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Citation: Cherepanova, O.; Petrova,
I.; Sannikov, S.; Mishchihina, Y.
Diagnostics and Description of a
New Subspecies of Calluna vulgaris
(L.) Hull from Western Siberia.
Horticulturae 2023,9, 386.
https://doi.org/10.3390/
horticulturae9030386
Academic Editor: Alberto Pardossi
Received: 19 December 2022
Revised: 6 March 2023
Accepted: 8 March 2023
Published: 16 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
horticulturae
Article
Diagnostics and Description of a New Subspecies of
Calluna vulgaris (L.) Hull from Western Siberia
Olga Cherepanova *, Irina Petrova, Stanislav Sannikov and Yulia Mishchihina
Botanical Garden, Ural Branch of the Russian Academy of Sciences, Ekaterinburg 620144, Russia;
irina.petrova@botgard.uran.ru (I.P.)
*Correspondence: botgarden.olga@gmail.com
Abstract:
The article presents the results of the study of fifty populations of common heather (Calluna
vulgaris (L.) Hull) collected throughout its range. A phased comparative analysis (genetic, biochemical,
anatomical, morphological, and ecological) was carried out with the estimation of indicators that
included two key populations—Zavodouspenskoe (Pritobolye, Western Siberia) and Luga (Baltic,
Eastern Europe). It was concluded that heather growing in Western Siberia should be identified as a
separate taxonomic group, giving it the status of a subspecies. The gene pool of Pritobolye populations
(including Zavodouspenskoe) is represented by the completely dominant (100%) monohaplotype
S, which is not found anywhere else. The heather plant growing in Zavodouspenskoe has a longer
lifespan. It is distinguished by larger linear leaf dimensions (length 2.06
±
0.09 mm), thicker cuticle
(4.77
±
0.33
µ
m), increased number of trichomes (18.98
±
0.56), and a reduced number of stomata
(13.60
±
0.63) than that growing in Luga. The new subspecies differs in biochemical composition:
twice less content of epicatechin (average 1.992
±
0.005 mg g
−1
), three times more myricetin (average
2.975
±
0.005 mg g
−1
), twice as much chlorogenic acid (average 2.763
±
0.004 mg g
−1
). An ecological
feature is that C. vulgaris does not grow in the swamps of Western Siberia and has a small population.
This species has a high horticultural potential and requires protection as its population in Western
Siberia continues to decline rapidly.
Keywords:
heather; population; insular isolate; genetics; biochemical composition; anatomical and
morphological features; coenoecology; divergence
1. Introduction
The shrub (C. vulgaris), which has always grown in the heathlands and damp places
of northern Europe, is increasingly found in various types of gardens. Modern heather
varieties vary in habit, corolla color, and foliage variability. Gardeners appreciate not only
the attractive variability of heather, long flowering, and high plasticity but also the ability
to easily tolerate pruning and bush formation.
For a long time, scientists considered heather to be the only species of the Calluna
genus. No intraspecific taxones, except different morphoecotypes: C. vulgaris f. alba,C. vul-
garis f. aurea,C. vulgaris f. foxii,C. vulgaris f. searlei,C. vulgaris f. serotina,C. vulgaris var.
hirsuta [
1
,
2
], have been distinguished in its structure yet. In our view, it is mainly due to
narrow regionality and insufficient development of quantitative population-based and
geographical approaches [
3
,
4
]. Heather, endowed with a wide range, contains features
that help it exist both in swamps and in arid places. Heather populations are also noted
far beyond their main range: New Zealand, Australia, and North America. Heather’s
colonization of these regions is the result of sailors and scientist expansion from the Euro-
pean territories [
5
,
6
]. Its specific xeromorphic features allowed it to gain a foothold in the
harsh conditions of poor soils and open areas of Australia [
7
–
9
]. Some small populations
growing sparsely in the preforest-steppe of Western Siberia can endure periods of drought
and retain seed germination for a long time, forming their reserve in soil. In most cases,
Horticulturae 2023,9, 386. https://doi.org/10.3390/horticulturae9030386 https://www.mdpi.com/journal/horticulturae
Horticulturae 2023,9, 386 2 of 14
seed germination begins only after the fire. Forest fire, burning the moss layer, opens the
possibility for the development of small seedlings of heather seeds without competition for
light, water, and mineral nutrition.
Pritobolye populations (Western Siberia), geographically isolated for many years
during the Pleistocene, have genetic features that distinguish these populations from their
nearest neighbors from the main part of the continuous range (Figure 1). Genetic features
are supported by a number of small but important ecological, anatomical, and biochemical
features. Geographic isolation confirms the originality of heather growth in the region of
the Tobol and Iset rivers [10–12].
Horticulturae 2023, 9, x FOR PEER REVIEW 2 of 16
drought and retain seed germination for a long time, forming their reserve in soil. In most
cases, seed germination begins only after the fire. Forest fire, burning the moss layer,
opens the possibility for the development of small seedlings of heather seeds without
competition for light, water, and mineral nutrition.
Pritobolye populations (Western Siberia), geographically isolated for many years
during the Pleistocene, have genetic features that distinguish these populations from
their nearest neighbors from the main part of the continuous range (Figure 1). Genetic
features are supported by a number of small but important ecological, anatomical, and
biochemical features. Geographic isolation confirms the originality of heather growth in
the region of the Tobol and Iset rivers [10–12].
Figure 1. Young heather plant in Botanical Garden of Ural Branch of the Russian Academy of Sci-
ences.
