Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 127-143, 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-579 Citation: E. Martin, T. Dirmenci, T. Ara- baci, T. Yazici, T. Özcan (2020) Karyo- type studies on the genus Origanum L. (Lamiaceae) species and some hybrids defining homoploidy. Caryologia 73(2): 127-143. doi: 10.13128/caryologia-579 Received: July 31, 2019 Accepted: April 27, 2020 Published: July 31, 2020 Copyright: © 2020 E. Martin, T. Dir- menci, T. Arabaci, T. Yazici, T. Özcan. 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. Karyotype studies on the genus Origanum L. (Lamiaceae) species and some hybrids defining homoploidy Esra Martin1, Tuncay Dirmenci2,*, Turan Arabaci3, Türker Yazici2, Taner Özcan2 1 Department of Biotechnology, Faculty of Science, Necmettin Erbakan University, Konya, Turkey 2 Department of Biology Education, Necatibey Education Faculty, Balıkesir University, Balıkesir, Turkey 3 Department of Pharmaceutical Botany, Faculty of Pharmacy, İnönü University, Malatya, Turkey *Corresponding author. E-mail: dirmenci@balikesir.edu.tr Abstract. In this study, chromosome numbers and structures of some Origanum L. taxa growing in Turkey were identified. Using the Image Analysis System, the comple- ments of plant accessions belonging to eight sections, namely Amaracus (Gleditsch) Vogel, Anatolicon Benth., Brevifilamentum Ietsw., Longitubus Ietsw., Chilocalyx (Briq.) Ietsw., Majorana (Miller) Ietsw., Origanum, and Prolaticorolla Ietsw. were determined, by classification with the cytogenetic method. The chromosome number of all taxa except O. sipyleum L. (2n = 28) and O. rotundifolium Boiss. (2n = 28) is 2n = 30. In addition, the hybrids and their parental species have 2n = 30 chromosome numbers. Also, the smallest chromosome length is 0.32 μm in O. sipyleum. The largest chromo- some length is 2.02 μm in O. minutiflorum O.Schwarz & P.H.Davis. The smallest total haploid length is 10.08 μm in O. vulgare subsp. hirtum (Link) A.Terracc. and the larg- est value is 22.00 μm in O. haussknechtii Boiss. The smallest mean length is 0.33 μm in O. vulgare L. subsp. hirtum and O. saccatum P.H.Davis. The largest mean length is 0.74 μm in O. sipyleum L. The chromosome numbers obtained in this study support the speciation of Origanum members via homoploid hybridization. Finally, the somatic chromosome numbers of 10 taxa (including 2 hybrids), chromosome measurements of 22 taxa (including 2 hybrids), and ideograms of 19 taxa (including 2 hybrids) were for the first time performed in this study. Keywords: chromosome, hybrid, karyotype, Lamiaceae, Origanum. INTRODUCTION The genus Origanum L. is placed in the family Lamiaceae, subfamily Nepetoideae, tribe Mentheae, subtribe Menthineae, contains 43 species and 20 hybrids (Ietswaart 1980, 1982; Govaerts et al. 2013; Dirmenci et al. 2018a, 2018b, 2019). Approximately, 21 species (24 taxa, including 13 endemic) and 128 Esra Martin et al. 13 hybrids are reported from the Turkey (Ietswaart 1982; Davis et al. 1988; Duman 2000; Dirmenci et al. 2018a, 2018b, 2019). Majority of the species are found in Medi- terranean basin and 75% of them are only found in East- ern Mediterranean region. Some species are distributed in the Middle East (Syria and Lebanon), North Africa (Algeria, Libya and Morocco) and the Canary Islands (Ietswaart 1980, 1982). The genus Origanum contains ten sections, eight of them are distributed in Turkey (Iet- swaart 1980). The species are mostly distributed along the Taurus Mountains in Turkey. Recently, the use of the Image Analysis System in karyotyping of plant taxa having small and indistin- guishable somatic chromosomes (Fukui 1986, 1998; Fukui and Iijima 1991; Iijima et al. 1991) has drawn the attentation for study chromosome morphology in Ori- ganum. Literature studies belonging to the genus Ori- ganum have revealed that there are too few karyotype analyses of this genus. The lack of sufficient data on the karyomorphology of the genus is probably due to the small size of its chromosomes. The chromosome num- bers are 2n = 28, 30 and 32 (Ietswaart 1980; Gill 1970, 1981, 1981a 1984; Saggoo 1983; Magulaev 1984; Ayy- angar and Vembu 1985; Krasnikov and Schaulo 1990; Wentworth et al. 1991; Khatoon and Ali 1993; Stepanov and Muratova, 1995; Dobea et al. 1997; Kıtıkı 1997; Balım and Kesercioğlu 1998; Albers and Pröbsting 1998; Lövkvist and Hultgård 1999; Yıldız and Gücel 2006; Dir- menci et al. 2018a, 2018b, 2019) in the genus Origanum. The hybridization is a widespread phenomenon among Origanum species. It usually occurs in the regions where the distribution of the species overlaps. The over- lappings can occur in natural habitat or botanical gar- dens. Because of interspecific hybridization, so far 19 hybrids have been identified in the genus. It was reported that the chromosome number of some hybrids was 2n = 30 (Bakha et al. 2017; Dirmenci et al. 2018a, 2018b, 2019). These results support the idea that the origin of the hybridization in Origanum is probably homoploidy. The main aims of this study are to contribute to the cytotaxonomy of Origanum with the following marks: 1- to define the karyotypes of some Origanum specimens for the first time, 2- to verify the homoploidy in the Ori- ganum