Cytogenetic characteristics of some Trollius L. species (Ranunculaceae) from Asian Russia

1 National Research Tomsk State University, 36 Lenin Ave., Tomsk 634050, Russia 2 Central Siberian Botanical Garden SB RAS, 101 Zolotodolinskaya St., Novosibirsk 630090, Russia 3 Altai State University, 61 Lenin Ave., Barnaul 656049, Russia 4 Amur Branch of Botanical Garden-Institute FEB RAS, Ignatievskoe Road, 2km, Blagoveshchensk 675000, Russia 5 Siberian Institute of Plant Physiology and Biochemistry SB RAS, 134 Lermontov St., Irkutsk 664033, Russia 6 Siberian Federal University, 79 Svobodny Pr., Krasnoyarsk, 660041, Russia Corresponding author E-mail: emitrenina@gmail.com Received: 12.11.2020. Accepted: 28.12.2020


Introduction
A cytogenetic approach is widely used to solve the problems of plant systematics and phylogeny, including the family Ranunculaceae Juss. (Yuan & Yang, 2006;Mlinarec, 2012;Baltisberger & Hörandl, 2016). Karyotypes (chromosome sets) of some taxa have not yet been studied despite a wide range of current chromosome research methods (Badaeva & Salina, 2013;Sharma & Sen, 2019). However, chromosome number, size, and morphology are important characteristics of the species.
should be proved.

Materials and Methods
All the studied plants have been collected in their natural localities. The vouchers are listed in Table 1. Trollius rhizomes were stored in wet moss before having young leaves. Karyotyping Newly formed 0.3-0.5 cm long leaves were excised and pretreated in 0.5 % aqueous colchicine solution for 3-4 h at room temperature. After that, they were fixed in a mixture of 96 % ethanol and glacial acetic acid (3:1). Leaves were stained with 1 % aceto-hematoxylin, and the squash method was employed for the investigation of the karyotype (Smirnov, 1968). Chromosomes were counted in 20-30 mitotic cells for each species. Mitotic metaphase chromosome plates were studied using an Axio Star microscope (Carl Zeiss, Munich, Germany) and photographed using an Axio Imager A.1 microscope (Carl Zeiss, Germany) with AxioVision 4.7 software (Carl Zeiss, Germany) and AxioCam MRc5 CCD-camera (Carl Zeiss, Germany) at 1000× magnification in the Laboratory for Ecology, Genetics and Environmental Protection (Ecogene) of National Research Tomsk State University. KaryoType software (Altinordu et al., 2016) was used for karyotyping, and Adobe Photoshop CS5 (Adobe Systems, USA) and Inkscape 0.92 (USA) were used for image editing. Karyotype formulas were based on measurements of mitotic metaphase chromosomes taken from photographs. We studied metaphase plates with the most condensed chromosomes. The measurements were performed on 3-5 metaphase plates. The symbols used to describe the karyotypes corresponded to those of Levan et al. (1964): m = median centromeric chromosome with an arm ratio of 1.0-1.7 (metacentric chromosome); sm = submedian centromeric chromosome with an arm ratio of 1.7-3.0 (submetacentric chromosome); st = subterminal centromeric chromosome with an arm ratio of 3.0-7.0 (subtelocentric chromosome); t = terminal centromeric chromosome with an arm ratio of 7.0-∞ (acrocentric or telocentric chromosome); T = chromosome without an obvious short arm, i.e., with an arm ratio of ∞. Additionally, we classified chromosomes with the intermediate value of the mean arm ratio: m/sm = metasubmetacentric chromosome and sm/st = submeta-subtelocentric chromosome. Mean values of the centromeric indices (CI), mean chromosome length (CL), and relative chromosome length (RL) for each chromosome pair, total haploid length (THL), and mean chromosome length of the set (MCL) were determined. Besides, we calculated the Coefficient of Variation of Chromosome Length (CVCL; Paszko, 2006), Coefficient of Variation of Centromeric Index (CVCI; Paszko, 2006), Mean Centromeric Asymmetry (MCA; Peruzzi & Eroğlu, 2013), and determined Stebbins asymmetry index (Stebbins, 1971). Flow cytometry Flow cytometry with propidium iodide (PI) staining was used to determine the absolute nuclear DNA content. Silica gel-dried leaf material was chopped with a sharp razor blade in a 1 ml cold nuclei extraction buffer composed of 50 mM Hepes, 10 mM sodium metabisulphite, 10 mM MgCl2, 0.5 % polyvinylpyrrolidone, 0.1 % bovine serum albumin, 0.3 % Tween 20, 0.2 % Triton X-100, 50 μg/ml RNase, 1 μg/ml β-mercaptoethanol, and 50 μg/ml propidium iodide (PI). The samples were filtered through 50 μm nylon membranes into sample tubes and incubated in the dark at 4 °C for 15 min. The samples were measured using a Partec CyFlow PA flow cytometer equipped with a green laser at 532 nm wavelength. The absolute nuclear DNA content, the 2C-value according to Greilhuber et al. (2005), was calculated as the ratio of the mean fluorescence intensity of the sample nuclei to that of an internal multiplied by the total nuclear DNA content of the standard. A possible effect of secondary metabolites on the binding of the intercalating dye was evaluated by measuring the fluorescence of Allium fistulosum L. leaf samples prepared as described above, but with the addition of the supernatant from Trollius, samples centrifuged without PI (Erst et al., 2020b;. The samples were measured three times at 10 min intervals. If the A. fistulosum peak Ukrainian Journal of Ecology, 10(6), 2020 showed no variation in the average values of the detection channels, the effect of secondary metabolites was considered negligible. Allium fistulosum L., 2C = 23.50 pg was used as an internal standard (Doležel et al., 1992;Ricroch et al., 2005;Smirnov et al., 2017). We used the Statistica 8.0 software (StatSoft, Inc.), Flowing Software 2.5.1 (Turku Centre for Biotechnology), and CyView software (Partec, GmbH) for data analyses. Flow cytometry was performed at the South-Siberian Botanical Garden, Altai State University (Barnaul, Russia).
Megaleranthis saniculifolia Ohwi, a representative of the tribe Adonidae, has a karyotype structure similar to Trollius. The karyotype formula of the species are 2n = 2x =16 = 2m + 12sm + 2st (Lee, Yeau, 1985). Another related species, Calathodes oxycarpa Sprague, has a more asymmetric karyotype without metacentric chromosomes (Yang, 2002). Q.-E. Yang noted that chromosomes of C. oxycarpa were larger than those of Trollius. Based on these data, he concluded that Calathodes was an independent genus in the tribe. Nevertheless, it was demonstrated in several studies that karyotypes of some Trollius consisted of heterobrachial chromosomes only. These include T. chinensis Bunge, T. europaeus L., and T. altaicus (Doroszewska, 1967;. W. Wang et al. (2010) used molecular and morphological analysis data to conclude that Calathodes is a segregate genus of the tribe Adonideae, and Megaleranthis is a member of the amended "Trollius". On the contrary, karyotypes of Adonis equally consist of isobrachial and heterobrachial chromosomes. For instance, the karyotype formula for A. vernalis L. (Schrager & Malakhova, 1981) and A. amurensis Regel & Radde (Volkova et al., 2020) was 2n = 2x =16 = 8m + 8sm, for A. brevistyla Franch. was 2n = 2x =16 = 8m + 2sm + 6st (Yang, 2001). Similar formulas were obtained for A. distorta Ten. (Del Grosso & Pogliani, 1971) and A. multiflora Nishikawa & Koji Ito (Ikeda et al., 2006). Consequently, the genus Adonis is clearly distinguished from Calathodes, Trollius, and Megaleranthis by chromosome complement. We estimated some karyotype asymmetry indices for seven Trollius species (Table 3). The quali-quantitative Stebbins asymmetry index (1971) was 3A for all karyotypes. It indicates that the proportion of chromosomes with an arm ratio < 2 was equal or less than 0.5, as well as the ratio between the largest and smallest chromosomes, was less than 2. Additionally, we calculated three parameters detecting interchromosomal and intrachromosomal karyotype asymmetries: CVCL -Coefficient of Variation of Chromosome Length, CVCI -Coefficient of Variation of Centromeric Index (Paszko, 2006), MCA -Mean Centromeric Asymmetry (Peruzzi & Eroğlu, 2013). CVCL is an index for estimating interchromosomal asymmetry, i.e., the degree of the Ukrainian Journal of Ecology, 10(6), 2020 difference between the chromosome lengths of a complement. It is a statistically correct parameter to show even a small variation among chromosome sizes in the complement (Peruzzi & Eroğlu, 2013).  Ukrainian Journal of Ecology, 10(6), 2020 The index value varied from 8.99 to 11.92 among the seven studied Trollius, i.e., interchromosomal asymmetry levels were similar to each other. MCA was used to estimate the intrachromosomal asymmetry. The lowest MCA level was obtained for T. riederianus (32.82) and T. austrosibiricus (33.77), followed by T. kytmanovii (34.68), which lack subtelocentric chromosomes. The rest four Trollius species have 1-2 pairs of subtelocentric chromosomes and, consequently, have higher MCA values (38.08-39.43). CVCI is an additional karyological parameter, not correctly referred to as the karyotype asymmetry. It is a measure of intrachromosomal heterogeneity. It can be used sometimes as an optional third parameter to reveal karyotype relationships among organisms, in addition to the asymmetry sensu stricto (Peruzzi & Eroğlu, 2013). The index value varied from 11.57 in T. austrosibiricus to 21.62 in T. lilacinus. The values of CVCI did not correlate with MCA in the studied Trollius species.

