Genetic divergence of eastern European and Tobol populations of Calluna vulgaris (L.) Hull.

Abstract

Analysis of geographic variation of chloroplast DNA in the eastern European and western Siberian (Tobol) groups of Calluna vulgaris populations isolated from each other for a long period of time has revealed distinct differentiation between them. It has been shown that three populations growing in the Russian Plain and Karelia share two haplotypes characteristic of central Europe, while three Tobol populations contain one haplotype F occurring in Sweden. These results confirm the hypothesis of genetic divergence between the Tobol and eastern European groups of C. vulgaris populations.

Full text

ISSN 10674136, Russian Journal of Ecology, 2013, Vol. 44, No. 2, pp. 118–122. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © S.N. Sannikov, I.V. Petrova, M.A. Polezhaeva, Yu.D. Mishchikhina, O.E. Cherepanova, O.S. Dymshakova, 2013, published in Ekologiya, 2013, No. 2,
pp. 110–114.
118
Studies in population biology have revealed wide
polymorphism in the ecogenetic population structure
of many plant species, which is explained by specific
differences in their centers of origin, routes and rates
of migration, reproductive isolation, and habitat con
ditions. Using the example of conifers in the Northern
Hemisphere, it has been shown that the level of poly
morphism in their insular populations is lower, while
the level of genetic differentiation between them is
higher in the marginal than in the central parts of their
ranges (Tigerstedt, 1973; Critchfield, 1984; Gullberg
et al., 1985; Moran, Bell, and Eldridge, 1988; Semer
ikov and Lascoux, 2003; Sannikov et al., 2011). In
general, however, specific ecogenetic features of mar
ginal isolates and their role in speciation have not been
studied sufficiently.
The group of endemic populations of heather (
Cal
luna vulgaris
[L.] Hull.) growing in the Tobol region,
in the southwest of Western Siberia, is an interesting
object for ecogeographic analysis of the role of long
term isolation in microevolutionary divergence of
plants. The ridges and foothills of the Urals separate
them from the main, European part of this monotypic
species for more than 500 km (Beijerink, 1940;
Hulten, 1958; Gorchakovskii, 1962). Scattered patches
and colonies of heather in this region are closely associ
ated with pine forests on sandy terraces above river
floodplains and occur in a zone 150–170 km wide
extending in the north–south direction from the
Konda River basin to the city of Kurgan (Gorchak
ovskii, 1962; Petrova et al., 2009).
Previous studies on
C. vulgaris
in Europe have pro
vided comprehensive data on its geographic and eco
logical ranges (Beijerink, 1940; Hulten, 1958; Gim
mingham, 1960, 1975), morphological and autecolog
ical features and seed reproduction (Nordhagen,
1937; Dommee, 1969; Gimmingham, 1975, 1988;
Herrera, 1987; Legg, Maltby, and Proctor, 1992;
Cumming and Legg, 1995), and differentiation into
ecotypes depending on altitude (Grant and Hunter,
1962; Dommee, 1968). Methods of allozyme and
chloroplast DNA (cpDNA) analysis have made it pos
sible to reveal geographic variation and differentiation
of populations in the direction from Pyrenean (proba
bly refugial) to Scandinavian habitats (Mahy et al.,
1997; Mahy, Ennos, and Jacquemart, 1999; Rendell
and Ennos, 2002).
The results of comparative studies on
C. vulgaris
in
geographically vicarious types of pine forests in the
Tobol region and Russian Plain have shed light on
cenoecogeographic features of its ecological range, the
structure of cenopopulations, and their heliophilia
and competitive relationships with the edificator
(
Pinus sylvestris
) tree stand (Petrova et al., 2009, 2010;
Sannikova et al., 2012). It has been shown that the
growth rate, abundance, and coverage of
C. vulgaris
under the canopy of similar pine forest types are higher
in the Russian Plain than in Western Siberia. More
over, this species does not grow at all on peat soils of
Genetic Divergence of Eastern European and Tobol Populations
of
Calluna vulgaris
(L.) Hull.
S. N. Sannikov
a
, I. V. Petrova
a
, M. A. Polezhaeva
b
, Yu. D. Mishchikhina
a
,
O. E. Cherepanova
a
, and O. S. Dymshakova
b
a
Botanical Garden, Ural Branch, Russian Academy of Sciences, ul. Vos’mogo Marta 202, Yekaterinburg, 620144 Russia
email: stanislav.sannikov@botgard.uran.ru
b
Institute of Plant and Animal Ecology, Ural Branch, Russian Academy of Sciences,
ul. Vos’mogo Marta 202, Yekaterinburg, 620144 Russia
Received September 1, 2011
Abstract
—Analysis of geographic variation of chloroplast DNA in the eastern European and western Sibe
rian (Tobol) groups of
Calluna vulgaris
populations isolated from each other for a long period of time has
revealed distinct differentiation between them. It has been shown that three populations growing in the Rus
sian Plain and Karelia share two haplotypes characteristic of central Europe, while three Tobol populations
contain one haplotype
F
occurring in Sweden. These results confirm the hypothesis of genetic divergence
between the Tobol and eastern European groups of
C. vulgaris
populations.
Keywords
:
Calluna vulgaris
, population, chloroplast DNA, Eastern Europe, Western Siberia, isolation,
genetic divergence
DOI:
10.1134/S1067413613020094