genus, 3- to present the chromosomal differences of the Origanum species, 4- to observe the chromosome structure of the hybrids and their parents in Origanum. MATERIALS AND METHODS Pla nt materia ls used in t his study were col- lected between 2013 and 2017 and collected speci- mens were deposited in Balıkesir University. Voucher numbers and collection information of the exam- ined specimens were given in the appendix section. The seeds were germinated at room temperature. All kar yological obser vations were carried out on root tips. Firstly, the root tips were pretreated for 16 h in α-monobromonaphthalene at 4°C, fixed in 3:1 abso- lute alcohol/glacial acetic acid, then hydrolyzed with 1 N HCl for 12 min at room temperature and stained with 2% aceto-orcein for 3 h at room temperature. Stained root tips were squashed in a drop of 45% ace- tic acid, and permanent slides were made by mounting in Depex (Martin et al. 2016). The five metaphase plates from each species were used to obtain chromosomal data using an Olympus microscope and the chromo- somal data were measured with software image analy- ses (Bs200ProP). Chromosome lengths were made by nomenclature following Levan et al. (1964). RESULTS Sect. Amaracus (Gleditsch) Benth. The chromosome numbers of Origanum aylini- ae Dirmenci & Yazıcı, O. boissieri Ietsw., O. saccatum P.H.Davis, and O. solymicum P.H.Davis are 2n = 30 (Fig- ure 1A-D, Table 1). The smallest chromosome length is 0.39 μm in O. saccatum (no. TD4522). The largest chro- mosome length is 1.94 μm in O. boissieri (no. TD4319). The smallest total haploid length is 10.13 μm in O. sac- catum (no. TD4522), and the largest value is 19.74 μm in O. boissieri (no. TD4319). The chromosome morphologies of Origanum aylin- iae, O. boissieri, O. saccatum, and O. solymicum are described for the first time in this study. Sect. Anatolicon Benth. The chromosome numbers of Origanum hypericifoli- um P.H.Davis is 2n = 30, and O. sipyleum L. has two dif- ferent counts as 2n = 28 and 2n = 30 (Figure 1E-F, Table 1). The smallest chromosome length is 0.32 μm in O. sipyleum (no. TD4308). The largest chromosome length is 2.00 μm in O. sipyleum (no. TD4517). The small- est total haploid length is 10.46 μm in O. sipyleum (no. TD4522) and the largest value is 20.98 μm in O. sipyle- um (no. TD4308). The chromosomes morphologies of Origanum hypericifolium are described for the first time. The chromosome number of Origanum sipyleum is mostly 2n = 30 in a biosystematic study performed by 129Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy Kıtıkı 1997 (Table 2). The diploid number of no. 4308 is 2n = 28 as different from the literature (Table 2). While the number of chromosomes of O. sipyleum is included in the literature, the chromosome morphologies of the species have been demonstrated for the first time in this study. Sect. Brevifilamentum Ietsw. The chromosome numbers of Origanum acutidens (Hand.-Mazz.) Ietsw. (no. TD4956a), O. brevidens Bornm. Dinsm. (no. TD4331), O. haussknechtii Boiss. (no. TD2824), O. husnucan-baseri H.Duman, Aytaç & A.Duran, (no. TD4528), and O. leptocladum Boiss. (no. TD4345), 2n = 30 (Figure 1G-K, Table 1). The chromo- Figure 1A-I. Somatic metaphase chromosomes of Origanum taxa: A) O. ayliniae (TD4516) B) O. boissieri (TD4319); C) O. saccatum (TD4732); D) O. solymicum (TD4347); E) O. hypericifolium (TD4357); F) O. sipyleum (TD4727); G) O. acutidens (TD4956a); H) O. brevi- dens (TD4331); I) O. haussknechtii (TA2824). Scale bar: 10 µm. 130 Esra Martin et al. some number of O. rotundifolium Boiss. (no. TD3943), 2n = 28 (Figure 1L, Table 1). The smallest chromosome length is 0.49 μm in O. leptocladum. The largest chromo- some length is 2.00 μm in O. haussknechtii. The smallest total haploid length is 14.67 μm in O. brevidens and the largest value is 22.00 μm in O. haussknechtii. The chromosomes morphologies of Origanum acutidens, O. brevidens, O. haussknechtii, O. husnu- can-baseri, O. leptocladum, and O. rotundifolium are described for the first time. Sect. Chilocalyx (Briq.) Ietsw. The chromosome numbers of Origanum bilgeri P.H.Davis (no. TD4343), O. minutiflorum O.Schwarz & P.H.Davis (no. TD4348) and O. vogelii Greuter & Burdet (no. TD4509) are 2n = 30 (Figure 1M-O, Table 1). The smallest chromosome length is 0.38 μm in O. bilgeri. The largest chromosome length is 2.02 μm in O. minutiflorum. The smallest total haploid length is 14.25 μm in O. bilgeri and the largest value is 19.01 μm in O. minutiflorum. Table 1. Chromosome counts and size of species and hybrids of Origanum determined in the study. Section Taxa Chromosome Number (2n) Chromosome length (µm) min-max Total haploid length (µm) Mean chromosome length (µm) Amaracus O. ayliniae TD 4516 30 0.83-1.72 18.35 0.61 O. boissieri TD 4319 O. boissieri TD 4501 30 30 0.78-1.94 0.47-1.61 19.74 14.48 0.65 0.48 O. solymicum TD 4347 O. solymicum TD 4520 30 30 0.45-1.59 0.40-1.26 16.22 12.16 0.54 0.40 O. saccatum TD 4342 O. saccatum TD 4522 O. saccatum TD 4732 30 30 30 0.53-1.75 0.39-1.12 0.60-1.46 17.17 10.13 14.77 0.55 0.33 0.49 Anatolicon O. hypericifolium TD 4357 30 0.34-1.36 13.80 0.40 O. sipyleum TD 4308 O. sipyleum TD 4352 O. sipyleum TD 4534 O. sipyleum TD 4517 O. sipyleum TD 4727 28 30 30 30 30 0.79-2.00 