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An integrative taxonomic approach reveals a new species of Eranthis (Ranunculaceae) in North Asia


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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 biochemical analyses, along with morphological data, was used to delimit this new species.
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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 (
Academic editor: M. Pellegrini|Received 3 December 2019|Accepted 6 February 2020|Published 4 March2020
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.
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.
Biochemistry, cytology, integrative taxonomic approach, morphology, phylogeny, Ranunculales, Russia
PhytoKeys 140: 75–100 (2020)
doi: 10.3897/phytokeys.140.49048
Copyright Andrey S. Erst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Andrey S. Erst et al. / PhytoKeys 140: 75–100 (2020)
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 (Partt 1997). Eranthis are perennial herbs with tuberous rhizomes, ba-
sal long-petiolate leaves with the blades divided into several or many palmate segments
(leaets) 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, bid
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 (Partt 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. albiora Franch., E. byunsanensis B.Y.Sun, E. lobulata W.T.Wang, E.
pinnatida 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 insuciently 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 dierent 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 ( 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. pinnatida 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 specied 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 conrmation 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 signicant bootstrap support for conicting 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)
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 dierent 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 dierences were considered signicant 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 signicant
intergroup dierences, 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× magnication 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 dierent
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
buer 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 eect 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 eect 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)
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 5ml.
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 identied by their
spectral characteristics (Bate-Smith 1962; Mabry et al. 1970). For identication 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 caeic 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. pinnatida. 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 signicant dierences 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, signicant dierences 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)
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 signied by the directions of the vectors indicating the gradients in the
character values, at owering, E. sibirica diered 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 diered 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 diered 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% condence 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.
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 ±
9.55 ± 0.14
3E. sibirica Irkutsk Province, Slyudyanka
42 6x2n = 30m + 12sm
(2 sat)
55.75±0.28 9.23±0.14
4E. sibirica Irkutsk Province, Burovschina
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) +
24.88±0.54 12.44±0.27
7E. tanhoensis Republic of Buryatia, Duliha
14 2x2n = 10m (2sat) +
24.97±0.43 12.49±0.22
8E. tanhoensis Republic of Buryatia,
Tolbazikha river
14 2x2n = 10m (2sat) +
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
14 2x2n = 10m (2sat) +
25.31±0.15 12.41±0,29
11 E. tanhoensis Republic of Buryatia,
Osinovka river (Tanhoi village)
14 2x2n = 10m (2sat) +
25.11±0.32 12.56±0.16
12 E. tanhoensis Republic of Buryatia, Mishiha
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) +
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 identied 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)
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 insignicant dierences 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 dierences
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) diered 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 diered 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 insignicant dierences 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 diversication 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)
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 signicance of these
substances for the taxonomy of Eranthis is still insucient. 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, caeic 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 specic 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 identied peaks are 1. chlorogenic acid, 2. gentisic acid, 3. caeic 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 prole of E. sibirica diered from that of E. tanhoensis in the
presence of caeic acid, orientin, vitexin and avone (peak 6: tR, min = 9.4, λmax, nm =
270, 310) in 70% ethanol leaf extracts (Fig. 4). Caeic 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 caeic acid,
which were characteristic of E. sibirica and quercetin, which was typical of E. tanhoensis.
e analysis of the data presented above allowed us to distinguish a new species from
specimens previously identied as E. sibirica.
Eranthis tanhoensis Erst, sp. nov.
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)
Figure 5. General habit of Eranthis tanhoensis. Scale bar: 1 cm.
A new species of Eranthis (Ranunculaceae) from North Asia 89
Figure 6. Morphological dierences 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 trid leaf-like segments (maximum length of segment dissection is 1.6cm 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)
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).
Anity. 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 dierences amongst the three species are presented in Table 2.
e new species diers 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 also2). Addition-
ally, all three species growing in Russia have dierent 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 specic 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., 01May
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 dierences 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
large hemispherical and
papillate trichomes
glandular and stellate
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)
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)
Figure 10. General distribution of Eranthis sibirica (dots) and E. tanhoensis (stars), based on herbarium
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 1372km2, 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)
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-
entic 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 signicant interspecic dierences. 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 signicant
interspecic dierences.
