Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 163-178, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-186 Citation: K. Saha, R.K. Sinha, S. Sin- ha (2020) Karyological studies in thir- teen species of Zingiberacaeae from Tripura, North East India. Caryologia 73(1): 163-178. doi: 10.13128/caryolo- gia-186 Received: March 11, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 K. Saha, R. Kumar Sinha, S. Sinha. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha Cytogenetics and Plant Biotechnology Laboratory, Department of Botany, Tripura Univer- sity, Suryamaninagar-799022, Tripura, India *Corresponding author. E-mail: saha.kshn@gmail.com Abstract. Tripura being a state in North East India belongs to Indo-Burma bio-diver- sity hotspot and is considered as a centre of origin of many species of Zingiberaceae. Alpinia calcarata, Alpinia malaccensis, Alpinia nigra, Amomum aromaticum, Amomum koenigii, Amomum maximum, Curcuma amada, Curcuma caesia, Curcuma longa, Cur- cuma picta, Hedychium coccineum, Hedychium coronarium and Hedychium thyrsiforme are found in wild state in different geographical locations of Tripura. Their karyotypes were analyzed both at interspecific and intraspecific levels. The somatic chromosome number of Alpinia spp. and Amomum spp. was found to be 2n = 4X = 48. The Cur- cuma spp. represented by C. amada had 2n = 42 chromosomes and three other species viz., C. caesia, C. longa and C. picta had 2n = 63 chromosomes having X=21, indicating that polyploidy is a common feature in this genus. The somatic chromosome number of Hedychium spp. was found to be 2n = 34 chromosomes having a basic no. X = 17. Chromosomal data based clustering pattern and their sub-grouping at intra-specific level validates the taxonomic status of these species. Gower’s similarity matrix is an indicator of cryptic changes leading genus specific karyotype conservatism. Keywords. Indo-Burma bio-diversity hotspot, Zingiberaceae, Karyotype, Polyploidy, Cryptic changes. INTRODUCTION The State of Tripura (20°56’-24°34’ North latitude and 91°10’-92°21’ East longitude) is situated in the Sub-Himalayan region of North East India and belongs to Indo-Burma biodiversity hotspot of the world (Myers et al. 2000; Mao et al. 2009). The diverse vegetation of this region includes a good num- ber of plants, which are endemic either to the state or to the North-Eastern region of India (Deb 1981). In India, the Zingiberaceae family includes 22 genera and about 200 species which are distributed in Andaman and Nicobar Island, Western Ghats and Eastern Himalaya region including North-Eastern India (Jain and Prakash 1995; Sabu 2006). Many natural useful products such as food, dyes, medicines, spices and condiments are obtained from the mem- bers of this family (Nayak 2002; Arambewela et al. 2004; Sahoo et al. 2014; Rahman and Islam 2015). The local people of South Asian countries also use 164 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha several plant products of Zingiberaceae in traditional medicines (Jayaweera 1982; Prakash and Mehrotra 1996; Gupta et al. 1999; Kala 2005; Bhuiyan et al. 2010; Roy et al. 2012; Sarangthem et al. 2013; Rajkumari and Sana- tombi 2018). Some species of Zingiberaceae are also high- ly valued as ornamental plants because of their showy scented flowers (Jatoi et al. 2007; Gao et al. 2008). Deb (1983) recorded nine genera having 24 different species of this family from this phyto-geographical region. Howev- er, Alpinia galanga, Hedychium ellipticum and Hedychium marginatum could not be found in specific localities as was earlier reported by Deb (1983). Probably, these spe- cies have eventually perished due to anthropological dis- turbances. In-spite of such habitat destruction, five more species viz., Curcuma caesia, Curcuma picta, Hedychium coccineum, Amomum aromaticum (Syn. A. jainii S. Tri- pati & Vedprakash), Amomum koenigii (Syn. Amomum corynostachyum. Wall.) and Amomum maximum are found in different locations which were not reported by Deb (1983). Thus, 13 different species belonging to four genera viz., Alpinia, Amomum, Curcuma and Hedychi- um of Zingiberaceae are now available in Tripura. These are Alpinia calcarata (Haw.) Roscoe, Alpinia malaccensis (Burm.f.) Roscoe, Alpinia nigra (Gaertn.) Burtt, Amo- mum aromaticum Roxb. (Syn. Amomum jainii S. Tri- pati and Vedprakash), Amomum koenigii J.F.Gmel. (Syn. Amomum corynostachyum Wall.), Amomum maximum Roxb., Curcuma amada Roxb., Curcuma caesia Roxb., Curcuma longa L., Curcuma picta Roxb. ex Skornick, Hedychium coccineum Buch.