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A new species of Eranthis (Ranunculaceae) from North Asia 75
An integrative taxonomic approach reveals a new
species of Eranthis (Ranunculaceae) in North Asia
Andrey S. Erst1,2, Alexander P. Sukhorukov3, Elizaveta Yu. Mitrenina2,
Mikhail V. Skaptsov4, Vera A. Kostikova1, Olga A. Chernisheva5,
Victoria Troshkina1, Maria Kushunina3, Denis A. Krivenko2,5, Hiroshi Ikeda6,
Kunli Xiang7,8, Wei Wang7,8
1 Central Siberian Botanical Garden, Siberian Branch of Russian Academy of Sciences, 101 Zolotodolinskaya
Str., 630090, Novosibirsk, Russia 2 Tomsk State University, 36 Lenin Ave., 634050, Tomsk, Russia 3 Lomo-
nosov Moscow State University, Leninskie Gory 1/12, 119234, Moscow, Russia 4 South-Siberian Botanical
Garden, Altai State University, 61 Lenin Ave., Barnaul, 656049, Russia 5 Siberian Institute of Plant Physio-
logy and Biochemistry, Siberian Branch of Russian Academy of Sciences, 132 Lermontov Str., 664033, Irkutsk,
Russia 6 e University Museum, e University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
7 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences,
100093, Beijing, China 8 University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China
Corresponding author: Andrey S. Erst (erst_andrew@yahoo.com)
Academic editor: M. Pellegrini|Received 3 December 2019|Accepted 6 February 2020|Published 4 March2020
Citation: Erst AS, Sukhorukov AP, Mitrenina EYu, Skaptsov MV, Kostikova VA, Chernisheva OA, Troshkina V,
Kushunina M, Krivenko DA, Ikeda H, Xiang K, Wang W (2020) An integrative taxonomic approach reveals a new species
of Eranthis (Ranunculaceae) in North Asia. PhytoKeys 140: 75–100. https://doi.org/10.3897/phytokeys.140.49048
Abstract
A new endemic species, Eranthis tanhoensis sp. nov., is described from the Republic of Buryatia and
Irkutsk Province, Russia. It belongs to Eranthis section Shibateranthis and is morphologically similar to
E. sibirica and E. stellata. An integrative taxonomic approach, based on cytogenetical, molecular and bio-
chemical analyses, along with morphological data, was used to delimit this new species.
Keywords
Biochemistry, cytology, integrative taxonomic approach, morphology, phylogeny, Ranunculales, Russia
PhytoKeys 140: 75–100 (2020)
doi: 10.3897/phytokeys.140.49048
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Copyright Andrey S. Erst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
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Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
76
Introduction
e genus Eranthis L. (Ranunculaceae) consists of eight to ten species distributed in
southern Europe and temperate Asia (Lee et al. 2012; Park et al. 2019). Most species
have narrow distributions and only one European species, E. hyemalis (L.) Salisb., has
been widely cultivated in gardens and become naturalised in Britain (Boens 2014) and
North America (Partt 1997). Eranthis are perennial herbs with tuberous rhizomes, ba-
sal long-petiolate leaves with the blades divided into several or many palmate segments
(leaets) that are entire or lobate; unbranched scapes carrying a solitary, bisexual and
actinomorphic ower supported by three verticillate leaf-like bracts forming an invo-
lucre; (4–)5–8 yellow, white or pink, caducous sepals; 5–10(–15) yellow or white, bid
petals shorter than sepals; nectaries located at the middle or upper part of the petals; >
10 stamens; and 3–10 follicles with several smooth seeds in each fruitlet (Partt 1997).
All species are early-blooming plants, with anthesis from March to May (depending on
the altitude), but E. hyemalis has been found at full anthesis in mid-January in gardens
(Sukhorukov, pers. obs. in Mainz, Germany, 2019 and Leiden, Netherlands, 2020).
On the basis of morphology, the genus has been divided into two sections: E. sect.
Eranthis and E. sect. Shibateranthis (Nakai) Tamura (Tamura 1987). e type section
is characterised by annual tubers, yellow sepals and emarginate or slightly bilobate
upper petal margins without swellings (nectaries), whereas the members of section
Shibateranthis have long-lived tubers, white sepals and bilobate or forked petal margins
with swellings (Tamura 1995). Molecular phylogenetic analysis. based on nrITS and
chloroplast trnL-trnF interspacer region, supports the subdivision of the genus into
these sections (Park et al. 2019). Furthermore, they are geographically separated, with
section Eranthis occurring in Europe (E. hyemalis) and SW & W Asia (E. cilicica Schott
& Kotschy, E. longistipitata Regel) and section Shibateranthis distributed in temperate
N & E Asia (E. albiora Franch., E. byunsanensis B.Y.Sun, E. lobulata W.T.Wang, E.
pinnatida Maxim., E. pungdoensis B.U.Oh, E. sibirica DC. and E. stellata Maxim.:
Park et al. 2019). Two additional species with yellow sepals, E. bulgarica (Stef.) Stef.
(Stefano 1963) and E. iranica Rukšāns & Zetterl. (Rukšāns and Zetterlund 2018),
have been described from Bulgaria and Iran, respectively, but have not yet been in-
cluded in molecular analysis.
Recent studies have revealed the genetic diversity, phylogeny and presumed origin
of some narrowly distributed Korean and Japanese species with further conclusions
about their taxonomic status (Lee et al. 2012; Oh and Oh 2019). e taxonomic
and genetic diversity of Eranthis in the Asiatic part of Russia is insuciently studied.
To date, only two species have been found in Russia: E. sibirica and E. stellata (both
belonging to sect. Shibateranthis) from South Siberia and Far East Russia (Malyshev
2005). High genetic polymorphism of E. sibirica across populations near Baikal Lake
was discovered only recently (Protopopova et al. 2015) and this fact has inspired us to
conduct a new study of Eranthis in the Asiatic part of Russia.
e aim of the present study was to investigate the morphological, molecular, bio-
chemical and cytogenetic heterogeneity of the Baikal populations to determine wheth-
A new species of Eranthis (Ranunculaceae) from North Asia 77
er any undescribed species were present there. e relationship between E. sibirica, E.
stellata and a new species, described and named below as Eranthis tanhoensis Erst, sp.
nov. is explored here.
