A Karyological Study of Some Dianthus L. Species (Caryophyllaceae) in Northeast of Iran

Feddes Repertorium (2014), 1–9

Research Paper

A karyological study of some Dianthus L. species (Caryophyllaceae) in Northeast of Iran

1 *, 2 1 MARYAM BEHROOZIAN , JAMIL VAEZI & MOHAMMAD REZA JOHARCHI

1 Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran 2 Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Keywords: Caryophyllaceae, Dianthus, polyploidy, karyotype, asymmetry index

* Corresponding author: Jamil Vaezi, Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Iran, E-mail: [email protected], Fax and Phone: +98-611-3331045

Accepted for publication: October 20th, 2013.

DOI 10.1002/fedr.201300011

Abstract The genus consists of 30 species in Iran. This study focuses on karyological investigation of Dianthus with high intra-populational and four taxa of Dianthus (Caryophyllaceae) including interspecific variation of morphological and D. crossopetalus, D. orientalis subsp. stenocalyx, genetic characters has been recognized as a D. orientalis subsp. gorganicus, and Dianthus sp. complex genus (BEHROOZIAN 2010). There- distributed in Northeast of Iran. The karyotype fore, morphological characters alone do not asymmetry/symmetry was evaluated using two provide effective clues for delimitation of Di- methods: 1) CVCL (coefficient of variation of chro- anthus species (BEHROOZIAN 2010). Additional mosome length) and CV (heterogeneity of the CI studies such as cytology and molecular ap- centromeric index); and 2) MCA (mean centromeric asymmetry). The karyotype asymmetry was also proaches could be useful for resolving taxo- used to investigate the relationships among the taxa. nomic problems in plants (SEN 1975; LARSON Results obtained from the current study revealed that et al. 2010; MARCUSSEN & BORGEN 2011; GAO there are two different ploidy levels (2n = 2x = 30 et al. 2012). The last decades, karyological and 2n = 4x = 60) among the investigated taxa. The investigations provided fundamental characters indices CVCL and MCA described karyotype asymme- for plant systematics and evolutionary analysis try correctly based on variation in chromosome (STACE 2000). Recently, scientists have devel- length. Diagram of CV vs. M seems to be appro- CL CA oped a variety of methods for karyotype priate for karyological delimitation and taxonomic relationships among the Dianthus taxa under study. asymmetry analysis (reviewed in ZUO & YUAN 2011; PERUZZI & EROĞLU 2013). Dianthus with seven ploidy levels (2x, 3x, 4x, 5x, 6x, 8x, and 12x; reviewed in WEISS et al. Introduction 2002 and BALAO et al. 2009), is one of the genera with the most extensive ploidy series Dianthus L. (Caryophyllaceae) comprises a known in the subfamily Caryophylloideae. group of annual or perennial species world- BALAO et al. (2010) suggested that different wide. ploidy levels within and between populations