-Ham. ex Sm., Hedychium coronarium J. Koenig. and Hedychium thyrsiforme Sm. Morphologically these species are quite distinct in their rhizome, floral and fruit characters (Deb 1983; Vanch- hawng and Lalramnghinglova 2016). Delineation of plant species based on morphological characters alone is not adequate (Larsen and Smith 1978) and so, for char- acterization of species at inter- and intra-specific levels cytological tools are now effectively used to understand the taxonomic relationship and evolutionary patterns between and within species (Yoshikane and Naohiro 1991; Joseph et al. 1999). Previously, cytological works on Zingiberaceae were mainly focussed on Curcuma spp. and various researchers from time to time (Raghavan and Venkatasubban 1943; Chakraborti 1948; Sato 1948; Sharma and Bhattacharya 1959; Sato 1960; Ramachan- dran 1961,1969; Fedorov 1969; Prana 1977; Prana et al. 1978; Nambiar 1979; Mandi 1990; Ardiyani 2002; Skor- nickova et al. 2007; Bhadra and Bandhyopadhyay 2015; Lamo and Rao 2017; Bhadra et al. 2018) reported the existence of different ploidy levels, ranging from diploid (2n = 42) to tetraploid (2n=84), having a basic number X=21. In addition, aneuploid cytotypes in Curcuma spp. had also been reported (Das et al. 1999, Sugiura (1931, 1936), Sato 1960). Cytological studies previously carried out in Alpinia spp. and Amomum spp. were mainly con- centrated on the determination of somatic chromosome numbers having 2n = 4X= 48 chromosomes (Raghavan and Venkatasubban 1943; Chakravorti 1948; Sharma and Bhattacharya 1959; Ramachandran 1969). But, from the literature it is evident that the detailed karyotype analy- sis in different species of Alpinia and Amomum was very meagre (Chen et al. 1988; Joseph 1998). In Hedychium spp. the occurrence of different ploidy levels (2n=34, 51 and 68) was reported in various cytological studies (Sharma and Bhattacharyya 1959; Bhattacharyya 1968; Ramachandran 1969; Mukherjee 1970; Mahanty 1970; Khoshoo 1979; Gao et al. 2008). Tripura being a part of Indo- Burma hotspot is considered as a centre of origin of many Indian species of Zingiberaceae but till date, cytological investigation of these species have not been carried out from this region. The present study is the first attempt to assess the karyotypic relationship in dif- ferent species of Zingiberaceae at inter- and intra- specif- ic level from the sub – Himalayan region of Tripura and for providing additional information to the chromosomal database of Zingiberean plants of Indian origin. MATERIALS AND METHODS Plant materials We examined two populations (described as Pop-I and Pop-II) of 13 different wild species of Zingiberaceae viz., Alpinia calcarata (Haw.) Roscoe, Alpinia malaccensis (Burm.f.) Roscoe, Alpinia nigra (Gaertn.) Burtt, Amomum aromaticum Roxb. (Syn. Amomum jainii S. Tripati and Vedprakash), Amomum koenigii J. F. Gmel. (Syn. Amo- mum corynostachyum Wall.), Amomum maximum Roxb., Curcuma amada Roxb., Curcuma caesia Roxb., Curcuma longa L., Curcuma picta Roxb. ex Skornick, Hedychium coronarium J. Koenig., Hedychium coccineum Buch.-Ham. ex Sm. and Hedychium thyrsiforme Sm. growing in differ- ent locations in Tripura. They were grown in the experi- mental garden, Department of Botany, Tripura University for future research. All the plant species were indepen- dently authenticated by the taxonomy experts. Herbarium of each species has been submitted in Tripura University Herbarium with respective voucher numbers (Table S1). Preparation of somatic chromosomes The somatic chromosome preparation was car- ried out with modified aceto-orcein staining technique 165Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India (Sharma and Sharma 1980). Young healthy root tips of each species were pre-treated in a mixture of saturated solution of para dichloro-benzene (p-DB) and 0.002M 8- hydroxyquinoline (1:1) at 12-150C for 6 hrs. The root tips were then washed with distilled water and kept in acidulated alcohol (mixture of 1NHCl and absolute ethyl alcohol in 1:1 ratio) for 1 hour. Thereafter, root tips were kept in 45% acetic acid for 20 minutes. After a thorough wash with distilled water, root tips were treated with 5% cellulase (Sigma Cat. No. 22178) and 5% pectinase (Sig- ma Cat. No. 17389) mixture (1:1) in citrate buffer (pH - 4.8) for 3 hours at 370C. Enzyme treated root tips were then washed with double distilled water and stained with 2% aceto-orcein : 1NHCl (9:1) mixture for over- night and finally squashed in 45% acetic acid. The well spread metaphase plate was captured using Zeiss make AXIO (Lab.A1) Microscope and Zen software was used for determining the length of short and long arm of individual chromosome of the species studied. Study of nucleoli in somatic cells Nucleolar staining was carried out following the technique of Fernandez-Gomez et al. (1969). Initially root tips were fixed in 10% Formol :1% Hydroquinone (1:1) solution for 2 hrs. These were thoroughly washed in distilled water and immersed in 2% silver nitrate solu- tion at 60ºC in dark for overnight. AgNO3 treated root tips were again kept in Formol-Hydroquinone (1:1) solu- tion for 1 hour and finally squashed in 45% acetic acid. Data analysis In preparing the numerical data of karyotype, three well spread metaphase plates of each species were com- pared. In cases, where the length and the arm ratio varied the mean was taken to calculate the value of centromeric Index (F%). The centromeric Index, TF%, Inter-chromosomal asymmetry index (A2), Coefficient of variation of chromosome length (CVCL), and Mean Cen- tromeric Asymmetry (MCA) were calculated by the fol- lowing formulae:Centromeric Index (F%) = S/(L+S)×100 (Levan et al. 1964);TF% = (∑S/∑CL) × 100 (Huziwara 1962);Inter-chromosomal asymmetry index (A2) = sCL/ XCL (Zarco 1986); Degree