Copyright notice: is dataset is made available under the Open Database License
( 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.
... Eranthis hyemalis (L.) Salisb. having its natural range in Europe is a type species of the genus, and some aspects of phylogeny and history of the Eranthis genus have already been described in several studies [1][2][3][4][5]. Until recently, only Eranthis sibirica DC. was known in South Siberia, being considered endemic to this region and a tertiary relict plant [6]. ...
... We actually aimed to: (i) describe phylogeographic patterns and phylogenetic relationships between the populations of Siberian Eranthis using a broadened sample, and (ii) explain the phylogenetic relationships between the Siberian Eranthis taxa and clarify the taxonomic status of E. tanhoensis. Looking ahead, and in a certain way predating the discussion of our findings, further in the text we will use the original name E. sibirica for all populations, specifying individually where specimens correspond to E. tanhoensis, according to A.S. Erst and colleagues [5]. ...
... N53.67042°, E100.65604° 610 m alt. * the geographic coordinates and altitude data were referenced by combined GPS/GLONASS positioning, datum WGS84 (GPSMAP 64st device, Garmin, Vancouver, KS, USA); ** localities correspond to E. tanhoensis according to A.S. Erst and colleagues [5]. *** the information about the collector is missing in the original voucher. ...
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Eranthis Salisb. (Ranunculaceae) is a herbaceous plant genus, including few species disjunctively distributed throughout the temperate zone from Southeastern Europe to Eastern Asia. Until recently, only Eranthis sibirica DC. was known in South Siberia, being considered endemic and tertiary relict. Not long ago, Eranthis tanhoensis Erst was also described in Siberia. We report here a reconstruction of the phylogenetic relationships between the Siberian Eranthis species based on nuclear (ITS) and plastid (trnL + trnL-trnF + trnH-psbA) DNA. The phylogeographic structure of Siberian Eranthis is distinguished by the presence of the two “eastern” and “western” supergroups, which most likely formed as a result of disjunction caused by active mountain uplifts during the late Neogene–early Quaternary and subsequent progressive Pleistocene cooling. The eastern supergroup combines lineage I, containing populations from the eastern Khamar-Daban Ridge, the Eastern Sayan Mountains, and the Tannu-Ola Ridge, and lineage II containing western Khamar-Daban populations. The western supergroup includes only lineage III, containing Western Sayan populations. Our data clearly show that E. tanhoensis is nested in the E. sibirica clade, thereby indicating that its description as a separate species is unjustified, as it compromises the monophyletic status of E. sibirica. Therefore, we suggest here to consider E. tanhoensis as a synonym of E. sibirica.
... Thus, in many cases, only the use of an integrative taxonomic approach makes it possible to accurately determine the taxonomic status of cyanobacteria with Nostoc-like morphology. An integrative taxonomic approach successfully applied to the identification of both flowering plants (Erst et al. 2020) and microorganisms (Mikhailyuk et al. 2018). For cyanobacteria, an integrative approach includes classical culturing and microscopic methods, obtaining pure strains in combination with molecular phylogenetic methods. ...
A new cyanobacterial species of the genus Desmonostoc is described according to the International Code of Nomenclature for algae, fungi, and plants. The new species Desmonostoc caucasicum sp. nov. is recorded in the mountain meadow subalpine soil from the Greater Caucasus, Russia. The analysis is based on morphological characters, the 16S rRNA gene phylogenetic analysis, and ITS secondary structure. Desmonostoc caucasicum differs from the other species of the genus in colony morphology, size of vegetative cells and heterocytes, and habitat type. The 16S rRNA gene sequence of the new strain displayed 95.3%-97.9% similarities to other species of the genus Desmonostoc. The phylogeny inferred by maximum likelihood and Bayesian inference placed D. caucasicum in the Desmonostoc clade, within the Nostocaceae. The novel strain formed an independent lineage within the clade. The D1-D1ʹ, Box-B and V3 helices obtained from the 16S-23S ITS region didn't fit those of any described species of Desmonostoc. Amplification of a fragment of the mcy gene involved in microcystin biosynthesis from D. caucasicum confirmed that it has this genetic determinant. An integrative taxonomic approach, based on morphological, 16S rRNA and mcy genes molecular analyses, ITS secondary structure, along with ecological data, is used to delimit this new species.