Genome size
The average absolute nuclear DNA content (2С-value) was initially determined for Trollius austrosibiricus, T. kytmanovii, T. ledebourii, and T. riederianus by flow cytometry. Specimens from other locations were additionally studied for T. altaicus, T. asiaticus, and T. chinensis (Table 1; Fig. 2). 2С-values for the three last species were close to our previous results . The nuclear DNA content in T. austrosibiricus, T. kytmanovii, T. ledebourii, and T. riederianus was higher than that in other studied Trollius and attained 9.36±0.05 pg, 9,09±0.32 pg, 9.08 ± 0.17 pg, and 8.92±0.16 pg, respectively. It should be noted that morphologically similar species, Trollius austrosibiricus, and T. chinensis, differed in the karyotype structure and the 2Сvalue. The second species showed a lower 2C-value that reached 8.78±0.20 pg and 8.87±0.26 pg in specimens from different locations. In conclusion, it should be noted that the absolute nuclear DNA content varied from 8.20±0.24 pg in Trollius farreri to 9.80±0.29 pg in T. lilacinus (Hegemone lilacina) . The 2С-value correlated with the total haploid length (THL) in all the studied Trollius except T. austrosibiricus, which can be due to the higher chromosome condensation in T. austrosibiricus or methodical aspects (Mártonfiová, 2013).

Conclusion
Ultimately, a similarity of karyotypes is typical of the genus Trollius. They have equal chromosome numbers and similar karyomorphological traits. Nevertheless, we revealed some variability of the absolute nuclear DNA content in the genus. The karyotype evolution was probably associated with small chromosome mutations, which insignificantly changed chromosomes shape. The genome size, in the meanwhile, had been changing.