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0014-8962 2 Feddes Repert., Weinheim (2014) of the genus are related to highly morphologi- an isolated and small population during field sam- cal variations. They declared that the evolution pling (Table 1). The individuals of this taxon seem of cytological features within the genus has to be morphologically intermediate between D. cri- taken place in conjunction with morphological nitus subsp. turcomanicus (SCHISCHK.) Rech.f. and D. orientalis subsp. stenocalyx. Their vegetative diversification. Furthermore, different ploidy habit is similar to the first subspecies while their levels have been reported for populations at the floral characters tend more to the second. Karyologi- subspecific level (WEISS et al. 2002). Karyolo- cal data of D. crinitus subsp. turcomanicus, which is gical studies on Dianthus are often restricted to published in previous study (JAFFARI & BEHROOZIAN just reporting of chromosome numbers, where- 2010), are included in the current study to reveal its as karyological analyses were rarely reported. karyological relationship with the unknown taxon. The aims of the present study are: 1) to report One individual of this unknown taxon, however, is somatic chromosome numbers; 2) to assess sampled to investigate its karyological characteris- karyotype asymmetry/symmetry; and 3) to tics. This taxon is named here after as Dianthus sp. in this study. reveal the karyotype relationships among some The seeds were germinated at 25 °C. Five to Dianthus species distributed in Northeast of ten root tips of each taxon were pretreated with Iran. 8-hydroxyquinoline (0.002 M) for three hours at room temperature. Then, they were fixed in Car- noy’s solution (3:1 absolute ethanol – glacial acetic Material and methods acid) for 24 h at 4 °C. The fixed root tips were hy- drolyzed in 1 N HCl at 60 °C for 5–7 min followed In order to study the karyological characteristics of by washing them in distilled water. Then they were Dianthus species, seeds were collected from various stained with aceto-orcein (1% w/v) for 2 h. Finally, regions of Northeast of Iran. A minimum of five the root tips were squashed in acetic acid 45%. The specimens were investigated for each species in- metaphase chromosomes were counted for at least cluded in the current study (except for D. sp., see 10 cells of each seedling root. The examined meta- below). Depending on the number of populations per phase plates were photographed using Olympus species, these individuals belong to one or more BX52 microscope with automatic camera. populations. At least five chromosome plates were Chromosome measurements including long and analyzed per specimen of species. No intraspecific short arms, total length of chromosome set, arm ratio karyological variability has been found. Therefore, index and relative chromosome length were per- one voucher specimen per taxon was presented in formed for four taxa using the Nutype software. Table 1. Voucher specimens are deposited in Fer- Chromosomes were identified according to the dowsi University of Mashhad Herbarium (FUMH) Levan method (LEVAN et al. 1964). The homologous (Table 1). chromosomes of diploid taxa were paired by their This study focuses on four taxa including similarity in size and shape. A number of parameters D. orientalis subsp. stenocalyx (BOISS.) Rech.f., were used to determine the karyotype asymmetry D. orientalis subsp. gorganicus Rech.f., the new and symmetry according to PASZKO (2006): CVCI = record D. crossopetalus (FENZL ex BOISS.) GROSSH [(SCI/xCI) × 100 (SCI = the standard deviation of the (GHAHREMANINEJAD et al. 2012), and an unknown centromeric index; xCI = the mean centromeric in- Dianthus taxon. The last taxon was collected from dex)], CVCL = A2 × 100 (A2 is proposed by ZARCO

Table 1 Voucher specimens included in the karyological study of four taxa of Dianthus

Voucher Species Location Longitude Latitude No. (E) (N) 35410 D. crossopetalus Khorassan shomali province, E. Jajarm, 56°45′3.6″ 37°06′32.4″ between Khorashah and Jorbat 44075 D. orientalis subsp. W. Bojnourd, Qorkhod protected area, 56°12′6.9″ 37°24′2.7″ stenocalyx Yakhtikalan pass 44111 D. orientalis subsp. Golestan province, NW of Islamabad 56°02′19.3″ 37°43′0.2″ gorganicus 44119 Dianthus sp. SW. Bojnourd, between Hessar-Hosseini 57°09′24.2″ 37°18′12.4″ and Rakhtian

Table 2 Karyotype formula according to LEVAN et al. (1964) used in the present work: SC – the shortest chromosome length; LC – the longest chromosome length; CL – mean length of chromosome; CI – mean centromeric index; SD – standard deviation; K.F. – karyotype formula. Karyological data of D. crinitus subsp. turcomanicus were used with permission from JAFFARI and BEHROOZIAN 2010

No. Species 2n Range Ratio CL (µm) CI (µm) K.F. SC-LS LC/SC Mean (±SD) Mean (±SD) 1 D. crossopetalus 30 1.94–3.45 1.78 2.61 (±0.37) 35.63 (±7.54) 10m + 20sm 2 D. orientalis subsp. 30 2.01–3.62 1.80 2.74 (±0.38) 40.30 (±6.57) 24m + 4sm + 2st stenocalyx 3 D. orientalis subsp. 60 1.81–3.84 2.12 2.73 (±0.44) 37.38 (±9.84) 24m + 28sm + 8st gorganicus 4 Dianthus sp. 60 2.16–3.35 1.77 2.67 (±0.29) 34.80 (±7.65) 24m + 32sm + 4st 5 D. crinitus subsp. 60 1.92–3.84 2.1 2.73 (±0.43) 47.11 (±5.54) 46m + 12sm + 2st turcomanicus

