Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 53-61, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-729 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: M. Hasaninejad, Z. Jamzad, Saeid Afsharzadeh, H. Saeidi (2021) Chromosome counts of eight Iranian endemic species of Nepeta L. (Lami- aceae). Caryologia 74(1): 53-61. doi: 10.36253/caryologia-729 Received: November 24, 2019 Accepted: April 26, 2021 Published: July 20, 2021 Copyright: © 2021 M. Hasaninejad, Z. Jamzad, S. Afsharzadeh, H. Saeidi. This is an open access, peer-reviewed article 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. Chromosome counts of eight Iranian endemic species of Nepeta L. (Lamiaceae) Maryam Hasaninejad1, Ziba Jamzad2, Saeid Afsharzadeh1,*, Hojjatol- lah Saeidi1 1 Department of Biology, Faculty of Biological Science and Technology, University of Isfa- han, Isfahan, Iran. 2 Research Institute of Forests and Rangelands, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran. *Corresponding author. E-mail: s.afshar@sci.ui.ac.ir Abstract. In this survey, the chromosome counts of eight Nepeta L. species were inves- tigated and the karyotypic diversity among these species was studied. The examined species belong to N. cephalotes Boiss. species group, namely N. eremokosmos Rech.f., N. gloeocephala Rech. f., cephalotes Boiss., N. pungens (Bunge) Benth., N. ispahanica Boiss., N. mahanensis Jamzad & Simonds, N. hormozganica Jamzad and N. denudata Benth. collected from different habitats in Iran. The ploidy levels, karyotype formula, chromo- some length range, total karyotype length, several karyotype asymmetries values and Stebbins classification were determined in this study. Results showed the same chromo- some number, 2n = 2x= 18 for all studied species. The basic chromosome number for the above mentioned species are x = 9. Also, the smallest chromosome length is 1.02 μm in N. mahanensis. The largest chromosome length is 2.3 μm in N. ispahanica. The chromosomes of species were metacentric or submetacentric. According to the Stebbins classification, these species were located into three classes 1A, 2A and 3A. The chromo- some numbers for six of studied species are reported here for the first time. Keywords: chromosome number, cytotaxonomy, endemics, Lamiaceae, karyotype, Iran, Nepeta L. INTRODUCTION The family Lamiaceae consists of 7173 species in 236 genera worldwide. Many of its species have a great importance due to their economic values (Harley et al. 2004). Nepeta L. (catmint) is a genus belonging to the subfam- ily Nepetoideae (Cantino et al. 1992). It is one of the largest genera within Nepetoideae, growing as annual, herbaceous perennial and fruticose plants (Rechinger 1982; Jamzad 2003a, 2012; Kaya and Dirmenci 2008). Endemic species, constituting valuable floristic elements are those which are confined to a particular geographic region. The narrow endemic species are not only scientifically interesting but also very important from conser- vation point of view. Therefore, the identification of endemic plants, their 54 Maryam Hasaninejad, Ziba Jamzad, Saeid Afsharzadeh, Hojjatollah Saeidi conservation and genetic resources are interesting for the scientific community (Ghaffari et al. 2005). In Iran, there are 165 endemic taxa of Lamiaceae including 42 endemic Nepeta species. Iran is one of the centers of diversity for the genus Nepeta (Jamzad et al. 2003b). Most species of the Lamiaceae have medicinal val- ues. There are numerous known species in the family that are used as analgesic drugs in traditional medicine (Uritu et al. 2018). Medicinal properties of the Lami- aceae species are often ascribed to their high content of volatile compounds (Khoury et al. 2016) and glandular hairs represent important sites for the synthesis of nat- ural bioactive compounds (Giuliani et al. 2020). Nepeta is an important genus in Lamiaceae and is specified by terpenoid-type compounds and phenolic constituents, which exert several activities such as an antimicro- bial, repellent against major pathogen vector mosqui- toes, insecticide, larvicide against Anopheles stephensi, cytotoxic anticarcinogen, antioxidant, anticonvulsant, analgesic, anti-inflammatory agent, and antidepressant, disclosing its importance in medicinal and