A high degree of xerophytization and a change in the species composition in the
communities of the preforest-steppe of Western Siberia may be a consequence of the
long-term determination of the aridity of the climate in this region. Heathers, despite
their origin, were able to develop several adaptations that allow them to survive in the
adverse conditions of hot summers. The low temperatures of the winter period in the
Trans-Urals are easily tolerated by heather under a high snow cover. We attribute the
fragmentation of the species range in Western Siberia to the lower frequency of fires than
in European barrens and, importantly, to the lower soil temperature in winter. The leaf
was able to adapt to the arid climate of Western Siberia by reducing the number of tri-
chomes and stomata, as well as by a longer period of reduced transpiration. Low autumn
and winter soil temperatures in Western Siberia are factors to which heather has no ad-
aptation mechanisms. The marginal isolates, growing in extreme environmental condi-
tions, namely protractedly isolated during Pleistocene relic, marginal, eastern, insular
Pritobolye populations, located in the west of Western Siberia, are of great interest for
revealing the process of intraspecific adaptive divergence of populations within the
heather range. In the last ten years, versatile research of genetic, morphological, ana-
tomic, and ecological peculiarities of the Pritobolye group of C. vulgaris populations was
performed in the Botanical Garden of Ural Branch of the Russian Academy of Sciences
Figure 1.
Young heather plant in Botanical Garden of Ural Branch of the Russian Academy of Sciences.
A high degree of xerophytization and a change in the species composition in the
communities of the preforest-steppe of Western Siberia may be a consequence of the
long-term determination of the aridity of the climate in this region. Heathers, despite
their origin, were able to develop several adaptations that allow them to survive in the
adverse conditions of hot summers. The low temperatures of the winter period in the
Trans-Urals are easily tolerated by heather under a high snow cover. We attribute the
fragmentation of the species range in Western Siberia to the lower frequency of fires
than in European barrens and, importantly, to the lower soil temperature in winter. The
leaf was able to adapt to the arid climate of Western Siberia by reducing the number
of trichomes and stomata, as well as by a longer period of reduced transpiration. Low
autumn and winter soil temperatures in Western Siberia are factors to which heather has
no adaptation mechanisms. The marginal isolates, growing in extreme environmental
conditions, namely protractedly isolated during Pleistocene relic, marginal, eastern, insular
Pritobolye populations, located in the west of Western Siberia, are of great interest for
revealing the process of intraspecific adaptive divergence of populations within the heather
range. In the last ten years, versatile research of genetic, morphological, anatomic, and
ecological peculiarities of the Pritobolye group of C. vulgaris populations was performed in
the Botanical Garden of Ural Branch of the Russian Academy of Sciences (RAS) based on
the ideas and approaches of Ural ecological-genetic science school of Schwartz–Timofeev-
Resovsky [
13
–
17
]. The results of the quantitative analysis of structure parameters and
geographic variation of Pritobolye populations, compared to other populations growing
Horticulturae 2023,9, 386 3 of 14
within the entire species range, demonstrated significant differences between them, which
enabled us to distinguish specific new taxon at the subspecies level—C. vulgais (L.) Hull ssp.
tobolica [
13
]. Previously, a subspecies of C. vulgaris in Western Siberia was not distinguished.
Due to the low or declining numbers of these heather populations and the many
threats they face, they have been assessed as critically endangered. Heather is listed in the
Red Books of several regions of Russia [
12
]. The introduction of C. vulgaris representatives
of these populations into culture can be considered as one of the steps towards their
conservation. In general, the results obtained by the authors of this work were considered
sufficient for the identification of a new specific taxon, the description of which is given
below. The work aimed to clarify the ecological, morphological, anatomical, biochemical,
and genetic differences between marginal populations and populations from the central
part. The article summarizes, and our work is based on new population data and the
parameters listed above.
2. Materials and Methods
The specificity of complex areographic, population-biological, genetic, anatomical,
morphological, and coenoecological features of the Pritobolye group of C. vulgaris popula-
tions was grounded in applying the generic approach “species in the range”.
As a result of long-term work (2009–2018), it was possible to carry out an analysis for
fifty populations of heather, which were collected throughout the entire range (Figure 2
and Supplementary Table S1). For correct comparison, we chose two main populations in
which ecological conditions are similar: Zavodouspenskoe (Pritobolye, Western Siberia)
and Luga (Baltic, Eastern Europe). These two populations are present in various anal-
yses and supplemented by others with similar ecological conditions to obtain the most
reliable results.
Horticulturae 2023, 9, x FOR PEER REVIEW 4 of 16
Figure 2. Geographic localization of population samples of Calluna vulgaris within the species
range. 1—Range border of C. vulgaris, 2—Borders of phylogenogeographic regions, 3—Localization
of C. vulgaris populations. NES—Northern-European-Scandinavian, WSP—Western-Siberian-
Pritobolye, WEA—Western-European-Atlantic, EEM—Eastern-European-Mediterranean,
SEE—Southern-Eastern-European.
Population codes: AI—Azorian insels, Ar—Arkhangelsk, Bd—Bordeaux,
Br—Bryansk, Bs—Belis, Chp—Cherepovets, Cv—Cevennes, Db—Dublin, Dj—Djerdap,
Dl—Dalarna, FI—Faeroes, Gm—Gomel, Ist—Istanbul, Kg—Kurgan, Kl—Kaliningrad,
Ki—Koimbra, Km—Kem, Kms—Komissarovo, Kr—Kirov, Kv—Katwijk,
Lb—Lueneburg, Lg—Luga, Lbl—Ljubljana, Ln—London, Mn—Minsk,
Mm—Murmansk, Mz—Mizun, Nch—Naroch, NN—Nizhny Novgorod, Nt—Nízke Ta-
try, Os—Oslo, Pz—Penza, Ps—Pskov, Rk—Reykjavik, Sm—Smolensk, Svl—Seville,
Sw-1—Sweden-1, Shm—Šumava, Tv—Tavda, Tm—Tyumen, Tr—Trentino,
Trb—Trabzon, Ur-1—Uraj-1, Ur-2—Uraj-2, Vb—Vyborg, VN—Veliky Novgorod,
Vr—Voronezh, YO—Yoshkar-Ola, Zuk—Zavodoukovsk, Zus—Zavodouspenskoe,
Vsh—Warsaw.
2.2. Genetic Analysis
The comparative analysis of the structure and differentiation of chloroplast DNA
parameters was conducted for 6 insular Pritobolye populations on the one hand and 50
local populations from the rest part of the range on the other hand (Figure 2, Supple-
mentary Table S1).
