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Research Article
Karyotype and genome size variation in white-flowered Eranthis sect. Shibateranthis (Ranunculaceae)
expand article infoElizaveta Yu. Mitrenina, Andrey S. Erst§, Lorenzo Peruzzi|, Mikhail V. Skaptsov, Hiroshi Ikeda#, Vyacheslav Yu. Nikulin¤, Wei Wang«
‡ National Research Tomsk State University, Tomsk, Russia
§ Laboratory of Herbarium, Central Siberian Botanical Garden, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| University of Pisa, Pisa, Italy
¶ Altai State University, Barnaul, Russia
# The University of Tokyo, Tokyo, Japan
¤ Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia
« State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany of the Chinese Academy of Sciences, Beijing, China
Open Access

Abstract

Comparative karyomorphological analyses of six out of the eight white-flowered species of Eranthis sect. Shibateranthis have been carried out. All studied specimens of E. byunsanensis, E. lobulata, E. pinnatifida, and E. stellata had a somatic chromosome number 2n = 16 with basic chromosome number x = 8. On the contrary, E. tanhoensis and E. sibirica had a basic chromosome number x = 7. The specimens of E. tanhoensis were diploid with 2n = 14, while the specimens of E. sibirica were polyploid with 2n = 42. Monoploid chromosome sets of the investigated diploid species had 4–5 metacentric chromosomes and 2–4 submetacentric/subtelocentric/acrocentric chromosomes. The highest level of interchromosomal asymmetry, estimated via CVCL, was found in E. byunsanensis and E. pinnatifida. The highest levels of intrachromosomal asymmetry (MCA) and heterogeneity in centromere position (CVCI) were found in E. lobulata and E. byunsanensis, while E. sibirica had the most symmetric karyotype. A multivariate PCoA analysis of basic karyotype parameters (2n, x, THL, CVCL, MCA, and CVCI) highlighted no overlap among species accessions, which was also confirmed by LDA. The average absolute monoploid DNA content (1Cx) of the 23 investigated samples of six Eranthis species varied from 9.26 ± 0.25 pg in E. sibirica to 15.93 ± 0.32 pg in E. stellata. Overall karyological affinity was highlighted between E. lobulata and E. stellata, on one side, and between E. byunsanensis and E. pinnatifida, on the other side. Interestingly, there was no significant correlation between total haploid (monoploid) chromosome length (THL) and 1Cx values in these species.

Keywords

Asia, chromosomes, Eranthis, genome size, karyotype, Ranunculaceae

Introduction

Chromosomal analysis is widely used in systematic and evolutionary studies of plants (Yuan and Yang 2006; Guerra 2012; Ilnicki 2014; Baltisberger and Hörandl 2016; Peruzzi et al. 2017). The main features of a karyotype are chromosome number, size and morphology of chromosomes (Astuti et al. 2017). Differences and similarities in karyotypes between taxa may reflect their evolutionary relationship (Shubert 2007; Peruzzi et al. 2009; Escudero et al. 2014; Baltisberger and Hörandl 2016). At present, it is appropriate to use the comparative analysis of karyotypes as part of an integrative approach to solving the issues of systematics and phylogeny (Astuti et al. 2017; Mráz et al. 2019; Erst et al. 2020b).

The genus Eranthis Salisb. belongs to Ranunculaceae Juss. tribe Cimicifugeae Torr. & A.Grey (Wang et al. 2009). This genus consists of ten to thirteen early flowering herbaceous perennial species distributed across Southern Europe, Western, Central and temperate Asia (Stefanoff 1963; Rukšāns and Zetterlund 2018; Park et al. 2019; Erst et al. 2020b). This genus generally exhibits a high level of endemism and it is distributed in both mainland and islands. Eranthis species seldom co-occur and the size of their distribution range usually varies significantly (Oh and Oh 2019). Eranthis is divided into two sections: E. sect. Eranthis and E. sect. Shibateranthis (Nakai) Tamura (Tamura 1987). The species belonging to the first section exhibit perennial tubers or tuberous rhizomes, yellow to orange sepals and yellow petals without pseudonectaries, whereas species of the second section have perennial tubers, white or slightly pink sepals and white petals with pseudonectaries (Tamura 1995; Zetterlund 2018; Park et al. 2019; Rukšāns and Erst et al. 2020b; Huang et al. 2021). The yellow-flowered E. sect. Eranthis includes five species distributed in Southern Europe (Eranthis bulgarica (Stef.) Stef., E. hyemalis (L.) Salisb.), Western Asia (E. cilicica Scott & Kotschy, E. iranica Rukšāns & Zetterl) and Central Asia (E. longistipitata Regel). The white-flowered E. sect. Shibateranthis includes eight species distributed in temperate Asia. Two species occur in Siberia (E. sibirica DC. and E. tanhoensis Erst), two in Tibet (E. albiflora Franch. and E. lobulata W.T.Wang), two in Korea (E. byunsanensis B.Y.Sun and E. pungdoensis B.U.Oh), one in Japan (E. pinnatifida Maxim.), and one is widespread and grows in China, Korea and the Far East of Russia (E. stellata Maxim.) (Oh and Oh 2019; Park et al. 2019; Erst et al. 2020b).