Materials and methods
Plant material
More than 300 herbarium specimens were collected during eld investigations in the
Republics of Khakassia and Buryatia and the Irkutsk Province during 2018 and 2019.
Fieldwork was conducted during dierent seasons to observe the species in both their
owering and fruiting stages. e specimens were deposited in the E and NS her-
baria (herbarium abbreviations according to iers 2019+). Revision of herbarium
materials was undertaken in the herbaria at IRK, LE, MW, NS, NSK, PE, VBGI and
VLA. Drawings of the new species, Eranthis tanhoensis, are based on images of the type
specimen (NS-0000948!) and paratype (NS-0000949!). e owering and fruiting
times and habitats are provided as cited on the collectors’ labels. Maps of records were
made with SimpleMappr (http://www.simplemappr.net). Conservation analysis was
performed using criteria from the International Union for the Conservation of Nature
(IUCN 2019). e Extent of Occurrence (EOO) and Area of Occupancy (AOO) of
each species were estimated using GeoCat (Bachman et al. 2011).
Molecular analysis
We sampled 15 individuals of E. tanhoensis and six of E. sibirica. Two individuals of E.
stellata and one each of E. pinnatida and E. longistipitata were also included. e details
of the samples are presented in Suppl. material 1: Table S1. Six nuclear and plastid DNA
regions (ITS, trnL-F, trnH-psbA, rps16, matK and rbcL) were included in the molecular
analysis. Total genomic DNA was extracted from silica gel-dried leaves or herbarium
specimens using DNeasy Mini Plant Kits (Tiangen Biotech, Beijing, China) following
the protocol specied by the manufacturer. Sequencing reactions were conducted using
BigDyeTM Terminators (Applied Biosystems Inc., Foster City, CA, USA). Sequences
were read using an automated ABI 3730xl DNA Analyzer. Geneious v8.0.4 (Kearse et
al. 2012) was used to evaluate the chromatograms for base conrmation and to edit con-
tiguous sequences. We rst used the Maximum Likelihood (ML) method to perform
non-parametric bootstrap analyses for each DNA region in RAxML v7.0.4 (Stamatakis
2006). No signicant bootstrap support for conicting nodes was evident amongst in-
dividual DNA regions (here considered to exceed 70%) and the six-locus datasets were
therefore combined for subsequent analyses. Phylogenetic analyses of the combined
dataset were conducted using ML and Bayesian Inference (BI) methods. RAxML was
conducted with the GTR + Γ substitution model for each region with the fast bootstrap
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
78
option using 1000 replicates. BI analysis was conducted in MrBayes v3.2.1 (Ronquist
et al. 2012). Data partitioning and nucleotide substitution models were determined
using PartitionFinder 2.1.1 (Lanfear et al. 2016). Two independent analyses, consist-
ing of four Markov Chain Monte Carlo chains were run, sampling one tree every 1000
generations for 10 million generations. Runs were completed when the average standard
deviation of split frequencies reached 0.01. e stationarity of the runs was assessed us-
ing Tracer v1.6 (Rambaut et al. 2014). After removing the burn-in period samples (the
rst 25% of sampled trees), a majority rule (> 50%) consensus tree was constructed.
Morphological analysis
e morphology of vegetative and reproductive structures was examined on well-de-
veloped specimens. For numerical analysis, 25 specimens at owering and 25 speci-
mens at fruiting stages were examined for each species (more than 150 specimens
altogether). For each species, we studied dierent populations from across the range,
including populations from the type localities of E. stellata and E. sibirica. As E. stellata
often does not produce basal leaves at owering, we studied this character in a limited
number of samples. e morphological characters were measured using AxioVision 4.8
software (Carl Zeiss, Munich, Germany).
e missing values in the original data table were restored using multidimensional
linear regression, in accordance with recommendations of Myers (2000) and Lee and
Carlin (2010). A one-way analysis of variance (ANOVA), according to Chambers et al.
(1992), was used to identify the distinguishing morphometric features of each species.
e dierences were considered signicant at P-value < 0.05. As multiple statistical test-
ing was performed, the calculated P-value was adjusted using the procedure proposed by
Benjamini and Hochberg (1995). e principal component analysis was used to visu-
alise the distribution of the analysed individuals over the space of morphometric char-
acters. is method was employed only for those characters that displayed signicant
intergroup dierences, according to the results of the ANOVA. For scale adjustment,
the logarithmic transformation of data was used. e results of the principal component
analysis were visualised using the Factoextra package (Kassambara and Mundt 2017).
Cytogenetic analysis
Somatic chromosomes were studied in 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 a 0.5% colchicine solution for 2–3 h at 15 °C. Roots were xed in a mixture of 96%
ethanol and glacial acetic acid (3:1). Root tips were stained with 1% aceto-haematoxylin
and the squash method was employed for investigation of the karyotype (Smirnov 1968).
Chromosomes were counted in 50–100 mitotic cells for each population. Mitotic
metaphase chromosome plates were observed using an Axio Star microscope (Carl
A new species of Eranthis (Ranunculaceae) from North Asia 79
Zeiss, Munich, Germany) and photographed using an Axio Imager A.1 microscope
(Carl Zeiss, Germany) with AxioVision 4.7 software (Carl Zeiss, Germany) and Ax-
ioCam MRc5 CCD–camera (Carl Zeiss, Germany) at 1000× magnication in the
Laboratory for Ecology, Genetics and Environmental Protection (Ecogene) of the Na-
tional Research Tomsk State University. KaryoType software (Altinordu et al. 2016)
was used for karyotyping, whereas Adobe Photoshop CS5 (Adobe Systems, USA) and
Inkscape 0.92 (USA) were used for image editing. Karyotype formulae were based on
measurements of mitotic metaphase chromosomes taken from photographs. e meas-
urements were performed on 5–10 metaphase plates. e symbols used to describe the
karyotypes followed those of Levan et al. (1964): m = median centromeric chromo-
some with arm ratio of 1.0–1.7 (metacentric chromosome); sm = submedian cen-
tromeric chromosome with arm ratio of 1.7–3.0 (submetacentric chromosome); st =
subterminal centromeric chromosome with arm ratio of 3.0–7.0 (subtelocentric chro-
mosome); t = terminal centromeric chromosome with arm ratio of 7.0–∞ (acrocentric
chromosome); T = chromosome without obvious short arm, i.e. with arm ratio of ∞.