0.53-1.59 0.39-1.52 0.32-1.11 0.49-1.08 20.98 14.19 13.32 10.46 11.26 0.74 0.47 0.44 0.34 0.37 Brevifilamentum O. brevidens TD 4331 O. haussknechtii TD 2824 O. husnucan-baseri TD 4528 O. leptocladum TD 4345 O. rotundifolium TD 3943 30 30 30 30 28 0.50-1.63 0.99-2.00 0.56-1.79 0.49-1.63 0.57-1.82 14.67 22.00 14.88 15.27 16.497 0.49 0.73 0.49 0.50 0.58 Chilocalyx O. bilgeri TD 4343 O. minutiflorum TD 4348 30 30 0.38-1.58 0.69-2.02 14.25 19.01 0.47 0.63 Longitubus O. amanum TD 4514-a 30 0.73-1.91 16.92 0.56 Majorana O. majorana TD 3984 O. majorana TD 4356 O. majorana TD 4346 30 30 30 0.50-1.58 0.43- 1.84 0.43-1.54 17.25 17.35 14.44 0.57 0.57 0.48 O. onites TD 4355 O. onites TD 4725 O. onites TD 4532 30 30 30 0.50-1.77 0.63-1.27 0.43-1.26 15.93 15.14 13.45 0.53 0.50 0.44 O. syriacum subsp. bevanii TD 4336 O. syriacum subsp. bevanii TD 4330 30 30 0.53- 1.58 0.49-1.13 16.26 11.02 0.54 0.36 Origanum O. vulgare subsp. hirtum TD 4733 O. vulgare subsp. hirtum TD 4722 O. vulgare subsp. hirtum TD 4359 30 30 30 0.58-1.14 0.49-1.22 0.40-1.07 13.22 13.32 10.08 0.44 0.44 0.33 O. vulgare subsp. vulgare TD 4688 30 0.56-1.18 12.73 0.42 Prolaticorolla O. laevigatum TD 4497 30 0.45-1.96 16.34 0.54 Hybrids O. × intermedium TD 4726 30 0.46-1.14 11.62 0.38 O. × adae TD 4518 30 0.72-1.40 15.47 0.51 131Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy Table 2. Chromosome counts of species and hybrids of Origanum according to references. Section Species Chromosome numbers (2n) References Amaracus O. ayliniae 30 (Dirmenci et al. 2018a) O. boissieri 30 (Dirmenci et al. 2018b, Kıtıkı et al., 1997) O. calcaratum. 30 (Von Bothmer, 1970) O. dictamnus 30 (Lepper, 1970) O. solymicum 30 (in this study; Kıtıkı et al., 1997) O. saccatum 30 (in this study; Kıtıkı et al., 1997) Anatolicon O. hypericifolium 30 (in this study) O. sipyleum 30 28 (Kıtıkı et al., 1997) (in this study) Brevifilamentum O. acutidens 30 (Dirmenci et al. 2019) O. brevidens 30 (in this study) O. haussknechtii 30 (in this study) O. husnucan-baseri 30 (in this study) O. leptocladum 30 (in this study) O. rotundifolium 28 (in this study) Chilocalyx O. bilgeri 30 (in this study) O. minutiflorum 30 (in this study) O. vogelii 30 (in this study ) Elongatispica O. elongatum 30 (Bastida and Talavera, 1994; Bakha et al., 2017) O. grosii 30 (Bakha et al., 2017) Longitubus O. amanum 30 (in this study; Lepper 1970) Majorana O. majorana 30 (in this study; Harriman, 1975; Májovský, 1978; Fernandes and Leitão, 1984; Balim and Kesercioğlu, 1998) O. onites 30 (in this study; Von Bothmer, 1970; Miege and Greuter, 1973; Ietswaart, 1980; Montmollin, 1986; Kıtıkı et al., 1997; Bakha et al., 2017) O. syriacum subsp. bevanii 30 (in this study; Balim and Kesercioğlu, 1998; Yildiz and Gücel, 2006) Origanum O. vulgare 28 (Magulaev, 1984) O. vulgare 30 (Skalinska et al., 1971; Krasnikov and Schaulo, 1990; Stepanov and Muratova, 1995; Lövkvist and Hultgård, 1999) O. vulgare 32 (Ayyangar and Vembu, 1985) O. vulgare subsp. hirtum 30 (Strid and Franzen, 1981; Markova and Goranova, 1995; Dirmenci et al. 2018b) O. vulgare subsp. gracile 30 (Astanova, 1981; Dirmenci et al. 2019) O.vulgare subsp. virens 30 (Fernandes and Leitão, 1984; Pastor et al. 1990; Bakha et al., 2017) O. vulgare subsp. viride 30 (in this study; Strid and Franzen 1981) O. vulgare subsp. viridulum 30 (Strid and Franzen, 1981; Markova and Goranova, 1995) O. vulgare subsp. vulgare 28 (Magulaev, 1984) O. vulgare subsp. vulgare 30 (in this study; Gill, 1981, 1981a; Saggoo, 1983; Bir and Saggoo, 1984; Gill, 1984; Krasnikov and Schaulo, 1990; Wentworth et al., 1991; Khatoon and Ali, 1993; Stepanov and Muratova, 1995; Dobea et al., 1997; Albers and Pröbsting, 1998; Lövkvist and Hultgård, 1999) O. vulgare subsp. vulgare 32 (Ayyangar and Vembu, 1985) Prolaticorolla O. compactum 30 (Bakha et al., 2017) O. laevigatum 30 (Balim and Kesercioğlu, 1998) Hybrids O. × adae 30 (Dirmenci et al. 2018a) O. × font-queri 30 (Bakha et al., 2017) O. × haradjanii 30 (in this study) O. × intermedium 30 (in this study) O. × munzurense 30 (Dirmenci et al. 2019) O. × sevcaniae 30 (Dirmenci et al. 2018b) 132 Esra Martin et al. The chromosome morphologies of O. bilgeri, O. minutiflorum, and O. vogelii are described for the first time. Sect. Longitubus Ietsw. Sect. Longitubus contains only one species. The chromosome number of Origanum amanum Post (no. TD4514a) is 2n = 30 (Figure 1P, Table 1). Iet- swaart (1980) reported that the chromosome number of O. amanum was 2n = 30 (Table 2). The chromosome Figure 1J-R. Somatic metaphase chromosomes of Origanum taxa: J) O. husnucan-baseri (TD4528); K) O. leptocladum (TA4345); L) O. rotundifolium (TD3943); M) O. bilgeri (TD4343); N) O. minutiflorum (TD4348); O) O. vogelii (TD4509); P) O. amanum (TD4514a); Q) O. majorana (TD3984); R) O. onites (TD4725). Scale bar: 10 µm. 133Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy lengths range from 0.73 to 1.91 µm. The chromosome morphologies of O. amanum are described for the first time. Sect. Majorana (Mill.) Benth. The chromosome numbers of Origanum majorana L. (no. TD3984), O. onites L. (no. TD4725), and O. syri- acum L. subsp. bevanii (Holmes) Greuter & Burdet (no. TD4336) are 2n = 30 (Figure 1Q-S, Table 1). The small- est chromosome length