The somatic chromosome number 2n = 2x = 16 has been reported in Eranthis in eight species from both sections: E. byunsanensis (Kim et al. 2011), E. cilicica (Langlet 1932), E. hyemalis (Colasante and Ricci 1974; Tak and Wafai 1996; Gömürgen 1998; Caparelli et al. 2007; Erst et al. 2020a), E. lobulata (Erst et al. 2019), E. longistipitata (Erst et al. 2019), E. pinnatifida (Kurita 1955), E. sibirica (Gnutikov et al. 2016, 2017), and E. stellata (Yuan and Yang 2006). According to another study, E. stellata from Russian Far East would have somatic chromosome number 2n = 14 (Starodubtsev 1985), and this number was recently found in E. tanhoensis (Erst et al. 2020b). Additionally, polyploid cytotypes have been revealed in the genus Eranthis, e. g., triploid E. hyemalis with 2n = 24 (Colasante and Ricci 1974), tetraploid E. sibirica that had 2n = 32 (Krogulevich 1976) chromosomes, and the same species was recently found actually hexaploid by Erst et al. (2020b). The karyotype has been analyzed for five species: E. pinnatifida (Kurita 1955), E. hyemalis (Gömürgen 1998), E. stellata (Yuan and Yang 2006), E. sibirica and E. tanhoensis (Erst et al. 2020b).

The genome size (absolute nuclear DNA content), estimated by flow cytometry, is an essential genome feature together with the chromosome number and karyomorphological parameters (Doležel and Bartoš 2005). Flow cytometry can be considered a quick and useful method for understanding taxonomic relationships (Mabuchi et al. 2005; Zonneveld 2010). However, the Plants DNA C-value DataBase (https://cvalues.science.kew.org) contains data on E. cilicica, E. hyemalis, and E. pinnatifida only. This study reports data on comparative karyotype analysis and genome size of six out of eight white-flowered species of Eranthis sect. Shibateranthis (Fig. 1): E. byunsanensis, E. lobulata, E. pinnatifida, E. sibirica, E. stellata, and E. tanhoensis.

Figure 1. 

The studied species of white-flowered Eranthis sect. Shibateranthis A E. stellata (photo by V.V. Yakubov) B E. sibirica (photo by A.S. Erst); C E. tanhoensis (photo by A.S. Erst) D E. lobulata (photo by K.-L. Xiang) E E. pinnatifida (photo by A.S. Erst) F E. byunsanensis (photo by H.J. Choi).

Methods

Plant samples

Plant material (tubers) of E. byunsanensis, E. lobulata, E. pinnatifida, E. sibirica, E. stellata, and E. tanhoensis was collected during field investigations in Russia, China, Japan and South Korea during 2018–2020. The list of the samples examined is presented in Table 1. Herbarium specimens were deposited in the E and NS herbaria (herbarium acronyms according to Thiers 2019, continuously updated).

Table 1.

Chromosome number, ploidy and genome size in white-flowered Eranthis sect. Shibateranthis.