Flow cytometry
Flow cytometry with propidium iodide (PI) staining was used to determine the abso-
lute DNA content. e relative DNA content in the nucleus (C-value) in representa-
tives of three Eranthis species – E. stellata, E. sibirica and E. tanhoensis from dierent
populations, was determined in this study. In total, more than 70 samples from 15
populations were studied (see Suppl. material 1: Table S1). Silica gel-dried leaf material
(0.5–1.0 cm2) was chopped with a sharp razor blade in a 1 ml cold nuclei extraction
buer composed of 50 mM Hepes, 10 mM sodium metabisulphite, 10 mM MgCl2,
0.5% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.3% Tween20, 0.2% Triton
X-100, 50 µg/ml RNase, 1 µg/ml β-mercaptoethanol and 50 µg/ml propidium iodide
(PI). e samples were ltered through 50 µm nylon membranes into sample tubes
and incubated in the dark at 4 °C for 15 min. Samples were measured using a Partec
CyFlow PA ow cytometer equipped with a green laser, at 532 nm wavelength. e ab-
solute nuclear DNA content, the 2C-value according to Greilhuber et al. (2005), was
calculated as the ratio of the mean uorescence intensity of the nuclei of the sample to
that of an external standard multiplied by the total nuclear DNA content of the stand-
ard. e possible eect of secondary metabolites on the binding of the intercalating
dye was evaluated by measuring the uorescence of Allium stulosum L. leaf samples
prepared as described above, but with the addition of the supernatant from Eranthis
samples, centrifuged without PI. e 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. stulosum peak, the eect of secondary metabolites was considered negligible.
e 1Cx-value (monoploid DNA content sensu Greilhuber et al. 2005) was calcu-
lated by dividing the 2C-value by the ploidy level of the species. e species, used as
external standards, were Zamioculcas zamiifolia Engl., 2С = 48.35 pg and Vicia faba
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
80
L. ‘Inovec’ 2С = 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 Bio-
technology) 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).
High-performance liquid chromatography (HPLC) analysis of individual phenolic
compounds in ethanol leaf extracts
In order to determine the composition of phenolic compounds, air-dried plant material
was mechanically ground to obtain a homogenous powder and then samples of ~0.2 g
were extracted three times using 70% aqueous ethanol solution for 30 min in a water
bath at 72 °C. Next, the combined extract was concentrated in porcelain dishes to 5ml.
e solutions were ltered and stored at 4 °C until analysis. Analysis of phenolic com-
ponents was performed using an Agilent 1200 HPLC system equipped with a diode
array detector and a ChemStation system for the collection and processing of chromato-
graphic data (Agilent Technology, Palo Alto, CA, USA). e separation was performed
on a Zorbax SB-C18 column (5 µm, 4.6 × 150 mm) at 25 °C. e methanol content of
the mobile phase in an aqueous solution of phosphoric acid (0.1%) varied from 50–52%
over 56 min (van Beek 2002). e eluent ow rate was 1 ml/min. Detection wavelengths
were 255, 270, 340 and 360 nm. Groups of phenolic substances were identied by their
spectral characteristics (Bate-Smith 1962; Mabry et al. 1970). For identication of the
phenolic components in plant extracts, standard samples of salicylic and chlorogenic
acids, quercetin, kaempferol, orientin (Sigma-Aldrich Chemie GmbH, Munich, Ger-
many), gentisic and caeic acids (Serva Heidelberg, Germany), hyperoside and vitexin
(Fluka Chemie AG, Buchs, Switzerland) were used. e samples were analysed twice.
Results and discussion
Molecular phylogenetic analysis
Bayesian and ML analyses of the combined dataset produced highly consistent topolo-
gies. Eranthis sibirica and the new species E. tanhoensis formed a sister clade of that of
E. pinnatida. e monophyly of each species, E. tanhoensis sp. nova, E. sibirica and E.
stellata, was strongly supported (Fig. 1).
Morphological analysis
e morphological analysis revealed that E. sibirica was not homogeneous across its dis-
tribution area. We compared 41 characters to distinguish E. sibirica, E. tanhoensis and
A new species of Eranthis (Ranunculaceae) from North Asia 81
Figure 1. ML tree inferred from the combined cpDNA and ITS data. e numbers above branches are
bootstrap values (BS > 50%) and numbers under branches are Bayesian posterior probabilities (PP > 0.50).
E. stellata (Suppl. material 1: Table S2). e basal and involucral leaves in Eranthis spp.
undergo changes at fruiting and, for this reason, the lengths of all leaves, their segments
and segment lobes were measured both at the owering and fruiting stages. In Suppl.
material 1: Table S2, an asterisk (*) indicates the characters used in the numerical analy-
sis. An ANOVA was conducted only for quantitative characteristics. As basal leaves
are often absent at the time of owering and there were no samples with basal leaves
in herbarium collections, there were limited data on these characteristics of E. stellata.
e ANOVA of morphometric characters showed signicant dierences amongst
the studied species in characters (1), (9), (16), (18), (22), (24), (29), (30), (31) and (32)
at the owering stage and (6), (10), (14), (17), (19), (23), (25), (40) and (41) at the
fruiting stage (Suppl. material 1: Tables S3, S4). In total, signicant dierences amongst
the species were found in 10 out of 15 morphometric parameters measured at owering
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
82
and in 9 out of 13 parameters at fruiting. e principal component analysis revealed
that the rst two main components accounted for 83.1% and 81.8% of the variance in
the entire data array of the parameters measured at owering and fruiting, respectively
and showed the best species discrimination. e highest variability of morphometric
characters was found at owering in E. sibirica (Fig. 2A) and at fruiting in E. tanhoensis
(Fig. 2B). As signied by the directions of the vectors indicating the gradients in the
character values, at owering, E. sibirica diered from E. tanhoensis by having lower
values for characters (18), (22), (24) and (31) and a higher value for character (9). At
fruiting, E. sibirica was characterised by having lower values for parameters (19), (17),
(23) and (25) and higher values for parameters (10), (40) and (29), in comparison with
those of E. tanhoensis. E. sibirica diered from E. stellata by having higher values for
characters (1), (16), (29), (30) and (32) at owering and (10) and (14) at fruiting. e
pattern of overlap between the species diered between owering and fruiting plants.