of karyotype asymmetry (A) = [∑ (L-S)/(L+S)]/n (Watanabe et al. 1999); Coefficient of variation of chromosome length (CVCL) = A2×100 (Paszko 2006); Mean Centromeric Asymmetry (MCA) = A×100 (Peruzzi and Eroglu 2013); (S = Length of short arm, L = Length of long arm, CL = Chromosome length, sCL- Standard deviation of chromosome length, XCL- Mean of chromosome length, n – haploid number of chromosome complement). Along with Stebbins asymmetry indices, the inter- and intra-chromosomal asymmetry indices were meas- ured statistically (Zarco 1986; Watanabe et al. 1999) and the relationship among the species was explained by means of bi-dimensional scattered plot (Peruzzi and Erog- lu 2013).To determine the karyological relationship among taxa UPGMA mediated dendrogram was constructed using the software Past 3.03 (Hammer 2013). In addition, multivariate Principal Co-ordinate Analysis (PCA) was also performed using different parameters of numerical data of karyotypes (Table 1) of the respective species and their populations (Peruzzi and Altinordu 2014). RESULTS Due to difficulty in obtaining suitable metaphase chromosome spreads with the conventional aceto-orcein staining technique, a new protocol has been developed to determine the somatic chromosome number and to analyze the detailed karyotype of all the taxa studied. The somatic chromosomes of Alpinia spp., Amomum spp., Curcuma spp. and Hedychium spp. could be classi- fied into three distinct morphological types. Type A: Short chromosomes (1.33 µm – 2.66 µm) bear two constrictions, primary and secondary, one is nearly median (m) to sub-median (sm) and the other is terminal (t) in position. Type B: Chromosomes are short in size and their length range from 0.96 µm – 2.79 µm. The position of centromere is sub-median (sm) to median (M) (F% >33.33%). Type C: Chromosomes are short in size and their length range from 1.86 µm – 2.13µm. The position of centromere is sub-median (sm) (F% <33.33%). Analysis of karyotypes Except in Alpinia nigra, which had two pairs of C type of submetacentric chromosomes, karyograms (Fig- ure S1) of rest of the 12 species showed the presence of different combinations of A and B types chromosomes as classified in the present study. The detailed analyses of the karyotypes of Pop-I and Pop-II of Alpinia spp., Amo- mum spp., Curcuma spp. and Hedychium spp. reveal the following data: Alpinia calcarata-Pop-I: Somatic chromosome number 2n=48 (Figure 1a); Number of chromosomes bearing secondary constric- 166 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha tion - 2; Range of chromosome length – (1.06 µm – 2.79 µm); Total chromosome length – 90.43 µm; Ratio of largest and smallest chromosome – 2.63:1; Mean arm ratio (L/S) –1.38; Karyotype formula - A2 (2m) B46 (6M +30m+10sm); Stebbins category – 1B; TF% - 42.61; Coeffi- cient of variation of chromosome length (CVCL) – 28.44; Mean centromeric asymmetry (MCA) – 30.00. Alpinia calcarata-Pop-II: Somatic chromosome count 2n=48 (Figure 1b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 2.79 µm); Total chromosome length – 91.20 µm; Ratio of largest and smallest chromosome – 2.63:1; Mean arm ratio (L/S) –1.39; Karyotype formula - A2 (2m) B46 (6M+34m+6sm) Stebbins category – 1B; TF% – 42.44; CVCL – 27.71; MCA – 30.23. Alpinia malaccensis-Pop-I: Somatic chromosome count 2n=48 (Figure 2a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.16 µm – 2.14 µm); Total chromosome length – 82.10 µm; Ratio of largest and smallest chromosome – 1.84:1; Mean arm ratio (L/S) – 1.33; Karyotype formula - A2 (2m) B46 (36m+10sm); Stebbins category – 1A; TF% – 43.57; CVCL – 18.17; MCA – 26.00. Alpinia malaccensis-Pop-II: Somatic chromosome number 2n=48 (Figure 2b); Number of chromosomes bearing secondary constriction - 2; Range of chromosome length – (1.16 µm - 2.26 µm); Total chromosome length – 85.74µm; Ratio of largest and smallest chromosome – 1.95:1; Mean arm ratio (L/S) –1.33; Karyotype formula - A2 (2m) B46 (4M+36m+6sm); Stebbins category – 1A; TF% – 43.65; CVCL – 17.79; MCA – 25.41. Table 1. Karyological parameters for cluster analysis and Gower’s similarity coefficient matrix. Name of the species and Population SCN HCL (µm) BCN TF% MCA CVCL Alpinia calcarata, Pop-I 48 45.22 12 42.61 30.00 28.44 Alpinia calcarata, Pop-II 48 45.60 12 42.44 30.23 27.71 Alpinia malaccensis, Pop-I 48 41.05 12 43.57 26.00 18.17 Alpinia malaccensis, Pop-II 48 42.87 12 43.65 25.41 17.79 Alpinia nigra, Pop-I 48 44.06 12 40.29 38.83 23.00 Alpinia nigra, Pop-II 48 44.14 12 40.27 38.91 22.45 Amomum maximum, Pop-I 48 33.06 12 44.75 20.76 18.61 Amomum maximum, Pop-II 48 32.93 12 44.79 20.84 17.82 Amomum aromaticum, Pop-I 48 30.61 12 45.39 18.45 21.75 Amomum aromaticum, Pop-II 48 30.57 12 45.52 17.94 20.93 Amomum koenigii, Pop-I 48 39.50 12 44.56 23.67 26.46 Amomum koenigii, Pop-II 48 39.32 12 44.28 22.87 26.48 Hedychium coronarium, Pop-I 34 20.72 17 47.32 10.72 15.91 Hedychium coronarium, Pop-II 34 20.78 17 47.24 11.06 15.11 Hedychium coccineum, Pop-I 34 26.40 17 46.68 13.27 15.51 Hedychium coccineum, Pop-II 34 26.46 17 46.47 14.11 15.40 Hedychium thyrsiforme, Pop-I 34 19.13 17 46.60 13.56 15.24 Hedychium thyrsiforme, Pop-II 34 19.70 17 46.61 13.69 14.88 Curcuma amada, Pop-I 42 31.05 21 47.02 11.91 12.52 Curcuma amada, Pop-II 42 32.35 21 46.96 12.17 12.77 Curcuma caesia, Pop-I 63 34.29 21 47.98 12.05 12.08 Curcuma caesia, Pop-II 63 34.37 21 48.11 11.35 12.08 Curcuma picta, Pop-I 63 44.67 21 47.96 12.23 13.16 Curcuma picta, Pop-II 63 44.43 21 47.94 12.34 12.66 Curcuma longa, Pop-I 63 44.16 21 47.01 16.78 11.18 Curcuma longa, Pop-II 63 44.04 21 47.13 17.23 11.01 SCN – Somatic chromosome number; HCL-Total chromosome length of the haploid complement; BCN- Basic chromosome number; TF% - Total Form percentage; MCA - Mean Centromeric Asymmetry; CVCL - Coefficient of variation of chromosome length. 167Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Figures. 1a-13b. Mitotic metaphase chromosomes of thirteen Zingiberaceae species from Tripura. 1- Alpinia calcarata; 2- A. malaccensis; 3- A. nigra; 4- Amomum maximum; 5- A. aromaticum; 6- A. koenigii; 7- Curcuma amada; 8- C. caesia; 9- C. picta; 10- C. longa; 11- H. coro- narium; 12- H. coccineum; 13- H. thyrsiforme (a – Pop - I; b – Pop - II); scale bars = 5µm. 168 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha Alpinia nigra-Pop-I: Somatic chromosome number 2n=48 (Figure 3a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.09 µm – 2.80 µm); Total chromosome length – 88.12 µm; Ratio of largest and smallest chromosomes – 2.57:1; Mean arm ratio (L/S) – 1.57; Karyotype formula – A2 (2sm) B42 (4M+26m+12sm) C4 (4sm); Stebbins category – 2B; TF% – 40.29; CVCL – 23.00; MCA – 38.83. Alpinia nigra-Pop-II: Somatic chromosome number 2n=48 (Figure 3b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.09 µm – 2.72 µm); Total chromosome length – 88.28 µm; Ratio of largest and smallest chromosome – 2.50:1; Mean arm ratio (L/S) – 1.57; Karyotype formula - A2 (2sm) B42 (4 M+26 m+ 12 sm) C4 (4 sm); Stebbins category – 2B; TF% – 40.27; CVCL – 22.45; MCA – 38.91. Amomum maximum-Pop-I: Somatic chromosome number 2n=48 (Figure 4a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 1.94 µm); Total chromosome length – 66.12 µm; Ratio of largest and smallest chromosome – 1.83:1; Mean arm ratio (L/S) –1.27; Karyotype formula - A2 (2m) B 46(14M+26m+6sm); Stebbins category – 1A; TF% – 44.75; CVCL – 18.61; MCA – 20.76. Amomum maximum-Pop-II: Somatic chromosome number 2n=48 (Figure 4b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 1.94 µm); Total chromosome length – 65.86 µm; Ratio of largest and smallest chromosome – 1.83:1; Mean arm ratio (L/S) –1.26; Karyotype formula - A2 (2m) B 46(16M+24m+6sm); Stebbins category – 1A; TF% – 44.79; CVCL – 17.82; MCA – 20.84.̀ Amomum aromaticum-Pop-I: Somatic chromosome number 2n=48 (Figure 5a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 2.10 µm); Total chromosome length – 61.22 µm; Ratio of largest and smallest chromosome – 1.98:1; Mean arm ratio (L/S) – 1.22; Karyotype formula - A2 (2m) B 46 (16M+30m); Stebbins category – 1A; TF% – 45.39; CVCL – 21.75; MCA – 18.45. Amomum aromaticum-Pop-II: Somatic chromosome number 2n=48 (Figure 5b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 2.10 µm); Total chromosome length – 61.14 µm; Ratio of larg- est and smallest chromosome – 1.98:1; Mean arm ratio (L/S) –1.21; Karyotype formula - A2 (2m) B 46(16M+30m); Steb- bins category – 1A; TF% – 45.52; CVCL – 20.93; MCA – 17.94. Amomum koenigii-Pop-I: Somatic chromosome number 2n=48 (Figure 6a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 2.39 µm); Total chromosome length – 79.00 µm; Ratio of largest and smallest chromosome – 2.25:1; Mean arm ratio (L/S) – 1.27; Karyotype formula - A2 (2m) B 46(12M+28m+6sm); Stebbins categorization – 1B; TF% – 44.56; CVCL – 26.46; MCA – 23.67. Amomum koenigii-Pop-II: Somatic chromosome number 2n=48 (Figure 6b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 2.39 µm); Total chromosome length – 78.64 µm; Ratio of largest and smallest chromosome – 2.25:1; Mean arm ratio (L/S) –1.28; Karyotype formula - A2 (2m) B 46(8M+32m+6sm); Stebbins category – 1B; TF% – 44.28; CVCL – 26.48; MCA – 22.87. Curcuma amada-Pop-I: Somatic chromosome number 2n=42 (Figure 7a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.35 µm – 1.91 µm); Total chromosome length – 62.10 µm; Ratio of largest and smallest chromosome – 1.41:1; Mean arm ratio (L/S) –1.13; Karyotype formula - A2 (2m) B 40 (10M+30m); Stebbins category – 1A; TF% – 47.02; CVCL – 12.52; MCA – 11.91. Curcuma amada-Pop-II: Somatic chromosome number 2n=42 (Figure 7b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.37 µm – 1.91 µm); Total chromosome length – 64.70 µm; Ratio of largest and smallest chromosome – 1.39:1; Mean arm ratio (L/S) – 1.14; Karyotype formula - A2 (2m) B 40(10M+30m); Stebbins category – 1A; TF% – 46.96; CVCL – 12.77; MCA – 12.17. Curcuma caesia-Pop-I: Somatic chromosome number 2n=63 (Figure 8a); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (0.96 µm – 169Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India 1.33 µm); Total chromosome length – 68.58 µm; Ratio of largest and smallest chromosome – 1.39:1; Mean arm ratio (L/S) –1.09; Karyotype formula – A3 (3m) B 60(30M+30m); Stebbins category – 1A; TF% – 47.98; CVCL – 12.08; MCA – 12.05. Curcuma