Species Voucher information 2n Ploidy level 1Cx ± SD (pg)
1 E. lobulata China, Sichuan Province, Jiuding Shan Mountain, 31°32'36.0"N, 103°51'12.0"E, 14 May 2018, L. Zhang 16 2x 13.87 ± 0.29
2 E. stellata* Russia, Primorsky Krai, Vladivostok City, Akademicheskaya Station, 43°11'25.9"N 131°55'31.7"E, 12 Apr 2018, V.V. Yakubov 16 2x 15.88 ± 0.31
3 E. stellata* Russia, Primorsky Krai, Vladivostok City, Malaya Sedanka River, 43°12'36"N, 131°59'24"E, 16 Apr 2019, V.Yu. Nikulin & A.Yu. Nikulin 16 2x 15.94 ± 0.34
4 E. stellata Russia, Primorsky Krai, Vladivostok City, forest in the vicinity of "13th km" railway station, 43°11'32"N, 131°55'49"E, 11 Apr 2019, V.Yu. Nikulin & A.Yu. Nikulin 16 2x 15.97 ± 0.31
5 E. stellata* Russia, Primorsky Krai, Vladivostok City, Russkiy Island, 42°59'05.0"N 131°51'51.5"E, 14 May 2019, V.Yu. Nikulin & A.Yu. Nikulin 16 2x 14.23 ± 0.23
6 E. stellata China, Jilin Province, Fusong County, Baishan City, Changbai Mt., 852 m alt., 42°06'55.5"N, 127°30'29.0"E, 29 Apr 2019, K. Xiang 16 2x 15.99 ± 0.91
7 E. tanhoensis* Russia, Republic of Buryatiya, Kabansky Raion, Bolshoi Mamai River, mixed forest, 51°23'30.1"N, 104°52'00.8"E, 20 Jun 2019, A.S. Erst, E.Yu. Mitrenina, D.A. Krivenko & O.A. Chernysheva 14 2x 12.44 ± 0.27
8 E. tanhoensis* Russia, Republic of Buryatia, Dulikha River, 51°32'04.9"N, 105°01'43.2"E, 1 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.49 ± 0.22
9 E. tanhoensis* Russia, Buryatia Republic, Kabansky Raion, Tolbazikha River, 51°26'21.06"N, 104°41'09.82"E, 2 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.38 ± 0.26
10 E. tanhoensis* Russia, Irkutsk Oblast, Slyudyansky Raion, Malye Mangaly River, 51°26'48.17"N, 104°34'16.62"E, 02 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.07 ± 0.06
11 E. tanhoensis* Russia, Irkutsk Oblast, Slyudyansky Raion, Semirechka River, 51°28'56.92"N, 104°19'43.47"E, 02 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.41 ± 0.29
12 E. tanhoensis* Russia, Buryatia Republic, Kabansky Raion, Osinovka River (Tankhoi Village), 51°33'06.2"N, 105°05'34.7"E, 01 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.56 ± 0.16
13 E. tanhoensis* Russia, Buryatia Republic, Kabansky Raion, Mishikha River, 51°37'32.6"N, 105°32'03.4"E, 01 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.07 ± 0.07
14 E. tanhoensis* Russia, Buryatia Republic, Kabansky Raion, Shestipalikha River, 51°32'46.4"N, 105°04'28.9"E, 01 May 2019, A.S. Erst, D.A. Krivenko, O.A. Chernysheva 14 2x 12.77 ± 0.09
15 E. sibirica* Russia, Irkutskaya Oblast', Slyudyanksky Raion, vicinity of Slyudyanka Town, mixed forest, 51°38'02.94"N, 103°41'13.90"E, 531 m alt., 02 May 2019, A.S. Erst, D.A. Krivenko & O.A. Chernysheva 42 6x 9.23 ± 0.14
16 E. sibirica* Irkutskaya Oblast', Slyudyanksky Raion, Burovschina River, 51°37'06.00"N, 103°49'16.17"E, 475 m, 20 Jun 2019, A.S. Erst, D.A. Krivenko, E.Yu. Mitrenina & O.A. Chernysheva 42 6x 9.27 ± 0.23
17 E. sibirica* Irkutskaya Oblast', Slyudyanksky Raion, Utulik River, 51°32'50"N, 104°02'45"E, 464 m alt., 20 Jun 2019, A.S. Erst, D.A. Krivenko, E.Yu. Mitrenina & O.A. Chernysheva 42 6x 9.22 ± 0.25
18 E. byunsanensis South Korea, Gyeonggi-do, Anyang-si, Suli-san, 37°21'42.8"N, 126°54'01.9"E, 190 m alt., 24 Mar 2019, H. Ikeda, H.-T. Im, K.-S. Chung, M. Fujii, M. Sakamoto & C. Hasekura, N°19032401 16 2x 10.75 ± 0.26
19 E. pinnatifida Japan, Saitama Prefecture, Chichibu-shi, Shiroku, near village, 35°57'24"N, 138°59'16"E, 340 m alt., 01 Apr 2019, A.S. Erst, T.V. Erst, H. Ikeda et al., N° 1 16 2x 9.87 ± 0.29
20 E. pinnatifida Japan, Mie Prefecture, Inabe-shi, Fujiwara-cho, Ogaito, forest, 35°10'11"N, 136°28'35"E, 180 m alt., 03 Apr 2019, A.S. Erst, T.V. Erst, H. Ikeda et al., N° 2 16 2x 9.80 ± 0.46
21 E. pinnatifida Japan, Mie Prefecture, Inabe-shi, Hokusei-cho, Betsumyo, 35°8'23"N, 136°28'20"E, 640 m alt., 04 Apr 2019, A.S. Erst, T.V. Erst, H. Ikeda et al., N° 5 16 2x 9.81 ± 0.10
22 E. pinnatifida Japan, Nagano Prefecture, Shiojiri-shi, Hideshio, near station, 36°2'58"N, 137°53'45"E, 825 m alt., 04 Apr 2019, A.S. Erst, T.V. Erst, H. Ikeda et al., N° 6 16 2x 9.80 ± 0.43
23 E. pinnatifida Japan, Nagano Prefecture, Shiojiri-shi, Motoyama, pine forest, 36°3'40"N, 137°53'50"E, 800 m alt., 04 Apr 2019, A.S. Erst, T.V. Erst, H. Ikeda et al., N° 7 16 2x 9.85 ± 0.27

Karyotype analysis

The comparative karyotype analysis was conducted for 22 populations: one of E. byunsanensis and E. lobulata, four of E. pinnatifida, three of E. sibirica, five of E. stellata, and eight of E. tanhoensis (Table 1). Somatic chromosomes of Eranthis were studied from root tip cells. Tubers were germinated in wet moss at ~ 15 °C for 2–4 weeks. Newly formed 1–2 cm long roots were excised and pretreated in 0.5% colchicine solution at 15 °C for 3–4 h. Roots were fixed in a mixture of 96% ethanol and glacial acetic acid (3:1). Root tips were stained with 1% aceto-haematoxylin, and the karyotypes were investigated by the squash method (Smirnov 1968). Chromosomes were counted in 30–100 mitotic cells for each population (a more detailed study was conducted for E. sibirica and E. tanhoensis). 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, Munich, Germany) with AxioVision 4.7 software (Carl Zeiss, Munich, Germany) and AxioCam MRc5 CCD-camera (Carl Zeiss, Munich, Germany) at 1000× magnification in the Laboratory for Ecology, Genetics and Environmental Protection (Ecogene), National Research Tomsk State University (Tomsk, Russia). KaryoType software (Altınordu 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 derived, based on measurements of the photographed mitotic metaphase chromosomes. The measurements were performed on 4–12 metaphase plates per population. We used 2–6 metaphase plates per population with the most condensed chromosomes to calculate mean karyomorphological parameters. The degree of chromosome condensation was estimated from the total haploid length of the chromosome set. The symbols used to describe the karyotypes corresponded to those coined by Levan et al. (1964): m = median centromeric chromosome with arm ratio (r) of 1.0–1.7 (metacentric chromosome); sm = submedian centromeric chromosome with arm ratio of 1.7–3.0 (submetacentric chromosome); st = subterminal centromeric chromosome with arm ratio of 3.0–7.0 (subtelocentric chromosome); t = terminal centromeric chromosome with arm ratio of 7.0 and more (acrocentric chromosome); T = chromosome without obvious short arm (telocentric chromosome). Mean values of arm ratio (r), centromeric indices (CI), mean chromosome length (CL), and relative chromosome length (RL) for each chromosome pair and total haploid length (THL) were determined. In addition, we calculated the Coefficient of Variation of Chromosome Length (CVCL; Paszko 2006), Coefficient of Variation of Centromeric Index (CVCI; Paszko 2006), and Mean Centromeric Asymmetry (MCA; Peruzzi and Eroğlu 2013).