... The genus Eranthis (Ranunculaceae), an early flowering perennial herb (Figure 1), provides an interesting case study for genetic differentiation and gene flow. This genus is composed of approximately 13 species, which are distributed from Europe to East Asia (Erst, Sukhorukov, et al., 2020;Erst et al., 2020b;Mabberley, 1993;Rukšāns, 2018;Tamura, 1990;Wang et al., 2001). This genus comprises relatively short plants (5-15 cm above ground) with tuberous rhizomes, palmately divided leaves and bracts, solitary flowers, and petaloid sepals (Oh & Ji, 2009;Sun et al., 1993). ...
Full-text available
Genetic differentiation between populations is determined by various factors, including gene flow, selection, mutation, and genetic drift. Among these, gene flow is known to counter genetic differentiation. The genus Eranthis, an early flowering perennial herb, can serve as a good model to study genetic differentiation and gene flow due to its easily detectable population characteristics and known reproductive strategies, which can be associated with gene flow patterns. Eranthis populations are typically small and geographically separated from the others. Moreover, previous studies and our own observations suggest that seed and pollen dispersal between Eranthis populations is highly unlikely and therefore, currently, gene flow may not be probable in this genus. Based on these premises, we hypothesized that the genetic differentiation between the Eranthis populations would be significant, and that the genetic differentiation would not sensitively reflect geographic distance in the absence of gene flow. To test these hypotheses, genetic differentiation, genetic distance, isolation by distance, historical gene flow, and bottlenecks were analyzed in four species of this genus. Genetic differentiation was significantly high, and in many cases, extremely high. Moreover, genetic differentiation and geographic distance were positively correlated in most cases. We provide possible explanations for these observations. First, we suggest that the combination of the marker type used in our study (chloroplast microsatellites), genetic drift, and possibly selection might have resulted in the extremely high genetic differentiation observed herein. Additionally, we provide the possibility that genetic distance reflects geographic distance through historical gene flow, or adaptation in the absence of historical gene flow. Nevertheless, our explanations can be more rigorously examined and further refined through additional observations and various population genetic analyses. In particular, we suggest that other accessible populations of the genus Eranthis should be included in future studies to better characterize the intriguing population dynamics of this genus. In our study, under the low probability of gene flow, genetic differentiation between populations was significantly high and genetic distance generally reflected geographic distance in the genus Eranthis. We suggest that significantly high level of genetic differentiation is due to the marker type used, genetic drift, and selection. We also suggest that the positive correlation between genetic distance and geographic distance might have resulted from historical gene flow or adaptation without historical gene flow.
... It considers a combination of data on phylogeography, morphology, population genetics, and ecology, as well as ontogenetical and behavioral traits, for species delimitation. Different traits are also used in plant taxonomy, but botanists usually do not designate this approach with any special term, though they sometimes mention an integrative taxonomy (Rouhan and Gaudeul, 2014;Erst et al., 2020). ...
... The aim of this study is to test the current two-species taxonomic hypothesis for Sardinia and Corsica by using an integrated approach [31,32], that involves molecular analyses, cypsela morpho-colorimetry, morphometry, and niche similarity tests. Santolina chamaecyparissus, traditionally considered as closely related, is also included in the study. ...
Full-text available
Santolina is a plant genus of dwarf aromatic shrubs that includes about 26 species native to the western Mediterranean Basin. In Corsica and Sardinia, two of the main islands of the Mediterranean, Santolina corsica (tetraploid) and S. insularis (hexaploid) are reported. Along with the cultivated pentaploid S. chamaecyparissus, these species form a group of taxa that is hard to distinguish only by morphology. Molecular (using ITS, trnH-psbA, trnL-trnF, trnQ-rps16, rps15-ycf1, psbM-trnD, and trnS-trnG), cypsela morpho-colorimetric, morphometric, and niche similarity analyses were conducted to investigate the diversity of plants belonging to this species group. Our results confute the current taxonomic hypothesis and suggest considering S. corsica and S. insularis as a single species. Moreover, molecular and morphometric results highlight the strong affinity between S. chamaecyparissus and the Santolina populations endemic to Corsica and Sardinia. Finally, the populations from south-western Sardinia, due to their high differentiation in the studied plastid markers and the different climatic niche with respect to all the other populations, could be considered as an evolutionary significant unit.