is 0.43 μm in O. majorana and O. onites (no. 4356, 4346, and 4532). The largest chromo- some length is 1.84 μm in O. majorana (no. 4356). The smallest total haploid length is 11.02 μm in O. syriacum subsp. bevanii (no. 4330), and the largest value is 17.35 μm in O. majorana (no. 4356). It was reported that the chromosome number of Origanum onites and O. majorana was 2n = 30 (Iet- swaart 1980; Kıtıkı 1997; Balım and Kesercioğlu 1998) (Table 2). The number of chromosomes we obtained overlapped with the literature. In addition to the chro- mosome numbers of taxa, chromosome measurements are also introduced to the scientific world. Sect. Origanum The chromosome numbers of Origanum vulgare L. subsp. gracile (K.Koch) Ietsw. (no. TD4821), O. vul- gare subsp. hirtum (Link.) A.Terracc. (no. TD4507), O. vulgare subsp. viridulum (Matrin-Dones) Nyman (no. TD4662a), and O. vulgare subsp. vulgare (no. TD4688) are 2n = 30 (Figure 1T-W, Table 1). The smallest chromo- some length is 0.40 μm in O. vulgare subsp. hirtum (no. TD4359). The largest chromosome length is 1.22 μm in O. vulgare subsp. hirtum (no. TD4722). The smallest total haploid length is 10.08 μm in O. vulgare subsp. hirtum (no. TD4359) and the largest value is 13.32 μm in O. vul- gare subsp. hirtum (no. TD4722). It was reported that the chromosome numbers of Origanum vulgare, O. vulgare subsp. hirtum, O. vulgare Figure 1S-X. Somatic metaphase chromosomes of Origanum taxa: S) O. syriacum subsp. bevanii (TD4336); T) O. vulgare subsp. gracile (TD4821); U) O. vulgare subsp. hirtum (TD4507); V) O. vulgare subsp. viridulum (TD4662a); W) O. vulgare subsp. vulgare (TD4688); X) O. laevigatum (TD4497). Scale bar: 10 µm. 134 Esra Martin et al. subsp. viride, and O. vulgare subsp. viridulum are 2n = 28, 30 and 32 (Ietswaart 1980; Strid and Franzen 1981; Gill 1981a, 1984; Saggoo 1983; Bir and Saggoo 1984; Magulaev 1984; Ayyangar and Vembu 1985; Krasnikov and Schaulo 1990; Wentworth et al. 1991; Khatoon and Ali 1993; Stepanov and Muratova 1995; Markova and Goranova 1995; Dobea et al. 1997; Kıtıkı 1997; Albers and Pröbsting 1998; Lövkvist and Hultgård 1999; Dir- menci et al. 2018b) (Table 2). The chromosome morphologies of Origanum vulgare subsp. gracile are described for the first time. In addi- tion, the detailed chromosome lengths are given for O. vulgare subsp. gracile, O. vulgare subsp. hirtum, O. vul- gare subsp. viridulum, and O. vulgare subsp. vulgare. Sect. Prolaticorolla Ietsw. Sect. Prolaticorolla contains only one species in Turkey. The chromosome number of Origanum laevigatum Boiss. is 2n = 30 (Figure 1X, Table 1). The chromosome lengths range from 0.45 to 1.96 µm (no. TD4497). The chromo- some number of O. laevigatum is given for the first time. The chromosome counts and morphologies of the hybrids and their parents Origanum × adae Dirmenci & Yazıcı (O. ayliniae × O. sipyleum) The chromosome number of Origanum × adae is 2n = 30 (Table 1). The chromosome lengths range from 0.72 to 1.40 µm. The total haploid length is 15.47 µm (no. TD4518). The chromosome lengths of O. ayliniae range from 0.83 to 1.72 µm. The total haploid length is 18.35 µm (no. TD4516). The chromosome lengths of O. sipyl- eum range from 0.32 to 1.11 µm. The total haploid length is 10.46 µm (no. TD4517). According to the karyological results, the chromo- some numbers of Origanum × adae, O. ayliniae, and O. sipyleum are similar with n = 15 for the haplotype (Fig- ure 2A-C). Karyological analyses support the idea that Origanum ×adae, is a natural hybrid, is generated from crossed homoploidy of O. ayliniae and O. sipyleum. The hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes) (Dirmenci et al. 2018a). The chromosome morphologies of Origanum ×adae and O. ayliniae, and O. sipyleum are described for the first time. Origanum × haradjanii Rech.f. (O. syriacum subsp. bevanii × O. laevigatum) The chromosome number of Origanum × haradja- nii is 2n = 30 (no. TD4335). The chromosome lengths of O. syriacum subsp. bevanii range from 0.53 to 1.58 µm. The total haploid length is 16.26 µm. (no. TD4336). The chromosome lengths of O. laevigatum range from 0.45 to 1.96 µm. The total haploid length is 16.34 µm (no. TD4497). According to the karyological results, the chromo- some numbers of Origanum × haradjanii, O. laevigatum, and O. syriacum subsp. bevanii are similar with n = 15 for the haplotype (Figure 2D-F). Karyological analyses support the idea that O. × haradjanii, is a natural hybrid, is generated from crossed homoploidy of O. syriacum subsp. bevanii and O. laevigatum. The hybrid is gener- ated by homoploid hybridization (all taxa have 2n = 30 chromosomes). The chromosome number of O. × haradjanii is first reported. In addition, the chromosome morphologies of O. syriacum subsp. bevanii and O. laevigatum are described for the first time. Origanum × intermedium P.H.Davis (O. onites × O. sipyleum) The chromosome number of Origanum × interme- dium is 2n = 30. The chromosome lengths range from 0.46 to 1.14 µm. The total haploid length is 11.62 µm (no. TD4726). The chromosome lengths of O. sipyleum range from 0.49 to 1.08 µm. The total haploid length is 11.26 µm (no. TD4727). The chromosome lengths of O. onites range from 0.49 to 1.08 µm. The total haploid length is 