To determine the karyological relationships among taxa, we carried out a multivariate PCoA (Principal Coordinate Analysis) using Gower's general coefficient of similarity, including six basic karyomorphological parameters (2n, x, THL, MCA, CVCL, and CVCI) in the data matrix (Peruzzi and Altınordu 2014), by plotting every single metaphase. Then, we also subjected the same data matrix to LDA (Linear Discriminant Analysis) to test the diagnosability of the six species on karyomorphological grounds. Finally, we tested the Spearman correlation between THL and 1Cx for each species, using mean data. To perform PCoA, LDA and correlation tests, the software Past 4.06b (Hammer et al. 2001; Hammer 2021), freely available online, was used.

Flow cytometry

Flow cytometry with propidium iodide (PI) staining was used to determine the absolute DNA content. In this study, we have determined this parameter in representatives of four Eranthis species: E. byunsanensis, E. lobulata, E. pinnatifida and E. stellata from 10 different populations (Table 1). Silica-gel-dried leaf material (0.5–1.0 cm2) 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 the external standard multiplied by the total nuclear DNA content of the standard. The 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 Eranthis samples centrifuged without PI. The samples were measured three times at 10 min intervals. If no variation in the average values of the detection channels was observed for the A. fistulosum peak, the effect of secondary metabolites was considered negligible. The 1Cx-value (monoploid DNA content sensu Greilhuber et al. 2005) was calculated by dividing the 2C-value by the ploidy level of the species. The species, used as external standards, were Zamioculcas zamiifolia Engl., 2C = 48.35 pg and Vicia faba L. "Inovec" 2C = 26.90 pg (Doležel et al. 1992; Skaptsov et al. 2016). 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 Laboratory for Bioengineering of the South-Siberian Botanical Garden, Altai State University (Barnaul, Russia).

Results

Karyotypes

Karyomorphometric data, microphotographs of metaphase plates and idiograms for the studied species are presented in Tables 2, 3 and Figs 2, 3.

Eranthis lobulata

Notes

The somatic and basic chromosome numbers in E. lobulata, endemic to China, are 2n = 16 and x = 8, respectively (Table 1; Fig. 2A). Five pairs of chromosomes (I–V) are metacentric, and three pairs (VI–VIII) are submetacentric, subtelocentric and acrocentric (Tables 2 and 3; Fig. 3). A pair of submetacentric chromosomes exhibits a secondary constriction. We also found a single small B chromosome in some cells. These Bs are metacentric, about 2.5 μm long. The karyotype formula of E. lobulata is 2n = 2x = 16 = 10m + 2smsat + 2st + 2t + 0–1B.

Table 2.

Karyomorphological parameters in white-flowered Eranthis sect. Shibateranthis.