1986), and PERUZZI and EROĞLU (2013): MCA = mula of 2n = 2x = 30. The chromosome number A × 100 (A is proposed by WATANABE et al. 1999). for Dianthus sp. and D. orientalis subsp. gor- CVCL and MCA indices provide the most suitable ganicus is 2n = 60 and they are tetraploid. Ka- methods for estimating karyotype asymmetry, while ryograms of the two last taxa are not shown in the CVCI did not describe karyotype asymmetry Figure 2 due to lacking information concerning correctly (ZUO & YUAN 2011; PERUZZI & EROĞLU 2013). Therefore, a bidimensional scatter plot was the origin (auto- or allopolyploidy) of these drawn with the couple of parameters CVCL and MCA taxa. to reveal karyotype relationships among the organ- isms included in the current study. In addition, an Karyotype asymmetry analysis idiogram was constructed for each taxa based on the average of the mean values calculated for the karyo- The highest and lowest ratio of longest to types using data taken from Table 2. shortest chromosomes is found in D. orientalis subsp. gorganicus and Dianthus sp., respectively (Table 2). The mean value of the total Results chromosome length in D. crossopetalus (2.61) and Dianthus sp. (2.67) was comparatively Chromosome numbers lower than that of the both subspecies of Karyotype characters, mitotic metaphase chro- D. orientalis (2.73 and 2.74). Dianthus cros- mosome, haploid idiogram and karyogram of sopetalus consists of metacentric and sub- the taxa investigated are shown in Tables 2 and metacentric chromosomes, while the remaining 3, and Figures 1 and 2. Detailed karyotypes and taxa contain metacentric, sub-metacentric, and idiograms of the taxa are reported here for the sub-telocentric chromosomes. In all examined first time. Both D. crossopetalus and D. orien- taxa, metacentric chromosomes are the most talis subsp. stenocalyx are diploid with a for- common (56.65% of all the chromosomes),

Table 3 Asymmetry indices used for the classification of Dianthus taxa: CVCL – coefficient of variation of chromosome length; CVCI – heterogeneity of the centromeric index; MCA – mean centromeric asymmetry. Karyologi- cal data of D. crinitus subsp. turcomanicus were used with permission from JAFFARI and BEHROOZIAN (2010)

No. Species CVCL CVCI MCA 1 D. crossopetalus 14.23 21.16 29 2 D. orientalis subsp. stenocalyx 14.01 16.31 19 3 D. orientalis subsp. gorganicus 16.22 25.91 33 4 Dianthus sp. 12.98 21.99 30 5 D. crinitus subsp. turcomanicus 13.86 11.76 5.8

Fig. 1 Somatic chromosomes of four taxa of Dianthus: A) D. crossopetalus; B) D. orientalis subsp. stenocalyx; C) D. orientalis subsp. gorganicus; and D) Dianthus sp. Karyotypes of the two first species are illustrated below each metaphasic plate

Fig. 2 Haploid idiograms of four taxa of Dianthus: A) D. crossopetalus; B) D. orientalis subsp. stenocalyx; C) D. orientalis subsp. gorganicus; and D) Dianthus sp. followed by submetacentric chromosomes crinitus subsp. turcomanicus and D. orientalis (36.32%), whereas subtelocentric chromosomes subsp. gorganicus show the lowest and highest are rare (6.25%). The detailed information of MCA, respectively. Furthermore, D. crossopeta- karyotype formulae is shown in Table 2. lus and D. sp. exhibit relatively high MCA. Asymmetry indices estimated on the basis Among all taxa investigated in the present of statistical data resolved the Dianthus karyo- study, D. sp. and D. orientalis subsp. gorgani- types into the range of symmetrical to lowest cus demonstrate the lowest and highest CVCL, asymmetrical values. Karyotype asymmetry respectively. also depends on both the relative variation in chromosome length (CVCL) and heterogeneity of the centromeric index (CVCI). Dianthus Discussion orientalis subsp. gorganicus is characterized by the highest value of both CVCL and CVCI, and The results obtained from the present study the remaining species were characterized by indicate that both subspecies of D. orientalis lower values of both CVCL and CVCI (Table 3). are identified with two different ploidy levels. In addition, the results indicate that the most Dianthus orientalis subsp. stenocalyx (2n = 2x asymmetrical karyotype belongs to D. orienta- = 30) has a widespread distributional range lis subsp. gorganicus with the highest karyo- from east to west of Iran, whereas D. orientalis type asymmetry of CVCL = 16.22 and MCA subsp. gorganicus (2n = 4x = 60) is distributed = 33. in narrow range in Northeastern Iran. JANAKI According to the scatter plot (Fig. 3) ob- AMMAL and SELIGMAN (1956) reported co- tained from the parameters CVCL vs. MCA, D. occurrence of diploid and tetraploid individuals

