genus Salvia L. (Lamiaceae) contains about 900 species distributed throughout the Old and New world growing in temperate and subtropical areas, with about 70 species reported in Flora Iranica. Cyto- logical studies of the Salvia have been mainly focused on chromosome number reports and karyotype analysis, while study of the chromosomes behavior in meiosis is very limited. Meiotic studies were performed in ten Salvia species of S. spinosa, S. reuterana, S. sclarea, S. ceratophylla, S. xanthocheiala, S. limbata, S. hypoleuca, S. staminea, S. nemorosa and S. verticillata showing 2n = 14, 20, 22 and 32 chromosome numbers indicating the role played by polyploidy and aneuploidy in Salvia species diversification. Among Salvia species, the highest value of rela- tive total, terminal and intercalary chiasmata occurred in S. verticellata while the lowest value of relative total and terminal chiasmata occurred in S. spinosa. Laggard chromosomes and chromosomes stickiness as well as frequent tripolar and multipolar cell formation due to anaphase I and II failure were observed. Potential unre- duced (2n pollen) pollen grains were formed due to meiotic irregularities. B-chromosomes of 0-2 were observed in the species of S. sclarea, S. nemorosa and S. reuterana. Key words: Chromosome pairing, Salvia, unreduced pollen grains. INTRODUCTION ment of cancer. Many species of the Lamiaceae are aromatic and are often used as herbs, spices, The genus Salvia L. (Lamiaceae) contains folk medicines and fragrances. In addition, Salvia about 900 species distributed throughout the species are grown in parks and gardens as orna- Old and New world growing in temperate and mental plants (ÖZDEMIR and SENEL 1999). subtropical areas (STANDLEY and WILLIAMS 1973; There have been about 70 Salvia species re- ÖZDEMIR and SENEL 1999). Western Asia and ported from Flora Iranica with 40% endemism Mediterranean regions have been considered (RECHINGER et al. 1982). These species show im- as the original centers of distribution for Salvia portant center of diversity in Flora Iranica regions (WU and LI 1982). with extensive morphological variation. Some Salvia species are herbaceous, suffruticose or species are very distinct while others show close shrabby perennials, rarely biennial or annual, affinity with others and some species are in the often strongly aromatic. These species are of sate of evolutionary flux (RECHINGER 1982). Inter- horticultural, commercial and medicinal values specific hybridization is suspected to be operative (BHATTACHARYA 1978). They contain monoter- in the genus leading to such a grate morphologi- pene with antiseptic characteristics (ÖZDEMIR cal diversity (RECHINGER 1982). and SENEL 1999) and the compounds obtained RECHINGER et al. (1982), in Flora Iranica state from these species decrease DNA synthesis in the that infra-generic taxa recognized in Salvia, are cell, an important feature in diagnosis and treat- unsatisfactory and produce a false impression of the genus. The genus Salvia is a vast genus with about 800 species distributed throughout the world and it is necessary to have a clear idea about the range of morphological variations in the genus as a whole in order to decide about *Corresponding author: e-mail firstname.lastname@example.org its classification. Therefore he suggests that