Species Chromosome pair CL (µm) r CI RL (%) Type
E. lobulata I 8.46 ± 0.42 1.07 ± 0.04 0.48 7.80 m
II 8.19 ± 0.31 1.16 ± 0.09 0.46 7.55 m
III 7.43 ± 0.30 1.17 ± 0.07 0.46 6.85 m
IV 7.38 ± 0.16 1.36 ± 0.10 0.42 6.80 m
V 7.00 ± 0.29 1.28 ± 0.05 0.44 6.45 m
VI 6.11 ± 0.15 2.05 ± 0.09 0.33 5.63 smsat
VII 5.05 ± 0.21 5.04 ± 0.51 0.17 4.66 st
VIII 4.62 ± 0.24 8.35 ± 0.84 0.11 4.26 t
E. stellata (pop. 2) I 9.61 ± 0.34 1.07 ± 0.04 0.48 7.84 m
II 9.29 ± 0.31 1.07 ± 0.04 0.48 7.58 m
III 8.85 ± 0.39 1.06 ± 0.03 0.49 7.22 m
IV 8.31 ± 0.42 1.06 ± 0.04 0.49 6.78 m
V 7.89 ± 0.16 1.33 ± 0.07 0.43 6.44 m
VI 6.21 ± 0.25 2.00 ± 0.19 0.33 5.06 sm
VII 6.13 ± 0.40 2.14 ± 0.18 0.32 5.00 smsat
VIII 5.01 ± 0.34 7.86 ± 0.38 0.11 4.08 t
E. tanhoensis (pop. 12) I 8.68 ± 0.36 1.09 ± 0.05 0.48 8.74 m
II 8.56 ± 0.41 1.23 ± 0.06 0.45 8.62 msat
III 8.16 ± 0.29 1.07 ± 0.05 0.48 8.21 m
IV 7.73 ± 0.35 1.07 ± 0.05 0.48 7.78 m
V 6.63 ± 0.46 1.37 ± 0.11 0.42 6.67 m
VI 5.72 ± 0.46 1.92 ± 0.14 0.34 5.76 sm
VII 4.19 ± 0.38 2.34 ± 0.15 0.30 4.22 sm
E. sibirica (pop. 15) I 9.51 ± 0.24 1.08 ± 0.04 0.48 2.88 m
II 9.47 ± 0.29 1.03 ± 0.02 0.49 2.87 m
III 9.20 ± 0.06 1.17 ± 0.03 0.46 2.78 m
IV 9.13 ± 0.13 1.10 ± 0.06 0.48 2.76 m
V 9.00 ± 0.11 1.05 ± 0.02 0.49 2.72 m
VI 8.91 ± 0.14 1.39 ± 0.12 0.42 2.70 m
VII 8.88 ± 0.07 1.20 ± 0.03 0.45 2.69 m
VIII 8.87 ± 0.16 1.05 ± 0.03 0.49 2.68 m
IX 8.67 ± 0.10 1.08 ± 0.05 0.48 2.62 m
X 8.47 ± 0.09 1.27 ± 0.09 0.44 2.56 m
XI 8.44 ± 0.15 1.07 ± 0.03 0.48 2.55 m
XII 8.14 ± 0.13 1.16 ± 0.02 0.46 2.46 m
XIII 7.71 ± 0.04 1.18 ± 0.09 0.46 2.33 m
XIV 7.46 ± 0.15 1.35 ± 0.15 0.43 2.26 m
XV 7.26 ± 0.21 1.70 ± 0.06 0.37 2.20 sm
XVI 7.10 ± 0.04 1.28 ± 0.03 0.44 2.15 m
XVII 6.89 ± 0.05 1.61 ± 0.05 0.38 2.08 m
XVIII 6.45 ± 0.31 1.70 ± 0.08 0.37 1.95 sm
XIX 5.36 ± 0.23 1.97 ± 0.09 0.34 1.62 sm
XX 5.24 ± 0.25 1.74 ± 0.03 0.37 1.59 smsat
XXI 5.08 ± 0.34 2.29 ± 0.14 0.30 1.54 sm
E. byunsanensis I 8.59 ± 0.19 1.05 ± 0.03 0.49 8.55 m
II 8.13 ± 0.31 1.06 ± 0.04 0.49 8.09 m
III 7.65 ± 0.13 1.07 ± 0.04 0.48 7.61 m
IV 6.18 ± 0.09 1.40 ± 0.05 0.42 6.15 m
V 5.68 ± 0.21 1.19 ± 0.05 0.46 5.65 m
VI 5.44 ± 0.19 5.22 ± 0.30 0.16 5.41 st
5.19 ± 0.08 1.74 ± 0.05 0.37 5.17 sm
VII 5.20 ± 0.13 5.64 ± 0.19 0.15 5.17 st
VIII 3.52 ± 0.07 4.06 ± 0.37 0.20 3.50 st
E. pinnatifida (pop. 21) I 9.24 ± 0.18 1.12 ± 0.02 0.47 8.58 m
II 8.63 ± 0.24 1.08 ± 0.06 0.48 8.02 m
III 8.25 ± 0.31 1.13 ± 0.03 0.47 7.66 m
IV 6.62 ± 0.12 1.37 ± 0.07 0.42 6.15 m
V 6.24 ± 0.26 2.77 ± 0.14 0.27 5.80 sm
VI 5.88 ± 0.18 2.38 ± 0.07 0.30 5.46 sm
VII 5.04 ± 0.11 1.95 ± 0.13 0.34 4.68 sm
VIII 3.92 ± 0.09 3.10 ± 0.29 0.24 3.64 stsat
Figure 2. 

Mitotic metaphase plates of white-flowered Eranthis sect. Shibateranthis A E. lobulata, 2n = 16 B E. stellata (pop. 2), 2n = 16 C E. stellata (pop. 6), 2n = 16 D E. tanhoensis (pop. 12), 2n = 14 E E. tanhoensis (pop. 10), 2n = 14+0–8B (arrows point at B chromosomes) F E. sibirica (pop. 15), 2n = 42 G E. byunsanensis, 2n = 16 (arrows point at the heteromorphic chromosome pair) H E. pinnatifida (pop. 21), 2n = 16 I E. pinnatifida (pop. 20), 2n = 16 (arrows point at heteromorphic chromosome pair). Scale bars: 10 μm. Microphotographs by E.Yu. Mitrenina.

Figure 3. 

Haploid idiograms of white-flowered Eranthis sect. Shibateranthis species. I–VIII – chromosome pairs; m – metacentric chromosome; sm – submetacentric chromosome; st – subtelocentric chromosome; t – acrocentric chromosome; T – telocentric chromosome; B – B chromosome.

Eranthis stellata

Notes

In all five studied populations of E. stellata from Primorsky Krai of Russia and Jilin Province of China, the somatic and basic chromosome numbers are 2n = 16 and x = 8, respectively (Table 1; Fig. 2B–C). Five pairs of chromosomes (I–V) are metacentric, two pairs (VI–VII) are submetacentric, and one pair (VIII) is acrocentric (Tables 2 and 3; Fig. 3). A pair of submetacentric chromosomes (VII) exhibits a secondary constriction. The karyotype formula of E. stellata is 2n = 2x = 16 = 10m + 2sm + 2smsat + 2t. No B was observed in this species. Here, we present the results of the karyomorphological analysis of E. stellata from the "Academicheskaya Station" population (pop. 2).

Table 3.

Karyotype parameters in white-flowered Eranthis sect. Shibateranthis.

