New chromosomal data, karyotype asymmetry and polyploid variations of some Gundelia (Asteraceae) species from Turkey

. The genus Gundelia is currently represented with 18 species, of which 16 are in Turkey. In genus Gundelia , the chromosomal data were reported from 12 species. In the present study, it is aimed to eliminate the deficiencies in the knowledge about chromosomal data of Gundelia species. In Genus Gundelia , only a single chromosome number had been detected as 2 n =18 so far. The chromosome numbers of four species were reported here for the first time: G. armeniaca , G. cappadocica , G. siirtica , and G. tehranica . In addition, the polyploidy in the genus was rare and G. anatolica was identified as the first polyploid species. All karyotypes except G. tehranica were symmetrical, consisting of metacentric and submetacentric chromosomes. Secondary constrictions were observed in the distal regions of the long arms of the longest metacentric and submetacentric chromosomes. Thus, the chromosomal data of all Turkish Gunde-lia species were completed. In conclusion, the present study presented new data into the karyological records relating the karyotype evolution and interspecific relations of genus Gundelia

Due to the monotypic genus of Gundelia, not many investigations have been studied with molecular techniques.In the first studies, the phylogenetic position of the genus Gundelia in the family was determined (Karis et al. 2001, Funk et al. 2004, Panero and Croizer 2016).According to Panero and Croizer (2016), the genus is a member of the subfamily Cichorioideae.After the new species started to be published, whether the species were phylogenetically supported became a matter of curiosity.Firstly, with limited species and samples, Vitek et al. (2010) and Tarıkahya-Hacıoğlu and Fırat (2017) were studied using the internal transcribed spacer marker.Finally, in 2021, an updated and expanded phylogeny based on DNA sequences of both ITS and ndhF genes was published by Ateş et al. (2021).
Gundelia genus has special inflorescence different from other genus in the Asteraceae family.The synflorescens of Gundelia as a whole in inflorescence is a composition of partial synflorescens (disseminules) in the axils of bracts.These partial synflorescens (disseminules) consist of 3-9 flowers and this number is an important characteristic structure for each species (Classen-Bockhoff et al. 1989).
Gundelia grows in the semi-humid to dry meadows, mountain (steppe) meadows, dry slope areas.The evidence showed that the center of diversity of Gundelia is Turkey, even if the genus is distributed in the area from Armenia to Egypt and from Turkey to Afghanistan (Figure 1).Gundelia is present everywhere in Turkey except the Black Sea, Marmara and coastal Aegean regions.The geographical boundaries play a decisive role in their distribution (Karis et al. 2001;Vitek 2018).
The species belonging to the genus Gundelia are called "Kenger" in Turkish, "Akub" in Arabic and "Kuub" in Persian, and there are some local dialectical differences.Young shoots are used as vegetables in dishes and pickles.The latex obtained from the cut shoots is dried and used as gum.Roasted fruits are consumed like coffee.It is also used as animal food in some regions.
Cytotaxonomy, one of the sub-branches of taxonomy, uses karyological parameters in the classification of organisms.In this context, chromosomal configurations are used to understand the relationships between species and based on the assumption that "closely related species show similar chromosomal configurations".Thus, karyotype evolution or interspecies relationships can be reconstructed by exploiting karyological similarities or variations.The chromosome number, chromosome structure and chromosomal behaviors stand out as important parameters of cytotaxonomy, and especially basic chromosome number (x), diploid chromosome number (2n) and karyotype asymmetry are the most preferred parameters (Eroğlu et al. 2020;Martin et al. 2020;Eroğlu et al. 2021;Kavcı et al. 2022).In genus Gundelia, the chromosomal data were reported from 12 species.All species were represented by only one base number (x = 9) and there were no reports of polyploidy (Al-Taey and Hossain 1984;Genç and Fırat 2019).
In Turkish species of Gundelia, the chromosomal data were reported from 12 species, which were G. anatolica, G. asperrima, G. cilicica, G. colemerikensis, G. dersim, G. glabra, G. komagenensis, G. mesopotamica, G. munzuriensis, G. rosea, G. tournefortii, and G. vitekii (Al-Taey and Hossain 1984;Genç and Fırat 2019).There was no record of the chromosome number of four species, which were G. armeniaca, G. cappadocica, G. siirtica, and G. tehranica.Due to the lack of some chromosome reports from Turkey, where there are many species of the genus, some cytotaxonomic knowledge is lacking.In this study, it is aimed to complete the missing chromosomal data in Turkish Gundelia species.

Plant material
Within the scope of this study, sixteen Gundelia taxa distributed in different localities of Turkey were evaluated karyologically.The evaluated samples were collected from natural habitats by Dr. Metin Armağan et al.Table 1 represents the collection information and distribution regions.Turkish Gundelia species whose chro-mosomal data were reported in previous studies were collected from different localities to investigate chromosomal variations.

Chromosome preparation
The plant seeds were germinated between moist Whatman papers and pretreated by α-monobromonaphthalene at 4°C for 16 h.Then, root tips were fixed by fixative solution (3 absolute alcohol and 1 glacial acetic acid -v:v) at 4°C for 24 h.The fixed root tips were stored in ethanol (70%) at 4°C.Then, root tips were hydrolyzed in 1 N hydrochloric acid at room temperature for 10 min and stained in aceto-orcein (2%).Then, squash preparations were prepared by acetic acid (45%).The preparations were frozen in liquid nitrogen, dried at room temperature, and stabilized with Depex medium (Eroğlu et al. 2020;Martin et al. 2020;Eroğlu et al. 2021;Kavcı et al. 2022).