RUSSIAN JOURNAL OF ECOLOGY Vol. 44 No. 2 2013
GENETIC DIVERGENCE OF EASTERN EUROPEAN AND TOBOL POPULATIONS 119
bogs in the Tobol region but commonly occurs on
waterlogged sphagnum substrates in the eastern Euro
pean part of its range, e.g., in the Carpathians and Bal
tic republics (Fedorchuk, Neshataev, and Kuznetsova,
2005; Petrova et al., 2009), as well as in western
Europe (Gimmingham, 1960, 1975). Taking into
account these cenoecological differences, strict and
longterm isolation by distance, and specific climatic,
soil, and biotic conditions in the arid continental
Transural region, we have put forward the hypothesis
concerning ecogenetic divergence of insular Tobol
populations from European populations of
Calluna
vulgaris
(Petrova et al., 2009).
This paper deals with a brief analysis of preliminary
results of a study performed to test this hypothesis.
OBJECTS AND METHODS
The comparative analysis of cpDNA variation was
performed between two groups of geographically iso
lated
C. vulgaris
cenopopulations (below, referred to
as populations), the Tobol group (populations from
Tavda, Tugulym, and Kurgan) and the eastern Euro
pean group (populations from Vyborg, Pskov, and
Kirs), growing under the canopy of climatically vicar
ious, topologically similar pine forests of cowberry–
heather–moss type (Petrova et al., 2009). Their geo
graphic locations are shown in Fig. 1.
In October, 18–55 samples of leafy shoots were
collected in each population from distinct, separately
growing plants (ramets) located no less than 30–50 m
from each other. Leaf tissues, fresh or frozen at
–70
°
С
,
were used in the study. Analysis of variation in
cpDNA, which is maternally inherited in
C. vulgaris
,
was performed by the PCR–RFLP method as
described (Rendell and Ennos, 2002), except that 6%
denaturing polyacrylamide gel was used instead of
nondenaturing 8% gel. On the whole, 213 individual
C. vulgaris
plants were analyzed in the six samples.
The pattern of variation in marker cpDNA frag
ments was analyzed to determine chloroplast haplo
types, their number, composition, and occurrence fre
quency in the populations studied. Hierarchical anal
ysis of cpDNA variation between the Tobol and
eastern European groups, between populations within
the groups, and between all samples included in the
study was performed by the AMOVA method
(Excoffier, Laval, and Schneider, 2006).
RESULTS AND DISCUSSION
Analysis was performed for four markers variable in
European populations of
C. vulgaris
: CS/
Alu
I,
FV/
Hae
III, K1K2/
Taq
I, and TabCD/
Taq
I (Rendell
and Ennos, 2002), but polymorphism was revealed
only in TabCD/
Taq
I (Table 1). Its variation is due to
two types of mutations, insertion and point replace
ment. Their combinations in all six populations
allowed us to determine the occurrence and frequency
of three chloroplast haplotypes corresponding to hap
lotypes
A
,
F
, and
G
(out of a total of 12) identified pre
viously in 23
C. vulgaris
population samples from west
ern and central Europe (Rendell and Ennos, 2002).
Thus, the total number of haplotypes in all
C. vulgaris
samples from the eastern part of its European range
and the Tobol region of Western Siberia proved to be
only onefourth of that recorded in the main, western
part of its range.
Analyzing the frequencies of chloroplast haplo
types, we revealed differences in their distribution
between the two phylogeographic groups of
C. vulgaris
populations: haplotypes
A
and
G
characteristic of cen
tral Europe proved to occur in different combinations
in populations of the eastern European group, while
all three populations from the Tobol region contained
only haplotype
F
, which was not identified in our sam
ples from the Russian Plain or southern Karelia (Fig. 1)
but was previously described in from Sweden (Rendell
and Ennos, 2002). Such a contrasting (alternative)
ratio of haplotypes is indicative of crucial differences
and, probably of a certain boundary between these
groups in the structure of the species gene pool. Hap
lotype
F
, which distinctly marks this boundary, can be
termed “differential,” by analogy with differential
plant species that make it possible to draw boundaries
between phytocenoses (Scamoni, 1956).
10
°
20
°
30
°
40
°
50
°
60
°
70
°
80
°
60
°
50
°
Vyborg
Pskov Kirs Tavda
Tug u l y m
Kurgan
AF G 12
Scale
1 : 20000 000
Fig. 1.
Geographic range and locations of population sam
ples and chloroplast DNA haplotypes (
A
,
F
,
G
) of
Calluna
vulgaris
in eastern Europe and the Tobol region of Western
Siberia. Tavda, Tugulym, and Kurgan are insular popula
tion groups in the Tobol region; (
1
) the boundary of the
European part of the range (according to Gorchakovskii,
1962); (
2
) isolated locations.