agricultural fields (Süntar et al. 2018). Species of this genus have been studied in the fields of morphology-anatomy (Kaya and Dirmenci 2008; Acar et al. 2011), palynology (Jamzad et al. 2000; Celenk et al. 2008; Moon et al. 2008). Chemical composition (Baser et al. 2000; Asgarpanah et al. 2014) and molecular phylog- eny (Jamzad et al. 2003b). The lack of sufficient data on the karyomorphology of the genus is probably due to the small size of its chromosomes (Esra et al. 2020). Many karyological data concerning chromosome numbers of the genus have already been reported as x = 6, 7, 8, 9, 11, 12, 15, 13, 17, 18. (IPCN, http://www.tropicos. org/Project/IPCN, Darlington and Wylie, 1955; Goldb- latt and Johnson 1979–2017; Chen et al. 2018) and there are a few reports from Iran (Aryavand 1977; Ghaffari and Kelich 2006; Kharazian et al. 2013; Payandeh et al. 2015; Akbarpur Mamagani et al. 2016; Hasaninejad et al. 2020). It should be admitted that the numerical variation in chromosome numbers within a genus is quite common. The chromosome numbers and karyotype studies are not only useful in predicting morphological similari- ties and diversity among species, but also, they are valu- able sources of taxonomic and biosystematic informa- tion. Regarding to the complexities in taxonomy of the genus Nepeta, the phylogenetic relationships of species and the chromosomal evolutionary trend may elucidate the systematics, and lead to a comprehensive infrage- neric classification of the genus. In this study, we aim to do a cytotaxonomic study of the genus, and follow up the process of chromosomal evolution and its use in the classification of this genus. Here we report part of our results on the chromosome counts of a natural spe- cies group, recognized previously as section Capituliferae Benth. p.p. (Bentham 1848) and group five (Jamzad et al. 2003b), with mostly Iranian endemic species. MATERIALS AND METHODS Seeds of 8 species were collected from different habi- tats of Iran are, as listed in Table 1. The voucher speci- mens of the examined species are preserved in the her- barium of the Research Institute of Forests and Range- lands of Iran (TARI). For mitotic studies, the seeds were germinated at 25 °C on wet filter paper in petri dishes. After ger- mination, roots of 0.5-1cm were selected for pre- treatment. Root tips were pretreated for 1 h in α-monobromonaphthalene at 4 °C, washed and fixed in Carnoy solution (3:1 absolute ethanol glacial acetic acid) overnight. The root tips were hydrolyzed for 5-8 minutes in 1N HCl at room temperature, washed and stained in 2% Hematoxylin for 1 h. Table 1. The voucher details of studied Nepeta species. No Species Geographical Location 1 N. cephalotes Boiss. Iran, Tehran, Jajroud highway towards Jajrood 1544 m, Golipour, 106883, TARI. 2 N. denudata Benth. Iran, Hamedan, near Razan, 1889 m, Golipour, 106879, TARI. 3 N. eremokosmos Rech.f. Iran, Semnan, Sorkhe, 1355 m, Golipour, 106880, TARI. 4 N. gloeocephala Rech. f. Iran, Yazd, Taft, Nasr Abad, Gilok village in the river, 2800m, Mirhoseini, 95002, TARI. 5 N. hormozganica Jamzad Iran, Hormozgan, N. Bandar Abbas, N. slop of M. Bokhon, 834 m, Ajani,105647, TARI. 6 N. ispahanica Boiss. Iran, Kerman, Rayen to the first Garow, Fazlabad village road, 2618 m, Golipour, 106881, TARI. 7 N. mahanensis Jamzad & Simonds Iran, Kerman East Silo Mahan-Before to Khaki-Asphalt Road, Hossein Abad 1980 m, Golipour, 106882, TARI. 8 N. pungens (Bunge) Benth. Iran, Chaharmahal va Bakhtiari, Shahrekord, Babahidar, the first road to the village of Sepidaneh, 2340 m, Ajani & Hasaninejad, 107079, TARI. 55Chromosome counts of eight Iranian endemic species of Nepeta L. (Lamiaceae) OLYMPUS BH-2 photomicroscope provided the clearest mitotic metaphase among 5 cells and measured by Micro Measure software 3.3. Karyotypes were prepared and chromosome pairs were classified according to Levan et al. (1964) and the metacentric and sub-metacentric chromosomes were symbolized using the letters m and sm, respectively. The chromosomes were arranged according to their lengths. The long arm (q), short arm (p), mean length of the chromosome (CL), and total chromosome length (TCL) were measured. Karyotype symmetry was determined according to Stebbins (1971) and total form percentage (TF, 