The chloroplast DNA (maternally inherited in heather) was extracted from the leafy
heather shoots taken from 8 to 37 samples in each population [18,19] and analyzed using
the PCR-RFLP method [3,20]. The universal chloroplast primers were used, which turned
out to be polymorphic in European populations of heather [21]. All these fragments were
sequenced with the help of a sequencer (Applied Biosystem-3130, St. Louis, MO, USA)
and edited using the software BioEdit [21]. Totally, 1500 heather specimens were ana-
lyzed in 50 local populations. The parameters F
ST
of the intra-group and inter-group
variation were estimated by ANOVA based on the haplotype frequencies and the num-
ber of mutations in them, followed by the N
ST
parameter [22].
Figure 2.
Geographic localization of population samples of Calluna vulgaris within the species range.
1—Range border of C. vulgaris, 2—Borders of phylogenogeographic regions, 3—Localization of
C. vulgaris populations. NES—Northern-European-Scandinavian, WSP—Western-Siberian- Prito-
bolye, WEA—Western-European-Atlantic, EEM—Eastern-European-Mediterranean, SEE—Southern-
Eastern-European.
2.1. Objects of Study
The central object of the study was a marginal, eastern group of insular populations,
protractedly isolated during the Pleistocene from the main European C. vulgaris range and
Horticulturae 2023,9, 386 4 of 14
located in Pritobolye, southwest of Western Siberia, Russia (Figure 2). Its contemporary
range included sandy above flood-plain terraces of the Tobol River and its confluence [
17
].
Throughout the Pleistocene (over 1.8 million years), we think that the Pritobolye heather
populations were reproductively and migratory separated from European populations.
Glaciers and cold soils in the region of the Ural ranges and foothills have become the
cause of isolation for heat-loving heather [
15
]. Insular populations of heather grew here,
sometimes dominating, in the substage of Pritobolye pine forests on the dry sandy soil of
cowberry-heather-green-moss pine forests and sometimes in other types of forests.
The second population of C. vulgaris (Luga) is located in the western part of the
continuous range of the Russian Plain of the southern taiga subzone in the middle part of
the slope of the Baltic sandy ridge in Eastern Europe. The material was collected in the
lingonberry-heather-green moss pine forest. The soil is sandy-podzolic, and groundwater
occurs at a depth of about 1.5 m. The stand is 110 years old; composition—10 C, absolute
density 19.8 m
2
ha
−1
, class II. Pine undergrowth, 15–20 thousand ind. ha
−1
; undergrowth is
absent. The moss layer (projective cover is about 70–75%) is dominated by the pleurocium
(Pleurozium schreberi), occasionally dicranum (Dicranum sp); lichens of the genus Cladonia—
10%. In some places, there is a dead cover of up to 20%. Heather (C. vulgaris)—29.8% and
lingonberry (Vaccinium vitis-idaea)—12% predominate in the grass-shrub layer; sedge (Carex
ericetorum) occurs singly.
Population codes: AI—Azorian insels, Ar—Arkhangelsk, Bd—Bordeaux, Br—Bryansk,
Bs—Belis, Chp—Cherepovets, Cv—Cevennes, Db—Dublin, Dj—Djerdap, Dl—Dalarna,
FI—Faeroes, Gm—Gomel, Ist—Istanbul, Kg—Kurgan, Kl—Kaliningrad, Ki—Koimbra,
Km—Kem, Kms—Komissarovo, Kr—Kirov, Kv—Katwijk, Lb—Lueneburg, Lg—Luga, Lbl—
Ljubljana, Ln—London, Mn—Minsk, Mm—Murmansk, Mz—Mizun, Nch—Naroch, NN—
Nizhny Novgorod, Nt—Nízke Tatry, Os—Oslo, Pz—Penza, Ps—Pskov, Rk—Reykjavik,
Sm—Smolensk, Svl—Seville, Sw-1—Sweden-1, Shm—Šumava, Tv—Tavda, Tm—Tyumen,
Tr—Trentino, Trb—Trabzon, Ur-1—Uraj-1, Ur-2—Uraj-2, Vb—Vyborg, VN—Veliky Nov-
gorod, Vr—Voronezh, YO—Yoshkar-Ola, Zuk—Zavodoukovsk, Zus—Zavodouspenskoe,
Vsh—Warsaw.
2.2. Genetic Analysis
The comparative analysis of the structure and differentiation of chloroplast DNA
parameters was conducted for 6 insular Pritobolye populations on the one hand and 50 local
populations from the rest part of the range on the other hand (Figure 2and Supplementary
Table S1).
The chloroplast DNA (maternally inherited in heather) was extracted from the leafy
heather shoots taken from 8 to 37 samples in each population [
18
,
19
] and analyzed using
the PCR-RFLP method [
3
,
20
]. The universal chloroplast primers were used, which turned
out to be polymorphic in European populations of heather [
21
]. All these fragments were
sequenced with the help of a sequencer (Applied Biosystem-3130, St. Louis, MO, USA) and
edited using the software BioEdit [
21
]. Totally, 1500 heather specimens were analyzed in
50 local populations. The parameters F
ST
of the intra-group and inter-group variation were
estimated by ANOVA based on the haplotype frequencies and the number of mutations in
them, followed by the NST parameter [22].
The results of this study are based on the ordination of all studied populations in a
three-dimensional coordinate field, a result of hierarchical analysis, and a comparative quan-
titative assessment of genetic distances was given (average FST values were determined).
2.3. Morphological and Anatomical Analysis
Comparative analyses and evaluation of morphological and anatomical differences
were carried out for two previously selected populations: Zavodouspenskoe (Pritobolye,
Western Siberia) and Luga (Baltic, Eastern Europe), located in the center of the species conti-
nental range (Figure 2). The main features of these relatively “standard” populations were
compared under similar conditions of ecotopic and phytogenic environments—under the
Horticulturae 2023,9, 386 5 of 14
canopy of Scots pine with close density (26.9 and 19.8 m
2
ha
−1
, respectively) in geographi-
cally vicarious topoecologically similar types of green moss pine forests on sandy-podzolic
soils [
23
]. To study the growth features of the heather root system, we dug up heather
plants of various age groups growing on sandy substrates in Western Siberia under natu-
ral conditions.