... Eranthis Salisb. belongs to the Cimicifugeae of Ranunculaceae and contains approximately ten species (Erst et al. 2020). Phylogenetic relationships within this genus have been elucidated recently (Xiang et al. 2021). ...
Full-text available
Flowers are an innovative characteristic of angiosperms, and elaborate petals usually have highly specialized structures to adapt to different living environments and pollinators. Petals of Eranthis have complex bilabiate structures with nectaries and pseudonectaries; however, the diversity of the petal micromorphology and structure is unknown. Petal development, micromorphology, structure and ultrastructure in four Eranthis species were investigated under SEM, TEM and LM. The results show that petals undergo 5 developmental stages, and accessory structure formation (stage 4) mainly determines the diversity of final mature petal morphology and pseudonectaries; the central depression formed in stage 2 will develop into nectary tissues. Petals are bilabiate and have hidden nectaries in nectary grooves; they consist of one layer of rounded and raised secretory epidermal cells and 3–14 layers of secretory cells with abundant plasmodesmata between cells. A large number of sieve tubes are distributed between the cells and extend to the epidermis; in addition, the vessel elements are located below the secretory area. Nectar is stored in the intercellular space between secretory parenchyma cells and escapes through microchannels or cell rupture. Pseudonectaries in all species of Eranthis except for E. hyemalis consist of smooth, ornamented epidermal cells and 9–12 layers of parenchyma cells with sparse cytoplasm, which may have the function of attracting pollinators.
... & A. Grey [3]. This genus consists of 10 to 13 early flowering herbaceous perennial species that are distributed across Southern Europe and Western, Central, and temperate Asia [4][5][6][7]. Species of this genus have rarely been the subject of phytochemical research. Examination of the literature indicates that triterpene glycosides and saponins have been studied (along with their biological activity) in E. cilicica Schott & Kotschy [8,9]. ...
Full-text available
Aqueous-ethanol extracts (70%) from the leaves of Eranthis longistipitata Regel. (Ranuncu-laceae Juss.)-collected from natural populations of Kyrgyzstan-were studied by liquid chromatography with high-resolution mass spectrometry (LC-HRMS). There was no variation of the metabolic profiles among plants that were collected from different populations. More than 160 compounds were found in the leaves, of which 72 were identified to the class level and 58 to the individual compound level. The class of flavonoids proved to be the most widely represented (19 compounds), including six aglycones [quercetin, kaempferol, aromadendrin, 6-methoxytaxifolin, phloretin, and (+)-catechin] and mono-and diglycosides (the other 13 compounds). In the analyzed samples of E. longistipitata, 14 fatty acid-related compounds were identified, but coumarins and furochromones that were found in E. longistipitata were the most interesting result; furochromones khelloside, khellin, visnagin, and cimifugin were found in E. longistipitata for the first time. Couma-rins 5,7-dihydroxy-4-methylcoumarin, scoparone, fraxetin, and luvangetin and furochromones methoxsalen, 5-O-methylvisammioside, and visamminol-3′-O-glucoside were detected for the first time in the genus Eranthis Salisb. For all the above compounds, the structural formulas are given. Furthermore, detailed information (with structural formulas) is provided on the diversity of chro-mones and furochromones in other representatives of Eranthis. The presence of chromones in plants of the genus Eranthis confirms its closeness to the genus Actaea L. because chromones are synthesized by normal physiological processes only in these members of the Ranunculaceae family.
Saponins have been studied for more than four decades and their relevance is due to their numerous biological and chemical activities. Indeed, saponins are attracting attention for their industrial exploitation in connection with their pharmacological properties. Saponins also find many applications in the food industry and in the cosmetics industry due to their foaming and emulsifying property. On the other hand, depending on the type of saponin, the species that ingests it and the context, they are more or less toxic to the body. This article describes a number of investigations works carried out on saponins to determine the impact of saponins on health.