For instance, E. tanhoensis was reliably distinguished from E. sibirica only at fruiting
(the ellipses enclosing the samples did not overlap; Fig. 2B). In addition to numerical
parameters, the new species was also distinguished by qualitative characters.
Cytogenetic analysis
e karyotypes of three related species, E. sibirica, E. tanhoensis and E. stellata, were inves-
tigated (Table 1), those of E. sibirica and E. tanhoensis being studied for the rst time. e
chromosomes of each species were medium or large in size (from 5 to 11–12 µm) and be-
longed to the R-type (Langlet 1932). e vouchers are listed in Suppl. material 1: Table S1.
Figure 2. Scatter point diagram in the space of the rst two main components for Eranthis sibirica (red
dots), Eranthis tanhoensis (green triangles) and Eranthis stellata (blue squares) A at owering and B at
fruiting stages. Ellipses enclose the regions of the space that contain each of the plant species with a 95%
probability (95% condence ellipses).
A new species of Eranthis (Ranunculaceae) from North Asia 83
Table 1. Chromosome numbers (2n), ploidy level (nx), karyotype formulas, and C-values (C ± SD) of
the three studied Eranthis species.
Voucher
number
Species Voucher information 2nnxKaryotype formulae 2C±SD, pg 1Cx±SD, pg
1E. sibirica Republic of Khakassia, Bolshoi
On river
28 4x2n = 20m (2 sat) +
2m/sm + 6 sm
38.83±1.03 9.71±0.26
2E. sibirica Irkutsk Province, Kuitun river 28 4x2n = 20m (2 sat) +
2m/sm + 6 sm
38.19 ±
0.28
9.55 ± 0.14
3E. sibirica Irkutsk Province, Slyudyanka
river
42 6x2n = 30m + 12sm
(2 sat)
55.75±0.28 9.23±0.14
4E. sibirica Irkutsk Province, Burovschina
river
42 6x2n = 30m + 12sm
(2 sat)
55.76±0.47 9.27±0.23
5E. sibirica Irkutsk Province, Utulik river 42 6x2n = 30m + 12sm
(2 sat)
55.31±0.45 9.22±0.25
6E. tanhoensis Irkutsk Province, Mamai river 14 2x2n = 10m (2sat) +
4sm
24.88±0.54 12.44±0.27
7E. tanhoensis Republic of Buryatia, Duliha
river
14 2x2n = 10m (2sat) +
4sm
24.97±0.43 12.49±0.22
8E. tanhoensis Republic of Buryatia,
Tolbazikha river
14 2x2n = 10m (2sat) +
4sm
24.77±0.52 12.38±0.26
9E. tanhoensis Irkutsk Province, Malye
Mangaly river
14 2x2n = 10m (2sat) +
4sm + 0–8B
24.15±0.11 12.07±0.06
10 E. tanhoensis Irkutsk Province, Semirechka
river
14 2x2n = 10m (2sat) +
4sm
25.31±0.15 12.41±0,29
11 E. tanhoensis Republic of Buryatia,
Osinovka river (Tanhoi village)
14 2x2n = 10m (2sat) +
4sm
25.11±0.32 12.56±0.16
12 E. tanhoensis Republic of Buryatia, Mishiha
river
14 2x2n = 10m (2sat) +
4sm + 0–4B
25.25±0.15 12.07±0.07
13 E. tanhoensis Republic of Buryatia,
Shestipalikha river
14 2x2n = 10m (2sat) +
4sm
25.53±0.18 12.77±0.09
14 E. stellata Primorsky Krai, Vladivostok,
Studencheskaya railway station
16 2x2n = 16 = 10m +
4sm (2sat) + 2t
31.76±0.61 15.88±0.31
15 E. stellata Primorsky Krai, Malaya
Sedanka river
16 2x2n = 16 = 10m +
4sm (2sat) + 2t
31.88±0.67 15.94±0.34
16 E. stellata Primorsky Krai, “13th
km” railway station
16 2x2n = 16 = 10m +
4sm (2sat) + 2t
– –
17 E. stellata Primorsky Krai, Russkiy Island 16 2x2n = 16 = 10m +
4sm (2sat) + 2t
28.47±0.46 14.23±0.23
Eranthis sibirica. Two cytotypes, with basic chromosome number x = 7, were re-
vealed. Eranthis sibirica from the Republic of Khakassia (1) and Irkutsk Province (2) were
tetraploid with 2n = 4x = 28 (Fig. 3A, B). ree populations from the Irkutsk Province
(3, 4 and 5) were hexaploid with 2n = 6x = 42 (Fig. 3C). Metacentric and submetacen-
tric chromosome types were present in all examined E. sibirica specimens. e karyo-
type formula of tetraploid plants was 2n = 20m(2sat) + 2m/sm + 6sm and 2n = 30m +
12sm(2sat) in hexaploid plants. No B chromosomes were identied in this species.