120
RUSSIAN JOURNAL OF ECOLOGY Vol. 44 No. 2 2013
SANNIKOV et al.
Parameters of intrapopulation cpDNA variation in
the Tobol and eastern European population groups,
respectively, are as follows: number of haplotypes
(
N
o
), 1 and 2; effective number of haplotypes (
N
e
), 1.0
and 1.39; index of intrapopulation variation (
H
), 0 and
0.27; i.e., these parameters obviously differ between
the groups.
A hierarchical analysis of the distribution of
cpDNA variation between the Tobol and eastern
European groups of
C. vulgaris
populations, between
populations within the groups, and between all the six
populations was performed using the AMOVA test
(Excoffier, Laval, and Schneider, 2006) for haplotype
frequencies (Table 2). The results confirmed distinct
differentiation between the two phylogeographic
groups with respect to the distribution pattern of vari
ation in chloroplast markers, especially in the variant
where haplotype kinship was not taken into account:
N
CT
= 0.443,
G
CT
= 0.719. The statistical significance
of these values was insufficient (
p
= 0.08–0.10)
because of a small number of samples per group. Dif
ferences between populations within the groups in the
two variants of analysis accounted for 26.0% and
13.1% of the total variance, and differences between
all populations, for 29.7% and 15.0% (
p
≤
0.001
); i.e.,
the proportion of explained variance was approxi
mately three times smaller than in the above case.
Thus, as follows from the difference revealed between
the eastern European and Tobol groups, the geo
graphic component obviously plays the dominant role
in differentiation of
C. vulgaris
populations with
respect to cpDNA structure.
Ordination of the samples in a threedimensional
space of principal coordinates based on
N
ST
as a mea
sure of genetic distance also provided fairly adequate
evidence for genogeographic differentiation between
European and Tobol populations of
C. vulgaris
(Fig. 2).
These results may be interpreted as follows. The
C.
vulgaris
range in the Miocene probably covered not
only Europe and East Atlantic islands but extended to
continental regions of northern Eurasia, up to central
Siberia. This follows from the occurrence of marginal,
insular
C. vulgaris
populations scattered over the
Azores, Faroe, and Shetland Islands, Iceland, and
Western Siberia and single habitats of this species in
Asia Minor, central Siberia, and central Kazakhstan
(Gimmingham, 1960; Gorchakovskii, 1962).
In the Eocene up to Oligocene, Siberian popula
tions of
C. vulgaris
remained isolated from European
populations for millions of years due to vast transgres
sions of the Kara and Tethys seas and, in the Pleis
tocene, by Ural glaciers and permafrost zones (up to
52
°
N). A long isolation of
C. vulgaris
, its exposure to
disruptive selection in new directions under specific
conditions of continental climate in the Transural
region, and the effects of mutation, genetic drift, and
other factors of microevolution provided for their
divergence from European populations accompanied
by segregation of the monomorphic “Transural” chlo
roplast haplotype
F
. It is noteworthy that this haplo
Table 1.
Haplotypes of
C. vulgaris
determined by restriction of four chloroplast DNA fragments
Haplotype CS/
Alu
I
band I,
307 bp
FV/
Hae
III
band I,
1354 bp
K1K2/
Taq
I
band I,
330 bp
K1K2/
Taq
I
band II,
313 bp
K1K2/
Taq
I
band III,
275 bp
TabC D /
Taq
I
band I,
464 bp
TabC D /
Taq
I
band II,
221 bp
A
2031012
F
2031002
G
2031001
Note: Restriction fragments are numbered in order of decreasing molecular weight; zero sign indicates the absence of restriction frag
ment at the expected position in the gel.
Table 2.
Hierarchical analysis (AMOVA) of variation and differentiation of the chloroplast genome in
C. vulgaris
popula
tions
Source of variation
Parameters of variation
with regard to haplotype kinship without regard to haplotype kinship
N
statistics proportion
of variance, %
pG
statistics proportion
of variance, %
p
Between groups*
N
CT
: 0.443 44.29 0.10
G
CT
: 0.719 71.93 0.08
Between populations within
group
N
ST
: 0.467 26.02 <0.001
G
SC
: 0.467 13.11 <0.001
Between all populations
N
ST
: 0.703 29.69 <0.001
G
ST
: 0.850 14.96 <0.001
* Phylogeographic groups: Tobol group (Tavda, Tugulym, Kurgan) and eastern European group (Vyborg, Pskov, Kirs).