Length and thickness of at least 5 leaves from the middle part of the three terminal
annual leading shoots taken from 20 specimens from each population were measured. From
the cuts of each leaf, fixed with a mixture of 95% ethanol and glycerol, we took 5 tissue
samples for epidermis maceration, which was performed in NaOH water solution. The
morphological and anatomical structure parameters of three to five leaves were measured
for each specimen using the binocular loupe Carl Zeiss Stemi 2000-C (Carl Zeiss Meditec AG,
Dresden, Germany), light microscope Carl Zeiss Scope A1 (Carl Zeiss Meditec AG, Dresden,
Germany) and software Axio Vizion Rel. 4.8 (Carl Zeiss Meditec AG, Dresden, Germany).
Main leaf features, representing the peculiarities of its adaptation to hydrothermal climate
parameters, included the number and location of stomas and trichomes and the thickness
of cuticle and epidermis.
The leaf elongation coefficient (LEC) was calculated by Equation (1):
LEC = L/Br, (1)
where L—is the leaf length (mm), and Br—is the leaf width (mm).
2.4. Chemophenotypic Analysis
The bioactive substance content was assessed by applying reversed-phase HPLC with
photometric detection. The extract of C. vulgaris was obtained according to the generally
accepted method [
24
]: 1 g of crushed heather shoots was transferred into a flask in a water
bath, 10 mL of 96% ethanol was added, and the mixture was extracted for 15 min. After
that, the extract was poured through the filter paper into a dark container with a tight lid.
Then 10 mL of 96% ethanol was again added to the residue. The procedure was repeated
three times. The resulting extract was evaporated to an air dry state. The determination of
the seasonal dynamics of the accumulation of biologically active substances was carried out
by reverse-phase HPLC with photometric detection on an Aligent 1100 instrument (Agilent
Technologies, Waldbronn, Germany) [
25
]. The following standards were used: chlorogenic
acid, oleic acid, quercetin, myricetin, and epicatechin (Dia-m, Moscow, Russia). To prepare
a standard sample with a concentration of 0.05 mg cm
−3
, 5 mg of the standard was placed
in a volumetric flask with a capacity of 100 cm3, and the volume was brought to the mark
with methanol. For analysis, the same extract as for determining the number of flavonoids
was used.
Conditions for chromatographic analysis. Column: octadecyl silica gel 5
µ
m, 250
×
4.6
(e.g., 32 Phenomenex Luna 5
µ
m C18(2)); mobile phase: acetonitrile—trifluoroacetic acid
solution pH 2.6 (40:60); mobile phase rate: 1.0 cm
3
min
−1
; column temperature: 30
◦
C;
detection: UV,
λ
= 365 nm. Injected sample volume: 10 mm
3
. The calculation of the
indicator components content (X) was carried out according to the calibration curve or
according to Equation (2):
X=C×S1×V×S2×m, (2)
where C is the concentration of the corresponding standard solution, mg cm
−3
; S
1
is the
peak area of the component in the analyzed sample; S
2
is the peak area of the component
in the standard sample; V is the total volume of the sample dilution, cm3; m—the mass of
the sample.
2.5. Ecological Features
In natural communities, C. vulgaris is one of the typical species of pine forest un-
derlayer [
26
]. The comparative analysis of such parameters as projective cover, growth,
vitality, and ecologic range of heather populations was performed for different types of
Horticulturae 2023,9, 386 6 of 14
geographically vicarious pine forests in Pritobolye (Zavodouspenskoe) and Baltic (Luga).
As a result of “coenopopulation-based and microecosystemic” regression analysis, the same
objects were used to compare the regional peculiarities of heather response to the tree stand,
root, and light competition indexes [27].
2.6. Statistical Analysis
The results were analyzed using the Statistica 10.0 portable (StatSoft, Tulsa, OK,
USA), Windows Excel, as well as specific programs Arlequin 3.5.1.2, Axio Vision Rel.
4.8 [
28
–
31
]. To assess the significance of the results, classical statistical parameters: mean
value (M), mean standard error (SE), and coefficient of variation were used. For in-depth
statistical analysis of nonparametric data, the ANOVA analysis (Statistica 10.0 portable)
was used. The results of statistical treatment are given in the relevant sections. The results
of the anatomical analysis were evaluated by several tests: Student’s t-test and ANOVA.
Significant differences are marked in the table with an asterisk.
3. Results and Discussion
3.1. Genetic Features
The most unbiased and systematic for the purpose of cross-regional population genetic
differentiation assessment were the gene pool differences between their phylogeographic
groups based on chloroplast DNA haplotype structure analysis in a 3-D coordinate space
within the whole species range (Figure 3). The 3-D analysis evidenced for subdivision of
the gene pool of 50 natural heather populations into five general phylogeographic groups,
the boundaries of which are marked in Figure 2: Eastern-European-Mediterranean (EEM),
Northern-European-Scandinavian (NES), Western-Siberian-Pritobolye (WSP), Southern-
Eastern-European (SEE), Western-European-Atlantic (WEA) (Figure 3). The two popula-
tions that were compared belong to different groups: Zavodouspenskoe (Pritobolye) is
included in WSP, while Luga (Baltic) is in SEE.
Horticulturae 2023, 9, x FOR PEER REVIEW 7 of 16
Figure 3. 3-D ordination of C. vulgaris populations. Population codes are given in Figure 2.
The calculation of heather populations’ intergroup genetic differentiation was car-
ried out based on population hierarchical analysis performed on the whole species level
(Table 1).
Table 1. Genetic distances (F
ST
) between groups of C. vulgaris populations.