Eranthis tanhoensis. We determined the chromosome numbers in specimens of eight
populations of E. tanhoensis. All plants studied were diploid, with 2n = 2x = 14 (Table
1 and Fig. 3D, E). Metacentric and submetacentric types of chromosomes were found
(Fig. 3D, E). e two populations examined (9, 12) were characterised by the presence
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
84
Figure 3. Mitotic metaphase chromosomes. A Eranthis sibirica (voucher 1 in Table 1), 2n = 28 B Eranthis
sibirica (voucher 2), 2n = 28 C Eranthis sibirica (voucher 3), 2n = 42 D Eranthis tanhoensis (voucher 11),
2n = 14 E Eranthis tanhoensis (voucher 9), 2n = 14 + 0–8 B (arrows point at B chromosomes) F Eranthis
stellata (voucher 14), 2n = 16. Scale bars: 10 µm.
A new species of Eranthis (Ranunculaceae) from North Asia 85
of B chromosomes. e maximum number of B chromosomes appeared to be eight
(9). B chromosomes in this species were represented by two types: small (2.3–2.5 µm)
metacentrics and dot-shaped 1.3–1.5 µm long chromosomes, which were obviously
acrocentric. e karyotype formula of E. tanhoensis was 2n = 10m (2sat) + 4sm + 0–8B.
Eranthis stellata. In all four studied populations of E. stellata, the basic chromosome
number was x = 8. is species was diploid with 2n = 2x = 16, which is typical of the
genus (Table 1; Fig. 3F). Five pairs of chromosomes were metacentric, two pairs were
submetacentric and one pair was acrocentric (Fig. 3F). e karyotype formula of E. stel-
lata was 2n = 10m + 4sm (2sat) + 2t. No B chromosomes were observed in this species.
e basic chromosome number x = 8 has been reported for the entire genus Eranthis
(Langlet 1932; Kurita 1955; Tak and Wafai 1996; Gömürgen 1997; Yuan and Yang 2006;
Kim et al. 2011; Marhold et al. 2019). Our results are consistent with previously pub-
lished data (Yuan and Yang 2006), with insignicant dierences in the karyotype formula.
However, we showed, for the rst time, that E. sibirica and E. tanhoensis are distinguished
from other species of the genus by the basic chromosome number x = 7. Such dierences
in basic chromosome numbers (x = 7 and x = 8) have been found in some other genera of
Ranunculaceae, for example, Anemone L. and Ranunculus L. (Rice et al. 2015). Our results
regarding the chromosome numbers in E. sibirica (2n = 28 and 2n = 42) diered from the
data reported by other researchers for this species (2n = 32: Krogulevich (1976) or 2n = 16:
Gnutikov et al. (2016, 2017)). Eranthis tanhoensis was found to have 2n = 14. Based on the
incongruence of the chromosome data with previous and recent analyses, we assume that
some populations of E. sibirica and E. tanhoensis may have diverse cytotypes. Both species
clearly diered from E. stellata by the absence of acrocentrics. All three species were char-
acterised by ve metacentrics and two submetacentrics per monoploid chromosome set.
Flow cytometry
e average absolute DNA content of hexaploid samples of E. sibirica was 2C = 55.33±
0.52 pg and that of tetraploid samples was 2C = 38.19 ± 0.28 pg. In diploid E. tan-
hoensis, the average absolute DNA content was 2C = 25.02 ± 0.28 pg. e average
absolute DNA content of diploid E. stellata was 2C = 31.47 ± 0.46 pg. e monoploid
DNA content of the E. sibirica cytotypes was similar: 1Cx = 9.55 ± 0.14 pg in tetra-
ploids and 1Cx = 9.25 ± 0.20 pg in hexaploids. e monoploid DNA content of E.
tanhoensis was 1Cx = 12.49 ± 0.16 pg and that of E. stellata was 1Cx = 15.77 ± 0.20 pg.
Tetraploids and hexaploids of E. sibirica exhibited insignicant dierences in DNA
content (9.25 pg for 6x and 9.55 for 4x), whereas diploids of E. tanhoensis showed a
higher 1Cx level (12.49 pg), which may indicate a relatively ancient diversication of
these species. Data on the 1Cx level of E. stellata (15.77 pg) indicated the independ-
ent or parallel evolution of genome size in this species. According to ow cytometry,
variations in 1Cx levels between diploid samples of E. tanhoensis and hexaploids and
tetraploids of E. sibirica were in accordance with the hypothesis of genome downsizing
in polyploid owering plants (Leitch and Bennett 2004).
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
86
HPLC analysis of individual phenolic compounds
Phenolic compounds are often used in chemotaxonomic studies owing to their wide dis-
tribution in plants, structural diversity and chemical stability (Braunberger et al. 2015;
Radušienė et al. 2018). ey have also been reported as promising chemotaxonomic
markers for Ranunculaceae (Hao 2018). However, data on the signicance of these
substances for the taxonomy of Eranthis is still insucient. Only a few studies of the
phytochemical characteristics of certain Eranthis species, considered as medicinal plants,
have been published (Djafari et al. 2018; Hao 2018; Watanabe et al. 2003, 2019).
Twenty four phenolic compounds were detected in 70% ethanol extracts of plant
leaves of the three Eranthis species (E. sibirica, E. stellata and E. tanhoensis) using HPLC
(Fig. 4). Phenolic acids (chlorogenic, gentisic, caeic and salicylic acids), avonols
(quercetin, kaempferol and hyperoside) and avones (orientin and vitexin) were identi-
ed amongst these compounds. All three species were very similar in the composition of
the phenolic compounds extracted from their leaves; however, there were specic com-
pounds for each taxon. e common compounds present in all studied plants were chlo-
rogenic acid, phenolic acids (Fig. 4, peak 3: tR, min = 10.0, λmax, nm = 250, 290 sh, 335;
peak 12: tR, min = 20.9, λmax, nm = 240, 290 sh, 335 and peak 23: tR, min = 44.3, λmax, nm
= 255, 300, 330) and avonols (Fig. 4, peak 9: tR, min = 15.1, λmax, nm = 255, 360 and
peak 15: tR, min = 32.7, λmax, nm = 270, 310, 365). Almost all plants contained kaemp-
Figure 4. HPLC chromatograms of 70% water-ethanol extracts of Eranthis leaves detected by HPLC-
DAD at 255 nm. e X-axis displays the retention time, min; Y-axis – the detector signal in optical
density units. e identied peaks are 1. chlorogenic acid, 2. gentisic acid, 3. caeic acid, 5. orientin,
8.vitexin, 10. hyperoside, 11. salicylic acid, 19. quercetin and 24. kaempferol.