Fig. 3 Scatter diagram of Dianthus taxa based on the karyotype parameters CVCL vs. MCA. Dianthus crossopetalus (plus sign), Dianthus sp. (cross sign), D. orientalis subsp. gorganicus (white circle), D. orientalis subsp. stenocalyx (dark circle), D. crinitus subsp. turcomanicus (triangle). Karyological data of D. crinitus subsp. turcomanicus were used with permission from JAFFARI and BEHROOZIAN (2010)

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.feddes-journal.com M. BEHROOZIAN et al.: A karyological study of some Dianthus L. species in Northeast of Iran 7 within a population of Dianthus monspes- versely, D. orientalis subsp. gorganicus with sulanus L. (2n = 2x = 30 and 2n = 4x = 60). the highest karyotype asymmetry shows obvi- Likewise, WEISS et al. (2002) reported occur- ously difference in morphological features of rence of tetraploid, pentaploid and hexaploid chromosomes as compared to those of the sub- cytotypes in two subspecies including Dianthus species D. orientalis subsp. stenocalyx. It was plumarius subsp. blandus (RCHB.) HAYEK also reported that increasing karyotype asym- (2n = 4x = 60, 2n = 5x = 75, and 2n = 6x = 90) metry could occur via a shift of the centromere and D. plumarius subsp. hoppei (Port.) Hegi position from median to subterminal or termi- (2n = 6x = 90). They also expressed that an nal chromosome and differences in the size of autopolyploidization model agrees with the individual chromosomes (LIU et al. 2006). occurrence of polyploids within diploid species According to the karyotype difference between such as D. monspessulanus, D. superbus L., D. orientalis subsp. stenocalyx and D. orien- D. acicularis FISCH. ex LEDEB., D. petraeus talis subsp. gorganicus, it seems, however, that WALDST. & KIT., and putatively diploid spe- the taxonomic position of D. orientalis subsp. cies D. arenarius L., and D. gratianopolitanus gorganicus could be considered as an inde- Vill. Moreover, the report of GATT et al. (1998) pendent species. Nonetheless, the exactly taxo- on significant production of unreduced pollen nomic position of this taxon should be investi- grains in diploid species D. knappii (PANT.) gated by further data. ASCH. & KANITZ ex BORBÁS further strength- There has been some uncertainty regarding ens this hypothesis. However, the occurrence of the taxonomic status of D. crossopetalus. In polyploidy and genome duplication as two Flora Kavkaza (GROSSHEIM 1930) this species major evolutionary phenomena seem to play is regarded as synonymous with D. crinitus prominent roles in speciation within Dianthus Sm. In Flora of Turkey (DAVIS 1967) this spe- (CAROLINE 1957; WEISS et al. 2002; BALAO cies is recognized as a variety of D. crinitus, et al. 2010). Results obtained from the scatter while in Flora Orientalis (BOISSIER 1975), diagrams (Fig. 3) showed that D. orientalis Flora of USSR (SHISHKIN 1936), Flora Iranica subsp. gorganicus could be identified by its (RECHINGER 1986), and ASSADI (1985), it is highly asymmetric karyotypes while D. orien- considered as an autonomous species. Dianthus talis subsp. stenocalyx separated from the other crinitus is a tetraploid species (2n = 2x = 60) taxa with the lowest asymmetry karyotype. with symmetrical karyotype (JAFFARI and BEHROOZIAN 2010), while results of the pre- Karyotypes and systematics sent study demonstrated that D. crossopetalus (2n = 2x = 30) could be assessed as an interme-