Species N Ploidy level 2n Karyotype formula THL CVCL MCA CVCI
E. lobulata 6 2x 16 10m + 2smsat + 2 st + 2t + 0–1B 54.24 (0.92) 19.68 (0.36) 28.15 (0.80) 38.05 (1.09)
E. stellata (pop. 2) 5 2x 16 10m + 2sm + 2smsat + 2t 61.30 (1.91) 20.77(0.84) 21.63(0.51) 31.69(0.83)
E. tanhoensis (pop. 12) 5 2x 14 8m + 2msat + 4sm + 0–8B 49.67 (2.02) 22.11 (1.10) 15.46 (0.76) 16.32 (1.24)
E. sibirica (pop. 15) 2 6x 42 32m + 8sm + 2smsat 165.24 (0.85) 17.55(0.87) 13.41(0.65) 12.87(0.18)
E. byunsanensis 4 2x 16 10m + 1sm + 5 st 50.26 (0.83) 25.69 (0.66) 26.43 (0.23) 37.18 (1.11)
E. pinnatifida (pop. 21) 5 2x 16 8m + 6sm + 2 stsat 53.82 (0.81) 25.81 (0.89) 25.16 (0.72) 24.90 (0.88)

Eranthis tanhoensis

Notes

In all eight studied populations of E. tanhoensis, Siberian endemic species, the somatic and basic chromosome numbers are 2n = 14 and x = 7, respectively (Table 1; Fig. 2D–E). Five pairs of chromosomes (I–V) are metacentric, two pairs (VI–VII) are submetacentric (Tables 2 and 3; Fig. 3). A pair of metacentric chromosomes (II) exhibited a secondary constriction. We found small B chromosomes in plants from two populations (pops 10 and 13). The maximum number of Bs in root tip cells appeared to be 8. They were represented by small metacentric and dot-shaped chromosomes, which are obviously telocentric. The karyotype formula of E. tanhoensis is 2n = 2x = 14 = 8m + 2msat + 4sm + 0–8B. Here, we present the results of the karyomorphological analysis of E. tanhoensis from the "Tanhoi Village" population (pop. 12).

Eranthis sibirica

Notes

The somatic chromosome number of E. sibirica, another endemic species from Siberia, is 2n = 42. The chromosome set of the species includes metacentric and submetacentric types of chromosomes. The karyotype formula of E. sibirica is 2n = 6x = 42 = 32m + 8sm + 2smsat. Here, we present the results of the karyomorphological analysis of E. sibirica from the "Slyudyanka Town" population (pop. 15) (Tables 2 and 3; Figs 2F and 3).

Eranthis byunsanensis

Notes

The chromosome set of the Korean endemic E. byunsanensis includes five pairs of metacentric chromosomes (I–V), one submetacentric (in the "pair" VI) and five subtelocentric chromosomes (in the "pair" VI and pairs VII–VIII) (Tables 2 and 3; Figs 2G and 3). The karyotype formula of E. byunsanensis is 2n = 2x = 16 = 10m + 1sm + 5st.

Eranthis pinnatifida

Notes

The Japanese endemic E. pinnatifida, unlike other related species, has four rather than five pairs of metacentric chromosomes (I–IV) and four rather than three pairs of submetacentric (V–VII) and subtelocentric chromosomes (VIII). The karyotype formula of the plants from three studied populations (pops 19, 21 and 22) is 2n = 2x = 16 = 8m + 6sm + 2stsat. These plants have secondary constrictions and small satellites at terminal regions of short arms of the pair VIII (Table 2; Figs 2H and 3). Specimens from the fourth population (pop. 20) have a pair of heteromorphic chromosomes (VIII) represented by one metacentric and one subtelocentric chromosome (Fig. 2I). The karyotype formula of these plants is 2n = 2x = 16 = 8m + 1msat + 6sm + 1stsat. In these plants, the secondary constriction in the metacentric homologue to the VIII pair is located in the pericentromeric region. Here we present the results of the karyomorphological analysis of E. pinnatifida from the "Inabe-shi" population (pop. 21).

Karyotype structure

The highest level of interchromosomal asymmetry, estimated via CVCL, was found in E. byunsanensis and E. pinnatifida. The highest levels of intrachromosomal asymmetry (MCA) and heterogeneity in centromere position (CVCL) were found in E. lobulata and E. byunsanensis, while E. sibirica had the most symmetric karyotype (Table 3). We analyzed 27 accessions (metaphase plates) by PCoA (cumulative variance explained by the first two axes: 81.31%). No overlap among species was evident (Fig. 4). Indeed, LDA correctly attributed objects (accessions) to the six species in 100% of cases (jackknifed).

Figure 4. 

PCoA (Coordinate 1, 65.31% of variance explained vs. Coordinate 2, 16% of variance explained) based on six karyological parameters of white-flowered Eranthis sect. Shibateranthis species.

Genome size

The absolute nuclear DNA content for 23 studied populations of six species of Eranthis is presented in Table 1. There was no significant correlation (r = 0.51, p = 0.29) between mean 1Cx values and total haploid (monoploid) chromosome length (THL) in these species. Indeed, for instance, while the 1Cx value is the smallest in the hexaploid E. sibirica, THL in this species is higher than in the diploid E. tanhoensis, sharing the same basic chromosome number x = 7 (Table 3).

Discussion

Karyotype structure in Eranthis

According to our results and other data (Kurita 1955; Tak and Wafai 1996; Gömürgen 1998; Yuan and Yang 2006), chromosome sets of different species of Eranthis share some common features, albeit showing some species-specific peculiarities, which allow a clear-cut distinction among species based on karyo-morphological features according to LDA (see also Fig. 4). The traits of the karyotype within each species are sufficiently stable. However, in some cases, polymorphism was observed in the chromosome morphology, for instance, in E. pinnatifida. The karyotypes of E. byunsanensis and E. lobulata were described here for the first time.