Phylogenogeographic
Groups
of
Populations
EEM
NES
WSP
SEE
WEA
Eastern
-
European
-
Mediterranean (EEM)
0.00000
Northern
-
European
-
Scandinavian (NES)
0.60004
0.00000
Western
-
Siberian
-
Pritobolye (WSP)
0.86929
0.63591
0.00000
Southern
-
Eastern
-
European (SEE)
0.43626
0.45641
0.90038
0.00000
Western-European-Atlantic (WEA) 0.53501 0.19462 0.64500 0.47764 0.00000
Max mean genetic divergence was noticed between WSP, protractedly isolated in
Pleistocene, marginal, eastern phylogeographic group of populations, and all the other
groups, especially from the neighboring EEM (0.870) and SEE (0.901), which were iso-
lated in Pleistocene by the vast Ural disjunction. The smallest pairwise intergroup dis-
tance was noted for WEA-NES (0.195), which indicates the free penetration of genetic
flows between these close groups. We observed a decrease in the pairwise intergroup
distance between population groups of the Iberian Peninsula (SEE) and populations of
the southern Mediterranean (EEM), populations of the Iberian Peninsula, and popula-
tions of the eastern Atlantic (WEA).
Figure 3. 3-D ordination of C. vulgaris populations. Population codes are given in Figure 2.
The most geographically and genetically separated population group of C. vulgaris
included six marginal eastern protractedly isolated Pritobolye populations located in the
southwest of Western Siberia. Their gene pool was represented by completely dominating
Horticulturae 2023,9, 386 7 of 14
(100%) monohaplotype S, which could not be found anywhere else within the whole heather
range. The origin of these haplotypes has been unknown yet, though we can assume that
the Pleistocene refugium of Pritobolye heather populations, as well as of P. sylvestris L., was
in Kazakhskiy Melkosopochnik where the relict heather location was noted [
15
,
17
]. The
sharp genetic border of the Pritobolye group of populations—with complete replacement
of monohaplotype S by monohaplotype D—can be noticed even in relation to the nearest
populations of the northern Zauralye (Uraj, Figure 2).
The calculation of heather populations’ intergroup genetic differentiation was car-
ried out based on population hierarchical analysis performed on the whole species level
(Table 1).
Table 1. Genetic distances (FST) between groups of C. vulgaris populations.
Phylogenogeographic Groups
of Populations EEM NES WSP SEE WEA
Eastern-European-Mediterranean (EEM) 0.00000
Northern-European-Scandinavian (NES) 0.60004 0.00000
Western-Siberian-Pritobolye (WSP) 0.86929 0.63591 0.00000
Southern-Eastern-European (SEE) 0.43626 0.45641 0.90038 0.00000
Western-European-Atlantic (WEA) 0.53501 0.19462 0.64500 0.47764 0.00000
Max mean genetic divergence was noticed between WSP, protractedly isolated in
Pleistocene, marginal, eastern phylogeographic group of populations, and all the other
groups, especially from the neighboring EEM (0.870) and SEE (0.901), which were isolated
in Pleistocene by the vast Ural disjunction. The smallest pairwise intergroup distance
was noted for WEA-NES (0.195), which indicates the free penetration of genetic flows
between these close groups. We observed a decrease in the pairwise intergroup distance
between population groups of the Iberian Peninsula (SEE) and populations of the southern
Mediterranean (EEM), populations of the Iberian Peninsula, and populations of the eastern
Atlantic (WEA).
3.2. Morphological Features
Table 2shows the results of the comparative assessment of C. vulgaris vegetative shoots
and leaves average morphoanatomical features. The analysis was carried out for typical
Pritobolye (Zavodouspenskoe) and continental Baltic (Luga) populations.
The pivotal differential morphological feature of the Pritobolye typical C. vulgaris pop-
ulation, which allowed us to taxonomically distinguish it from the typical Baltic population
of this species, was the linear dimensions of the leaves. Their average length with high
reliability (p
≤
0.00001) differed 1.6 times from the European population. At the same time,
the average thickness of leaves in the Western Siberian heather population was twofold
more than in the typical Eastern European population (Table 2). The difference in leaf
elongation coefficient between Pritobolye and Baltic populations was also significant. We
did not note significant differences in the vascular system. Each leaf has one vein, along
which the stomata are arranged in dense groups. The vein is displaced towards the center
of the leaf when the leaf margins are closed, which reduces transpiration. There are less
significant differences in the length and annual growth of the apical shoot, as well as in life
expectancy and leaf growth, which were mainly determined by the stage of ontogenesis
and interspecific competition.
Horticulturae 2023,9, 386 8 of 14
Table 2.
Leaf morphological and anatomical parameters of C. vulgaris in typical Pritobolye (Zavodous-
penskoe) and Baltic (Luga) populations.
Parameter
Typical C. vulgaris Populations
Zavodouspenskoe Luga TST p*
Morphological features
Leaf length, mm 2.06 ±0.09 11.26 ±0.03 −18.2810 0.00001
Leaf thickness, mm 0.76 ±0.02 0.37 ±0.03 −10.9994 0.00001
Leaf elongation coefficient (LEC) 0.39 ±0.02 0.30 ±0.02 2.0930 0.05
Anatomical features
Stoma number, pc. 13.60 ±0.63 19.69 ±1.14 4.3012 0.0002
Trichome number, pc. 18.98 ±0.56 15.65 ±0.75 −2.8235 0.008
Cuticle thickness, µm 4.77 ±0.33 3.97 ±0.28 2.024 0.06
Upper epidermis cells
Height, µm 38.08 ±2.56 26.61 ±1.44 2.069 0.05
Length, µm 56.54 ±1.56 58.78 ±2.4 2.035 0.03
Thickness, µm 25.51 ±1.22 29.39 ±4.46 2.447 0.05
Area, µm21126.43 ±61.40
1195.19
±
56.37
1.991 0.06
Perimeter, µm 166.37 ±5.23 180.48 ±5.02 2.035 0.03
LEC 0.453 ±0.01 0.490 ±0.06 2.018 0.05
Palisade tissue cells
Length, µm 32.02 ±0.23 26.19 ±1.14 −4.9125 0.00004
Thickness, µm 13.82 ±0.05 11.82 ±0.55 −3.5992 0.001
Area, µm2402.25 ±8.50 290.85 ±19.87 −3.9669 0.0005
Perimeter, µm 112.23 ±7.99 67.99 ±4.98 −2.7204 0.01
LEC 0.432 ±0.003 0.460 ±0.03 2.101 0.05
Spongy parenchyma cells
Length, µm 20.62 ±0.44 16.76 ±1.06 −2.8758 0.007
Thickness, µm 15.12 ±0.29 13.29 ±1.48 1.973 0.05
Area, µm2252.68 ±6.96 192.74 ±17.48 −3.1006 0.004
Perimeter, µm 59.19 ±0.89 54.99 ±3.25 1.977 0.06
LEC 0.738 ±0.01 0.86 ±0.15 2.011 0.05
1Data is presented as M ±SE. * Significance level of differences between studied C. vulgaris populations.