The CVCL vs. MCA diagram is rather preferable diate karyotype asymmetry when compared than scatter diagrams based on other indices to the other taxa (Table 3). In addition, the because it is better suited to demonstrate karyo- karyological position of D. crossopetalus in type relationships among taxa, especially when scatter diagram (Fig. 3) is obviously discrimi- chromosome size variation is negligible (PE- nated from that of D. crinitus. As a result, RUZZI & EROĞLU 2013). Dianthus orientalis based on the karyotype asymmetry analyses subsp. gorganicus and Dianthus sp. displayed a (JAFFARI & BEHROOZIAN 2010 and the results high asymmetry index value with a high level of the current study) and morphometric investi- of karyotype asymmetry (Table 3). In the gation (unpublished results), our karyological other hand, D. orientalis subsp. stenocalyx and interpretation on the taxonomic position of D. crossopetalus showed a low asymmetry D. crossopetalus is that this taxon is probably index value indicating greater karyotype sym- an autonomous species rather than a variety or metry and stabilized genome (SHAMURAILAT- synonym of D. crinitus. PAM et al. 2012). Two subspecies of D. orien- Dianthus sp. with an unknown taxonomic talis were not grouped together in the scatter position is morphologically intermediate be- plot (Fig. 3). Dianthus orientalis subsp. steno- tween D. crinitus subsp. turcomanicus and calyx with the lowest karyotype asymmetry D. orientalis subsp. stenocalyx. Results of this (among the taxa included in the present study) study show that this species has its own karyo- is separated from the other taxa (Fig. 3). Con- type characteristics (Fig. 3). Although karyo-

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.feddes-journal.com 8 Feddes Repert., Weinheim (2014) type features comparably provide valuable GHAHREMANINEJAD, F.; JOHARCHI, M. & VITEK, E. insights into taxonomic relationship (SILJAK- 2012: New plant records for Khorassan pro- YAKOVLEV & PERUZZI 2012) but we suggest vince, Iran, V, with complementary notes to that more investigations are necessary to clarify its flora. – Ann. Naturhist. Mus. Wien, B 114: 59–94. its exactly taxonomic position. GREIHUBER, J. & SPETA, F. 1976: C-banded karyotypes in the Scilla hohenackeri group, S. persica Acknowledgments and Puschkinia (Liliaceae). – Pl. Syst. Evol. 126: 149–188. The authors thank Dr. Jaffari and Dr. Darvish for GROSSHEIM, A. A. 1930: Dianthus crossopetalus in their suggestions and technical helps. Flora Kavkaza, 2: 428. Tiflis, Narodnyi Komis- Financial support of this work is provided by sariat Zemledeliya SSR Armenii, Baku, Izd. Az. research center for plant sciences of Ferdowsi Uni- Otd. Zak. Fil. Ak. Nauk. versity of Mashhad. HUZIWARA, Y. 1962: Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosome of Aster. – Amer. J. Bot. 49: References 116–119. JAFFARI, A. & BEHROOZIAN, M. 2010: A cyto- ARANO, H. 1963: Cytological studies in sub family taxonomic study on Dianthus species in northeast Carduoideae (Compositae) of Japan. IX. The of Iran. – Asian J. Plant Sci. 9(1): 58–62. karyotype analysis and phylogenic consideration JANAKI AMMAL, E. K. & SELIGMAN, R. 1956: Notes of Pretya and Ainsliaea. – Bot. Mag. Tokyo 79: on occurrence of chromosome races in Dianthus 32–39. monspessulanus in Northern Italy. – J. Roy. Hort. ASSADI, M. 1985: The genus Dianthus L. (Caryo- Soc. 77: 221–223. phyllaceae) in Iran. – Iranian J. Bot. 3(1): 9–54. LARSON, S. R.; CULUMBER, C. M.; SCHWEIGERT, BALAO, F.; SORIGUER, R. C.; TALAVERA, M.; HER- R. N. & CHATTERTON, N. J. 2010: Species de- RERA, J. & TALAVERA, S. 2009: Distribution and limitation tests of endemic Lepidium papilliferum diversity of cytotypes in D. broteri as evidenced and identification of other possible evolutionarily by genome size variation. – Ann. Bot. 104: 965– significant units in the Lepidium montanum com- 973. plex (Brassicaceae) of western North America. – BALAO, F.; VALENTE, L. M.; VARGAS, P.; HERRERA, Conserv. Genet. 11: 57–76. J. & TALAVERA, S. 2010: Radiative evolution of LAVANIA, U. C. & SRIVASTAVA, S. 1992: A simple polyploidy races of the Iberian carnation Dian- parameter of dispersion index that serves an thus broteri (Caryophyllaceae). – New Phytol. adjunct to karyotype asymmetry. – J. Biosci. 17: 187(2): 542–551. 179–182. BEHROOZIAN, M. 2010: Genetic polymorphism in- LEVAN, A. K.; FREDGA, K. & SANDBERG, A. A. vestigation between and within populations of 1964: Nomenclature for centromic position on Dianthus L. using RAPD technique and com- chromosomes. – Hereditas 52: 201–220. paring with ploidy levels of populations in LIU, H.; YAN, G.; SHEN, F. & SEDGLEG, R. 2006: Khorassan Razavi province. MSc dissertation, Karyotype in Leucodendron (Protaceae): evi- Mashhad branch, Azad Islamic University (in dence of the primitiveness of the genus. – Bot. J. Persian with summary in English). Linn. Soc. 151: 387–394. BOISSIER, E. 1975: Dianthus L. in Flora Orientalis, MARCUSSEN, T. & BORGEN, L. 2011: Species deli- 1: 479–515. A. Asher & CO B.V. Amsterdam. mitation in the Ponto-Caucasian Viola sieheana CAROLINE, R. C. 1957: Cytological and hybridi- complex, based on evidence from allozymes, zation studies in the genus Dianthus. – New morphology, ploidy levels, and crossing experi- phytol. 56: 81–97. ments. – Pl. Syst. Evol. 291: 183–196. DAVIS, P. H. 1967: Dianthus in Flora of Turkey, 2: PASZKO, B. 2006: A critical review and a new 99–131, Edinburg University Press. England. proposal of karyotype asymmetry indices. – Pl. GAO, Y. D.; ZHOU, S. D.; HE, X. Y. & WEN, J. 2012: Syst. Evol. 258: 39–48. Chromosome diversity and evolution in tribe PERUZZI, L. & EROĞLU, H. 2013: Karyotype asym- Lilieae (Liliaceae) with emphasis on Chinese metry: again, how to measure and what to meas- species. – J. Plant Res. 125: 55–69. ure? – Comp. Cytogen. 7(1): 1–9. GATT, M.; HAMMETT, K. R. W.; MARKHAM, K. R. & RECHINGER, K. H. 1986: Caryophyllaceae II, Flora MURRAY, B. G. 1998: Yellow pinks: intraspecific Iranica, no. 163 – Graz. hybridization between Dianthus plumarius and ROMERO ZARCO, C. 1986: A new method for related species with yellow flowers. – Sci. estimating karyotype asymmetry. – Taxon 35: Hortic. 7: 207–218. 526–530.