100 × ΣS/C) (Huziwara 1962). RESULTS There was no difference between basic chromosome numbers of the eight studied species and they were x = 9. The details of each species are as follow: Nepeta cephalotes is an Irano-Turanian endemic spe- cies and grows in central and northwest of Iran. This species showed a diploid chromosome number 2n = 2x = 18 (Figure 1A) and the basic chromosome number of x = 9. Karyotype consisted of 9 pairs of submetacentric chromosomes (Tables 2, 3; Figure 2A). The chromosome length ranged from 1.14 to 2.07μm. The chromosome number of this species is reported here for the first time. Nepeta denudata is an endemic perennial species, with a distribution range in central, northeast, and west of Iran. The results showed that this species is also dip- loid with chromosome number of 2n = 18 (Figure 1B). The karyotype was formed of eight pairs of submetacen- tric and one pair of metacentric chromosomes (Tables 2, 3; Figure 2B). The mean length of chromosome varied from 1.1 to 1.9μm. The chromosome number of this spe- cie is reported here for the first time. Nepeta eremokosmos is a narrow endemic species. It grows in a limited geographical area in central Iran. The studied specimens showed a diploid chromosome num- ber of 2n = 2x = 18 in this taxon (Figure 1 C) and basic chromosome number of x = 9. Karyotype in this taxon consisted of 9 pairs of submetacentric chromosomes Table 2. Karyotype formula according to Levan et al. (1964) of the studied Nepeta species: 2n– Chromosome number; x– Basic chromo- some number; PL– Ploidy level; KF– Karyotype formula R– range; SC– The shortest chromosome length; LC– The longest chromosome length. No Species 2n x PL KF R (SC–LC) (μm) 1 N. cephalotes 18 9 2x 9sm 1.14-2.07 2 N. denudata 18 9 2x 8sm+m 1.17-1.90 3 N. eremokosmos 18 9 2x 9sm 1.31-1.99 4 N. gloeocephala 18 9 2x 6m+3sm 1.05-1.98 5 N. hormozganica 18 9 2x 5m+4sm 1.15-1.73 6 N. ispahanica 18 9 2x 7sm+2m 1.47-2.30 7 N. mahanensis 18 9 2x 9m 1.02-1.74 8 N. pungens 18 9 2x 7sm+2m 1.11-2.12 Table 3. Karyomorphological parameters of studied Nepeta species: AR– arm ratio; CI– Mean centromeric index; p– Mean length of the short arm; q– Mean length of the long arm; TCL– The total chromosome length of the haploid complement; CL– Mean length of the chro- mosome; TF– Total form percentage and Stebbins– Classification of Karyotypes in relation to their degree of asymmetry according to Steb- bins (1971). No Species AR (L/S) (μm) CI (μm) P mean (μm) q mean (μm) TCL CLmean (μm) TF(%) Stebbins 1 N. cephalotes 2.18 0.32 0.47 1.10 14.47 1.61 31.41 3A 2 N. denudata 2.12 0.33 0.50 1.06 13.98 1.55 32.01 3A 3 N. eremokosmos 1.91 0.35 0.56 1.07 14.61 1.62 34.37 2A 4 N. gloeocephala 1.58 0.36 0.59 0.93 13.71 1.52 38.81 2A 5 N.hormozganica 1.67 0.33 0.52 0.87 12.58 1.40 37.47 1A 6 N. ispahanica 1.83 0.35 0.64 1.18 16.44 1.83 35.28 2A 7 N. mahanensis 1.34 0.43 0.58 0.78 12.29 1.37 42.79 1A 8 N. pungens 1.89 0.35 0.57 1.08 14.86 1.65 34.60 2A 56 Maryam Hasaninejad, Ziba Jamzad, Saeid Afsharzadeh, Hojjatollah Saeidi Figure 1A-H. Somatic chromosomes of Nepeta (A- N. cephalotes; B- N. denudata; C- N. eremokosmos; D- N. gloeocephala; E- N. hormoz- ganica; F- N. ispahanica; G- N. mahanensis; H- N. pungens). Scale bars: 10 µm. 57Chromosome counts of eight Iranian endemic species of Nepeta L. (Lamiaceae) Figure 2A-H. Idiograms of the karyotypes of Nepeta (A- N. cephalotes; B- N. denudata; C- N. eremokosmos; D- N. gloeocephala; E- N. hor- mozganica; F- N. ispahanica; G- N. mahanensis; H- N. pungens). Scale bars: 10 µm. 58 Maryam Hasaninejad, Ziba Jamzad, Saeid Afsharzadeh, Hojjatollah Saeidi (Tables 2, 3; Figure 2C). The chromosome length is 1.3 to 1.9 μm. This is the first chromosome count for this species. Nepeta gloeocephala is an endemic species found in few localities in central Iran. Chromosome number of 2n = 2x = 18 and somatic chromosome count in this species showed an x = 9 (Figure 1D). Karyotype was included 6 pairs of metacentric and 3 pairs of submeta- centric chromosomes in this specie (Tables 2, 3; Figure 2D). The chromosome length varied from 1.05 to 1.98 μm. This is the first chromosome number reported for this taxon. Nepeta hormozganica is an annual species from Saharo-Sindian region, growing in south Iran. The dip- loid