11.26 µm (no. TD4725). According to the karyological results, the chromo- some numbers of Origanum × intermedium, O. onites, and O. sipyleum are similar with n = 15 for the haplo- type (Figure 2G-I). Karyological analyses support the idea that O. × intermedium, is a natural hybrid, is gener- ated from crossed homoploidy of O. onites and O. sipyle- um. The hybrid is generated by homoploid hybridization (all taxa have 2n = 30 chromosomes). The chromosome number and morphologies of Ori- ganum × intermedium are described for the first time. Origanum × sevcaniae Dirmenci, Arabacı & Yazıcı (O. vogelii × O. vulgare subsp. hirtum) The chromosome number of Origanum × sevcaniae is 2n = 30 (no. TD4508). According to the karyological results, the chromosome numbers of O. × sevcaniae, O. vogelii and O. vulgare subsp. hirtum are similar with n = 15 for the haplotype (Figures 2J-L). Karyological analy- ses support the idea that Origanum × sevcaniae, is a nat- ural hybrid, is generated from crossed homoploidy of O. vogelii and O. vulgare subsp. hirtum. The hybrid is gener- ated by homoploid hybridization (all taxa have 2n = 30 chromosomes) (Dirmenci et al. 2018b). 135Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy Figure 2A-L. Somatic metaphase chromosomes of hybrids and their parents: A) O. × adae (TD4518); B) O. ayliniae (TD4516); C) O. sipyl- eum (TD4517); D) O. × haradjanii (TD4335); E) O. syriacum subsp. bevanii (TD4336); F) O. laevigatum (TD4497); G) O. × intermedium (TD4726); H) O. onites (TD4725); I) O. sipyleum (TD4727); J) O. × sevcaniae (TD4508); K) O. vogelii (TD4509); L) O. vulgare subsp. hir- tum (TD4507). Scale bar: 10 µm. 136 Esra Martin et al. Figure 3A-N. Ideograms of Origanum taxa: A) O. amanum (TD4514a); B) O. bilgeri (TD4343); C) O. boissieri (TD4319); D) O. brevidens (TD4331); E) O. haussknechtii (TA2824); F) O. husnucan-baseri (TD4528); G) O. hypericifolium (TD4357); H) O. leptocladum (TD4345); I) O. majorana (TD3984); J) O. minutiflorum (TD4348); K) O. rotundifolium (TD3943); L) O. saccatum (TD4732); M) O. solymicum (TD4347); N) O. vulgare subsp. vulgare (TD4688). Scale bar: 10 µm. 137Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy The ideograms, which were drawn by Software Image Analysis (Bs200ProP) loaded on a personal com- puter are given in Figure 3A-N and Figure 4A-F. DISCUSSION Counting of chromosomes has been a very useful approach (particularly at the generic level) for research- ers investigating evolutionary relationships (Levin and Wilson 1976; Stace 1991; Goldblatt 2007; Guerra 2008; Stuessy 2009; Contreras and Ruter 2011). Indeed, the chromosome numbers can affect inbreeding depres- sion and the potential for introgression of traits through interspecific hybridization, among other factors that can alter breeding strategy (Fehr 1991; Contreras and Ruter 2011). Measuring of chromosome size correlated with evo- lutionary age provides to estimate genome size using the chromosomal data (Mehra and Bawa 1972; Contreras and Ruter 2011). According to the Index to Plant Chromosome Num- bers (IPCN, http://w w w.tropicos.org/Project/IPCN) (Goldblatt and Johnson 1979-2017) and Chromosome Counts Database (CCDB, version 1.45, http://ccdb.tau. ac.il/home/) (Rice et al. 2015), there are the chromosome numbers of more than 1500 taxa of 140 genera belonging to Lamiaceae family (Chen et al. 2018). In Lamiaceae, the chromosome numbers between genera and even species are highly variable from 2n = 10 to 2n = 240. Allopoly- ploidy and autopoliploidy are the main reasons for varia- tions. The basic numbers are x = 5, 7, 8, and 10. Secondly, basic numbers can be assumed to be x = 13, 14, 15, 17, and 19 (Singh 1995; Mabberley 1997). Singh (1995) considered that x = 17 arised as the result of the combination of x = 8 and x = 9 (Miura et al. 2005). Extensive cytological stud- ies have revealed the presence of diploid, tetraploid, hexa- ploid and octoploid species in the family Lamiaceae. In family, the diversification may be attributed to the pres- ence of polyploidy and aneuploidy (Rather et al. 2018). In the genus Origanum, it was reported that the main diploid numbers were 2n = 28, 30, and 32 and the Figure 4A-F. Ideograms of hybrids and their parents: A) O. × adae (TD4518); B) O. ayliniae (TD4516); C) O. sipyleum (TD4517); D) O. × intermedium (TD4726); E) O. onites (TD4725); F) O. sipyleum (TD4727). Scale bar: 10 µm. 138 Esra Martin et al. basic number was x = 15 (IPCN, http://www.tropicos. org/Project/IPCN) (Goldblatt and Johnson 1979-2017) and Chromosome Counts Database (CCDB, version 1.45, http://ccdb.tau.ac.il/home/) (Rice et al. 2015). The chro- mosome counts of the investigated species in present and previous studies are given in Table 2. In Table 1 and Table 2, the chromosome number of all sections is 2n = 30 except some Origanum sipyl- eum specimens (because some of the specimens have 2n = 30) in sect. Anatolicon and O. rotundifolium in the sect. Brevifilamentum with 2n = 28. The smallest chro- mosome length is 0.32 μm in Origanum sipyleum (no. TD4517). The largest chromosome length is 2.02 μm in O. minutif lorum (no. TD4348). The smallest total haploid length is 10.08 μm in O. vulgare subsp. hirtum (no. TD4359) and the largest value is 22.00 μm in