SEN, S. 1975: Cytotaxonomy of Liliales. – Feddes WATANABE, K.; YAHARA, T.; DENDA, T. & KOSUGE, Repert. 86(5): 255–305. K. 1999: Chromosomal evolution in the genus SHAMURAILATPAM, A.; MADHANAV, L.; YADAV, Brachyscome (Asteraceae, Astereae): statistical R. R.; BHAT, K. V. & RAO, S. R. 2012: Chro- tests regarding correlation between changes in mosome diversity analysis in various species of karyotype and habit using phylogenetic infor- Vigna Savi from India. – Nucleus 55(2): 107– mation. – J. Plant Res. 112: 145–161. 114. WEISS, H.; DOBES, C. H.; SCHNEEWIESS, G. M. & SHISHKIN, B. K. 1936: Dianthus. – In: V. L. KO- GRIEMLER, J. 2002: Occurrence of Tetraploid and MAROV (ed.), Flora of the U.S.S.R, Vol. 6: 804– Hexaploid Cytotypes between and within Popu- 861. Moskva & Leningrad. lations in Dianthus Sect. Plumaria (Caryo- SILGAK-YAKOVLEW, S. & PERUZZI, L. 2012: Cyto- phyllaceae). – New Phytol. 156: 85–94. genetic characterization of endemics: past and ZUE, L. & YUAN, Q. 2011: The difference between future. – Plant Biosyst. 146: 694–702. the heterogeneity of the centromeric index and STACE, C. A. 2000: Cytology and cytogenetics as a intrachromosomal asymmetry. – Pl. Syst. Evol. fundamental taxonomic resource for the 20th and 297: 141–145. 21th centuries. – Taxon 49: 451–477.