Cytogenetic Studies in Some Species of Melica L. and Milium L. in Iran

Masoud Sheidai* and Khaled Moghaddam

Shahid Beheshti University, GC, Faculty of Biological Sciences, Tehran, Iran

Received August 22, 2008; accepted July 20, 2009

Summary Cytogenetical studies were performed on 15 populations of 7 species and subspecies of the genus Melica as well as 4 populations of 2 species of the genus Milium growing wild in Iran. The meiotic analysis of Melica and Milium species studied showed the occurrence of a post pachytene diffuse stage possibly due to adaptation to adverse environmental conditions. Melica species studied possess 2n2x18 chromosome number. The chromosome numbers of M. persica subsp. persica and M. persica subsp. inaequiglumis have been reported for the ﬁrst time. The ANOVA test performed among Melica species and populations studied, revealed a signiﬁcant difference in the amount of total, terminal and intercalary chiasmata as well as amount of rod bivalents (p0.01), indicating partly their genetic differences. Two populations of Milium vernale showed the presence of 2n2x14 and 2n2x18, while two populations of M. schmidtianum possessed 2n2x14 chromosome number, which is new for M. chmidtianum.

Key words Cytogenetic, Melica, Milium, Iran.

The genus Melica L. is the largest genus of the family Meliceae (Gramineae, Pooideae), comprised of about 80 species of perennial rhizomatous grasses, distributed all over the world except tropical regions and Australia (Clayton and Renovize 1986), mainly grown in North temperate, Southern Africa and South America and are mesophytic to xerophytic; shade species and species of open habitats. The Melica species are bisexual, with bisexual spikelets; with hermaphrodite florets; exposed- cleistogamous, or chasmogamous. The inflorescence is few spikeleted to many spikeleted; a single raceme, or paniculate; open, or contracted; with capillary branchlets, or without capillary branchlets. The genus Melica includes phylogenetically old forms in East Asia and many forms (in a con- tinuous evolution) in the (semi) arid regions of West Eurasia, North and South America. The genus chromosome base number is x9 and diploid and tetraploid levels have been reported. The somatic chromosome number of 2n14 has been rarely reported in this genus (Dallwitz and Watson 1992). Due to the presence of a large number of species in the genus Melica, controversy exists about taxonomic treatment of the genus and different authors have considered different number of subgenera and sections for this genus (for example see: Stebbins 1982, 1986, Tzvelev 1976, Torres 1980). Polyploidy and inter-specific hybridization is considered to be of high importance in the evolution of Gramineae (Stebbins 1982, 1986), however the genus Melica is an exception by possessing a very homogenous group of mainly diploid species with the rare occurrence of change in the basic chromosome number and a very low level of inter-specific hybridization (Teresa and Bibsy 2000). Many species of the genus Melica are of limited distribution and are of less fodder value due to the presence of prussic acid which is harmful to the cattle. However two species of M. cupani and M. ciliata are known to be of forage value. Controversy also exists about the number of Melica species growing in Iran, as Parsa (1950)

* Corresponding author, e-mail: [email protected], [email protected] 186 M. Sheidai and K. Moghaddam Cytologia 74(2) reported 5 species from Iran while Bor (1970) in Flora Iranica reported 8 species from the country including 2 sub-species for M. persica, 3 sub-species for M. jacquemontii and 2 varieties for M. ciliata. Therefore according to Bor (1970), in total 12 species, sub-species and varieties of Melica grow in Iran. Recently, Assadi (1996) has reported M. picta from Iran. The genus Milium L. (Aveneae, Poaceae) is comprised of 3–7 annual or perennial stolonifer- ous, or caespitose species, mainly grown in the North temperate regions of the old world and also the east part of North America. They are commonly adventive, mesophytic and xerophytic; shade species and species of open habitats. The species are bisexual, having bisexual spikelets with hermaphrodite florets. The inflorescence is paniculate; open; with capillary branchlets; espatheate; not comprising ‘partial inflorescences’ and foliar organs. The chromosome base number of the genus Milium is x4, 5, 7, and 9 and diploid, tetraploid and hexaploid levels have been reported (Clayton and Renovize 1986, Tzvelev 1976, Simon and Tomas 1991, Dallwitz and Watson 1992). Parsa (1950) reported 1 species of M. vernale with 2 varieties from Iran while Bor (1970) in Flora Iranica reported 4 species from the country. The present report is a part of biosystematic study of two genera of Melica and Milium concerned with cytogegetic analysis of 15 populations of 7 species and subspecies of the genus Melica as well as 4 populations of 2 species of the genus Milium growing wild in Iran for the first time. According to our knowledge, almost all the meiotic reports of the present study regarding the chiasma frequency and distribution as well as chromosome pairing are new to science. The chromosome number of Melica picta and M. persica subsp. persica and a new ploidy level (2n2x14) in Milium schmidtianum have been reported for the first time.

