Karyotypes and genome size of Adonis amurensis and Adonis apennina (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 Katanov Khakass State University, 90 Lenin Ave., Abakan 655017, Russia 5 Amur Branch of Botanical Garden-Institute FEB RAS, Ignatievskoe Road, 2 km, Blagoveshchensk 675000, Russia 6 Siberian Institute of Plant Physiology and Biochemistry SB RAS, 134 Lermontov St., Irkutsk 664033, Russia 7 Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation, 8 Izmailovsky Ave., 105043 Moscow, Russia Institute of Botany of the Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China Corresponding author Email: emitrenina@gmail.com Received: 06.01.2021. Accepted 11.02.2021


Introduction
The genus Adonis L. is composed of perennial and annual herbaceous plants included in the tribe Adonideae T. Duncan&Keener under the subfam. Ranunculoideae of the Ranunculaceae Juss. family (Tamura, 1991;Luferov, 2004;Nishikawa & Kadota, 2006;Ren et al., 2009). According to various authors, the genus includes 40-50 (Poshkurlat, 2000) or 47-50 species (Luferov, 2018) distributed mainly in the extratropical zones of Eurasia. Approximately 26−30 species grow in the northern temperate zone, including Asia, Europe, and North America, and some annual plants are known to be distributed from Southwest Asia to North Africa, as well as along the shores of the Mediterranean (Meusel et al., 1965;Cronquist, 1981;Wang, 1994a;1994b;Son et al., 2016). In Russia, nine species are known, which include six perennials and three annuals. This genus's representations are found on plains and uplands in forest, meadow, steppe cenoses, limestone outcrops, and rocky outcrops (Luferov, 2020). The genus Adonis is characterized by rhizomatous perennials or annual herbaceous plants with erect flowering shoots. Leaves basal and cauline (cauline often absent at flowering time), proximal leaves petiolate, distal leaves sessile; cauline leaves alternate. Leaf blade 1-3-pinnately dissected, segments narrowly linear, margins entire or with the occasional tooth. Inflorescences terminal, flowers solitary; bracts absent. Flowers bisexual, radially symmetric; sepals 5-8, not persistent in fruit, nearly colorless or green, plane, obovate, 6-22 mm, apex erose; petals 3-20, distinct, yellow, red or white, often striped or basally darkened with black, purple, or blue, plane, oblanceolate, 8-35 mm; nectary absent; stamens 15-80; filaments filiform; Ukrainian Journal of Ecology, 11(1), 2021 staminodes absent between stamens and pistils; pistils ca. 20-50, simple; ovule 1 per pistil; style present. Fruits achenes, aggregate, sessile, nearly globose, sides veined or rugose; persistent style terminal, straight or strongly curved (Parfitt, 1993). The somatic chromosome numbers are known for most species of Adonis (Rice et al., 2015). The basic chromosome number for the genus is x = 8. Perennial species tend to have the diploid number of chromosomes 2n = 16 (Shlangena, 1976). However, there are also polyploid races such as Adonis amurensis Regel et Radde (Kurita, 1955;Nishikawa & Ito, 1979;Nishikawa, 1989). Annual species tend to have higher numbers of chromosomes, such as 2n = 32; 48 (Shlangena, 1976). There are less data on the structure of chromosome sets, although many species have been studied in detail. In this study, we present data on chromosomal sets of two species of Adonis growing in Russia: Adonis amurensis from Amur Oblast' and Adonis apennina L. from Altai Republic, Khakassia Republic, and Irkutsk Oblast. We appear to be the first to provide data on the nuclear DNA content (2C-values) of these species. Adonis amurensis Regel et Radde is referred to the section Adonanthe W.T. Wang, subsection Amurenses (Poschkurl.) M.H. Hoffm. and characterized by 15 cm tall at flowering, up to 30-40 cm high at fruiting time, with a short rhizome and numerous blackbrown adventitious roots. Shoots erect or ascending, simple or branching, with 3-6 scaly leaves up to 3 cm long, single-flowered, occasionally multi-flowered, straight or geniculate stems; imparipinnate, 4-pinnatifid with narrow-lanceolate acute or obtuse segments, shortly pubescent basal leaves; smaller, 3(2) order dissection upper leaves: 5 (rarely up to 7), greenish-gray, lavender sepals 4; 5-12 (sometimes up to 15), 1.2-2.5 cm long, 0.3-0.8 cm wide, oblong-elliptical, rounded at the apex and narrowed at the base, yellow petals; 3.5-5.0 mm long, greenish-brown, densely pubescent. Stylodia is located almost at the ovary's apex or shifted to the dorsal suture stamens (Fig. 1A). This species is distributed in Russian Far East: Amur Oblast, Jewish Autonomous Oblast', Khabarovsk and Primorsky Territories, Sakhalin Island, southern Kuril Islands: Kunashir, Shikotan, Iturup, North-East China, Korea Peninsula, North Japan: Hokkaido. -Deciduous forests, glades, meadows, rocky outcrops (Luferov, 2004). Adonis apennina L. is referred to the section Adonanthe W.T. Wang, subsection Vernales Poschkurl. and characterized by thick short rhizomes and black-brown adventitious roots; erect or ascending, simple or branching, up to 15 cm high at flowering, up to 30-40 cm high at fruiting, with 3-6 scaly leaves up to 3 cm long, one-flowered or multi-flowered stems (extremely rarely); straight or slightly curved, rounded, slightly ribbed stem; imparipinnate, 2-or 3-pinnatifid, with narrow lanceolate, acute or obtuse segments; upper leaves are smaller, second-order dissection basal leaves; 5 (rarely up to 7), greenish-gray, lavender sepals; 5-12 (sometimes up to 15), 2.0-3.0 cm long, 0.5-1.0 cm wide, obovate or rounded and narrowed at the base, mostly overlapping, yellow petals; numerous, 3.5-5.0 mm long, greenish-brown, densely pubescent stamens; almost at the apex of the ovaries, often more or less displaced to the dorsal suture stylodia (Fig. 1B). This species is distributed in the north-eastern part of European Russia, West, Middle and East Siberia, Russian Far East: south-west of Amur Oblast', Middle Asia, Mongolia, China. -Dry meadows, forest glades, and among forbs (Poshkurlat, 2000). In some publications, Adonis apennina is referred to as Adonis sibirica Patrin ex Ledeb. (Wang, 1994b), but the first name is nomenclature priority (Sennikov, 1998).