O. haussknechtii (no. TA2824). The smallest mean length is 0.33 μm in O. vulgare subsp. hirtum and O. saccatum (no. TD4359; TD4522, respectively). The largest mean length is 0.74 μm in O. sipyleum (no. TD4308). The chro- mosome lengths range from 0.75 to 6.00 μm. The chro- mosome lengths were measured between 0.33 and 0.74 in this karyological study of the genus Origanum. In the genus Origanum, the centromeric position could not be clearly observed because the chromosomes were general- ly very small compared to family members. On the other hand, total chromosome length could be measured. The chromosome number as 2n = 30 is typical in some genera of Lamiaceae family (Clinopodium L., Micromeria Benth., Satureja L., Thymus L. etc.). Some Thymus species have the same chromosome number (Jalas & Kaleva 1967; Vaarama 1948). In addition, some species have different basic numbers as x = 6, 7, 9, 14, 21, 27, and 24 in the genus (Darlington and Wylie 1955; Vaarama 1948). The secondary basic numbers, namely x = 14 and 15 probably originate from x = 7. The most common numbers are 2n = 28, 30, 56, and 60, while 2n = 84 and 90 are rarely. Jalas (1967) showed that although the chromosome numbers of distinct Thymus subsec- tions were different, both subsections Vulgares L. and Piperella Willk. had the same chromosome number with 2n = 30. In genus Micromeria s.str., the chromosome num- ber of most species was reported as 2n = 30 (Martin et al. 2011). On the other hand, Micromeria s.l. has various somatic chromosome numbers as 2n = 20, 22, 26, 30, 48, 50, and 60. Similarly, Clinopodium s.str. has various somatic chromosome numbers as 2n = 18, 20, 22, 24, and 48 (IPCN, http://www.tropicos.org/Project/IPCN) (Goldblatt and Johnson 1979-2017) and Chromosome Counts Database (CCDB, version 1.45, http://ccdb.tau. ac.il/home/) (Rice et al. 2015). Morton (1962) reported that the basic numbers were x = 6, 7, 8, 9, 10, 11, and 15 in Satureja. Harley and Brighton (1977) reported that the genus Mentha had var- ious chromosome counts ranging from x = 6 to x = 54. This means that we can see diploid-octoploid members in this genus. As mentioned before, the genus Origanum is a mem- ber of the Mentheae tribe and the Menthinae subtribe (Harley et al. 2004). The chromosome numbers of Men- thinae subtribe vary from 2n = 12 (Hyssopus) to 2n = 144 (Mentha). The chromosome numbers were 2n = 30 in most of the Thymbra L., Satureja, and Micromeria (s.str.) species belonging to the tribe Menthinae. In addi- tion, the chromosome numbers of some species in Thy- mus, Mentha and Prunella L. were 2n = 30 (Harley et al. 2004). Ietswaart’s hypothesis with regard to the origin of the genus Origanum suggests that Origanum might have emerged in the Pliocene, and that formerly, the tribe Sat- urejeae genera (Clinopodium sl., Micromeria, Satureja, Thymus, etc.) were probably the main genera from which Origanum could be derived (see Ietswaart 1980: pp. 7-14 and 21-24, Figure 2). When we analyzed the clos- est genera mentioned in the discussion part, homoploid hybridization was the main ploidy type in Origanum. The emergence of other sections has been diversified by hybridization between the main sections and hybridi- zation of some Thymus and Origanum species (in part including Satureja, Micromeria s.str. and Clinopodium sl. species) (Ietswaart 1982). According to this hypothesis, the genera mentioned above are important regarding chromosome number compatibility. However, Thymbra, Prunella, and Clinopodium (partly) are morphological- ly very different from Origanum species. On the other hand, Satureja, Thymus, Micromeria s.str., and Clino- podium (partly) are morphologically closer. It may have been derived from one or more of these genera and then continued to speciation via hybridization in time. The compatibility of chromosome numbers between species supports Ietswaart’s hypothesis (1980). Although the hybridization is widespread, the gen- eration of a unique and isolated hybrid lineage is prob- ably very rare. New hybrid lineages must establish repro- ductive isolation and a unique ecological niche to over- come genetic swamping and competition from parental species (Goulet et al. 2017). A new hybrid lineage may be formed through allopolyploidy or homoploid hybrid speciation. Allopolyploid lineages may be formed by the fusion of unreduced gametes, genome doubling follow- ing hybridization, or via a triploid bridge (Ramsey and Schemske 1998; Goulet et al. 2017). Homoploid hybrid speciation forms a new unreproductive hybrid lineage 139Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy with the same ploidy level with its parents (Goulet et al. 2017). It should be shown that both the hybrid ori- gin of the species and the hybridization having repro- ductive isolation to verify a homoploid hybrid spe- ciation (Schumer et al. 2014). While homoploid hybrid speciation is often concentrated on the demonstration of genetic divergence of hybrids and their origin and a pronounced ecological separation, the number of exam- ples showing the strong link between hybridization and isolated species is very few (Stebbins 1947; Chapman and Burke 2007; Schumer et al. 2014; Yakimowski and Riese- berg 2014). Hybridization is a common phenomenon