Saponins have been studied for more than four decades and their relevance is due to their numerous biological and chemical activities. Indeed, saponins are attracting attention for their industrial exploitation in connection with their pharmacological properties. Saponins also find many applications in the food industry and in the cosmetics industry due to their foaming and emulsifying property. On the other hand, depending on the type of saponin, the species that ingests it and the context, they are more or less toxic to the body. This article describes a number of investigations works carried out on saponins to determine the impact of saponins on health.
Full-text available
Baikalsky State Nature Biosphere Reserve is situated in the central part of the Khamar-Daban Range (Southern Baikal, Siberia), in three administrative districts of Republic of Buryatia (i.e. Kabansky District, Dzhidinsky District and Selenginsky District), Russia. In general, this territory has been relatively well studied by botanists, but until now there was no detailed information about the flora of the Reserve with precise geographic localities. Moreover, some records in the Baikalsky Reserve's flora were published without references to documenting herbarium specimens. The dataset contains 39,238 unique occurrences of 875 taxa (854 species, 14 subspecies, five varieties and two species aggregates) from the Baikalsky Reserve and its buffer zone. All the data were acquired during the field studies by the author in 2009–2021, when 152 taxa (17.3% of all the taxa included into the dataset) were first recorded by the author from the study area. Herbarium vouchers are preserved in the Moscow University Herbarium (MW). This dataset is the first attempt at creating a database of vascular plants of the Baikalsky Reserve and its buffer zone, based on modern research. These data will provide the background for the updated check-list of the Baikalsky Reserve's flora.
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The relationships among species in the genus Eranthis Salisb. were investigated using single nucleotide polymorphisms (SNPs) of the nuclear DNA internal transcribed spacer 1, 2 region (nrITS) and the chloroplast trnL-trnF interspacer region (cpIS). Phylogenetic relationships based on the nrITS and cpIS were inferred with posterior probabilities with STRUCTURE analysis and the neighbor-joining method. Two major clades from nrITS and cpIS were consistent with species with yellow sepals in E. hyemalis, E. cilicica, E. longistipitata and the hybrid E. ×tubergenii; and white sepals in E. sibirica, E. longituba, E. albiflora, E. stellata, E. pungdoensis, E. byunsanensis, and E. pinnatifida. The phylogenetic tree of nrITS formed more subclades than the tree of cpIS, which suggested that nrITS SNPs are useful molecular markers for phylogenetic studies in the genus Eranthis. Only the SNPs of cpIS in E. pungdoensis accessions had a deletion at positions 259–420, and the posterior probability values (PPVs) assigned E. pungdoensis to population 4, which suggested that E. pungdoensis is different from E. byunsanensis. Therefore, E. byunsanensis and E. pungdoensis are considered to be true-to-type based on q-values because the PPVs were greater than 0.9 in both species based on the STRUCTURE analysis of nrITS SNPs. Significant genetic variation in E. stellata collected in Goesan-kun and Mt. Mugap, Korea indicated a potential gene flow among closely related E. byunsanensis, E. pinnatifida, E. sibirica, and E. stellata that could be due to geographic proximity in their distributions. E. stellata from Mt. Mugap showed mixed PPVs for E. stellata and E. byunsanensis, therefore, E. byunsanensis might be a possible hybrid origin for E. stellata collected from Goesan-kun and Mt. Mugap.