The chromosomes of Eranthis belong to the Ranunculus-type (Langlet 1932). The karyotypes of E. lobulata and E. stellata are similar. Both species show a chromosome pair with a very small, not always visible, short arm. In the other four studied species of Eranthis, no chromosome of this type was found. The secondary constrictions in E. lobulata and E. stellata are localized in the short arms of submetacentric chromosome pairs. Different localization of secondary constrictions in these species (Fig. 3) is possibly due to a paracentric inversion. Previously, the karyotype of E. stellata from China (Jilin Province) was studied by Yuan and Yang (2006). These authors described its formula as 2n = 2x = 16 = 10m + 2sm + 2st + 2T. In contrast to our data, they assigned pair VII to subtelocentric rather than submetacentric chromosome type. Our data show that the arm ratio of this chromosome pair is 2.14 ± 0.18 (Table 2), congruent with a sm chromosome-type. They also did not find a short arm in pair VIII and referred it to T-type (telocentric chromosomes). We found short arms in this VIII chromosome pair, which led us to classify it as chromosomes of t-type (acrocentric chromosomes).

Two species, endemic to Siberia, E. sibirica and E. tanhoensis, show atypical dysploid basic chromosome number for Eranthis (x = 7) and exhibit hexaploid (2n = 42) and diploid (2n = 14) cytotypes, respectively (Erst et al. 2020b). Since there are different definitions of the term "basic chromosome number (x)" concerning polyploids (Peruzzi 2013), we clarify that, in the study, we mean, "x" as "chromosome number found in the gametes of their diploid relatives", according to Darlington (1958). A recent phylogenetic study (Xiang et al. 2021) found that E. tanhoensis and E. sibirica are closely related species that formed separate groups with basic chromosome number x = 7 within the North Asian clade of Eranthis. The same basic chromosome number x = 7 with 2n = 14 was previously reported in the genus Eranthis for E. stellata (Starodubtsev 1985), albeit this author does not provide any microphotograph of the metaphase plate. We re-analyzed plants from the same area (pop. 3; Russia, Primorsky Krai, Malaya Sedanka River), but we found a somatic chromosome number 2n = 16. At the same time, previous studies on E. sibirica reported 2n = 16 (Gnutikov et al. 2016, 2017) and 2n = 32 (Krogulevich 1976) chromosomes. However, the diploid plants described in these studies apparently refer to the recently described species E. tanhoensis. Some populations of this species show B chromosomes that researchers may have identified as regular chromosomes. In addition, a pair of metacentric chromosomes show large satellites, which, when using the squash method, are sometimes detached and can be misidentified as small telocentric chromosomes. Based on the large amount of material analyzed and on careful analysis of chromosome morphology, we conclude that the basic chromosome number of the studied populations of E. tanhoensis and E. sibirica is x = 7. However, we do not rule out the possible occurrence of different cytotypes in plants from Siberia.

The karyotypes of the two related species, endemic to Korea and Japan, also show peculiar features. Eranthis byunsanensis has a heteromorphic pair of chromosomes (VI). Unfortunately, we had material from a single population of this species. Therefore, we cannot conclude whether this feature is characteristic of the whole species or just a heterozygous chromosomal mutation. Eranthis pinnatifida has another feature that distinguishes it from other diploid species: four pairs of isobrachial chromosomes and four pairs of heterobrachial chromosomes. Our results concerning this species are consistent with the data published by Kurita (1955). Among the four E. pinnatifida populations studied, one population (pop. 20) shows a heteromorphic pair of chromosomes. In this case, we are sure that this mutation is just a polymorphic variant.

Carta et al. (2020) estimated x = 7 as the most likely ancestral basic chromosome number in Ranunculaceae. However, we hypothesize that, in Siberian species, E. sibirica and E. tanhoensis, the basic chromosome number evolutionarily reduced from x = 8 to x = 7 and not vice versa. This hypothesis is because most of the tribe Cimicifugeae members (i.e., Actaea, Anemonopsis, Beesia, Cimicifuga, Souliea and closely related Helleborus; Wang et al. 2009) have x = 8 (Rice et al. 2015). In addition, it has been established that Eranthis originated in East Asia and then dispersed to the west Qinghai-Tibetan Plateau and Mediterranean regions (Xiang et al. 2021). East Asian Eranthis species (i.e., E. byunsanensis, E. lobulata, E. pinnatifida, and E. stellata) have x = 8. According to a recent phylogenetic study (Xiang et al. 2021), E. sibirica and E. tanhoensis are a derived group within the North Asian clade of Eranthis with non-canonical basic chromosome number x = 7 for the tribe Cimicifugeae.