Materials and methods Plant material Meiotic studies were performed in 15 populations of 7 species and subspecies of the genus Melica as well as 4 populations of 2 species of the genus Milium growing wild in Iran. The Melica species studied are: 1–M. persica Kunth subsp. persica (2 populations), 2–M. persica Kunth subsp. inaequiglumis (Boiss.) Bor (2 populations), 3–M. jacquemontii Decne subsp. jacquemontii (1 population), 4–M. jacquemontii Decne subsp. canescens (Regel) Bor (1 population), 5–M. jacquemontii Decne subsp. hohenackeri (Boiss.) Bor (2 populations), 6–M. ciliata L. var. ciliata (2 populations), and 7–M. picta C. Koch. (1 population). The Milium species studied are: 8–M. vernale L. (2 populations), and 9–M. schmidtianum C. Koch. (2 populations). The voucher specimens are deposited in the Herbarium of Shahid Beheshti University (HSBU) and TARI.

Cytological preparation and meiotic analysis Young flower buds were collected from 10 randomly selected plants of each species/population and fixed in glacial acetic acid : ethanol (1 : 3) for 24 h. Flower buds were washed and preserved in 70% ethanol at 4°C until used (Sheidai et al. 2002). Cytological preparations used the squash tech- nique and 2% aceto-orcein as the stain. Fifty to 100 pollen mother cells (PMCs) were analysed for chiasma frequency and distribution at diakinesis metaphase stage and 500 PMCs were analysed for chromosome segregation during the anaphase and telophase stages. The Pearson correlation test was performed for meiotic characteristics among populations studied. The analysis of variance (ANOVA) followed by the Least Significant Test was performed among populations and species studied to detect significant difference in the relative chiasma frequency and distribution as well as chromosomes association (Sheidai et al. 2006). In order to group the species studied based on similarity in their meiotic characteristics, different clustering methods of UPGMA (unweighted paired group with arithmetic average) and WARD 2009 Cytology of Melica and Milium 187

Fig. 1. Representative meiotic cells in Melica and Milium species and populations studied. AMeiotic cell showing n9 in Vardavard population of M. persica subsp. persica. BMeiotic cell showing n9 and one B-chromosome (arrow) in Mazandaran population of M. ciliata var. ciliate. CMeiotic cell showing n9 in Gilan population of Milium vernale. DMeiotic cell showing n9 in Tehran population of M. persica subsp. persica. EMeiotic cell showing n9 in M. jacquemontii subsp. jacquemontii. FMeiotic cell showing n7 in Mazandaran population of Milium vernale. GMeiotic cell showing n9 in Gajereh population of M. jacquemontii subsp. hohenackeri. HMeiotic cell showing n7 in Ardebil population of M. schmidtianum. IMeiotic cell showing n9 in M. picta. JMeiotic cells showing a single quadrivalent (arrow) in Arasbaran population of M. schmidtianum.