in the genus Origanum (Dirmenci et al. 2018a, 2018b, 2019). The sections Amaracus, Majorana, and Origanum (O. vulgare s.l.) can be considered as the ancestral sections in the genus. Later, speciation via homoploid hybridiza- tion has an important role in speciation in the genus. This is important evidence that species hybridize easily in different sections, or that species in one section have intermediate characteristics between two different sec- tions. According to Ietswaart (1980) and our morpholog- ical observations, sect. Anatolicon species have transition characters between the species of sect. Amaracus and Origanum (Figures 5A-C, 6A-F). Thus, sect. Amaracus is characterized by its branches. The firstorder branches are usually present, but the second-order branches are seldom present. Leaves are usually leathery, spikes large and usually nodding (Figure 5A), bracts partly purple, calyces 1 or 2-lipped, corollas saccate and all stamens greatly exserted from corolla (Figure 6A, D). In the sect. Anatolicon, branches of the first order are always present, while the second-order branches are some- times present. Leaves may be leathery or not. Spikes are medium sized, usually nodding, bracts partly or slightly purple (5B), calyces usually 2-lipped, corollas not saccate and all stamens exserted from corolla (Figure 6B, E). The first- and second-order branches of the sect. Origanum are always present, while the third-order branches are usually present. Leaves are usually herbaceous, spikes dense and small to medium-sized, bracts green or pur- ple (5C), calyces actinomorphic and with 5 equal teeth, corollas not saccate and stamens subincluded or slightly protruding (Figure 6E, F). These morphological transitions are important evi- dence that the genus may have intrageneric speciation via hybridization. Besides, recent studies and the chro- mosome numbers of hybrids and ancestors occurring in this study are the cytological evidence supporting this hypothesis. Origanum hybrids originate via homoploidy. The chromosome numbers of Origanum × adae, O. × Figure 6. Flower and calyx of O. ayliniae (A, D), O. sipyleum (B, F), O. vulgare subsp. hirtum (C, F) (figures A, D, B, F from Dirmenci et al 2018a and C, F from Dirmenci et al. 2018b). Figure 5. Inflorescence of O. ayliniae (A), O. sipyleum (B), O. vulgare subsp. vulgare (C) (photo by Tuncay Dirmenci). 140 Esra Martin et al. intermedium, O. × haradjanii, O. × munzurense Sorg- er & Kit Tan, O. × sevcaniae, O. × font-queri and their parents were 2n = 30 (Bakha et al. 2017; Dirmenci et al. 2018a, 2018b, 2019). Finally, it is clear that the cy tologic f indings obtained in this study combined with the previously obtained morphological, palynological and molecu- lar studies (continuing) will contribute significantly to answering the questions about the origin and diversity of the genus Origanum. 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APPENDIX Origanum acutidens; Tunceli: Between Ovacık and Tunceli, 21-22. km, Aşağıtorunoba village, Mun- zur stream, near bridge, c. 1200 m, Dirmenci 4956a, T.Arabacı & M.Açar; Origanum amanum; Osmaniye, Düziçi district, Düldül mountain, between Mando Taşı and Hüseyinoluk Çeşme, 03.10.2015, Dirmenci 4514a & T.Arabacı; Origanum ayliniae: Aydın: Kuşadası, Dilek Yarımadası Nationa l Park, Radar, 3922 f t, 08.10.2015, Dirmenci 4516; Origanum bilgeri; Antalya: Gündoğmuş, between Hanboğazı and Oğuz yayla, 1. km, 4775 ft, 24.10.2014, Dirmenci 4343; Origanum bois- sieri: İçel: 15 km Çamlıyayla district to Saimdibi place, 6045 ft, 18.09.2014, Dirmenci 4319, T.Arabacı & T.Yazıcı; Origanum brevidens: Osmaniye: 1-2 km from Yarpuz to Yağlıpınar, 5030 ft, 19.09.2014, Dirmenci 4331, T.Arabacı & T.Yazıcı; Origanum haussknechtii; Erzincan, 15. km from Kemaliye to Arapkir, 1000 - 1100 m, 22.08.2013, T.Arabacı 2824; Origanum husnucan-baseri; Antalya: between Alanya and Hadim, 1-2 km to Gökbel Yayla, 4500 ft, 09.10.2015, Dirmenci 4528, T.Arabacı & T.Yazıcı; Origanum hypericifolium: Denizli: Honaz district, Honaz mountain, İncekara stream, 1300 m, 07.11.2014, Dirnmenci 4357 & T.Yazıcı; Origanum laevigatum: Osmaniye: Düziçi, above Kuşçu village, 03.10.2015, Dir- menci4497 & T.Arabacı; Origanum leptocladum; Kara- man: between Ermenek and Kazancı, above Görmeli vil- lage, 1. km, 870 m, 24.10.2014, Dirmenci 4345, T.Arabacı & T.Yazıcı; Origanum majorana; Mersin: Güzeldere, 252 m, 13.07.2013, Dirmenci 3984, T.Arabacı & T.Yazıcı; İçel: between Anamur and Gazipaşa, 15-20. km, 300 m 26.10.2014, Dirmenci 4356 & T.Yazıcı; Karaman: Erme- nek, 2 km from Kazancı to Abanoz Yayla, 1238 m, 24.10.2014, Dirmenci 4346, T.Arabacı & T.Yazıcı; Ori- ganum minutiflorum: Antalya: Kemer, Üçoluk, above Tülek, 4470 ft, 25.10.2014, Dirmenci 4348 & T.Yazıcı; Origanum onites: Denizli: Taşocağı, 527 m, 26.10.2014, Dirmenci 4355 & T.Yazıcı; ibid, 11.10.2015, Dirmenci 4532 & T.Yazıcı; Denizli: between Buldan and Güney, 13 from road disjunction to Güney, Dirmenci 4725 & T.Yazıcı; Denizli: Origanum rotundifolium: Artvin: between Artvin and Ardanuç, 600-700 m, 27.08.2013, Dirmenci 3943, B.Yıldız & T.Arabacı; Origanum sac- catum: Antalya, 1 km from Gündoğmuş to Çayırözü village, 3715 ft, 24.10.2014, Dirmenci 4342, T.Arabacı & T.Yazıcı; Antalya: 