RUSSIAN JOURNAL OF ECOLOGY Vol. 44 No. 2 2013
GENETIC DIVERGENCE OF EASTERN EUROPEAN AND TOBOL POPULATIONS 121
type was previously found in
C. vulgaris
from north
eastern Sweden (Rendell and Ennos, 2002), where it
had probably been preserved since the Paleogene. The
extreme impoverishment of
C. vulgaris
haplotype
spectrum in insular pine forests of the Tobol region
may be due to the effect of a series of population bot
tlenecks experienced by the species in this region dur
ing the Pleistocene.
It appears in first approximation that populations
of the Tobol group have already diverged from eastern
European populations to the subspecies level, which,
in particular, is confirmed by their distinctive arealog
ical and cenoecological features (Petrova et al., 2009).
A similar situation is observed, for example, with mar
ginal Pleistocene isolates of
Larix kurilensis
ssp.
glabra
Dyl. in Kamchatka, in which monomorphic mito
chondrial haplotypes are different from those in
neighboring marginal populations of
L. cajanderi
Mayr (Polezhaeva, 2009).
CONCLUSIONS
The analysis of geographic variation and differenti
ation of
C. vulgaris
chloroplast DNA haplotypes in the
Tobol and eastern European groups of populations
isolated from each other for a long time has revealed
distinct genetic divergence between these groups.
Populations of the eastern European group from
the Russian Plain and southern Karelia share two
chloroplast haplotypes (
A
and
G
) characteristic of cen
tral Europe, whereas populations of the Tobol group
have only one (monomorphic) haplotype
F
, which
may be regarded as a differential haplotype. The pro
portion of total variance in the chloroplast haplotype
frequencies explained by genogeographic differences
between the population groups is three times higher
than that explained by differences between popula
tions within the group and between all populations
included in analysis.
Thus, preliminary results of comparative genogeo
graphic analysis of marginal
C
.
vulgaris
populations
from the Tobol region (Western Siberia), which have
been long isolated by distance from the main part of
the species range in Europe, confirm the hypothesis of
their genetic divergence from European populations.
ACKNOWLEDGMENTS
This study was supported by the Russian Founda
tion for Basic Research (project no. 120401482a)
and research program of the Presidium of the Russian
Academy of Sciences (project nos. 12P41060 and
12P41062).
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