406 MASOUD , ALIJANPOO and KHAYYAMI the only natural supra-specific categories to be MATERIALS AND METHODS recognized are species-groups, which are often small, not infrequently monotypic groups of Plant material - Plant materials collection was clearly related species. He finally arranged the done in Central Alborze region during 2009 and species in the four major informal groups based 2010. Young flower buds could be obtained for in largely on stamen structure within 3 of which, eleven populations of ten Salvia species growing subdivisions based on habit for, leaf, calyx and wild in this region namely S. spinosa L., S. reuterana corolla characters have been recognized. Boiss., S. sclarea (Mönch) Benth., S. ceratophylla L., Various cytological studies performed on the S. xanthocheiala Boiss. ex. Benth., S. limbata C. A. genus Salvia indicate surprisingly diverse chro- Mey., S. hypoleuca Benth., S. staminea Monthbr. & mosome numbers in the genus. The genus seems Auch. ex. Benth., S. nemorosa L., and S. verticillata to be polybasic, with different groups of species L. The voucher specimens are deposited in Her- in different parts having polyploid origins. The barium of Shahid Beheshti University (HSBU), de- Mediterranean group seems to be characterized tails of which have been given in Table 2. by x = 7 (AFZAL-RAFII 1976), those in Europe and Cytological studies - Meiotic studies were per- Russia by x = 11 (PATUDIN et al. 1975), and those formed on young flower buds collected from at studied in California by x = 16 (EPLING et al. least 10 randomly selected plants from each spe- 1962). Studies made by PALOMINO et al. (1986), cies and population. Minimum 100 metaphase/ show that Salvia subgenus Calosphace is charac- diakinesis pollen mother cells (PMCs) and 500 terized by x = 11 and lower numbers. anaphase and telophase cells were analysed for Cytological studies of the Salvia have been data collection. Pollen satiability as a measure mainly focused on chromosome number reports of fertility was determined by staining minimum and karyotype analysis (for example ÖZDEMIR 1000 pollen grains with 2 % acetocarmine: 50 % and SENEL 1999; YILDIZ and GÜCEL 2006; PALO- glycerin (1:1) for about ½ hr. Round complete MINO et al. 1986; AL-TURKY et al. 2000; FOLEY et pollens which were stained were taken as fer- al. 2008; AFZAL-RAFII 1976; 1980; 1981; PATUDIN tile, while incomplete, shrunken pollens with no et al. 1975; MIZIANTI et al. 1981; HAQUE 1981; stain were considered as infertile. . BORGEN 1980; DÍAZ-LIFANTE et al. 1992; LEE Statistical analyses - χ2 test was performed 1967), while study of the chromosomes behavior to detect a significant difference in chiasma fre- in meiosis is very scare (BAHATTACHARYA 1978; quency and chromosome pairing among the spe- GHAFFARI and CHARIAT-PANAHI 1985; ESTILAI cies and populations studied. T-test analysis was and HASHEMI 1990). We hope by studying mei- performed to study size difference between re- otic peculiarities of Salvia species, a better un- duced and unreduced pollen grains. At least 50 derstanding of species relationships would be larger pollen grains and 50 smaller pollen grains obtained in future. were randomly measured for t-test analysis. TABLE 1 — Meiotic characteristics in Salvia species studied. Species Locality 2n TX IX TOX RB RD PF A1L A2L M1ST S. nemorosa 1 Khojier 14 6.22 2.13 8.35 2.70 3.39 90.50 5.26 0.00 0.00 S. nemorosa 2 Gachsar 14 5.36 2.45 7.86 3.23 2.05 99.90 3.18 0.40 5.26 S. hypoleuca Khojier 22 11.22 1.42 12.64 3.44 5.89 98.70 18.50 0.00 8.66 S. ceratophylla Sorkhehesar 22 8.35 4.35 12.76 5.11 3.03 98.30 7.60 0.00 12.38 S. limbata Khojier 22 10.85 2.84 13.70 4.56 4.69 99.80 15.87 7.14 0.00 S. verticellata Gachsar 16 16.94 6.12 23.06 7.76 6.24 95.50 6.00 5.60 3.61 S. sclarea Poloor 22 8.77 3.61 12.39 4.65 2.90 99.50 2.00 0.00 2.00 S. staminea Abali 22 7.59 2.15 9.73 2.88 3.78 99.00 6.77 0.00 2.18 S. reuterana Khojier 20 11.05 4.65 15.63 6.67 2.00 97.33 10.60 0.00 3.40 S. spinosa Khojier 20 5.74 2.57 8.26 3.43 2.05 98.73 10.00 0.00 1.00 S. xanthochilla Poloor 22 6.62 4.62 11.24 5.06 1.79 97.30 3.70 0.00 2.36 Abbreviations: TX = Terminal chiasmata, IX = Intercalary chiasmata, TOX = Total chiasmata, RB = Ring bivalents, RD = Rod bivalents, PF = Pollen fertility percentage, A1L = Anaphase I laggards percentage, A2L = Anaphase II laggards percentage and M1ST = Metaphase I stickiness percentage.