A new species of Eranthis (Ranunculaceae) from North Asia 87
ferol, phenolic acids (Fig. 4, peak 14: tR, min = 25.4, λmax, nm = 255, 300 and peak 16: tR,
min = 34.5; λmax, nm = 250, 290, 330) and avonols (Fig. 4, peak 13: tR, min = 22.3, λmax,
nm = 255, 360 and peak 18: tR, min = 38.7, λmax, nm = 255, 305, 360). Eranthis sibirica
leaves contained about 15 to 20 phenolic compounds, whereas, in E. stellata leaves, their
number varied from 16 to 18. Phenolic compounds were less diverse in E. tanhoensis
leaves than in leaves of other species, whose numbers varied from 13 to 16 substances.
e chromatographic prole of E. sibirica diered from that of E. tanhoensis in the
presence of caeic acid, orientin, vitexin and avone (peak 6: tR, min = 9.4, λmax, nm =
270, 310) in 70% ethanol leaf extracts (Fig. 4). Caeic acid, orientin and avone (peak 6)
were generally absent from leaves of E. tanhoensis, whereas vitexin was found in some sam-
ples in trace amounts. e leaves of E. tanhoensis from almost all the studied populations
contained quercetin, which was not detected in E. sibirica. Distinguishing compounds in
leaf extracts of E. stellata were gentisic acid, phenolic acid (Fig. 4, peak 4: tR, min = 7.1,
λmax, nm = 250, 300) and avone (Fig. 4, peak 21: tR, min = 42,2; λmax, nm = 210, 310),
which were absent from the two other species. Vitexin, hyperoside and salicylic acids were
not found in E. stellata leaves. All samples of E. stellata contained orientin and caeic acid,
which were characteristic of E. sibirica and quercetin, which was typical of E. tanhoensis.
Taxonomy
e analysis of the data presented above allowed us to distinguish a new species from
specimens previously identied as E. sibirica.
Eranthis tanhoensis Erst, sp. nov.
urn:lsid:ipni.org:names:77206949-1
Figs 5, 6A–D, 7B
Type. R, Republic of Buryatia, Kabansky district, Osinovka River near Tanhoi
village, 51°33'06.2"N, 105°05'34.7"E, 458 m a.s.l., 01 May 2019, A.S. Erst, D.A.
Krivenko, & O.A. Chernysheva s.n. (holotype, NS-0000948!, isotypes TK, IRK, E).
Description. Herb perennial, 12.0–23.0 cm long at owering and 18.0–40.0 cm
long at fruiting. Tubers subglobose, not or slightly branching, 1.2–3.3 cm diam., pro-
ducing thin brous roots. Basal leaf single, long-petiolate, green; petioles 5.0–6.0 cm
long at owering and 23–25 cm at fruiting; blades 2.5–3.8 × 2.5–3.5 cm at owering
and 7.5–12 × 7.5–12 cm at fruiting, deeply palmately divided into 5 segments (maxi-
mum length of segment dissection 2.3 cm at owering (3.5 cm at fruiting)); leaf blade
segments rounded or widely rhombic, 0.8–2.5 × 0.4–1.8 cm at owering (1.7–8.5 ×
1.2–7.5 cm at fruiting), unlobed or dissected into 1–2 lobes at both owering and
fruiting stages; segment of basal leaves with 5–19 acute teeth at apex at owering, 6–25
teeth at fruiting. Involucre present, 1.1–5.5 cm diam. at owering (7–11 cm at fruiting
stage); involucral bracts (cauline leaf) sessile, laciniate, similar to basal leaf, divided into
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
88
Figure 5. General habit of Eranthis tanhoensis. Scale bar: 1 cm.
A new species of Eranthis (Ranunculaceae) from North Asia 89
Figure 6. Morphological dierences amongst A–D Eranthis tanhoensis E–H Eranthis sibirica; and I–LEr-
anthis stellata A, E , I ower position B, F, J owers C , G, K involucral bracts and follicles D, H, L basal leaves.
5 trid leaf-like segments (maximum length of segment dissection is 1.6cm at ower-
ing (4.0 cm at fruiting)); segments rounded or widely rhombic, 1.1–3.0 × 0.5–2.5 cm
at owering (3.3–6.4 × 1.4–5.3 cm at fruiting), unlobed or dissected into 2 lobes both
Figure 7. Petals. A Eranthis sibirica B Eranthis tanhoensis C Eranthis stellata.
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
90
at owering and fruiting stages; each segment with 5–21 teeth (at both owering and
fruiting stages), acute at the apex. Pedicels 0.5–1.5 cm long, elongated in fruiting (3.5–
5.5 cm long), densely covered with papillate and large hemispherical trichomes. Flowers
bisexual, actinomorphic, solitary, erect, 2–4 cm diam. Sepals 4–7, deciduous in fruit,
white or light pink at margin, at, narrowly obovate or elliptic, 1.1–2.6 × 0.5–1.3 cm.
Petals 5–15 × 0.6–0.8 cm long, bicoloured, white, tubular, two-lipped with bilobate or
forked lips, each lobe of abaxial lip acute at the apex and with globular yellow swellings
(nectaries: Fig. 7B). Stamens 36–45, 0.7–1.1 cm long; laments liform, white; an-
thers white. Follicles 3–10, 0.8–1.4 cm long, on short (0.3–0.5 mm) stalks, divergent
towards the end of fruiting; stylodium 0.1–0.3 mm long, straight or slightly curved.