3.3. Morphological and Anatomical Features
Works devoted to heather leaves anatomical structure study are rare. Heather has a
specific leaf structure, characteristic of xerophytic plants, which reduces the variability of
traits. The small size of the heather leaf makes it difficult to study [
28
,
29
,
31
]. Throughout
its range, heather exhibits weak plasticity, which may be due to the high specialization of
the leaf. Due to a combination of anatomical and ecophysiological features, heather could
be referred to as mesoxerophytes [11,32–34].
As the climate continentality in Zauralye increased, the xeromorphic features in leaf
anatomical structure intensified, compared to heather growing in the European part of
the range. It is true that the number of stomas bordering the channel on the lower side of
the leaf decreased by 30%, p
≥
0.0002 (Table 2), and on the upper epidermis, there were
almost no stomas (Figure 4), or they were very rare. The trichomes number increased
(1.2 times), as well as epidermis cell height (1.4 times), with a simultaneous decrease in
their length and thickness. Moving deeper into the continent of Eurasia in the eastern
direction, heather plants are forced to adapt to the increasing dryness of atmospheric air,
which is expressed in an increase in cuticular wax productivity (Table 2, Figure 4). An
interesting feature is observed as we move deeper into the continent—a decrease in the
tortuosity of epidermal cells. This can be seen from the decrease in the perimeter of the
cell with relatively stable linear parameters of the epidermal cells. In Zavodouspenskoe,
palisade chlorenchyma cells are larger than in Luga. At the same time, the elongation of
the cell does not differ significantly, which indicates balanced changes in the elements. The
area of spongy parenchyma cells is 1.3 times larger in Zavodouspenskoe than in Luga. The
remaining linear dimensions of this type of cell do not change. The growth of mesenchymal
cell linear dimensions led to the overall leaf linear dimensions rise. In general, almost all
studied parameters specifying the sizes and forms of heather palisade parenchyma and
Horticulturae 2023,9, 386 9 of 14
lacunose parenchyma in Pritobolye isolates differed from the ones in Baltic populations
(Table 2).
Horticulturae 2023, 9, x FOR PEER REVIEW 9 of 16
Figure 4. Schematic C. vulgaris leaf morphologic structure and cross-section view: (a,c)—Pritobolye
type; (b,d)—Baltic type.
Table 2. Leaf morphological and anatomical parameters of C. vulgaris in typical Pritobolye (Za-
vodouspenskoe) and Baltic (Luga) populations.
Parameter
Typical
C. vulga
ris
Populations
Zavodouspenskoe
Luga
T
ST
p
*
Morphological features
Leaf length, mm
2.0
6 ± 0
.09
1
1.2
6 ± 0
.03
−
1
8.2810
0.00001
Leaf thickness, mm
0.7
6 ± 0
.02
0.3
7 ± 0
.03
−
1
0.9994
0.00001
Leaf elongation coefficient (LEC)
0.3
9 ± 0
.02
0.3
0 ± 0
.02
2.0930
0.05
Anatomical features
Stoma number, pc.
13.6
0 ± 0
.63
19.6
9 ± 1
.14
4.3012
0.0002
Trichome number, pc.
18.9
8 ± 0
.56
15.6
5 ± 0
.75
−
2
.8235
0.008
Cuticle thickness, μm
4.7
7 ± 0
.33
3.9
7 ± 0
.28
2.024
0.06
Upper epidermis cells
Height, μm
38.0
8 ± 2
.56
26.6
1 ± 1
.44
2.069
0.05
Length, μm
56.5
4 ± 1
.56
58.7
8 ± 2
.4
2.035
0.03
Thickness, μm
25.5
1 ± 1
.22
29.3
9 ± 4
.46
2.447
0.05
Area, μm
2
1126.4
3 ± 6
1.40
1195.1
9 ± 5
6.37
1.991
0.06
Perimeter, μm
166.3
7 ± 5
.23
180.4
8 ± 5
.02
2.035
0.03
LEC
0.45
3 ± 0
.01
0.49
0
±
0
.06
2.018
0.05
Palisade tissue cells
Length, μm
32.0
2 ± 0
.23
26.1
9 ± 1
.14
−
4
.9125
0.00004
Thickness, μm
13.8
2 ± 0
.05
11.8
2 ± 0
.55
−
3
.5992
0.001
Area, μm
2
402.2
5 ± 8
.50
290.8
5 ± 1
9.87
−
3
.9669
0.0005
Perimeter, μm
112.2
3 ± 7
.99
67.9
9 ± 4
.98
−
2
.7204
0.01
LEC
0.43
2 ± 0
.003
0.46
0
±
0
.03
2.101
0.05
Spongy parenchyma cells
Length, μm
20.6
2 ± 0
.44
16.7
6 ± 1
.06
−
2
.8758
0.007
Thickness, μm 15.12 ± 0.29 13.29 ± 1.48 1.973 0.05
Area, μm
2
252.6
8 ± 6
.96
192.7
4 ± 1
7.48
−
3
.1006
0.004
Perimeter, μm
59.1
9 ± 0
.89
54.9
9 ± 3
.25
1.977
0.06
LEC
0.73
8 ± 0
.01
0.8
6 ± 0
.15
2.011
0.05
1
Data is presented as M ± SE. * Significance level of differences between studied C. vulgaris popu-
lations.
The statistically significant characteristics that form the special structure of the
Pritobolye heather leaf include the height of the upper epidermis cells, the perimeter of
a b
c d
Figure 4.
Schematic C. vulgaris leaf morphologic structure and cross-section view: (
a
,
c
)—Pritobolye
type; (b,d)—Baltic type.