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 standard 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 Adonis samples centrifuged without PI (Erst et al., 2020b;Mitrenina et al., 2020). The samples were measured three times at 10 min intervals. If the A. fistulosum peak 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).
Ukrainian Journal of Ecology, 11(1), 2021  Fig. 2). The obtained average lengths of chromosomal arms were used to draw idiograms. The chromosomes were divided into two groups according to the morphology (metacentric and submetacentric ones), and within the group, they have been arranged as the chromosome length decreases. In general, our results are consistent with those presented in other studies. The karyotype of A. amurensis was previously studied for plants with 2n = 24 from Japan (Kurita, 1955). It was found Ukrainian Journal of Ecology, 11(1), 2021 that the karyotype equally consists of isobrachial and heterobrachial chromosomes. The difference in the karyotype formulas presented in this study and the manuscript by M. Kurita is due to different chromosome nomenclature. M. Kurita classified heterobrachial chromosomes as subtelocentric, while we classified them as submetacentric ones. The karyotype of Adonis amurensis with 2n = 16 from Primorsky Krai and Sakhalin Island has been recently described (Volkova et al., 2020). The karyotype formula is similar to our result, but there are some differences in some chromosomes' relative length. All submetacentric chromosomes are shorter than metacentric ones in the karyotypes of plants from the Primorsky Krai and Sakhalin Island. While in karyotype plants from Amur Oblast' the shortest metacentric chromosome (IV pair) is shorter than several submetacentric chromosomes (V-VII pairs). These differences are likely related to the level of condensation of the chromosomes studied. We studied more compact chromosomes because the total length of the set in our study was lower than that in another research. The chromosome sets of Adonis apennina were previously studied for Kemerovo, Tomsk, and Krasnoyarsk Oblast' for plants with 2n = 16 (Schrager & Malakhova, 1978;Schrager, 1980). The plants from the Altai Republic, Khakassia Republic, and Irkutsk Oblast' have been investigated in this study. There are no significant differences in the karyotype structure between the populations studied and the previous works' results. There are slight variations in the relative length of chromosomes and centromeric indices. Up to four satellite chromosomes were presented in all the investigated karyotypes. Satellites were small and single or double. They were not found in all cells related to the level of chromosome compaction of the metaphase plates studied. In the studied populations of Adonis apennina and A. amurensis we have not detected polymorphism on the morphology of satellite chromosomes, as previously shown (Schrager, 1980;Volkova et al., 2020). In addition to the basic karyomorphological parameters, we estimated some karyotype asymmetry indices (Table 3). The qualiquantitative Stebbins asymmetry index (1971) was 2A for Adonis amurensis and 3A for A. apennina. The chromosome number with an arm ratio < 2 was five pairs in A. amurensis and four pairs in A. apennina. Three parameters detecting interchromosomal and intrachromosomal karyotype asymmetries, CVCL -Coefficient of Variation of Chromosome Length, CVCI -Coefficient of Variation of Centromeric Index (Paszko, 2006), and MCA -Mean Centromeric Asymmetry (Peruzzi & Eroğlu, 2013), were close in A. apennina from two investigated populations. Insignificant differences are related to the degree of condensation of chromosomes of the plates studied. The intrachromosomal and interchromosomal asymmetries' parameters were lower in A. amurensis, which means that this species has a more symmetric karyotype than A. apennina. In general, other representatives of the genus Adonis also equally include isobrachial and heterobrachial chromosomes in the set. For instance, the karyotype formula for A. vernalis L. (Schrager & Malakhova, 1981) was 2n = 2x =16 = 8m + 8sm, and for A.
Ukrainian Journal of Ecology, 11(1), 2021 brevistyla Franch. it 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). This is probably the general pattern of karyotype in the genus Adonis. Differences between species relate primarily to the degree of asymmetry of heterobrachial chromosomes. An additional parameter characterizing the chromosome set is the genome size (nuclear DNA content, or 2C-value). We have determined absolute nuclear DNA content for most of the studied populations of both species (Table 1; Fig. 3). The values varied slightly between the plants of diverse populations within the species. Absolute nuclear DNA content in Adonis amurensis was higher (from 20.05±0.92 to 20.71±1.20 pg) than that in A. apennina (from 16.96±0.50 to 17.67±0.62 pg), even though the total chromosome length of the set was close. This is obviously due to higher condensation of the A. amurensis chromosomes compared to A. apennina.

Conclusion
Thus, the Adonis apennina chromosome set is conservative in both chromosome morphology and nuclear DNA content over at least a significant part of the areal. Adonis amurensis is a more polymorphic species, at least in its ploidy level.