Notes. Turczaninow (1842) described the species E. uncinata Turcz., growing at
higher altitudes and distinguished from E. sibirica by the number of petals (5–6, not
strictly 5), by the shape of the stylodium (recurved rather than straight), smaller ow-
ers and more dissected leaf blades. However, our studies have shown that these mor-
phological characters are variable and all variations can be found both in the foothill
and alpine plants. Shipchinskiy (1937) merged E. uncinata with E. sibirica. However,
he described two varieties: E. sibirica DC. var. nuda Schipcz. with glabrous pedicels
(= E. sibirica var. sibirica) and E. sibirica DC. var. glandulosa Schipcz. with glandular-
pubescent pedicels. ese varieties were not validly published under ICN Article 39.1
(Turland et al 2018). Nakai (1937) attributed E. sibirica and E. uncinata to the genus
Schibateranthis Nakai (≡ Eranthis sect. Schibateranthis (Nakai) Tamura).
Anity. e new species belongs to E. sect. Shibateranthis (Nakai) Tamura and it
is sister to E. sibirica, according to the results of molecular phylogenetic analysis (Fig.
1). E. tanhoensis is morphologically similar to E. sibirica and E. stellata (Figs 5–9) in
having white sepals, tubular two-lipped petals with bilobate or forked lips, apically
acute lobes with abaxial lip and globular yellow swellings (nectaries) at the top or in
the central part. e dierences amongst the three species are presented in Table 2.
e new species diers from other related species by dense glandular pubescence of
the ower stems, rounded or widely rhombic (not obovate or lanceolate) leaf blade seg-
ments, acute, rather than rounded teeth apices of the basal and stem leaves, a large num-
ber of teeth and width of the segments of the basal and stem leaves (see also2). Addition-
ally, all three species growing in Russia have dierent distribution patterns (Figs 10, 11).
Phenology. Flowering time: April–early May; fruiting time: late May–June.
Distribution (Fig. 10): Eranthis tanhoensis is endemic to southern Baikal (Khamar-
Daban range of the Republic of Buryatia and Irkutsk Province).
Habitat and ecology. Eranthis tanhoensis can be found at 350–2400 m a.s.l., where
it grows in r, Siberian pine, spruce and birch forests, on riverbanks, beside streams (up
to 1500 m a.s.l.) and in subalpine meadows (at higher altitudes).
Etymology. e specic epithet of the new species is derived from the type local-
ity, Tanhoi village, Republic of Buryatia, Russia.
Additional specimens examined. R: Republic of Buryatia: Kabansky
district, Osinovka river (Tanhoi village), 51°33'06.2"N, 105°05'34.7"E, 458 m a.s.l.,
20 Jun 2019, A.S. Erst, D.A. Krivenko, E.Yu. Mitrenina & O.A. Chernysheva s.n. (NS-
A new species of Eranthis (Ranunculaceae) from North Asia 91
0000949!); Kabansky district, Mishikha river, 51°37'46.7"N, 105°32'05.2"E, 480 m
a.s.l., 01 May 2019, A.S. Erst, D.A. Krivenko & O.A. Chernysheva 31 (NS-0000950!);
Kabansky district, Mishikha river, 51°37'46.7"N, 105°32'05.2"E, 480 m a.s.l., 01May
2019, A.S. Erst, D.A. Krivenko & O.A. Chernysheva 31a (NS-0000951!); Kabansky
district, Mishikha river, 51°37'32.6"N, 105°32'03.4"E, 478 m a.s.l., 20 Jun 2019, A.S.
Erst, D.A. Krivenko, E.Yu. Mitrenina & O.A. Chernysheva s.n. (NS-0000952!); Kabansky
district, Dulikha river, 51°32'04.9"N, 105°01'43.2"E, 461 m a.s.l., 01 May 2019, A.S.
Erst, D.A. Krivenko & O.A. Chernysheva 14 (NS-0000953!); Kabansky district, Dulikha
river, 51°32'04.9"N, 105°01'43.2"E, 461 m a.s.l., 20 Jun 2019, A.S. Erst, D.A. Krivenko,
E.Yu. Mitrenina & O.A. Chernysheva (NS-0000954!); Kabansky district, Shestipalikha
river, 51°32'46.4"N, 105°04'28.9"E, 465 m a.s.l., 01 May 2019, A.S. Erst, D.A.
Krivenko & O.A. Chernysheva s.n. (NS-0000955!); Kabansky district, Shestipalikha river,
51°32'46.4"N, 105°04'28.9"E, 465 m a.s.l, 21 Jun 2019, A.S. Erst, D.A. Krivenko, E.Yu.
Mitrenina & O.A. Chernysheva (NS-0000956!); Kabansky district, Tolbazikha river,
Table 2. Morphological dierences among E. sibirica, E. tanhoensis, and E. stellata.
Character E. sibirica E. tanhoensis E. stellata
Leaf colour at owering green green coppery or green
Teeth at the apex of basal leaf segments rounded acute rounded
Maximum dissection of the basal leaf
segments (at owering), cm
1.0 2.3 0.4–?
Maximum dissection of the basal leaf
segments (at fruiting), cm
2.3 3.5 1.3
Number of teeth on the segments of the
basal leaf (at fruiting)
3–12 6–25 3–5
Apex of involucral leaves rounded acute rounded
Width of the involucral leaf segments (at
fruiting), cm
0.4–1.2 1.4–5.3 0.5–2.3
Maximum dissection of the involucral
leaf segments (at owering), cm
1.6 1.6 1.0
Maximum dissection of the involucral
leaf segments (at fruiting), cm
2.1 4.0 1.7
Number of teeth on the segments of the
involucral leaf (at owering)
1–5 5–21 3–9
Number of teeth on the segments of the
involucral leaf (at fruiting)
2–5 5–21 3–8
Flower position erect erect recurved
Scape pubescence glabrous or with papillate
trichomes
large hemispherical and
papillate trichomes
glandular and stellate
trichomes
Sepal number 5–7 4–7 5–8
Shape of petals narrow urn-shaped broadly urn-shaped funnelform
Swellings (nectaries) position at the apex at the apex in medium part
Apex colour of adaxial lip yellow yellow white
Apex colour of abaxial lip yellow yellow white
Margin colour between abaxial and
adaxial lips
white yellow white
Stamen colour white white violet, pink or white
Stylodium length, cm 0.2–0.5 0.1–0.3 0.2–0.4
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
92
Figure 8. General habit of Eranthis sibirica. Scale bar: 1 cm.
