Karyotypic studies in eight populations of Zhumeria majdae Rech. f. & Wendelbo from Iran

Abstract

The karyotype asymmetry/symmetry of a taxon (eight populations) from different geographic sites of Zhumeria genus is presented. The ploidy level between and within populations was 4x. According to intra-chromosomal asymmetry, population Z₂ had the most symmetrical karyotype and population Z₇ had the most asymmetrical evolutionary karyotype among the populations. In terms of the Stebbins system, the karyotype of populations can be classified in 1A, 2A and 2B classes. The results of analysis of variance revealed significant differences between the populations based on nine karyotypic traits (p < 0.05 and p < 0.01). Cluster analysis showed that the populations of Zhumeria majdae have been grouped in separate clusters. The results seemed to provide enough genetic evidence to identify different populations and useful data to clarify the interspecific relationships. Detailed karyotype analysis allows us to group the different populations and to postulate relationships among them.

Keywords:

• Chromosome
• diversity
• karyotype
• principle components analysis
• Zhumeria majdae

Introduction

Zhumeria is a genus of flowering plant in the Lamiaceae family, first described in 1967. It contains just one known species, Zhumeria majdae Rech. f. & Wendelbo, endemic to Iran, known locally by the name of mohrekhosh, and it has a limited geographical range, very sharp slope of the mountains at 520–1450 m altitude in Bandar-Abbas (Hormozgan province) in the south of Iran (Rechinger and Wendelbo 1967; Rechinger 1982).

Zhumeria is unusual within the broader Salvia clade in that, in addition to the two fertile stamens, two large staminodes are easily identified in the corolla, and it is monotypic genus of Lamiaceae (Bokhari and Hedge 1976). It is a perennial fragment shrub, evergreen and without any spine.

Research by Walker and Sytsma (2007) showed that Z. majdae could be placed as sister to one of the two groups in clade III of Salvia genus. They informally recognize within the larger tribe Mentheae a lineage that would correspond to a sub tribe consisting of the genera Salvia, Dorystaechas, Meriandra, Zhumeria, Perovskia, Rosmarinus, Lepechinia and Melissa (Walker and Sytsma 2007; Will and Claßen-Bockhoff 2014).

The leaves have been used for many years as a curative for stomach aches, as an antiseptic, as a carminative especially in infants and for treatment of painful menstruation (Safa et al. 2013).

The extract of aerial parts of the plant has shown anti-nociceptive effects in mice and rats (Hosseinzadeh et al. 2002). The ethanol extract of Z. majdae has shown potent anti-leishmanial and anti-plasmodial activity in vitro (Moein et al. 2008). Antibacterial activity of the essential oil of Z. majdae has been previously reported only against two bacterial strains of S. aureus and E. coli (Soltanipoor 2007). The aim of the present work was to study mitosis of eight populations of Z. majdae in all points of distribution in Iran, trying to reveal the chromosome numbers and basic cytogenetic information.

Materials and methods

In this study, we used root tip meristems from seedlings obtained by the germination of ripe seeds collected from eight natural populations on wet filter paper in Petri dishes at 22°C. The studied populations are listed in (Table 1).

Table 1. Ecological characteristics of Zhumeria majdae localities.

Code	Localities	Height (m)	Geographical coordinates	Vegetative type	Slope side
Z1	Geno Mount	720	27° 23′ 23ʺ56° 14′ 30ʺ	Gymnocarpus decander-Convolvulus spinosus	SouthWest
Z2	Keshar Mount	850	27° 21′ 49ʺ55° 54′ 0.8ʺ	Platychaete aucheri-Gymnocarpus decander	NorthEast
Z3	Tangezagh Mount	1400	27° 55′ 24ʺ55° 57′ 55ʺ	Convolvulus spinosus-Ebenus stellata	South
Z4	Sarchahan Mount	1100	27° 56′ 59ʺ55° 56′ 31ʺ	Gymnocarpus decander–Ebenus stellata	North
Z5	Sirmand Mount	1450	27° 55′ 24ʺ55° 57′ 55ʺ	Convolvulus spinosus-Ebenus stellata	South
Z6	Finu Mount	1250	27° 55′ 24ʺ55° 57′ 55ʺ	Convolvulus spinosus-Ebenus stellata	East
Z7	Sarchahan Mount	810	27° 58′ 18ʺ55° 56′ 34ʺ	Cymbopogon olivieri–Anvillea garcinii	North
Z8	Geno Mount	520	27° 22′ 09ʺ56° 11′ 00ʺ	Euphorbia larica-Cymbopogon olivieri	SouthWest