The statistically significant characteristics that form the special structure of the Pri-
tobolye heather leaf include the height of the upper epidermis cells, the perimeter of the
palisade tissue cells, and the area of spongy cells of parenchyma (based on Student’s t-test).
The statistical discrepancies found during the evaluation of the morphological and anatom-
ical features using the Student criterion were confirmed with nonparametric tests for two
independent samples (Mann–Whitney).
3.4. Chemotypic Analysis
Heather contains many biologically active components, allowing for expanding its
application scope. We have focused on the most revealing components found in the green
parts of the plant [35].
The chlorogenic acid and myricetin content in the tissues of the Pritobolye heather is
significantly higher (by 2–8 times), and epicatechin is two times lower than in the European
part of the range (Table 3). There is an increase in the content of almost all components,
except for epicatechin, in the direction from west to east of the range. This can be associated
with an increase in continentality, which is expressed in increased adaptability (possibly,
an increase in the activity of the photosynthetic apparatus) to more severe environmental
conditions. Thus, the chemotypic test could be used as an additional diagnostic method
to prove the specificity of features of the distinguished taxon C. vulgaris (L.) Hull ssp.
tobolica Sannikov.
Table 3. Biochemical composition of C. vulgaris extract.
Biologically Active
Substances, mg g−1
Typical C. vulgaris Populations
Zavodouspenskoe Luga
Chlorogenic acid 2.614 ±0.005 11.911 ±0.005 *
Oleic acid 0.198 ±0.005 0.208 ±0.004
Quercetin 2.982 ±0.004 1.194 ±0.005 *
Myricetin 2.361 ±0.005 0.451 ±0.005 *
Epicatechin 2.004 ±0.004 2.954 ±0.004
1Data is presented as M ±SE. * Significant differences between the studied C. vulgaris populations at p≤0.01.
Horticulturae 2023,9, 386 10 of 14
3.5. Ecologic Range
The most pronounced and reliable coenoecologic differences between the Pritobolye
Western-Siberian (Zavodouspenskoe) and Baltic Eastern-European (Luga) groups of
C. vulgaris populations were determined in peculiarities of their regional topoecologic
ranges. With the advancement to the east deep into the continent of Eurasia, heather is
more often found under the canopy of pine forests and on the outskirts of raised bogs,
avoiding flowing mesotrophic bog complexes. Its range is becoming more and more scat-
tered, largely reflecting the range of pine forests growing on sandy terraces in Western
Siberia [10–17].
In a generalized profile of topoecologically similar types of Pritobolye pine forests, the
maximum heather projective cover in the cowberry-heather-green moss pine forest (30.4%)
was almost the same as in a geographically vicarious type of forest in the Baltics (29.8%)
(Figure 5). However, in the edaphic “dry” Pritobolye lichen pine forest, this parameter
(7.5%) was 2.5 times less, and in the “wet” polytric pine forest (3.5%), contrariwise, this
was 2.5 times more than in the Baltics (18.8 and 1.7%, respectively) [12,36].
Horticulturae 2023, 9, x FOR PEER REVIEW 11 of 16
(a) (b)
Figure 5. Average projective cover and C. vulgaris shoot length in geographically vicarious types of
pine forests of (a) Pritobolye, Western Siberia, and (b) Baltic, Eastern Europe. 1—Projective cover;
2—Leading shoot length. Data is presented as M ± SE. Types of pine forests: cl—cowberry-lichen;
chg—cowberry-heather-green moss; cbg—cowberry-bilberry-green moss; bg—bilberry-green
moss; p—polytric; llg—ledum-leatherleaf-bog moss.
C. vulgaris growing in Western Siberia, adapted to the increased dryness of atmos-
pheric air and severe winter temperatures, forms a spherical shape of a bush with suffi-
cient illumination (
Figure 6
).
Figure 5.
Average projective cover and C. vulgaris shoot length in geographically vicarious types of
pine forests of (
a
) Pritobolye, Western Siberia, and (
b
) Baltic, Eastern Europe. 1—Projective cover;
2—Leading shoot length. Data is presented as M
±
SE. Types of pine forests: cl—cowberry-lichen;
chg—cowberry-heather-green moss; cbg—cowberry-bilberry-green moss; bg—bilberry-green moss;
p—polytric; llg—ledum-leatherleaf-bog moss.
C. vulgaris growing in Western Siberia, adapted to the increased dryness of atmospheric
air and severe winter temperatures, forms a spherical shape of a bush with sufficient
illumination (Figure 6).
Summarizing our results, we can identify some adaptive traits for heather from
Pritobolye. Probably, heather mastered the territories of Western Siberia, dispersing from
the regions of Northern Kazakhstan [
4
,
14
]. As dry conditions increase, the leaf reduces
the number of stomata and trichomes and increases the thickness of the cuticle, which we
consider to be an adaptation to decreasing humidity. Most often, heather growing in the
main part of its range (Meadows) tends to open areas. It shows higher heliophilicity than
heather in the Pritobolye. The accumulation of biologically active substances in plants
depends on many factors. The concentration of biologically active substances can change
during ontogenesis or under the pressure of environmental conditions. Thus, the content
of secondary metabolites changes during the growing season and strongly depends on
the altitude of the population, illumination, transpiration level, and the quality of water
Horticulturae 2023,9, 386 11 of 14
exchange. For example, the amount of synthesized quercetin depends on the number of
chloroplasts in the cell [11].
Horticulturae 2023, 9, x FOR PEER REVIEW 12 of 16
Figure 6. General view of (a) C. vulgaris 20-years-old adult (origin from seed) growing in Pritobol-
ye, Western Siberia (Zavodouspenskoe); (b) Enlarged part of an annual shoot with leaves and in-
florescences (3D- visualization).