caesia-Pop-II: Somatic chromosome number 2n=63 (Figure 8b); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (0.96 µm – 1.33 µm); Total chromosome length – 68.73 µm; Ratio of largest and smallest chromosome – 1.39:1; Mean arm ratio (L/S) – 1.08; Karyotype formula – A3 (3m) B 60(33M+27m); Stebbins category – 1A; TF% – 48.11; CVCL – 12.08; MCA – 11.35. Curcuma picta-Pop-I: Somatic chromosome number 2n=63 (Figure 9a); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (1.10µm – 1.67 µm); Total chromosome length – 89.34 µm; Ratio of largest and smallest chromosome – 1.52:1; Mean arm ratio (L/S) – 1.10; Karyotype formula – A3 (3m) B60 (42M+18m); Stebbins category – 1A; TF% – 47.96; CVCL – 13.16; MCA – 12.23. Curcuma picta-Pop-II: Somatic chromosome number 2n=63 (Figure 9b); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (1.10 µm – 1.63 µm); Total chromosome length – 88.86 µm; Ratio of largest and smallest chromosome – 1.48:1; Mean arm ratio (L/S) – 1.10; Karyotype formula – A3 (3m) B60 (42M+18m); Stebbins category – 1A; TF% – 47.94; CVCL – 12.66; MCA – 12.34. Curcuma longa-Pop-I: Somatic chromosome number 2n=63 (Figure 10a); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (1.17 µm – 1.70 µm); Total chromosome length – 88.32 µm; Ratio of largest and smallest chromosome – 1.45:1; Mean arm ratio (L/S) –1.13; Karyotype formula – A3 (3m) B60 (15M+45m); Stebbins category – 1A; TF% – 47.01; CVCL – 11.18; MCA – 16.78. Curcuma longa-Pop-II: Somatic chromosome number 2n=63 (Figure 10b); Number of chromosomes bearing secondary constric- tion - 3; Range of chromosome length – (1.18 µm – 1.70 µm); Total chromosome length – 88.08 µm; Ratio of largest and smallest chromosome – 1.44:1; Mean arm ratio (L/S) – 1.13; Karyotype formula – A3 (3m) B60 (15M+45m); Stebbins category – 1A; TF% – 47.13; CVCL – 11.01; MCA – 17.23. Hedychium coronarium-Pop-I: Somatic chromosome number 2n=34 (Figure 11a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 1.65 µm); Total chromosome length – 41.14 µm; Ratio of larg- est and smallest chromosome – 1.56:1; Mean arm ratio (L/S) –1.18; Karyotype formula - A2 (2m) B 32(18M+14m); Steb- bins category – 1A; TF% – 47.32; CVCL – 15.91; MCA – 10.72. Hedychium coronarium-Pop-II: Somatic chromosome number 2n=34 (Figure 11b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.06 µm – 1.60 µm); Total chromosome length – 41.56 µm; Ratio of largest and smallest chromosome – 1.51:1; Mean arm ratio (L/S) –1.18; Karyotype formula - A2 (2m) B32 (16M+16m); Stebbins category – 1A; TF% – 47.24; CVCL – 15.11; MCA – 11.06. Hedychium coccineum-Pop-I: Somatic chromosome number 2n=34 (Figure 12a); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.32 µm – 2.08 µm); Total chromosome length – 52.80 µm; Ratio of largest and smallest chromosome – 1.58:1; Mean arm ratio (L/S) –1.22; Karyotype formula - A2 (2m) B32 (18M+14m); Stebbins category – 1A; TF% – 46.68; CVCL – 15.51; MCA – 13.27. Hedychium coccineum-Pop-II: Somatic chromosome number 2n=34 (Figure 12b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (1.32 µm – 2.08 µm); Total chromosome length – 52.92 µm; Ratio of largest and smallest chromosome – 1.58:1; Mean arm ratio (L/S) – 1.22; Karyotype formula - A2 (2m) B32 (18M+14m); Stebbins category – 1A; TF% – 46.47; CVCL – 15.40; MCA – 14.11. Hedychium thyrsiforme-Pop-I: Somatic chromosome number 2n=34 (Figure 13a); Number of chromosomes bearing secondary constriction - 2; Range of chromosome length – (0.96 µm – 1.44 µm); Total chromosome length – 38.26 µm; Ratio of largest and smallest chromosome – 1.50:1; Mean arm ratio (L/S) –1.21; Karyotype formula - A2 (2m) B32 (12M+20m); Stebbins category – 1A; TF% – 46.60; CVCL – 15.24; MCA – 13.56. 170 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha Hedychium thyrsiforme-Pop-II: Somatic chromosome number 2n=34 (Figure 13b); Number of chromosomes bearing secondary constric- tion - 2; Range of chromosome length – (0.96 µm – 1.44 µm); Total chromosome length – 39.40 µm; Ratio of largest and smallest chromosome – 1.50:1; Mean arm ratio (L/S) –1.21; Karyotype formula - A2 (2m) B32 (12M+20m); Stebbins category – 1A; TF% – 46.61; CVCL – 14.88; MCA – 13.69. DISCUSSION Somatic chromosome number 2n=48 having a basic number X=12 was found to be constant in Alpinia calcar- ata, Alpinia malaccensis and Alpinia nigra as was report- ed by earlier researchers (Raghavan and Venkatasubban 1943; Chakravorti 1948, 1952; Ramachandran 1969; Chen 1988; Joseph 1998). The different proportions of metacen- tric and submetacentric chromosomes present in the somatic chromosome complements of these three species are in partial agreement with the reports of Joseph (1998). At intra-specific (Pop-I and Pop-II) level, the kar- yotype of each species is more or less homogeneous hav- ing one pair of chromosomes bearing secondary constric- tion, a characteristic of the karyotype itself. On the con- trary, at inter-specific level, their karyotypes differ though the chromosomes are mostly metacentric and submetacentric in nature. According to Stebbins categori- zation, the karyotype of A. malaccensis, A. calcarata and A. nigra falls under category 1A, 1B and 2B, respectively. Therefore, the