38 km from Alanya to Hadim, Kuşkayası, 3814 ft, 09.10.2015, Dirmenci 4522 T.Arabacı & T.Yazıcı; ibid., Dirmenci 4732 & T.Arabacı; Origa- num sipyleum: Denizli: 5 km from Serinhisar to Deni- zli, 1066 m, 19.08.2014, Dirmenci 4308; ibid., 26.10.2014, Dirmenci 4352; Denizli: Taşocağı, 1808 ft, Dirmenci 4534 & T.Yazıcı; Aydın: Kuşadası, Dilek Peninsuna National Park, Radar, 3922 ft, 08.10.2015, Dirmenci 4517; Denizli: Denizli: between Buldan and Güney, 13 from road disjunction to Güney, Dirmenci 4727 & T.Yazıcı; Origanum solymicum: Antalya: Kemer, 4 km from Kesmeboğazı to Kuzdere village, 1470 m, 25.10.2014, Dirmenci 4347 & T.Yazıcı; Antalya: Kemer, 7 km from Kesmeboğazı to Karçukuru Yayla, 1506 ft, 09.10.2015, Dirmenci 4520 & T.Yazıcı;; Origanum syriacum sub- sp. bevanii: Hatay: between Antakya and Samandağ, around St. Symeone church, 20.09.2014, Dirmenci 4336, T.Arabacı & T.Yazıcı; Osmaniye: Düziçi, between Kuşçu village and Düldül mountain, 19.09.2014, Dirmenci 4330 & T.Arabacı; Origanum vogelii; Niğde: Ulukışla, Horoz village, Fenk Boğazı, 4800 ft, 02.10.2015, Dirmenci 4509 & T.Yazıcı; Origanum vulgare subsp. gracile: K.Maraş: Göksun, between Yeşilköy and Kınıkkoz villages, 400- 450 m, 04.08.2017, Dirmenci 4821 & T.Arabacı; Tunceli: 20-21 km from Ovacık to Tunceli, c. 1200 m, 11.08.2017, Dirmenci 4958, T.Arabacı & M.Açar; Origanum vulgare subsp. hirtum: Antalya: Antalya: between Alanya and Hadim, 1 km to Gökbel Yayla, Dirmenci 4733, T.Arabacı & T.Yazıcı; Denizli: Taşocağı, 527 m, Dirmenci 4722 & T.Yazıcı; Denizli: Honaz district, North face of Honaz mountain, Arpacık Yayla road, 4160 ft, 07.11.2014, Dir- menci 4359 & T.Yazıcı; Niğde: Ulukışla, Horoz village, 143Karyotype studies on the genus Origanum (Lamiaceae) species and some hybrids defining homoploidy Fenk Boğazı, 4800 ft,, 02.10.2015, Dirmenci 4507 & T.Yazıcı; Origanum vulgare subsp. viridulum: Giresun: 33 km from Şebinkarahisar to Tamdere, nort of Eğribel pass, Dirmenci 4662a & T.Arabacı; Origanum vulgare subsp. vulgare: 22 km from Şavşat to Ardahan, Dirmen- ci 4688 & T.Arabacı; Origanum × adae: Kuşadası, Dilek Yarımadası National Park, Radar, 3922 ft, 08.10.2015, Dirmenci 4518; Origanum × haradjanii: Hatay: between Antakya and Samandağ, around St. Symeone church, 20.09.2014, Dirmenci 4335, T.Arabacı & T.Yazıcı; O. × intermedium: Denizli: Denizli: between Buldan and Güney, 13 from road disjunction to Güney, Dirmenci 4726 & T.Yazıcı; Origanum × munzurense: Tunceli: Between Ovacık and Tunceli, 21-22. km, Aşağı Toruno- ba village, Munzur stream, near bridge, c. 1200 m, Dir- menci 4957a, Arabacı & Açar; Origanum × sevcaniae: Niğde: U Ulukışla, Horoz village, Fenk Boğazı, 4800 ft, 02.10.2015, Dirmenci 4508 & T.Yazıcı. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 2 - 2020 Firenze University Press The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) Shaimaa S. Sobieh*, Mona H. Darwish Gene flow patterns reinforce the ecological plasticity of Tropidurus hispidus (Squamata: Tropiduridae) Fernanda Ito, Danielle J. Gama-Maia, Diego M. A. Brito, Rodrigo A. Torres* The technique of Plant DNA Barcoding: potential application in floriculture Antonio Giovino1,*, Federico Martinelli2,*, Anna Perrone3 Cytogenetic of Brachyura (Decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species Alessio Iannucci1, Stefano Cannicci1,2,*, Zhongyang Lin3, Karen WY Yuen3, Claudio Ciofi1, Roscoe Stanyon1, Sara Fratini1 Comparison of the Evolution of Orchids with that of Bats Antonio Lima-de-Faria Identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection Mehdi Zahravi1,*, Panthea Vosough-Mohebbi2, Mehdi Changizi3, Shahab Khaghani1, Zahra-Sadat Shobbar4 Populations genetic study of the medicinal species Plantago afra L. (Plantaginaceae) Saeed Mohsenzadeh*, Masoud Sheidai, Fahimeh Koohdar A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum using EMA-giemsa and fluorochrome banding Timir Baran Jha1,*, Partha Sarathi Saha2, Sumita Jha2 Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii Hook.f. and Elymus nutans Griseb. (Triticeae: Poaceae) from Western Himalaya, India Harminder Singh2, Jaswant Singh1,*, Puneet Kumar2, Vijay Kumar Singhal1, Bhupendra Singh Kholia2, Lalit Mohan Tewari3 Population genetic and phylogeographic analyses of Ziziphora clinopodioides Lam., (Lamiaceae), “kakuti-e kuhi”: An attempt to delimit its subspecies Raheleh Tabaripour1,*, Masoud Sheidai1, Seyed Mehdi Talebi2, Zahra Noormohammadi3 Induced cytomictic crosstalk behaviour among micro-meiocytes of Cyamopsis tetragonoloba (L.) Taub. (cluster bean): Reasons and repercussions Girjesh Kumar, Shefali Singh* Karyotype diversity of stingless bees of the genus Frieseomelitta (Hymenoptera, Apidae, Meliponini) Renan Monteiro do Nascimento1, Antonio Freire Carvalho1, Weyder Cristiano Santana2, Adriane Barth3, Marco Antonio Costa1,* Karyotype studies on the genus Origanum L. (Lamiaceae) species and some hybrids defining homoploidy Esra Martin1, Tuncay Dirmenci2,*, Turan Arabaci3, Türker Yazici2, Taner Özcan2 Determination of phenolic compounds and evaluation of cytotoxicity in Plectranthus barbatus using the Allium cepa test Kássia Cauana Trapp1, Carmine Aparecida Lenz Hister1, H. Dail Laughinghouse IV2,*, Aline Augusti Boligon1, Solange Bosio Tedesco1