(minimum spherical cluster method) as well as ordination based on principal coordinate analysis (PCO) were performed (Sheidai and Noormohammadi 2005). In order to avoid the chaining effect of clustering methods, the Neighbor joining method was also performed on cytogenetic data. The Euclidean and taxonomic distances were used as dissimilarity coefﬁcient in cluster analysis of cytological data (Sheidai et al. 2006). SPSS Ver. 9 (1998) and NTSYS Ver. 2.2 (1998) were used for numerical analyses. 188 M. Sheidai and K. Moghaddam Cytologia 74(2) Terminal Univalent/cell, Mazandaran and Gilan Quadrivalent, TXN Quadrivalent, Draband and Boomehen populations Ploor and Gajereh populations respec- Milium vernale Rod bivalent/cell, IN Rod bivalent/cell, Univalent, IV Univalent, hohenackeri inaequiglumis subsp. subsp. Rod bivalent, I Rod bivalent, M. persica Ring bivalent/cell, RD Ring bivalent/cell, species studied. Arasbaran population, vernal 1, 2 M. jacquemontii Milium Ring bivalent, RD Ring bivalent, M. picta and , hoh 1, 2 Melica canescens subsp. Total chiasmata, RB Total Total chiasmata/bivalent, RBN chiasmata/bivalent, Total Meiotic characteristics in M. jacquemontii Tehran and Vardavard populations respectively, ina 1, 2 populations respectively, and Vardavard Tehran , can Table 1. Ardebil and Arasbaran populations respectively. persica Intercalary chiasmata, TOX jacquemontii subsp. Mazandaran and Arasbaran populations respectively, picta Mazandaran and Arasbaran populations respectively, subsp. M. schmidtianum ciliata Intercalary chiasmata/bivalent, TOXN Intercalary chiasmata/bivalent, var. TX IX TOX RB RD I IV TXN IXN TOXN RBN RDN IN IVN Melica persica Terminal chiasmata, IX Terminal M. jacquemontii M. ciliata n Quadrivalent/cell). Sp per 1per 2ina 1 9ina 2 9jac 9can 15.33 9hoh 1 14.61hoh 2 9 0.27 15.87cilia 1 9 0.56 9 14.23cilia 2 15.63 9 0.37picta 11.29 9 15.17 1.26 17.23vernal 1 16.39 9 6.87 16.23vernal 2 0.32 15.85 6.94 15.49 7 9 0.15 16.40scmid 1 0.52 7.60 2.13 9 16.24scmid 2 11.62 0.77 7.00 1.86 7 17.38 0.29 16.88 12.92 16.42 1.40 7 0.00 0.45 5.88 16.62 17.33 1.17 0.08 8.85 16.69 8.47 0.22 0.25 0.00 9.81 0.00 16.70 8.49 3.11 0.33 16.73 0.06 0.00 8.13 0.15 0.50 13.14 16.67 0.42 0.00 8.30 1.70 0.51 17.67 0.02 0.36 0.00 1.62 0.85 0.00 6.19 7.96 0.06 10.22 1.76 0.67 0.03 9.00 17.09 0.00 0.00 1.58 0.06 0.04 0.00 0.80 1.02 0.00 6.25 0.04 0.06 6.38 1.74 0.00 0.00 1.25 0.14 1.69 0.00 1.91 0.02 0.04 1.82 0.72 1.80 0.00 0.60 0.76 0.00 1.76 0.04 1.72 0.77 1.82 0.02 0.00 0.00 0.06 0.06 0.84 1.80 0.00 0.24 0.00 0.09 1.29 0.78 0.21 0.03 1.93 1.85 1.82 1.88 0.00 0.16 0.05 0.01 0.00 1.93 1.85 0.65 0.13 0.01 1.85 0.98 0.03 0.03 0.94 1.40 0.00 1.86 2.39 0.04 0.94 0.35 0.000 0.01 0.90 0.02 1.88 1.85 0.06 0.06 0.000 0.92 0.05 1.96 0.06 0.00 0.000 0.09 0.00 0.88 0.88 0.01 1.46 0.000 0.07 2.44 1.00 0.00 0.000 0.00 0.11 0.11 0.89 0.000 0.01 0.91 0.000 0.00 0.000 0.00 0.00 0.10 0.000 0.09 0.00 0.000 0.01 0.000 0.000 0.00 0.000 0.000 0.001 populations respectively, scmid populations respectively, IVN Species name: per 1, 2 jac respectively, cilia 1, 2 tively, chiasmata/bivalent, IXN chiasmata/bivalent, (Abbreviations: TX (Abbreviations: 2009 Cytology of Melica and Milium 189

Table 2. ANOVA test for meiotic characteristics among the Melica species and varieties (meiotic characters as in Table 1).

Sum of Squares df Mean Square F Sig.