reduction in size of some of the chromo- somes in relation to other has occurred in chromosome complements of A. calcarata and A. nigra. Moreover, the presence of two pairs of submetacentric chromosomes having F% < 33.33 in Pop-I and Pop-II of A. nigra indi- cates that their karyotypes are slightly asymmetric. Thus, in Alpinia spp. a tendency from symmetric to asymmet- ric karyotype is observed. The somatic chromosome count 2n=48 is the distinctive character of Amomum spe- cies which corroborates previous findings (Sharma and Bhattacharya 1959; Chen et al. 1988). Due to limited cytological studies in these three species, little informa- tion was available regarding the detailed chromosomal architecture. In general, chromosomes are short in size where one pair of metacentric chromosomes possessed secondary constriction. The karyotype of A. aromaticum, A. koenigii and A. maximum at inter-specific and intra- specific levels exhibit gross similarity in the types of chromosomes present, number of chromosomes bearing secondary constriction, TF%, total chromosome length and L/S arm ratio. The arm ratio L/S and TF% are the indicators of symmetric type of karyotype. According to Stebbins (1971) categorization, the karyotype of A. aro- maticum and A. maximum falls under 1A while that of A. koenigii belonging to category 1B indicates minor deviation in the size of largest to smallest chromosome ratio. The identical karyotype formula A2B46 without any pair of acrocentric or telocentric chromosomes having arm ratio L/S ˃2:1 suggests accentuated chromosomal homology. In Curcuma complex, the somatic chromo- some number of C. amada was found to be 2n=42 with a basic number X=21 which corroborates previous reports (Sharma and Bhattacharya 1959; Ramachandran 1961, 1969; Islam 2004; Joseph 1998; Bhadra and Bandhyopad- hyay 2015; Lamo and Rao 2017). At intra-specific level, the karyotype of Pop-I and Pop-II of C. amada is almost identical and symmetrical (1A) in nature indicating the absence of acrocentric or telocentric chromosomes. Based on asymmetry index value, Bhadra and Bandyopadhyay (2015, 2018) reported that the karyotype of C. amada falls under Stebbins category 1B which may be due to its occurrence in different eco-climatic zone. The somatic chromosome number 2n=63 of C. caesia and C. longa is in agreement with previous reports (Ramachandran 1961, 1969; Joseph et al. 1999; Nair and Sasikumar 2009; Nair et al. 2010; Lamo and Rao 2014; Bhadra and Bandyopad- hyay 2015; Lamo and Rao 2016, 2017). The karyotype of these two species are identical both at inter- and intra- specific levels. According to Stebbins formula, their kary- otypes belong to category 1A which is not in agreement with the report of Bhadra and Bandyopadhyay (2015, 2018). The somatic chromosome count of 2n=63 chromo- somes of C. picta is a first report and the karyotypes of Pop-I and Pop-II of C. picta exhibit gross similarity in the types of chromosomes present, number of chromo- somes with secondary constriction, TF%, total chromo- some length and mean arm ratio (L/S). According to the degree of asymmetry, the karyotype of C. picta belongs to the category 1A. There are different views regarding the basic number of Curcuma spp. According to one school, the different species of Curcuma have a basic number X=21 (Sharma and Bhattacharya 1959; Ramachandran 1961, 1969; Islam 2004; Joseph 1998; Chen et al. 2013; Bhadra and Bandhyopadhyay 2015; Lamo et al. 2016) which has been derived from a combination of X=12 and X=9 (Ramachandran 1961; Lamo and Rao 2017). The oth- er group (Sato 1960; Skornickova et al. 2007) proposed that the basic number of Curcuma is X=7 suggesting, thereby, all the species of Curcuma studied, having somatic chromosome number 2n=63 are nonaploid and C. amada with 2n=42 chromosomes is a hexaploid spe- cies. Apparently, there is no conflict between X=7 and X=21 in Curcuma complex. But the absence of cytotypes 171Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India in multiple of X such as 2X, 3X, 4X and 5X in natural populations and/or cultivars of Curcuma species suggests that the basic number X=21 is deep seated in Curcuma spp. from which the diploid and triploid species have eventually evolved in the due course of evolution. The occurrence of 2X, 3X, 4X, 5X and 6X cytotypes is not uncommon in flowering plants and in the monocot genus Dioscorea, valid cytotypes having 2X, 3X, 4X, 5X etc. are found in the natural population (Muthamia et al. 2014). In the light of this knowledge, the acceptance of X=7 in Curcuma complex is questionable. Moreover, if X= 7 is accepted as basic number in Curcuma spp., then their natural cytotypes having 9X, 12X and 15X chromosomes would have more copy number of genes. In such a situa- tion, the regulation of dosage compensation having excess copy of genes in their cytotypes is beyond any elu- cidation. The somatic chromosome count 2n=34 chromo- somes found in Pop-I and Pop-II of Hedychium coccine- um and H. coronarium having a basic number X=17 sup- ports previous findings (Mukherjee 1970; Mahanty 1970; Gao et al. 2008). Sharma and Bhattacharya (1959) report- ed a cytotype of H. thyrsiforme having 2n = 24 chromo- somes with basic number X=12. In the present investiga- tion, the presence of 34 chromosomes recorded in most of the somatic cells (modal number) of H. thyrsiforme indicates that the diploid chromosome number is, indeed 2n=34 as was reported by Mahanty (1970). This also sug- gests that all the three Hedychium species growing in this region are stabilized with a basic chromosome number X=17. It is imperative that the karyotypes of the six indi- viduals studied are almost identical and have the same karyotype formula A2B32, but the absence of acrocentric and telocentric chromosomes infers the symmetric nature of karyotypes justifying their inclusion under Stebbins category 1A. Karyotype conservatism without any structural alteration leading to the formation of acro- centric or telocentric chromosomes in these species hav- ing a basic number X=17 reveals a karyotype stasis. Infor- mation regarding the number of chromosomes having secondary constriction in all the taxa of Zingiberaceae studied by previous researchers is minimum (Bhattachar- yya 1957; Mandi 1990; Joseph et al. 1999; Islam 2004) and in some of the previous investigations there was no refer- ence with regard to the number of chromosomes possess- ing secondary constriction. For resolving this problem, Ag-NOR study (Figure S2) has been carried out for the first time. There are different views regarding the maxi- mum number of nucleoli per cell and the number of chromosomes bearing secondary constriction present in somatic chromosome complements of flowering plants (Sharma and Ghosh 1954; Sato 1980). However, the coin- cidence between the number of chromosomes possessing secondary constriction and the maximum number of nucleoli per cell indicates the relationship between the presence of secondary constriction and the ability of those chromosomes to form the maximum number of nucleoli in all the taxa studied. The presence of three nucleoli in triploid species of Curcuma is also an indica- tor of such a relationship. The inter- and intra chromo- somal asymmetry based on Stebbins quali-quantitative method reveals that the karyotypes of most of the species are symmetrical in nature. A progressive asymmetry is, however, observed in Amomum koenigii (1B), Alpinia cal- carata (1B) and Alpinia nigra (2B). Undoubtedly, Stebbins (1971) quali-quantitative method is the determinant of evaluation of the karyotype asymmetry index but based on this data alone, it would not be possible to ascertain the genetic relationship between different taxa of the same family. The bi-dimensional (Figure 14) scatter plot (Peruzzi and Eroglu 2013) drawn against Mean Centro- meric Asymmetry (MCA) and Coefficient of Variation of Chromosome Length (CVCL) clearly indicates that the two tribes Alpinieae (represented by Alpinia spp. and Amomum spp.) and Zingibereae (represented by Curcuma spp. and Hedychium spp.) of Zingiberaceae (Angiosperm Phylogeny Group – 2009) have distinct karyotypes in which Alpinia spp. and Amomum spp. show comparative- ly high inter-chromosomal and intra-chromosomal asymmetry indices (Zarco 1986; Watanabe et al.1999; Paszko 2006) than those of Curcuma spp. and Hedychium spp. The dendrogram reveals that Alpinia spp., Amomum spp., Curcuma spp. and Hedychium spp. with their respec- tive populations formed four separate clusters (Figure 15). Figure 14. Bi-dimensional scattered plot against MCA (x axis) and CVCL (y axis). 172 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha The PCA illustrates no overlap and thus, the distinctive position of each genus with respect to other is evident (Figure 16). The karyotype data, therefore, validates the taxonomic position of Alpinia spp., Amomum spp., Cur- cuma spp. and Hedychium spp. The chromosomal data of Alpinia spp., Amomum spp., Curcuma spp., and Hedychi- um spp. suggest that they might have originated from a common ancestry but eventually through gradual chang- es, evolved as separate species in the due course of time. Gower’s (1971) similarity matrix points towards a possible rearrangement of the chromosomal architectures in 13 different taxa studied. Such chromosomal rearrange- ments are possibly associated with cryptic changes main- taining a high syntenic value. CONCLUSION The present study has been focussed on t he detailed kar yot y pe ana lysis of two populations of Alpinia calcarata, A. malaccensis, A. nigra, Amomum aromaticum, A. koenigii, A. maximum, Curcuma ama- da, C. caesia, C. longa, C. picta, Hedychium coccineum, H. coronarium, and H. thyrsiforme of Zingiberace- ae, collected from different geographical locations of Tripura. The somatic chromosome number of Alpinia and Amomum is found to be 2n = 48 having a basic number of X=12 chromosomes. In Hedychium spp. the diploid chromosome number is 2n=34 with basic num- ber X=17 chromosomes. The present findings reveal that in majority of the somatic cells of Curcuma caesia, Curcuma longa and Curcuma picta, 63 chromosomes are present indicating their triploid nature. In con- trast, diploid somatic chromosome number (2n=42) is recorded in Curcuma amada. Stebbins quali-quantita- tive analysis indicates that except in Alpinia nigra, the