CONTRIBUTION TO CYTOLOGY OF GENUS SALVIA L . ( LAMIACEAE ) IN IRAN 407 RESULTS AND DISCUSSION al. (1981) and GHAFFARI and CHARIAT-PANAHI (1985), S. hypoleuca, S. ceratophylla, S. limbata Ploidy level and chiasma frequency - Details and S. sclarea and S. xanthocheila showed 2n = 22 of cytological characteristics are presented in Ta- chromosome number, supporting the reports bles 1-3, Fig. 1. The species studied had 2n = 14, of AFZAL-RAFII, (1980; 1981), as well as ÖZ- 20, 22 and 32 chromosome numbers. S. nemoro- DEMIR and SENEL (1999), S. verticillata showed sa showed 2n = 14 in both populations studied 2n = 16 supporting the reports of AFZAL-RAFII, supporting the earlier reports of MIZIANTI et (1980) and LÖVKVIST and HULTGÅRD (1999), S. 10 µm Fig. 1 — Representative meiotic cells in Salvia species studied. A = Meiocyte showing 2n = 22 in S. ceratophylla (arrow indicated quarivalent). B = Meiocyte showing 2n = 20 in S. spinosa. C-E = Meiocytes showing 2n = 22 in S. limbata, S. scalrea and S. hypoleuca respectively. F = A polyploid meiocyte in S. limbata. G and H = Meiocytes showing B- chromosomes in S. reuterana and S. nemorosa (arrows) respectively. I and J= Micronuclei formation in S. hypoleuca and S. reuterana respectively. K and L = Tripolar cells in S. scalrea and S. spinosa. M-O = Potential unreduced pollen grains (larger pollen grains) in S. reuterana, S. xanthochilla and S. spinosa respectively. The bar corresponds to 10 µm.
408 MASOUD , ALIJANPOO and KHAYYAMI reuteriana showed 2n = 20, supporting the re- and rod bivalents in metaphase of meiosis-I with ports of AFZAL-RAFII, (1981), as well as GHAFFARI almost high pollen fertility (>0.90%). In two mei- and CHARIAT-PANAHI (1985). S. spinosa showed otic cells of S. ceratophylla, one ring quadrivalent 2n = 20 which seems to be new to science. was observed which due to low frequency were The occurrence of different basic chromo- not included in final analysis. Such quadrivalents some numbers in a single species has been re- may be formed due to heterozygote transloca- ported in the genus Salvia. For example in S. tions among two pairs of chromosomes. aegyptiaca L., 2n = 12, 26 and 28 have been re- Among Salvia species, the highest value of ported by HAQUE (1981), BORGEN (1980), and relative total, terminal and intercalary chiasmata DÍAZ-LIFANTE et al. (1992), in S. chanroenica ssp. occurred in S. verticellata (2.12, 0.76 and 2.88 glomerifolia Chung, 2n = 14 and 16 have been re- respectively) while the lowest value of the total ported by LEE (1967), in S. nemorosa L. 2n = 14 and terminal chiasmata occurred in S. spinosa and 16 were reported by MIZIANTI et al. (1981) (0.88 and 0.57 respectively). The lowest value of and GHAFFARI and CHARIAT-PANAHI (1985). All intercalary chiasmata occurred in Kojier popu- these data indicate the role played by polyploidy lation of S. nemorosa (0.30). χ2 test performed and aneuploidy in Salvia species diversification. showed no significant difference for chiasma The chromosome number so far reported in frequency and distribution among Salvia species Salvia species, fall into different aneuploid series indicating that during species diversification no starting from x = 7 to 11. According to BAHAT- significant change has occurred in the number of TACHARYA (1978), base numbers 7 and 8 appear genes controlling chiasma formation. However to be the primitive numbers from which second- since variation in chiasma frequency and local- ary base numbers became established and again ization is genetically controlled (QUICKE 1993), diversified in different directions, out of which minor genetic changes leading to meiotic varia- base number 11 is common in many species. FU- tions is considered as a mean for generating new JITA (1970), considered Salvia as the most primi- forms of recombination influencing the variabil- tive genus in the family Labiateae with the highest ity within natural populations in an adaptive way primary basic number 11 although Ajuga is often (REES and DALE 1974). considered as the most primitive genus. However, The correlation test showed no significant according to BAHATTACHARYA (1978), due to cyto- correlation between relative total, terminal and logical numerical instability, the presence of highly intercalary chiasmata as well as ring and rod irregular cytological behavior and presence of ad- bivalents with change in chromosome number vanced karyotype in the Salvia species, they are re- (ploidy level) of the species studied, indicating cent and advanced members of a complex group. the presence of a control