TX Between Groups 259.476 10 25.948 4.523 .001 Within Groups 1875.758 327 5.736 Total 2135.234 337 IX Between Groups 46.946 10 4.695 5.876 .001 Within Groups 261.235 327 .799 Total 308.180 337 TOX Between Groups 114.199 10 11.420 3.756 .001 Within Groups 994.310 327 3.041 Total 1108.509 337 RING Between Groups 378.362 10 37.836 1.448 .158 Within Groups 8545.937 327 26.134 Total 8924.299 337 ROD Between Groups 74.835 10 7.484 4.423 .001 Within Groups 553.320 327 1.692 Total 628.155 337 UNI Between Groups .244 10 2.442E-02 .487 .898 Within Groups 16.398 327 5.015E-02 Total 16.642 337

Results and discussion Chiasma frequency and chromosome pairing Data with regard to chromosome number, ploidy level, chiasma frequency and distribution, as well as chromosome pairing, is presented in Table 1 (Fig. 1). The Melica species and populations studied possess 2n2x18 chromosome number. Gohil and Koul (1986) reported the gametic chromosome number of n9 (2n18) for the species of M. persica, while no report is available for the subspecies of this species. Therefore the chromosome numbers of 2n18 are new for 2 subspecies of M. persica subsp. persica and M. persica subsp. inaequiglumis. The meiotic behavior of these 2 subspecies is also described for the first time. In the 4 populations of M. persica subsp. persica and M. persica subsp. inaequiglumis studied, the chromosomes formed mainly ring and rod bivalents in metaphase of meiosis-I in both subspecies while some univalents were formed in the Vardavard population of M. persica subsp. persica (0.08). The highest value of total and terminal chiasmata occurred in the Darband population of M. persica subsp. inaequiglumis (16.23 and 15.87 respectively), while the value of intercalary chiasmata occurred in the Boomehen population of M. persica subsp. inaequiglumis (1.26). Some amount of anaphase-II chromosome stickiness and late separation was observed in populations of M. persica subsp. inaequiglumis. The ANOVA test revealed a significant difference in the amount of terminal and intercalary chiasmata (p0.05), both between the two populations of M. persica subsp. persica and among the subspecies of persica and inaequiglumis, indicating partly their genetic differences. The Pearson correlation test performed for meiotic characteristics among populations of M. persica subsp. persica and M. persica subsp. inaequiglumis showed a positive significant correlation between total and terminal chiasmata (r0.95, p0.01) and also between terminal chiasmata and the number of ring bivalents (r0.69, p0.01), indicating that with an increase in the number of terminal chiasmata more bivalents are formed, which in turn leads to a better chromosome segregation and normal meiosis. The low amount of univalents observed did not reduce the pollen fertility of the species studied as all show a high pollen fertility of 0.98%. 190 M. Sheidai and K. Moghaddam Cytologia 74(2)

Fig. 2. NJ clustering of the Melica species and populations based on meiotic data (species name as in Table 1).

Fig. 3. PCO ordination of the Melica species and populations based on meiotic data (species name as in Table 1).

The populations of 3 subspecies of M. jacquemontii subsp. jacquemontii, M. jacquemontii subsp. canescens and M. jacquemontii subsp. hohenackeri, possessed 2n2x18 chromosome number, supporting the previous somatic chromosome number reports of Tzvelev (1986) for the same species. These populations showed mainly bivalent formation along with some amount of univalents. The highest value of total and terminal chiasmata occurred in the Mazandaran population of M. jacquemontii subsp. canescens (17.38 and 17.23 respectively), while the value of intercalary chiasmata occurred in Tehran population of the M. jacquemontii subsp. hohenackeri (0.77). The ANOVA test revealed a significant difference in the amount of terminal and intercalary chiasmata (p0.05), among these populations. The Pearson correlation test performed for meiotic characteristics among populations of M. jacquemontii showed a positive significant correlation between total and terminal chiasmata (r0.95, p0.01) and a significant negative correlation between these 2 characteristics and the number of rod bivalents and univalents (p0.01). 2009 Cytology of Melica and Milium 191