Summarizing our results, we can identify some adaptive traits for heather from
Pritobolye. Probably, heather mastered the territories of Western Siberia, dispersing from
the regions of Northern Kazakhstan. [4,14]. As dry conditions increase, the leaf reduces
the number of stomata and trichomes and increases the thickness of the cuticle, which we
consider to be an adaptation to decreasing humidity. Most often, heather growing in the
main part of its range (Meadows) tends to open areas. It shows higher heliophilicity than
heather in the Pritobolye. The accumulation of biologically active substances in plants
depends on many factors. The concentration of biologically active substances can change
during ontogenesis or under the pressure of environmental conditions. Thus, the content
of secondary metabolites changes during the growing season and strongly depends on
the altitude of the population, illumination, transpiration level, and the quality of water
exchange. For example, the amount of synthesized quercetin depends on the number of
chloroplasts in the cell [11].
Heather leaf anatomy was studied in European populations in the 20th century, but
with only a descriptive purpose [33,34,37]. There were no comparable analyzes of heather
a
b
Figure 6.
General view of (
a
)C. vulgaris 20-years-old adult (origin from seed) growing in Prito-
bolye, Western Siberia (Zavodouspenskoe); (
b
) Enlarged part of an annual shoot with leaves and
inflorescences (3D- visualization).
Heather leaf anatomy was studied in European populations in the 20th century, but
with only a descriptive purpose [
33
,
34
,
37
]. There were no comparable analyzes of heather
leaves population variability. There are a few works describing the high adaptability
of heather due to changes in soil conditions and the accumulation of heavy metals [
38
].
Heather growing in Western Siberia has a greater thickness of leaves than heather growing
in the Russian Plain [
17
]. Most likely, the increase in the content of quercetin is associated
with an increase in the number of chloroplasts in leaf cells, the number of which, in
turn, also rises with an increase in the leaf linear dimensions, which changes following
illumination decrease in the lower forest layer.
Botanic description of the typical full-grown specimen of C. vulgaris (L.) Hull ssp.
tobolica Sannikov (tribe Ericeae; subfamily Ericoideae; family Ericaceae; order Ericales [
39
])
Evergreen semifrutex (Figure 6) of about 50–60 cm height growing under the canopy of
pine tree stands (P. sylvestris), maximum 90 cm height on the open site. Shoots are intensely
Horticulturae 2023,9, 386 12 of 14
branched, mostly lodging, heavy pubescent being young, shoot leafage—from 2 to 4 years.
The leaves are imbricated in four rows, triangular small of 1.0–3.5 mm in length, squamose,
downy, sagittal at the bottom, and sessile, with two awl-shaped outgrowths (Figure 6a,b).
The stomas are mostly located on the abaxial side of the leaf in the channel and are covered
with tight rows of trichomes, but there are almost no stomas on the adaxial side, or they
are very rare. The flowers are gathered in a paniculate inflorescence consisting of falsely-
racemose branches ending with foliated heads; sepals and lobes are quadripartite and
membranous; the color of lobes varies from pale pink to purple (Figure 6b). The corolla is
shorter than the cups and remains on the fruits. The number of stamens is 8, and the pistil
has a four-blade snout and protrudes from the cup. The fruit represents a dry pubescent
small boll (up to 1.5 mm in length). The seeds are small (0.8–1.2 mm in length), and the
color varies from light brown to almost black.
Typus: herbarium sample is stored in the herbarium department of the Institute of
Plant and Animal Ecology of Ural Branch of the Russian Academy of Sciences. International
acronym of herbarium collection SVER0922190 (https://herbarium.ipae.uran.ru/about.
html, accessed on 15 May 2018).
Distribution: The endemic scattered patches populations of heather growing in the
river Tobol region on sandy terraces in the southwest of Western Siberia.
Similar Species: Calluna vulgaris (L.) Hull: a small shrubby plant usually growing
less than 50 cm tall. Its tiny scale-like leaves are arranged in four vertical rows along
the branches; it is pink or pale purple, bell-shaped, flowers have four petals that are
joined together at the base, its tiny round capsules are hairy and have four compartments
(Salisbury, Trans. Linn. Soc. London. 6: 317. 1802).
4. Conclusions
As a result of the comprehensive study of Calluna vulgaris populations in Western
Siberia, significant differences were established between them and populations in all other
parts of the species range. Our results are consistent with S. Wright’s island model of
microevolutionary divergence.
The most pronounced differences between the typical Pritobolye C. vulgaris popula-
tion (Zavodouspenskoe) and continental East-European population (Luga) were found
in the chloroplast DNA structure (revealing unique S haplotype) as well as in leaf linear
dimensions (twice bigger) and xeromorphic features rise, which probably evidenced for
adaptation to dry climate of Pritobolie, Western Siberia [
12
,
13
]. The divergence in other
anatomic leaf parameters as well as in chemotypic features (bioactive substances content) in
shoot tissues was less obvious. We revealed some alternate differences in the coenoecologic
heather range in Pritobolye, where the heather was completely absent on peat moss bogs,
unlike the European range.
Heather Pritobolsky can be recommended for breeding more drought-resistant va-
rieties with high endurance, compactness of the vegetative part, a good growth rate of
annual shoots, and a high rate of vegetative offspring formation.
In general, obtained research results are sufficient to distinguish specific taxon C. vul-
garis (L.) Hull ssp. tobolica Sannikov in the infrastructure of C. vulgaris species. This species
has a high horticultural potential and requires protection as its population in Western
Siberia continues to decline rapidly.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/horticulturae9030386/s1. Table S1: List of fifty studied populations of C. vulgaris with their
geographic coordinates.
Author Contributions:
Conceptualization S.S. and I.P.; methodology, O.C.; software, O.C.; validation
S.S. and I.P.; formal analysis O.C.; writing—original draft preparation, data curation, resources, Y.M.;
writing—review and editing, O.C. and I.P.; visualization O.C.; supervision S.S.; project adminis-
tration S.S.; funding acquisition I.P. All authors have read and agreed to the published version of
the manuscript.
Horticulturae 2023,9, 386 13 of 14
Funding:
The work was carried out within the framework of the state task of the Botanical Garden of
the Ural Branch of the Russian Academy of Sciences.
Data Availability Statement: All results are included within the article.
Acknowledgments:
The authors express their thanks to L. Paule (Technical University in Zvolen,
Slovakia) for collecting and providing the number of C. vulgaris samples from Europe.
Conflicts of Interest: The authors declare no conflict of interest.
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