over the mean value of Data with regard to chiasma frequency and chiasmata/bivalent. chromosome pairing has been given in Tables 1 Meiotic abnormalities - Almost in all the spe- and 2. The species studied mainly formed ring cies studied, laggard chromosomes were ob- TABLE 2 — Relative meiotic data and size of pollen grains in Salvia species studied. Species Voucher No. TXN IXN TOXN RBN RDN NP UP S. nemorosa 8500980 0.89 0.30 1.19 0.39 0.48 27.18 41.91 S. nemorosa 2 8500990 0.77 0.35 1.12 0.46 0.29 27.18 41.92 S. hypoleuca 8500981 1.02 0.13 1.15 0.31 0.54 25.64 37.62 S. ceratophylla 8500982 0.76 0.40 1.16 0.46 0.28 30.92 52.63 S. limbata 8500983 0.99 0.26 1.25 0.41 0.43 27.52 34.53 S. verticellata 8500984 2.12 0.76 2.88 0.97 0.78 20.21 38.12 S. sclarea 8500985 0.80 0.33 1.13 0.42 0.26 30.91 42.59 S. staminea 8500986 0.69 0.20 0.88 0.26 0.34 37.87 45.88 S. reuterana 8500987 1.10 0.47 1.56 0.67 0.20 35.49 49.54 S. spinosa 8500988 0.57 0.26 0.83 0.34 0.21 28.48 38.10 S. xanthochilla 8500989 0.60 0.42 1.02 0.46 0.16 26.24 36.82 Abbreviations: TXN = Terminal chiasmata/bivalent, IXN =Intercalary chiasmata/bivalent, TOXN = Total chiasmata/biva- lent, RBN =Ring bivalent/cell, RDN = Rod bivalent/cell, NP = Size of normal pollen grains (µm), UP = Size of unreduced pollen grains. (µm).
CONTRIBUTION TO CYTOLOGY OF GENUS SALVIA L . ( LAMIACEAE ) IN IRAN 409 served during anaphase I and II and (Table 2). 1-2% of pollen grains in the species studied. The highest value of anaphase-I laggards (18.50) The mean diameter of normal (reduced) pol- occurred in S. hypoleuca followed by S. limbata len grains ranged from 20.21 in S. verticellata to (15.00), while S. sclarea showed the lowest value 37.87 µm in S. staminea, while the size of poten- of the same (2.00). Laggard chromosomes would tial unreduced (2n) pollen grains ranged from lead to micronuclei formation observed in most 34.53 in S. limbata to 52.63 µm in S. ceratophylla. of the species (Fig. 1, I and J). T-test analysis revealed a significant difference The chromosomes stickiness occurred from (p 90% (Table 2), therefore it seems that meiotic tion. To our knowledge this is the first report on abnormalities may be partly the reason for some the occurrence of unreduced pollen grain for- degree of pollen sterility observed in Salvia spe- mation in the genus Salvia. cies. However it has been suggested that infertil- B-chromosomes - B-chromosomes (Bs) of ity in polyploids is not solely due to the produc- 0-2 were observed in the species of S. sclarea, tion of aneuploid gametes formed by improper S. nemorosa and S. reuterana (Fig. 1, D, G and segregation of chromosomes during anaphase/ H). B-chromosomes were round in shape and telophase stages, the genetic factors may also did not pair with the A-chromosomes or among bring about pollen sterility as evidenced in tet- themselves. These chromosomes are also known raploid strains of rye (HAZARIKA and REES 1967) as accessory chromosomes reported in about and Avena sativa cultivars (BAPTISTA-GIACOMELLI 1300 plants species and 500 animals species et al. 2000). (CAMACHO et al. 2000). They can reduce the Other meiotic irregularities observed were growth and vigor of the plants when present tripolar and multipolar cell formation due to in high number; but in low number may ben- anaphase I and II failure (Fig.1, K and L) and efit the plant possessing them. One of the well meiotic cells with double the chromosome num- known effects of B-chromosomes is the change ber, possibly due syncyte formation and absence they bring about on chiasma frequency, thereby of anaphase segregation (Fig. 1, F). Bahattacha- affecting the genetic changes of the gametes and rya (1978) also reported meiotic irregularities in the next generation as reported in several grass two varieties of S. splendens Ker-Gawl (2n = 22), species including Festuca (JAUHAR and CRANE leading to formation of tripolar cells and pollen 1990), Aegilops (SHEIDAI et al. 2002), Avena grains with size variation and polyspory. (SHEIDAI et al. 2003). However we did not have Unreduced pollen grain formation - The occur- many cells possessing B-chromosomes to anal- rence of large pollen grains (possibly 2n pollen yse statistically the effect of B-chromosomes on grains) was observed along with smaller (normal) chiasma frequency in Salvia species. According pollen grains in all the species studied (Fig.1, to our knowledge this is the first report on the M-O). The large pollen grains comprised about occurrence of B-chromosomes in Salvia.
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