Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(4): 9-14, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2295 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Asadi-Corom, F., Asadi, F., & Mirzaie-Nodoushan, H. (2023). Evalua- tion of the evolutionary process within Populus caspica species from Hyr- canian forests by karyotype analysis. Caryologia 76(4): 9-14. doi: 10.36253/ caryologia-2295 Received: August 30, 2023 Accepted: February 04, 2024 Published: March 14, 2024 Copyright: © 2023 Asadi-Corom, F., Asa- di, F., & Mirzaie-Nodoushan, H. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. ORCID FA-C: 0000-0002-4295-107X FA: 0000-0001-6920-1781 HM-N: 0000-0002-1021-9220 Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis Fereshteh Asadi-Corom1,*, Farhad Asadi2, Hossein Mirzaie-Nodoushan1 1 Biotechnology Department, Research Institute of Forests and Rangelands of Iran, Agri- cultural Research, Education and Extension Organization (AREEO), Tehran, I.R. Iran 2 Research Division of Natural Resources, Mazandaran Agricultural and Natural Resourc- es Research and Education Center, AREEO, Sari, I.R. Iran *Corresponding author. E-mail: asadikaram@ut.ac.ir Abstract. Caspian poplar (Populus caspica Bornm.) is distributed exclusively in the Hyrcanian forests. Hyrcanian forests are the final remnants of Tertiary temperate deciduous forests in Western Eurasia and worldwide. This species plays a significant ecological role in the protection of the natural environment in Hyrcanian forests. In this research, chromosome number and karyotype details of 11 populations of the species were investigated for the first time, using fresh root cuttings collected from mature trees in different parts of the forest, located in the northern parts of Iran. Pre- treatment, fixation, hydrolyzing, and staining were conducted by α-bromonaphthalene, carnoy’s solution, 1N HCl, and hematoxylin agent, respectively. Chromosomal data were analyzed according to a nested model based on a completely randomized design. Chromosome numbers of all of the populations were the same as 2n = 38, which mostly were medium region and sub-metacentric types. Significant differences were observed between the provinces and populations, based on chromosome length grand means, arm ratios and centromere indices. The results demonstrated that structural rearrangement has occurred within the studied populations and indicated an active evolutionary process within and between populations of the species due to natu- ral hybridization. Also, these results showed that artificial inter-specific hybridization between the P. caspica and its relative species can be employed to broaden the ecologi- cal zone of the species. Keywords: asymmetry indices, chromosome number, karyotype, Populus caspica. INTRODUCTION Hyrcanian forests inscribed on the UNESCO World Heritage List contain the final remains of tertiary wide-leaved forests in western Eurasia and world- wide (UNESCO World Heritage Centre 2019; Bayranvand et al. 2017; Alipour et al. 2023). Hyrcanian forests are located between the north of the Alborz Moun- tain chains and the Caspian sea (Iran and Caucasus) (Sabeti, 1994). In Iran, which accounts for most of the hotspots of Iranian-Anatolian biodiversity, 30 http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2295 https://doi.org/10.36253/caryologia-2295 https://doi.org/10.36253/caryologia-2295 http://www.fupress.com/caryologia http://www.fupress.com/caryologia https://orcid.org/0000-0002-4295-107X https://orcid.org/0000-0001-6920-1781 https://orcid.org/0000-0002-1021-9220 mailto:asadikaram@ut.ac.ir 10 Fereshteh Asadi-Corom, Farhad Asadi, Hossein Mirzaie-Nodoushan percent of vascular plant species are endemic (Noroozi et al. 2018). In flora of Iran only two species, Populus caspica Bornm., and P. euphratica, are native and the others (P. alba, P. deltoides, P. nigra and hybrid species (P. canaden- sis)) are widely cultivated at different parts of Iran (Maas- soumi et al. 2011). Caspian poplar (P. caspica), a member of the Salicaceae family, is classified as endangered in Iran (Jalili and Jamzad, 1999; Alipour et al. 2021). Many species including P. caspica have survived periods of glaciation in the Hyrcanian forests (Qin et al. 2017; Mohammadi et al. 2019). Poplars are so noteworthy in agroforestry indus- try because of their fast growth, desirable figure, as well as in providing wood, fiber, fuel-wood, and other forest products (Stettler, 2009; Evans, 2014). Natural habitats of P. caspica have largely been destroyed through environ- mental conditions and human activities like planting non- indigenous species, and agricultural usage (Khoshravesh et al. 2009). Habitat degradation has impacted the restoration of P. capsica due to unsuitable seedbed conditions (Asadi and Mirzaie-Nodoushan, 2011). Comparative genomics and sequencing have been done in several poplar species that demonstrated high genetic diversity and frequent interspecific hybridization among the species (Li et al. 2023). Other karyological studies showed that in Populus genus, with a basic hap- loid chromosome number of 19, diploidy is predominant. Due to the small chromosomes, karyotype information has been reported for several Populus species (Islam-Fari- di et al. 2009; Liu et al. 2021). Hence, in recent years fluo- rescence in situ hybridization (FISH) has been employed for identifying chromosomes in poplars to compare the karyotype and similarity of chromosome structure among different species (Xin et al. 2020; Kim et al. 2020). Apart from P. euphratica, there is no information on the number of chromosomes and karyotypic indices of the Populus species, including P. caspica, in Iran. Therefore, this study was undertaken to provide cytological infor- mation on the species based on conventional methods that can be useful as a guide in future breeding programs and evaluation of evolutionary process. MATERIALS AND METHODS Plant materials were obtained from trees in 11 dif- ferent parts of two provinces, Gilan and Mazandaran, located in north part of Iran. Root tip meristems, col- lected from cutting grown under hydroponic conditions, were pre-treated with 0.5% α-bromonaphthalene for 1 hour in refrigerator, then fixed in a mixture of ethanol alcohol and glacial acetic acid (3:1 v/v) for 16 hours. The fixed samples were washed 2–3 times and preserved in 70% ethanol. Root tips were hydrolyzed with 1N HCl solution at 60°C for 6 minutes, stained in hematoxylin reagent for 2 hours at 60°C, and finally squashed in 45% acetic acid (v/v) (Mirzaie-Nodoushan and Asadi-Corom, 2002). Somatic chromosomes were photographed using digital camera and the chromosomes were measured via Ideokar 1.2 software (Ghader Mirzaghaderi and Karim Marzangi, 2015). Based on the centromere position nomenclature of chromosomes was described (Levan et al. 1964). Along with chromosomal dimensions several chromosomal parameters, such as Arm ratio, r-value, Relative length of chromosome, Form percentage of chromosome, centromeric index (CI=S/TL) were cal- culated. As well as, asymmetry indices were calculated using Intra-chromosomal asymmetry index (A1) (Zarco, 1986), Inter-chromosomal asymmetry index (A2) (Zarco, 1986), Symmetry index (S%) (Watanabe et al. 1999), Total form percentage (TF%) (Huziwara, 1962), differ- ence of range relative length (DRL) and Stebbins class asymmetry index (Stebbins, 1971). Chromosomal data were analyzed using a nest- ed model based on a completely randomized design , regarding the provinces, populations and chromosomes as the three nested factors with three replications of well-spread metaphasic plates. In this case provinces are considered as factor A, populations as factor B, nested within factor A, which is shown in statistical point of view as, populations (A), and chromosomes as factor C, nested within factor B, (chromosomes (B A)). Duncan multiple range test was carried out for classifying the populations by SAS 9.4 software. Cluster analysis was performed in order to classify the populations based on chromosomal measures and karyotypic indices using Ward method, by JMP 13.2.0 software. RESULTS The chromosome counting revealed that all