chromosome number of 2n = 18 was counted in this species (Figure 1E). Five chromosome pairs were meta- centric and four pairs were submetacentric (Tables 2, 3; Figure 2E). The chromosome length was in the range of 1.1 to 1.7 μm. The chromosome number of this species is reported here for the first time. Nepeta ispahanica is a regional endemic annual species growing in west, northeast, central, south, and southeast of Iran. It is also distributed in Afghanistan. The studied specimens showed a diploid chromosome number of 2n = 2x = 18 (Figure 1F) and basic chromo- some number of x = 9. N. ispahanica had 7 pairs of sub- metacentric and 2 pairs of metacentric chromosomes (Tables 2, 3; Figure 2F). The chromosome length ranged from 1.4 to 2.2 μm. This is the first chromosome count for this species. Nepeta mahanensis is a narrow endemic annual spe- cies. This species grows in a limited geographical area in Kerman province. Chromosome number in this species was 2n = 18 (Figure 1G). The karyotype was formed of 9 pairs of metacentric chromosomes (Table 2, 3; Figure 2G). The chromosome length varied from 1.02 to 1.74 μm. This is the second report on the chromosome num- bers of this species. The result of this study is in agree- ment with the previous report conducted by Payandeh et al. (2015) for N. mahanensis (x = 9; 2n = 18). Nepeta pungens is a regional endemic species with wide distribution in central, northwest, west, northeast and southwest of Iran, Afghanistan, Turkmenistan and Central Asia. Nepeta pungens (2n = 2x = 18) had 7 pairs of submetacentric and 2 pair of metacentric chromo- somes (Figure 1 H). The chromosome length was in the range of 1.1 to 2.1μm. This is the second report on the chromosome numbers of this species (Table 2, 3; Figure 2 H). However, the result of this study was not in agree- ment with the previous report conducted by Kharazian et al. (2013) for the N. pungens (x = 11; 2n = 22). DISCUSSION According to the Index to Plant Chromosome Num- bers (IPCN, http://w w w.tropicos.org/Project/IPCN) (Goldblatt and Johnson 1979-2017), in Lamiaceae, the chromosome numbers vary from 2n = 10 to 2n = 240 in different genera and species. Alloplipoid and autop- loid changes can be an important reason for this diver- sity. Extensive cytological studies of the different gen- era, including Thymus L., Ajuga L., Lamium L., Salvia L., Scutellaria L. and Elsholtzia Willd. had revealed the presence of diploid, tetraploid, hexaploid and octaploid species in the family Lamiaceae (Rather et al. 2018). The chromosome numbers together with other fac- tors can alter breeding strategy in plants (Fehr 1991; Contreras and Ruter 2011). Genome size can be esti- mated by measuring chromosomal data. Therefore, chro- mosome size is directly related to evolution (Mehra and Bawa 1972; Contreras and Ruter 2011; Esra et al. 2020). The results of our study show that the examined species have 2n = 18 chromosome numbers and the basic chromosome numbers are x = 9. Different researchers have suggested x = 8, 9 and 17 as the most common primary and secondary base num- bers for the genus Nepeta (Gill 1972, 1979; Aryavand 1977; Saggoo 1983; Bir and Saggoo, 1984; Hasaninejad et al. 2020). The previous studies support the results of our study (Kaczmarek 1957; Gill 1979, 1984; Ghaffari and Kelich 2006; Saggoo et al. 2011; Kharazian et al. 2013; Payandeh et al. 2015; Akbarpur Mamagani et al. 2016; Hasaninejad et al. 2020), reporting the base chromo- some number, x = 9 for Nepeta as a common number. The studied species in this research had small chro- mosomes according to the classification of Lima-De- Faria (1980), with mean chromosome lengths (CLm) ranging from 1.37 to 1.83 μm (Table 3). Whereas Baden (1983) argued that the karyotype details studies are dif- ficult because of the small size of chromosomes. Although the chromosome number of all stud- ied Nepeta species was the same (2n = 18), their karyo- type formulas were different, 9 sm of N. cephalotes and N. eremokosmos and 9m of N. mahanensis and 8sm+m, 6m+3sm, 5m+4sm, 7sm+2m and 7sm+2m of N. denu- date, N. gloeocephala, N. hormozganica, N. ispahanica and N. pungens, respectively. Baden (1983) reported the metacentric and sub- metacentric karyotype formula for N. sibthorpii Benth. and Kharazian et al. (2013), suggested the metacentric, sub-metacentric and metacentric point karyotype for- mula, which confirms our results. N. cephalotes is distinguished by having the high- est AR and the lowest CI values, and N. mahanensis by 59Chromosome counts of eight Iranian endemic species of Nepeta L. (Lamiaceae) having lowest AR, TCL and CL values, N. isphanica by having the highest TCL and CL values; N. denudata by having the lowest AR value (Table 2). It was found that all studied Nepeta species are in classes 1A, 2A and 3A based on Stebbins classification. 