A new species of Eranthis (Ranunculaceae) from North Asia 93
Figure 9. General habit of Eranthis stellata. Scale bar: 1 cm.
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
94
Figure 10. General distribution of Eranthis sibirica (dots) and E. tanhoensis (stars), based on herbarium
materials.
Figure 11. General distribution of Eranthis stellata, based on herbarium materials and data in the litera-
ture (Oh and Oh 2019; Park et al. 2019).
A new species of Eranthis (Ranunculaceae) from North Asia 95
51°26'21.06"N, 104°41'09.82"E, 471 m a.s.l., 02 May 2019, A.S. Erst, D.A.
Krivenko & O.A. Chernysheva s.n. (NS-0000957!); Kabansky district, Tolbazikha river,
51°26'21.06"N, 104°41'09.82"E, 471 m a.s.l., 20 Jun 2019, A.S. Erst, D.A. Krivenko,
E.Yu. Mitrenina & O.A. Chernysheva s.n. (NS-0000958!); Irkutsk Province: Slyudyansky
district, Semirechka river, 51°28'56.92"N, 104°19'43.47"E, 470 m a.s.l., 02 May 2019,
A.S. Erst, D.A. Krivenko & O.A. Chernysheva 048 (NS-0000959!); Slyudyansky district,
Semirechka river, 51°28'56.92"N, 104°19'43.47"E, 470 m a.s.l., 21 Jun 2019, A.S. Erst,
D.A. Krivenko, E.Yu. Mitrenina & O.A. Chernysheva s.n. (NS-0000960!).
Preliminary conservation status. Although the species seems to have a small dis-
tribution area in southern Baikal Lake, the populations observed in 2018 and 2019
consisted of numerous individuals producing viable fruits and no threats to the habi-
tats were observed in the eld studies. e EOO of E. tanhoensis was estimated for an
area of more than 1372km2, while the AOO was 72 km2. Preliminary conservation
status, according to IUCN’s Extent of Occurrence criteria indicates the species as En-
dangered (EN) (IUCN 2019).
Key to the Eranthis species growing in Asiatic Russia
1 Maximum dissection of basal leaf segments ~0.4 cm long at owering stage,
1.3 cm long at fruiting stage; scape with stellate hairs; involucral leaves green
or coppery at owering; maximum dissection of the involucral leaves 1.7 cm
long at fruiting; owers recurved; petals narrowly funnelform, swellings (nec-
taries) located in medium part of adaxial lip lobes, apex of abaxial and adaxial
lips white; anthers violet, pink or white ........................................E. stellata
– Maximum dissection of basal leaf segments at least 1.0 cm long at owering,
2.3 cm long at fruiting stage; scape without stellate hairs; involucral leaves
green at owering; maximum dissection of the involucral leaves 2.1 cm long
or more at fruiting; owers erect, petals urn-shaped, swellings (nectaries) lo-
cated at the apex of adaxial lip lobes, apex of abaxial and adaxial lips yellow;
anthers white ..............................................................................................2
2 Apex of basal and involucral leaves rounded; maximum dissection of basal
leaf segments 1.0 cm long at owering and 2.3 cm long at fruiting; segments
of involucral leaves at fruiting 0.4–1.2 cm wide; maximum dissection of the
involucral leaves at fruiting 2.1 cm long; each segment of involucral leaves
with 1–5 teeth; scape glabrous or papillate; petals narrowly urn-shaped, mar-
gins between abaxial and adaxial lips white .................................. E. sibirica
– Apex of basal and involucral leaves acute; maximum dissection of basal leaf
segments 2.3 cm long at owering and 3.5 cm long at fruiting; segments of
involucral leaves at fruiting 1.4–5.3 cm wide; maximum dissection of the in-
volucral leaves at fruiting 4.0 cm long; each segment of involucral leaves with
5–21 teeth; scape papillate and with large hemispherical glands; petals broadly
urn-shaped, margins between abaxial and adaxial lips yellow ..... E. tanhoensis
Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
96
Acknowledgements
We thank Mark Newman, Marco Pellegrini, Andriy Noviko, Colin Pendry,
Christoph Dobeš and Johannes Walter for discussion of some parts of the manu-
script and valuable comments, the sta of the herbaria visited, as well as Valentin
Yakubov for the images of Eranthis stellata and Roman Annenkov for preparing
Fig. 3. We are indebted to Natalya Pridak for all the black and white drawings.
e samples of E. longistipitata were kindly provided by Evgeny Boltenkov. e
research was supported by the Russian Foundation for Basic Research, grant 18-
34-20056 mol_a_ved. e work of Alexander Sukhorukov and Maria Kushunina
was also supported by a Moscow State University (MSU) Grant for Leading Sci-
entic Schools “Depository of the Living Systems” in the framework of the MSU
Development Programme.
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Supplementary material 1
Tables S1–S4
Authors: Andrey S. Erst, Alexander P. Sukhorukov, Elizaveta Yu. Mitrenina, Mikhail
V. Skaptsov, Vera A. Kostikova, Olga A. Chernisheva, Victoria Troshkina, Maria Kus-
hunina, Denis A. Krivenko, Hiroshi Ikeda, Kunli Xiang, Wei Wang
Data type: measurement.
Explanation note: Table S1. List of samples characters used in molecular (M), cy-
togenetical (C) and biochemical (B) analyses. Table S2. Morphological characters
of Russian Eranthis species. An asterisk indicates characters used in the numerical
analysis. Table S3. e results of the variance analysis for plant characters in the
owering stage. e values in parentheses are adjusted P-values; the characters in
bold are those without signicant interspecic dierences. Table S4. e results
of the variance analysis for plant characters in the fruiting stage. e values in pa-
rentheses are adjusted P-values; the characters in bold are those without signicant
interspecic dierences.
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/phytokeys.140.49048.suppl1
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