of the studied populations were diploid, containing a total of 38 chromosomes (2n = 38) with a single pair carrying the satellites, located on the short arms of the chromo- somes (Fig. 1). Chromosome length grand mean and arm ratio (AR) showed a significant difference (p≤0.05) between the provinces, while arm ratios and centromere indices were different (p≤0.01) between the populations (Table 1). Size of the chromosomes among the studied populations varied from 0.65 μm (Tash12) to 2.90 μm (Tash2) and from 0.67 μm to 2.32 μm in Mazandaran and Gilan populations, respectively (Table 2). The karyotype formula of the studied populations is presented in Table 1. In both provinces, medium region 11Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis (m) type chromosomes were the dominant type, espe- cially in Gil22 and Tash13. That’s why their karyotypes are symmetrical. All populations possessed one to seven chromosomes of sub-metacentric (sm) type. Sub-termi- nal region (st) type was observed only in two popula- tions of Mazandaran. The most asymmetrical karyotype was found in the Konesi2 population of the same prov- ince (21m+14sm+3st). In this population, medium point and terminal point types of chromosomes were also found in several single plants (23m+4sm+9st+2T+sat; 1M+34m+1sm+2st) (Table 3). Also based on chromo- some characteristics and karyotypic indices, the plant populations were clustered in three groups. The konesi2 population, with the most asymmetrical karyotype, was clustered into a single group. (Fig. 2). DISCUSSION In most modern poplars, the cell nucleus typically contains two sets of 19 (2n = 38) chromosomes (Chen et al. 2005; Shou-Gong et al. 2005) that agree with the results obtained by this research. Triploids with three sets of chromosomes (2n = 57) (Peto, 1983) and tetra- ploids (2n = 76) have also been identified in section Populus (Einspahr et al. 1964; Every and Wiens 1971). According to IPCN and literature surveys, triploidy in P. nigra and P. canadensis (Shou-Gong et al. 2005; Chen et al. 2005) and aneuploidy in two varieties of P. alba have been reported (IPCN, http://www.tropicos.org/Project/ IPCN). The presence of only one couple carrying one pair of satellite, represents the basic profile of this spe- cies same as other species of poplars. Chromosome numbers and chromosome rear- rangements are the major source of karyotype evolution and closely related species maintain a similar chromo- some number. Despite the similar chromosome number in the studied populations, a structural diversification was observed in the studied populations. Difference in the karyotypic formula within the species indicates that chromosome structural changes have occurred. Pericen- tric inversions is one of the most common mechanisms related to karyotypic variation (Molina and de Freitas Bacurau, 2006; Carbone et al. 2014). They can shift the position of the centromere within a chromosome and cause the arm ratio to change. Inverted chromosomes Figure 1. Mitotic metaphase chromosomes of the studied popula- tions of Populus caspica in Iran (Arrows are pointing to satellites; Bar=100 μm). Table 1. Mean squares resulted from nested model analysis of variance of chromosome parameters of Populus caspica populations. Source of variation DF L (μm) S (μm) TLM (μm) AR r-Value RL% F% CI A: provinces 1 0.37* 0.004ns 0.0.07* 0.97* 0.002ns 0.00ns 0.006ns 0.001ns B: populations (A) 9 0.16ns 0.11ns 0.48ns 1.31** 0.06** 1.68ns 0.06ns 0.009** C: Chromosomes (B A) 198 0.13** 0.07** 0.39** 0.14ns 0.01ns 0.1** 0.4** 0.002ns Error 418 0.007 0.004 0.01 0.21 0.02 0.02 0.02 0.002 CV% 13.65 14.46 10.16 31.98 17.04 5.84 12.52 11.27 L= length of the longest chromosome, S= length of the shortest chromosome, TLM = grand mean of chromosome length, AR= L/S, r-Val- ue= S/L, RL%= relative length of chromosome, F%= form percentage of chromosome, CI= centromeric index. **: significant difference at 1% level, *: significant difference at 1% level, ns: no significant difference. http://www.tropicos.org/Project/IPCN http://www.tropicos.org/Project/IPCN 12 Fereshteh