Vouchers are deposited in RIFR gene bank (Research Institute of Forest and Rangelands in Iran). Root tip meristems obtained from seedlings were pretreated with 0.7% saturated α-bromo naphthalene at 4°C for 3.5 h, fixed in 10% formaldehyde and 1% chromic acid (1:1) for at least 15 h at room temperature, then root tips were rinsed for 2 h in distilled water. Hydrolysis was carried out with 1 N NaOH at 60°C for 10 min, dyed with hematoxylin-iron for 4 h and squashed in a droplet of 45% acetic acid and lactic acid (10:1) (Wittmann 1965; Hesamzadeh Hejazi and Rasouli 2006; Hesamzadeh Hejazi and Ziaei Nasab 2010). The preparations were observed with an optical microscope (BH2 Olympus supplemented digital color video camera) at a magnification of 2000 × . The best metaphase plates were selected and measured by Micromeasure 3.3 software (Reeves and Tear 2000). In each mitotic metaphase (at least 10 plates) the arm length of each chromosome was measured. The parameters such as long arm (LA), short arm (SA), total length (TL), relative length percentage (RL %), arm ratio (AR), centromeric index (CI), and value of relative chromatin (VRC) were estimated in each metaphase plate to characterize the karyotypes numerically (Martinoli and Ogliotti 1970; Hesamzadeh Hejazi and Ziaei Nasab 2009, 2010). Karyotype asymmetry was estimated by four different methods namely, total form percentage (TF %) (Huziwara 1962); difference of relative length (DRL); intra-chromosomal asymmetry index (A₁) and inter-chromosomal asymmetry index (A₂) (Romero Zarco 1986). Both indices (A₁ and A₂) are independent to chromosome number and size. Dispersion index (DI) (Lavania and Srivastava 1992) and also karyotypic evolution by using the symmetry classes of Stebbins (SC) (Stebbins 1971) has been determined.

The karyotype formula was determined according to classification of Levan (Levan et al. 1964). For each population, karyograms were adjusted based on length of chromosome size (arranged large to small). In order to determine the variation between populations, one-way balanced ANOVA was performed on normal data and parameter means were compared by Duncan’s test. The principal components analysis (PCA) was performed to evaluate the contribution of each karyotypic parameter to the ordination of species. Clustering was performed using Ward’s cluster analysis method after calculation of Cophenetic correlation coefficient (r) to examine karyotype similarity among populations. FCM analysis was used to reconfirm the ploidy level of various populations. To determine the standard peak of diploid cells (2C DNA) in FCM analysis, at least 10 leaves were collected from diploid salvia reuteriana (2n = 2x = 20) plants grown in the greenhouse.

Results

This study reveals a detailed picture of the chromosome features in Z. majdae. Pictures of the mitotic metaphases and karyograms of the populations are presented in Figure 1. The somatic chromosome numbers (2n), ploidy levels, ranges of chromosome length, symmetry index percentage, intra and inter-asymmetry indices, difference of range relative length, total form percentage, symmetry classes, total karyotype length and karyotype formula of the taxa and populations investigated are summarized in Table 2. The somatic chromosome number and details of the karyotypes of the studied populations, revealed that Z. majdae populations possessed one ploidy level (2n = 4x = 40).

Figure 1. Mitotic metaphase of Zhumeria majdae populations accompanied by karyograms (Z₁–Z₈).

Figure 2. Scatter plot of eight populations for the first two principal components.

Table 2. Somatic chromosome number (2n), ploidy levels, ranges of chromosome length, arm ratio (AR), Romero Zarco asymmetry indices (A₁, A₂), dispersion index (DI), difference of range relative length (DRL), total form percentage (TF%), Stebbins symmetry classes (SC), total karyotype length (TKL) and karyotype formula (KF) (m: metacentric, sm: submetacentric).