Karyotype analysis
Well-spread ten metaphase plates were used to detect for the chromosome numbers of all species.Detailed chromosomal measurements of four species whose chromosome number was investigated for the first time were made by Karyotype software.The following parameters and formulae were used to chromosome characterizations karyotype analysis: short arm length of chromosome (p), long arm length of chromosome (q), total chromosome length (p + q), total haploid length (THL), mean haploid length (MHL), relative length (RL) = [(p + q) / THL] × 100, and centromeric index (CI) = [(p) / (p + q)] × 100.The karyotype formulae were detected based on centromere position (Levan et al. 1964) and the monoploid ideograms were drawn.
The following formulae were used to determine the intrachromosomal asymmetry (M CA ) and interchromosomal asymmetry (CV CL ): M CA = [mean (q t − p t ) / (qt + pt)] × 100; q t , total length of long arms and p t , total length of short arms (Peruzzi and Eroğlu 2013).CV CL = (S CL / X CL ) × 100; S CL , standard deviation in a chromosome set and X CL , mean chromosome length in a chromosome set (Paszko 2006).

RESULTS
Chromosome records of 16 species are herein provided (Figure 2), four of which are reported for the first time (G. armeniaca, G. cappadocica, G. siirtica, and G. tehranica), one presents polyploidy for the first time (G.anatolica), and twelve agree previous reports.Table 2 shows the chromosome numbers of present and previous reports.Except for polyploidy (Figure 3), the only one chromosome number detected was 2n = 18.
Detailed chromosomal data and monoploid ideograms of the four species, whose chromosome numbers were reported for the first time were given in Table 3 and Figure 4.The smallest chromosome length among the species was 3.94 μm, in G. siirtica.The largest chromosome length was detected in G. tehranica, with 8.65 μm.The smallest total haploid length was 44.37 μm, in G. siirtica, and the highest value was 51.71 μm, in G. armeniaca and G. cappadocia.
All species except G. tehranica had median (m) and submedian (sm) chromosomes, but not subtelocentric (st) and telocentric (t) chromosomes.Two different karyotype formulae were observed, which were 14m + 4sm and 12m + 2sm + 2st.Secondary constrictions were observed in the distal regions of the long arms of the longest metacentric and submetacentric chromosomes (Figure 4).
A basic chromosome number of x = 9 dominates in genus Gundelia and the genus is monobasic.The absence of basic number variations in genus Gundelia indicated that the mechanism of dysploidy probably did not occur in the karyotype evolution of the genus.Because dysploidy causes basic number variations by fusion of metacentric chromosomes or reciprocal translocations (Eroğlu et al. 2020;Martin et al. 2022).In addition, the polyploidy in the genus was rare and G. anatolica was identified as the first polyploid species.
Fifteen species had metacentric and submetacentric chromosomes and only one species had subtelocentric chromosomes, whereas no telocentric (t) chromosomes were observed.Two different karyotype samples were observed, which were m-sm and m-sm-st including secondary constrictions.Thus, five chromosome types were determined according to the positions of the primary and secondary constrictions: (i) metacentric (ii) metacentric with secondary constriction in the distal region     of the long arm, (iii) submetacentric, (iv) submetacentric with secondary constriction in the distal region of the long arm, (v) subtelocentric.In twelve Gundelia species, Genç and Fırat (2019) reported that the secondary constrictions at short or long arms of submetacentric chromosomes and in the distal region of long arm of the longest metacentric chromosome.
In intrachromosomal asymmetry, all karyotypes were symmetric.The most symmetric and asymmetrical karyotypes were the karyotypes of G. siirtica and G. tehranica, respectively.In interchromosomal asymmetry, all karyotypes were symmetric.The most symmetric and asymmetrical karyotypes were the karyotypes of G. armeniaca and G. tehranica, respectively.Genç and Fırat (2019) reported that G. rosea and G. tournefortii had the relatively high intrachromosomal asymmetry and low intrachromosomal asymmetry, respectively; also, G. vitekii and G. anatolica had the high interchromosomal and low interchromosomal asymmetry, respectively.
In the present study, it was recorded only one chromosome number (2n = 18) excluding polyploidy (2n = 36), the first report for diploid numbers of four species, the first report of polyploidy for the genus, and the same chromosome count with previous report in the twelve species.Thus, the chromosomal data of all Turkish Gundelia species were completed.In conclusion, the present study presented new data into the karyological records relating the karyotype evolution and interspecies relations of genus Gundelia.In addition, the dysploidy and polyploidy mechanisms probably did not have an important role in the speciation of genus.

Figure 1 .
Figure 1.The distribution map of genus Gundelia.

Table 1 .
Collection information and voucher specimens of Gundelia taxa.

Table 2 .
The chromosome numbers of Turkish Gundelia in present and previous studies.All species were studied in this study.Turkish Gundelia species whose chromosomal data were reported in previous studies were collected from different localities to investigate chromosomal variations.

Table 3 .
The detailed chromosomal data and asymmetry indices of species whose chromosome number was reported for the first time (KF: karyotype formula, SC: the shortest chromosome length, LC: the longest chromosome length, RL: relative length, CI: centromeric index, THL: total haploid length, MHL: mean haploid length, M CA : mean centromeric asymmetry, CV CL : coefficient of variation of chromosome length).