Asadi-Corom, Farhad Asadi, Hossein Mirzaie-Nodoushan have the potential to contribute to asymmetrical biva- lents (Singh, 2017). In fact, chromosomal rearrangements are often the main source of karyotypical evolution and would indicate an active evolutionary process within and between populations of the species (Mirzaie-Nodoushan et al. 2006; Xin et al. 2020). In Hyrcanian forest, the nat- ural hybridization and large-scale interspecific hybridi- zation between P. caspica and other cultivated poplar species, such as European P. alba, P. nigra, P. deltoids (North American poplar), is documented using cpDNA (chloroplast DNA) and ITS (Internal Transcribed Spacer) fragments (Yousefzadeh et al. 2019). Inter-chromosomal translocation is another factor for chromosomal rear- rangements and diversity of karyotypic parameters in P. caspica as a result of cross-pollination (Fig. 2) while this result is contrary to previous study by Xin et al. (2020). By chromosome painting probes they demonstrated that no chromosomal rearrangements on any of the 19 chro- mosomes among some species of Populus including P. euphratica and P. deltoids have occurred. On the other hand, the potential for intact or largely partial chromo- some transfer between poplar hybrids has been proposed by Liu et al. (2021) using labeled telomeres, rDNA, and repetitive sequences as probes, that supports the findings in the present study. The existing variation within the species based on chromosomal parameters was remarkable. As it was mentioned earlier, sexual and clonal reproduction of P. caspica is limited and the species is endangered in the area of its habitation in the country. This impor- Table 2- Means of mitotic features of the studied populations of Populus caspica. Provinces Populations L (μm) S (μm) TLM (μm) AR r-Value RL% F% CI Gilan Gil13 0.59d 0.43d 1.02g 1.43b 0.74bcd 2.63a 1.11abc 0.42ab Gilan Gil23 0.66bc 0.47c 1.13d 1.47b 0.74bcd 2.63a 1.09bc 0.42b Gilan Gil22 0.67bc 0.51b 1.18bc 1.34b 0.78abc 2.63a 1.14ab 0.43ab Gilan Gil31 0.66bc 0.52b 1.18c 1.31b 0.80a 2.63a 1.16a 0.44a Gilan GilP 0.64c 0.46c 1.10de 1.47b 0.73cd 2.63a 1.09bc 0.41b Mazandaran Konesi1 0.61d 0.44cd 1.05fg 1.46b 0.74abcd 2.63a 1.11abc 0.42ab Mazandaran Konesi2 0.69b 0.46c 1.15cd 1.87a 0.70d 2.63a 1.05c 0.40c Mazandaran KonesiP 0.61d 0.46c 1.07ef 1.40b 0.77abc 2.63a 1.14ab 0.43ab Mazandaran Tash12 0.60d 0.45cd 1.05fg 1.39b 0.77abc 2.63a 1.13ab 0.43ab Mazandaran Tash13 0.69b 0.53ab 1.23b 1.35b 0.79ab 2.63a 1.15ab 0.44ab Mazandaran Tash23 0.76a 0.55a 1.31a 1.42b 0.75abc 2.63a 1.11abc 0.42ab L= length of the longest chromosome, S= length of the shortest chromosome, TLM =grand mean of chromosome length, AR= L/S, r-Value= S/L, RL%= relative length of chromosome, F%= form percentage of chromosome, CI= centromeric index, Similar letters within each col- umn, indicate no significant difference between the populations at 5% level. Table 3. Karyotypic parameters of the studied populations of Populus caspica. Provinces Populations Stebbins FK A1 A2 S% TF% DRL% Gilan Gil1 1B 34m+4sm 0.26 0.09 33.77 42.05 3.34 Gilan Gil23 2B 32m+6sm 0.26 0.05 32.09 41.55 3.59 Gilan Gil22 1B 37m+1sm (2sat) 0.22 0.08 34.33 43.49 3.40 Gilan Gil3 1B 32m+6sm (2sat) 0.20 0.06 32.68 43.96 3.48 Gilan GilP 1B 34m+4sm (2sat) 0.27 0.06 30.68 41.58 3.78 Mazandaran Konesi1 1B 32m+6sm 0.26 0.09 33.42 42.01 3.44 Mazandaran Konesi2 2B 21m+14sm+3st (2sat) 0.30 0.13 28.28 39.93 4.08 Mazandaran KonesiP 2B 34m+3sm+1st 0.23 0.11 32.53 43.21 3.26 Mazandaran Tash12 1B 35m+3sm 0.23 0.11 34.26 42.88 3.25 Mazandaran Tash13 1B 36m+2sm 0.21 0.08 32.31 43.59 3.54 Mazandaran Tash2 1B 31m+7sm (2sat) 0.25 0.05 26.04 42.00 4.31 KF= karyotypic formulae, A1= Intra-chromosomal asymmetry index, A2= Inter-chromosomal asymmetry index, S%= Symmetry index, TF%= Total form percentage, DRL%= Differences between the maximum and minimum relative length of the chromosomes; sat= satellite. 13Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis tant point should be regarded as a major restriction of the species. 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