karyotype of other 12 species is symmetric in nature. The accentuated chromosome homology at intra-spe- cies level is the intrinsic characteristic of their karyo- types. The inter-chromosomal and intra-chromosomal asymmetry indices, based on statistically based meth- od, suggest that Alpinia spp. and Amomum spp. have higher inter- and intra-chromosomal asymmetry in comparison to Curcuma spp. and Hedychium spp. The dendrogram and PCA derived data clearly validate the taxonomic position of these four genera investigated. Gower’s similarity matrix endorses the cryptic changes leading to karyotype conservatism at species level of each genus. ACKNOWLEDGEMENTS KS is grateful to UGC, New Delhi for providing BSR Fellowship. DISCLOSURE OF STATEMENT The authors declare that they have no conflict of interest. Figure 15. UPGMA dendrogram based on Gower’s similarity matrix of six karyological parameters. 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Chro- mosomal evolution in the genus Brachyscome (Aster- aceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit using phylo- genetic information. J Plant Res. 112:145-161. Yoshikane I, Naohiro N. 1991. Karyotypes of Fragaria nubicola and F. daltoniana. Cytologia 56:453-457. Zarco CR. 1986. A new method for estimating karyotype asymmetry. Taxon 35: 526–530. 176 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha SUPPLEMENTARY FILE (APPENDICES) Figure S1. Karyogram of thirteen Zingiberaceae species from Tripura. 1- Alpinia calcarata; 2- A. malaccensis; 3- A. nigra; 4- Amomum maximum; 5- A. aromaticum; 6- A. koenigii; 7- Curcuma amada; 8- C. caesia; 9- C. picta; 10- C. longa; 11- H. coronarium; 12- H. coccineum; 13- H. thyrsiforme (a – Pop - I; b – Pop - II); scale bars = 5µm. 177Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Figure S2. Ag- impregnated somatic cells of thirteen Zingiberaceae species. 1- Alpinia calcarata; 2- A. malaccensis; 3- A. nigra; 4- Amomum maximum; 5- A. aromaticum; 6- A. koenigii; 7- Curcuma amada; 8- C. caesia; 9- C. picta; 10- C. longa; 11- H. coronarium; 12- H. coccineum; 13- H. thyrsiforme (a – Pop - I; b – Pop - II). 178 Kishan Saha, Rabindra Kumar Sinha, Sangram Sinha Table S1. Details of plant samples collected from different geographical locations of Tripura. Name of the species Herbarium Voucher No. Locations and altitudes Alpinia calcarata TUH – 460 (Pop-I) Suryamaninagar, (34 mt.) 23045.0’44.74’’ N, 91015.0’54.29’’E TUH – 2001 (Pop-II) Baramura, (110 mt.) 23049.0’0.70’’ N, 91031.0’2.33’’E Alpinia malaccensis TUH – 466 (Pop-I) Debtamura, (48.0 mt.) 23033.0’2.50’’N, 91038.0’5.40’’E TUH – 2002 (Pop-II) Atharamura, (134 mt.) 23029.0’54.10’’N, 91037.0’34.30’’E Alpinia nigra TUH – 2049 (Pop-I) Mohanpur, (30.0 mt.) 23049.0’28.50’’N, 91026.0’45.0’’E TUH – 2003 (Pop-II) Chabimura, (110 mt.) 23033.0’18.20’’N, 91037.0’0.98’’E Amomum aromaticum TUH – 2055 (Pop-I) Tulamura,(46 mt.) 23023.0’11.3’’N, 91026.0’10.7’’E TUH – 2004 (Pop-II) Unakoti, (200 mt.) 24023.0’11.3’’ N, 9204.0’13.0’’E Amomum maximum TUH – 1407 (Pop-I) Atharamura foot Hills, (45 mt.) 23053.0’2.10’’N, 91042.0’12.5’’E TUH – 2005 (Pop-II) Jampui Hills, (191 mt.) 23059.0’4.63’’ N, 92017.0’50.6’’E Amomum koenigii TUH- 470 (Pop-I) Dhuptali, (40 mt.) 23023.0’11.3’’ N, 91026.0’15.1’’E TUH- 2006 (Pop-II) Baramura,(120 mt.) 23050.0’4.0’’ N, 91036.0’38.96’’E Curcuma amada TUH – 465 (Pop-I) Suryamaninagar, (21 mt.) 23045.0’44.27’’ N, 91015.0’53.38’’E TUH – 2007 (Pop-II) Howaibari,(105 mt.) 23049.0’0.60’’ N, 91034.0’04.61’’E Curcuma longa TUH – 1407 (Pop-I) Belkum Para, (48 mt.) 23053.0’2.1’’ N, 91043.0’13’’E TUH – 2008 (Pop-II) Jampui Hills, (163 mt.) 2402.0’30.21’’ N, 92016.0’42.34’’E Curcuma caesia TUH- 466 (Pop-I) Suryamaninagar, (30 mt.) 23045.0’43.13’’ N, 91015.0’52.17’’E TUH- 2009 (Pop-II) Sepahijala, (30 mt.) 23039.0’35.74’’ N, 91018.0’8.54’’E Curcuma picta TUH – 459 (Pop-I) Kamarikhala, (47 mt.) 23033.0’3.0’’ N, 91038.0’5.30’’E TUH – 2010 (Pop-II) Baramura foot Hills (102 mt.) 23048.0’41.24’’ N, 91030.0’54.24’’E Hedychium coronarium TUH – 458 (Pop-I) College Tilla, (25 mt.) 23049.0’46.72’’ N, 91017.0’36.28’’E TUH – 2011 (Pop-II) Baramura, (105 mt.) 23049.0’0.10’’ N, 91033.0’3.54’’E Hedychium coccineum TUH – 461 (Pop-I) Paikhola, (45 mt.) 23022.0’27.8’’ N, 91030.0’48.30’’E TUH – 2012 (Pop-II) Karbook, (106 mt.) 23021.0’38.90’’ N, 91042.0’12.15’’E Hedychium thyrsiforme TUH- 507 (Pop-I) Jampui Hills, (120 mt.) 23059.0’4.7’’ N, 92017.0’56.0’’E TUH- 2013 (Pop-II) Manpui, (290 mt.) 2408.0’5.9’’ N, 92016.0’39.0’’E Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 1 - 2020 Firenze University Press Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat High genetic diversity and presence of genetic structure characterise the endemics Ruta corsica and Ruta lamarmorae (Rutaceae) Marilena Meloni1, Caterina Angela Dettori2, Andrea Reid3, Gianluigi Bacchetta2,4,*, Laetitia Hugot5, Elena Conti1 Cytogenetic effects of C6H4 (CH3)2 (xylene) on meristematic cells of root tips of Vicia faba L. and mathematical analysis Cihangir Alaca1, Ali Özdemir1, Bahattın Bozdağ2, Canan Özdemir2,* Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. 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