3A species are more asymmetric or more advanced than class 1A species. Thus, N. cephalotes and N. denudate are more symmetric and N. hormozganica and N. mahan- ensis are more asymmetric. This study suggested that TF% varied from 31.41 to 42.79. N. mahaensis was dis- tinguished by having the highest TF%, N. cephalotes by having the lowest TF% (Table 3). Kharazian et al. (2013) reported 2n = 22 for N. pun- gens, which is in line with the previously reported base numbers (Chen et al. 2018). In our study, the chromo- some number of N. pungens was counted 2n = 18, which is contrary to the previous reports. In this case of vari- ability, Gill (1979) reported the intra-specific races for some of Nepeta species, or the case may be incorrect identification of the studied specimen. Moreover, N. mahanensis was reported with 2n = 18 by Payandeh et al. (2015). In our report, the basic chro- mosome number is x = 9, which is fully in agreement with the results of our study for this species. All studied species are either Iranian or regional endemics and showed chromosome numbers of x = 9. Srivastava (2012) believed that, there is a probability of base number x = 9 at the phylogenetic root of the Nepe- ta, but annual species are considered to be the most evolved species in the genus (Jamzad et al. 2003b). As it is shown here for four annual Nepeta species (N. ispa- hanica, N. mahanensis and N. hormozganica), the base number is x = 9, which does not support Sirvastava’s idea. Previous literatures indicate that the genus Nepeta has a heterogeneous set of chromosome numbers. Con- sidering the close phylogenetic relationship among the studied species (Jamzad et al. 2003b), it may be inferred that the similar chromosome numbers approve their close phylogenetic relationships. Future comprehensive cytotaxonomic studies and inferring the results on the Nepeta phylogenetic tree may elucidate the evolutionary trends in the genus and lead us to better understanding of the evolutionary values of chromosome numbers. Most frequent count of the base chromosome num- ber in Nepeta is x = 9. Whereas, in most species of Lamiaceae, the base chromosome numbers are different. The chromosome number as 2n = 30 is typical in some genera including Origanum, Clinopodium L., Micromer- ia Benth., Satureja L., Thymus etc. (Esra et al. 2020). In genus Caryopteris Bunge the chromosome number was reported as 2n = 26 and x = 16 in genus Chelonopsis Miq. was (Chen et al. 2018). Huang et al. (1996) reported that the basic chromosome number was x = 8 in Erio- phyton Benth.. Phlomoides Moench is known to have a base chromosome number of x = 11, which is distinct from the base number x = 10 in Phlomis L. (Fang et al. 2007). Scutellaria is one of the largest genera within Lamiaceae that also has a complex chromosomal varia- tion as at least 14 different chromosome numbers have been found for the genus 2n = 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 44, 60, 88. The basic chromosome num- ber x = 13 in East and Southeast Asia, x = 12 in America and x = 11 in North Africa and Eurasia are predominat- ing (Ranjbar and Mahmoudi 2013). Whereas all studied species were homoploid, but according to previous studies (Gill 1972; Bir and Saggoo 1979, 1984; Saggoo 1983; Chen et al. 2018; Hasaninejad et al. 2020), aneuploidy and dysploidy changes had role in taxa evolution. Variation in the chromosome numbers is one of the important factors in the process of evolu- tion (Srivastava 2012). However, all these species were not affected by chromosome number variation. The results of this study provided a considerable contribution to the cytotaxonomic data of the genus Nepeta. 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