Two populations of M. ciliata var. ciliata possessed 2n2x18 chromosome number and showed mainly bivalents along with a low amount of univalent formation in the metaphase of meiosis-I. The ANOVA test did not show any significant difference in the chiasma frequency and distribution as well as chromosome pairing between these two populations. The correlation test showed a positive significant correlation between total and terminal chiasmata (r0.96, p0.01), between terminal chiasmata and the number of ring bivalents (r0.55, p0.01) and also between total chiasmata and the number of ring bivalents (r0.72, p0.01). The species of M. picta possessed 2n2x18 chromosome number supporting the earlier report of Tutin (1980). This species formed mainly bivalents along with a low amount of univalents in the metaphase of meiosis-I. The ANOVA test performed for meiotic characteristics among the Melica species and populations studied (Table 2) revealed a significant difference in the amount of total, terminal and intercalary chiasmata as well as amount of rod bivalents (p0.01), indicating partly their genetic differences. A significant increase in the amount of intercalary chiasmata may bring about new genetic combinations as the genes located in the middle part of chromosomes also become involved in crossing over and genetic recombination. Such genetic changes may be of use for both local adapta- tions and species diversification (Hazarika and Rees 1967, Coucoli et al. 1975) and has been reported in populations of different grass species like Aegilops, Lolium and Festuca (Rees and Dale 1974, Rees and Jones 1977). The Pearson correlation test performed for meiotic characteristics among the Melica species and populations studied showed a positive significant correlation between total and terminal chiasmata (r0.95, p0.01) on one hand and between total and terminal chiasmata with the number of ring bivalents (p0.01) on the other. A significant negative correlation was observed between these meiotic characteristics and the number of rod bivalents and univalents (p0.01). The presence of 0-1 B-chromosome was observed in the Mazandaran population of M. ciliata var. ciliata (Fig. 1, B). The Bs observed were much smaller than the A-chromosomes, round in shape and did not pair with the A-chromosomes or among themselves (Fig. 1). B-chromosomes are accessory chromosomes occurring in more than 1300 species of plants and almost 500 species of animals (Camacho et al. 2000). B-chromosome has been reported in M. cupani (Romero et al. 1985), but to our knowledge this is the first time of a report of B-chromosomes in M. ciliata. 2 populations of Milium vernale showed the presence of 2 different chromosome numbers; the Javaherdeh population possessed 2n2x14 while the Asalem population possessed 2n2x18, supporting the earlier reports (Kozuharov and Petrova 1991, Goukasian and Nazarova 1998). Ben- nett and Thomas (1991) have reported 2n8 and 2n10 for this species. Both populations studied showed a normal chromosome pairing and segregation. The Ardebil and Arasbaran populations of M. schmidtianum possessed 2n2x14 chromosome number, while Davlianidze and Mosulischvili (1984), reported 2n6x42 for this species. Therefore we are reporting a new ploidy level for M. schmidtianum. Both the Arasbaran and Arde- bil populations of M. schmidtianum showed mainly bivalent formation in metaphase of meiosis-I, while univalents and a single quadrivalent occurred only in the Arasbaran population (Table 1). Quadrivalent formation may indicate the occurrence of heterozygote translocation in this diploid species. 2 populations of M. schmidtianum varied significantly in the number of total and terminal chiasmata (p0.05) with the Arasbaran population possessing a higher values for both meiotic characteristics. The Arasbaran population of M. schmidtianum also differed significantly from populations of Milium vernale in its relative meiotic characteristics of total and terminal chiasmata per bivalent (Table 1). Grouping of the Melica species by different clustering methods based on cytogenetical characteristics are presented in Figs. 2 and 3; all producing similar results. In all analyses, M. persica 192 M. Sheidai and K. Moghaddam Cytologia 74(2) subsp. persica shows a close relationship to M. persica subsp. inaequiglumis, while M. jacquemontii subsp. canescens, M. jacquemontii subsp. hohenackeri, M. ciliata var. ciliata, and M. picta show similarity and are placed close to each other. However, M. jacquemontii subsp. jacquemontii is placed far from the other species studied. The grouping obtained based on cytogenetic characteristics mainly agrees with the results obtained from phenetic and cladistic analysis of morphological characters of the Melica species studied (unpublished data) and taxonomic treatment of the genus in Flora Iranica (Bor 1970). The only exception is that M. jacquemontii subsp. jacquemontii shows an affinity with the other subspecies of M. jacquemontii in morphological characters but in cytogenetic clustering it stands far from them. Therefore the present finding may indicate the use of cytological data in taxonomy and phylogeny of the genus Melica.

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