Population	2n	Ploidy level	Chromosome length range	AR	A1	A2	DI	DRL	TF%	SC	TKL (μm)	KF
Z1	40	4x	1.58–3.58	1.55	0.31	0.21	8.07	3.49	39.77	2A	46.95	16 m+4sm
Z2	40	4x	1.85–3.70	1.33	0.22	0.146	6.70	2.60	43.21	1A	53.49	20 m
Z3	40	4x	1.58–3.58	1.46	0.29	0.155	6.42	2.98	41.12	1A	53.91	18 m+2sm
Z4	40	4x	2.07–4.05	1.63	0.33	0.136	5.05	2.77	39.36	2A	58.45	17 m+3sm
Z5	40	4x	1.82–3.82	1.58	0.32	0.137	5.69	2.64	39.45	2A	52.61	16 m+4sm
Z6	40	4x	1.76–3.88	1.46	0.28	0.21	8.38	3.47	41.10	1A	51.40	18 m+2sm
Z7	40	4x	1.70–3.70	1.81	0.39	0.171	6.73	3.14	36.72	2A	54.74	10 m+10sm
Z8	40	4x	1.32–3.70	1.69	0.36	0.197	7.61	3.59	37.56	2B	49.76	12 m+8sm

Total karyotype length, roughly indicative of the DNA content, ranges from 46.95 to 58.45 μm in all populations. Also size of the chromosomes among the populations varied from 1.32 μm in population Z₈ to 4.05 μm in population Z₄. Most of the chromosomes were mainly “m” type chromosomes (centromeres at median region). Another type of chromosome was “sm” type chromosomes (centromeres near median region).

Among the studied populations, the highest TF% value (43.21) was estimated in the Z₂ population and the lowest TF% value (36.72) was estimated in the Z₇ population, the lowest of TF% value is one of the main reasons to make karyotype asymmetric in Z₇ population. In view of the fact that lower DRL value indicated more karyotype symmetry, the Z₂ and Z₈ populations, with DRL 2.60 and 3.59 values, respectively, had the most symmetric and asymmetric karyotypes.

Intrachromosomal asymmetry index (A₁) showed sharp differences between the chromosome arms in the different populations. In general, based on intrachromosomal asymmetry (A₁ and TF %), population Z₇ had the most asymmetric karyotype and population Z₂ had the most symmetrical karyotype in all of the populations.

In terms of the Stebbins system, the karyotype of populations seizes 1A, 2A and 2B classes which are considered mainly primitive classes in this system. In this study, Z. majdae (Z₆) had a higher DI value, which is associated with an enhanced order of karyotypic specialization. Z. majdae (Z₆) had the highest A₂ values; therefore its karyotype was more asymmetric than the other populations in the same symmetry classes (Tables 2 and 4).

To analyze the variability of the karyotypes among populations, length of chromosome, long and short arms of chromosome, arm ratio values, difference of range relative length, total form percentage and asymmetry indexes (A₁, A₂) were compared by one-way analysis of variance based on completely randomized design (CRD). Also, the Duncan test was carried out to test differences between each pair of means. The results of variance analysis revealed significant differences between the populations based on seven karyotypic characteristics (p < 0.05 and p < 0.01) (Table 3). The Duncan test applied to the chromosome morphometric traits showed a highly significant difference among all examined populations (Table 4). So, mean values of chromosome total length varied from 2.35 μm in populations Z₁ to 2.92 μm in populations Z₄. The mean values of chromosome long arms varied from 1.52 μm in populations Z_2, and Z₆ to 2.27 μm in population Z₁. Also the mean values of chromosome short arms was different from 0.93 μm in populations Z₁ and Z₈ to 1.15 μm in populations Z₂ and Z₄.

Table 3. The results of analysis of variance for karyotypic data based on CRD design.

	Mean of squares
SOV	DF	TL	LA	SA	AR	CI	LA%	SA%	DRL	VRC	TF%	A1	A2	DI
Population	7	0.089	0.023	0.022 **	0.067 *	0.0012	0.032 *	0.032 *	0.040	0.088	12.812*	0.008*	0.003 **	0.147
Error	16	0.040	0.013	0.005	0.019	0.0006	0.011	0.011	0.052	0.040	4.237	0.0028	0.0006	0.064
%C.V.		7.56	8.77	6.73	8.82	6.18	3.42	5.18	12.48	7.56	5.17	13.26	14.53	9.71

**Significant at 1% level of probability; *significant at 5% level of probability.

Notes: Df: degree of freedom, TL: total length of chromosome, LA: long arm, SA: short arm, AR: arm ratio, CI: centromeric index, LA%: long arm percentage, SA%: short arm percentage, DRL: difference of relative length, VRC: value of relative chromatin,TF%: total form percentage, A1: intrachromosome asymmetry index, A2: interchromosome asymmetry index, DI: dispersion index.