The karyotypes of the two related species E. stellata and E. tanhoensis, with 2n = 16 and 2n = 14 chromosomes, respectively, are similar concerning five metacentric (I–V) and two submetacentric (VI–VII) chromosome pairs and differ by the presence of acrocentric pair (VIII) in E. stellata. It is well known that the basic chromosome number can change (dysploidy) due to chromosome rearrangements, fusion or fission of some chromosomes of the set and chromosome loss (Shubert 2007; Guerra 2008; Escudero et al. 2014). Dysploidy can establish powerful crossing barriers between sympatric taxa, as it disturbs regular chromosome pairing and bivalent formation at meiosis, drastically reducing hybrid fertility. These processes can result in the formation of new species (Grant 1981; Levin 2002; Baltisberger and Hörandl 2016). Such restructuring is known, for example, in the evolution of Arabidopsis thaliana (2n = 10) from A. lyrata (2n = 16) (Koch and Kiefer 2005). A similar case of descendant dysploidy was revealed for other Brassicaceae (Lysak et al. 2006) and plants from other families (Levin 2002). For Ranunculaceae, a decrease in the basic chromosome number from x = 8 to x = 7, caused by chromosome rearrangements, is known within Ranunculus (Baltisberger and Hörandl 2016) and Anemone (Mlinarec et al. 2012).

The shift to x = 7 in Eranthis possibly led to reproductive isolation of the populations with a new cytotype and, ultimately, speciation. We assume that further isolation of E. tanhoensis and E. sibirica was associated with polyploidization of the latter species. However, the type of polyploidy (i.e., autopolyploidy or allopolyploidy) has to be determined for this species. The karyotype of E. sibirica is similar to that of E. tanhoensis in chromosome morphology (metacentric and submetacentric chromosomes only), and they differ from the karyotypes of other related species. The organization of E. sibirica karyotype with 2n = 42 seems functionally diploid. The chromosomes are grouped in pairs (Fig. 3) and not in groups of 6. It is known that the size and shape of homologous chromosomes may change in the course of the diploidization process following polyploidization, i.e., due to the genome downsizing. Repetitive DNA sequences, both non-coding and coding, gene duplicates may be eliminated from the genome, resulting in changes in the karyotype parameters (Leitch and Bennett 2004; Mandáková and Lysak 2018; Wang et al. 2021).

A distinguishing feature of E. tanhoensis is the presence of small Bs in some of its populations (pops 11 and 14). Sporadic Bs were previously detected in individual cells only in E. lobulata (Erst et al. 2019). In some representatives of E. tanhoensis, up to 8 Bs could be observed in many cells. Bs are often found in representatives of Ranunculaceae and other families (Rice et al. 2015). However, their origin and possible adaptive and/or evolutionary roles are still poorly understood (Datta et al. 2016; Dhar et al. 2019). It is generally accepted that Bs are formed from A chromosomes in different ways. The most convincing case was the fully documented origin of a nascent B in trisomic Plantago lagopus L. from a supernumerary. This origin was associated with chromosome fragmentation, specific DNA sequence amplification, the addition of telomeric repeats and centromeric misdivision (Dhar et al. 2002). Bs could also escape as small centric fragments following unequal translocation and a reduction in chromosome number (Jones et al. 2008). Bs in E. tanhoensis may be preserved fragments of the lost ancestral pair VIII. The presence of Bs in the genome increases the adaptive capabilities of the population to adverse environmental conditions (Datta et al. 2016), which can be quite relevant for plants growing in this climatic zone.

Genome size of Eranthis

The Kew list of DNA C-values contains only one C-value for white-flowered Eranthis (i.e., 1C = 8.20 pg) determined by Zonneveld et al. (2005) for E. pinnatifida. In the present study, we determined the genome size for six white-flowered Eranthis species. According to our data, the Japanese E. pinnatifida has an average 1Cx = 9.80 ± 0.33 pg. It is the lowest absolute nuclear DNA content among the studied diploids. A lower 1Cx value (9.26 ± 0.25 pg) was found only in the polyploid E. sibirica. Remarkably, a closely related diploid species, such as E. tanhoensis, shows 1Cx = 12.48 ± 0.25 pg. According to the genome downsizing theory, an increase in the ploidy level leads to a decrease in the size of the monoploid genome. The loss of DNA in polyploids is a widespread phenomenon occurring in many plant groups (Shaked et al. 2001; Leitch and Bennett 2004; Adams and Wendel 2005). In the present study, Eranthis stellata exhibited the highest 1Cx-value of 15.93 ± 0.32 pg and the highest total haploid length as well. However, it is interesting to note that, in this system, we found no significant correlation between 1Cx and THL, as otherwise commonly found in plants (Levin 2002; Peruzzi et al. 2009), where this correlation typically exceeds r = 0.8. This inconsistency could be explained by different condensation degrees of the studied chromosomes. Nonetheless, it also may suggest differences in chromosomes width and volume (Kramer et al. 2021), not addressed in this study.

Conclusions

In this study, the comparative karyomorphological analyses and genome size determination of six white-flowered species of Eranthis sect. Shibateranthis from different populations have been carried out. The chromosome complements of E. lobulata and E. byunsanensis were determined for the first time. Karyotypes of studied Eranthis are shown to have both common features and species-specific features related to chromosome number, size and morphology. All the studied species can be distinguished based on their karyotype structure. They have the basic chromosome numbers x = 8 and x = 7, diploid and polyploid cytotypes. Additionally, E. tanhoensis and E. lobulata have small supernumerary chromosomes in the root tip cells. The monoploid genome size (C-value) determined by flow cytometry varies more than 1.5 times in the studied species.

Acknowledgements

We thank the associated editor and two anonymous reviewers for constructive comments which greatly improved this manuscript. This study was supported by Russian Science Foundation (research project No. 19-74-10082). The authors are grateful to R. V. Annenkov for compiling Figs 2 and 3 and D. A. Krivenko, O. A. Chernysheva, T. N. Veklich, A. Yu. Nikulin, V. V. Yakubov, L. Zhang and K. Xiang for collecting plant materials.

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