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The temporal and spatial origins and evolution of the genus Eranthis have not been previously studied. We investigated the speciation and establishment histories of four Eranthis species: Eranthis byunsanensis, E. pungdoensis, E. stellata, and E. pinnatifida. The sampling localities were Korea, Japan, Jilin in China, and the area near Vladivostok in Primorskiy, Russia. We used 12 chloroplast microsatellite loci (n = 935 individuals) and two chloroplast noncoding regions (rpl16 intron, petL‐psbE intergenic spacer; n = 33 individuals). The genetic diversity, genetic structure, phylogenetic relationships of the four species were analyzed, and their ancestral areas were reconstructed. The high genetic diversity of the Jeju island population of E. byunsanensis and Russian populations of E. stellata indicated these species’ northward and southward dispersal, respectively. The genetic structure analyses suggest that the populations in these four species have limited geographical structure, except for the Chinese E. stellata population (SCP). The phylogenetic analyses suggest that E. byunsanensis and E. pinnatifida are sister species and that Chinese SCP may not belong to E. stellata. The ancestral area reconstruction revealed that the most recent common ancestor of the four species existed in the current Chinese habitat of E. stellata. This study shows that E. byunsanensis and E. pinnatifida originated from a southern Eranthis species and speciated into their current forms near Jeju island and near western regions of Japan, respectively, during the Miocene. E. stellata may have dispersed southward on and near the Korean peninsula, though its specific origin remains unclear. Interestingly, the Chinese E. stellata population SCP suggests that the Chinese population might be most ancient among all the four Eranthis species. E. pungdoensis may have allopatrically speciated from E. byunsanensis during the Holocene. The Korean peninsula and the surrounding areas can be considered interesting regions which provide the opportunity to observe both northern‐ and southern‐sourced Eranthis species.
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Phytochemical analysis of the tubers of Eranthis cilicica was performed as part of our continuous study on the plants of the family Ranunculaceae, which resulted in the isolation of eleven new cycloartane glycosides (1–11) and one new oleanane glycoside (13), together with one known oleanane glycoside (12). The structures of the new compounds were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis followed by either X-ray crystallographic or chromatographic analysis. The aglycone (1a) of 2 and its C-23 epimer (8a), and the oleanane glycosides (12 and 13) showed cytotoxic activity against HL-60 leukemia cells with IC50 values ranging from 10.6 μM to 101.6 μM. HL-60 cells were much more sensitive to 8a (IC50 14.8 μM) than 1a (IC50 101.1 μM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. Compound 12 was revealed so as to partially induce apoptotic cell death in HL-60 cells, as was evident from morphology of HL-60 cells treated with 12.
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The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant (EHL) is a Type II Ribosome Inactivating Protein (RIP). Type II RIPs have shown anti-cancer properties and have great potential as therapeutic agents. Similarly, colloidal gold nanoparticles are successfully used in biomedical applications as they can be functionalised with ligands with high affinity and specificity for target cells to create therapeutic and imaging agents. Here we present the synthesis and characterization of gold nanoparticles conjugated with EHL and the results of a set of initial assays to establish whether the biological effect of EHL is altered by the conjugation. Gold nanoparticles functionalised with EHL (AuNPs@EHL) were successfully synthesised by bioconjugation with citrate gold nanoparticles (AuNPs@Citrate). The conjugates were analysed by UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Zeta Potential analysis, and Transmission Electron Microscopy (TEM). Results indicate that an optimal functionalisation was achieved with the addition of 100 µL of EHL (concentration 1090 ± 40 µg/mL) over 5 mL of AuNPs (concentration [Au0] = 0.8 mM). Biological assays on the effect of AuNPs@EHL were undertaken on Caenorhabditis elegans, a free-living nematode commonly used for toxicological studies, that has previously been shown to be strongly affected by EHL. Citrate gold nanoparticles did not have any obvious effect on the nematodes. For first larval stage (L1) nematodes, AuNPs@EHL showed a lower biological effect than EHL. For L4 stage, pre-adult nematodes, both EHL alone and AuNPs@EHL delayed the onset of reproduction and reduced fecundity. These assays indicate that EHL can be conjugated to gold nanoparticles and retain elements of biocidal activity.
The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses — the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
This chapter describes S functions for tree-based modeling. Tree-based models provide an alternative to linear and additive models for regression problems and to linear logistic and additive logistic models for classification problems. The models are fitted by binary recursive partitioning whereby a dataset is successively split into increasingly homogeneous subsets until it is infeasible to continue. The implementation described in this chapter consists of a number of functions for growing, displaying, and interacting with tree-based models. This approach to tree-based models is consistent with the data-analytic approach to other models, and consists primarily of fits, residual analyses, and interactive graphical inspection.