Table 4. Mean of parameters of chromosomes analysis of Zhumeria majdae populations.

Population	TL	LA	SA	AR	CI	LA%	SA%	TF%	DRL	VRC	A1	A2	DI
Z1	2.35 c	2.27 a	0.93 c	1.55 abc	0.401 ab	3.01 abc	1.99 abc	39.77 abc	3.49 a	2.35 c	0.31 abc	0.21 a	8.07 ab
Z2	2.67 abc	1.52 b	1.15 a	1.33 c	0.432 a	2.85 c	2.15 a	43.21 a	2.60 a	2.67 abc	0.22 c	0.14 6c	6.70 abc
Z3	2.69 abc	1.59 b	1.11 ab	1.46 bc	0.412 ab	2.95 bc	2.05 ab	41.12 ab	2.98 a	2.69 abc	0.29 bc	0.155 bc	6.42 abc
Z4	2.92 a	1.77 ab	1.15 a	1.63 ab	0.413 ab	3.03 abc	1.97 abc	39.36 abc	2.77 a	2.92 a	0.33 ab	0.136 c	5.05 c
Z5	2.63 abc	1.59 b	1.04 abc	1.58 abc	0.395 ab	3.03 abc	1.97 abc	39.45 abc	2.64 a	2.63 abc	0.32 ab	0.137c	5.69 bc
Z6	2.58 abc	1.52 b	1.06 abc	1.46 bc	0.413 ab	2.95 bc	2.05 ab	41.10 ab	3.47 a	2.58 abc	0.28 bc	0.21 a	8.38 a
Z7	2.74 ab	1.73 ab	1.004 bc	1.81 a	0.369 b	3.16 a	1.83 c	36.72 c	3.14 a	2.74 ab	0.39 a	0.171abc	6.72 abc
Z8	2.49 bc	1.55 b	0.93 c	1.69 ab	0.383 b	3.12 ab	1.87 bc	37.56 bc	3.59 a	2.49 bc	0.36 ab	0.197 ab	7.61ab

Notes: TL: total length of chromosome, LA: long arm, SA: short arm, AR: arm ratio, CI: centromeric index, LA%: long arm percentage, SA%: short arm percentage, TF%: total form percentage, DRL: difference of relative length, VRC: value of relative chromatin, A₁: intrachromosome asymmetry index, A₂: interchromosome asymmetry index, DI: dispersion index.

Using principal components analysis (PCA), the first two independent components accounted for about 84.55% of total variation. The first component emphasized short arm, arm ratio, total form percentage, intrachromosome asymmetry index, long arm and short arm percentage value which had the highest coefficients of Eigen vectors and were important characters for classification of populations with about 59.4% of total variation. Interchromosome asymmetry index was important trait in the second component (25.15%) (Table 5).

Table 5. Eigenvectors from the first two principal components for seven karyotype parameters to classify eight populations of Zhumeria majdae.

Parameters	First component	Second component
SA	− 0.33177	0.32032
AR	0.38151	0.23097
LA%	0.39115	0.18718
SA%	−0.39115	−0.18718
TF%	− 0.39115	−0.18718
A1	0.38538	0.20230
A2	0.19214	−0.40611
Eigen value	5.93	2.5156
Percentage of variance	59.39	25.1558
Cum percentage of variance	59.39	84.5465

Note: The bold values are significance in each component.

Grouping of studied populations was based on their karyotypic traits (Figure 3). The results showed that populations of Z. majdae have been grouped in separate cluster.

Figure 3. Dendrogram of eight populations of Zhumeria majdae by analyzing seven karyotypic parameters using Ward’s cluster analysis method. Cophenetic correlation r = 0.88.

By cutting the dendrogram resulting from cluster analysis by Ward’s method with cophenetic correlation coefficient (r = 0.88) with a metric distance of 3.18, the populations were classified into four groups. The highest metric distance (5.67) was obtained between Z₁ and Z₂, which implies the least affinity between them. The lowest metric distance (1.59) was obtained between Z₃ and Z₅, which implies the least karyotypic difference between them (Figure 3). The diagram of population dispersion, based on the first two components, showed that the populations separated in four groups, which completely fits with results obtained through the grouping analysis by Ward’s method (Figure 2).

Population of Z₂ is classified as a separate group. Two populations of Z₇ and Z₈, two populations of Z₁ and Z₆ and three populations of Z₃, Z₄, Z₅ are grouped together in the same cluster.

The population of Z₂ had the lowest value of A₁ with 0.22 and the highest TF% with 43.21%. The populations of Z₇ and Z₈ had the highest values of A₁ (0.28–0.31) and the lowest values of TF% (36.72–37.56). The populations of Z₁ and Z₆ were grouped together in the same cluster that had the highest value of A₂, with 0.21. The populations of Z₃, Z₄ and Z₅ were grouped together in the same cluster, although some of the factors were statistically significant (Table 4). The ranges of A₁, A₂ and TF% values were 0.29–0.33, 0.136–0.155 and 39.36–41.12 respectively (Table 4 and Figure 2).

Discussion

In this study, chromosome numbers and detailed measurements of eight populations of the Zhumeria genus were determined for the first time in Iran. The karyotype concept has been used in characterizing and distinguishing chromosomes of different populations. Mitotic karyotype analyses are also helpful in studying evolutionary problems. Bokhari and Hedge (1976) reported that one population of Z. majdae located in Hormozgan province in Iran has 2n = 40 or 42. Our study indicated that the chromosome numbers in different populations of Hormozgan and Fars province are tetraploid (2n = 4x = 40). To reconfirm the ploidy level, flow cytometric analysis was performed using a PA-1 (Partec, GmbH/CyFlow space-Munster) flow cytometer. All the studied populations fitted a 4x ploidy level in compare with salvia reuteriana as a standard plant (2n = 2x = 20). By using A₁ and A₂ parameters, we can determine the more asymmetric karyotype among the populations which have the similar Stebbins classes of symmetry. The populations which are classified as 1A group also showed the lowest value of A₁ in the range of 0.22–0.29 and the highest value of TF%, ranging from 41.10 to 43.21. In order to refine the measure of karyotype asymmetry we used the DI, which has the potential to decipher even minor karyotypic variations. The DI index plays an important role in arranging the species within the same class of karyotype asymmetry in an advancing order of specialization by permitting further gradations, as depicted by species arrangement within sections. The results of variance analysis between the populations revealed significant differences based on karyotypic characteristics. This result indicated the occurrence of quantitative changes in chromosome size of the studied populations. A significant effect of chromosomal traits proved karyotypic variation between populations. This shows the importance of chromosome study to distinguish the state of evolution and affinity between different populations.

This study indicates that populations of a specific species in within itself will show variety. The important factors in separation of populations were inter and intra chromosome asymmetry factors (A1, A2 and TF% values). Thus, these studies could greatly help us in classification and taxonomic studies. The present study shows the change in chromosomal traits as one of the mechanism of inter and intra-species diversification in the Zhumeria genus, as well as the earlier cytological reports (Hesamzadeh Hejazi and Ziaei Nasab 2009; Javadi et al. 2009; Hesamzadeh Hejazi and Ziaei Nasab 2010; Hesamzadeh Hejazi 2011; Kalvandi et al. 2012; Ziaei Nasab et al. 2012; Salehi et al. 2014). In this research we found that there are differences in position of centromere and total length of chromosomes, while there are no morphological differences among populations in Zhumeria genus. So, karyotype study may show variations which do not appear in the morphology of the plant.

Investigation of different Zhumeria populations showed that they grow in locations with varying climate, height, soil, slope, vegetative type and geographical characteristics. For this reason some of the populations are separated or are grouped together.

The essential oils from eight populations studied in this paper also have been chemically investigated. The results showed that there are about 25 different volatile components in total. The amount of components varied between populations (data not shown). So it seems the different components of essential oils in different populations of Z. majdae are variable due to hybridization and geographical location.

The results of the molecular markers used among the eight populations also showed a high diversity as well as the amounts of components of essential oils (data not shown).

The variation of climate and soil in Iran provides a suitable field for plant variations. One of the clearly detectable genetic variations in Z. majdae is the structural changes of chromosomes. These genomic differences could be used for breeding purposes.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the biotechnology department of Research Institute of Forests and Rangelands (RIFR), Iran.

Acknowledgements

The authors are grateful to the Hormozgan Agricultural and Natural Resources Research Center for providing seedlings and seeds.