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The results of studying the polymorphism and genetic structure of populations of D. salina and D. incarnata growing in Zabaykalsky krai and Buryatia are represented according to the data of allozyme analysis of eight genetic loci (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, and IDH). The specificity of the allelic structure of loci SKDH, PGM, and IDH is established, for which D. salina and D. incarnata reliably differ from each other. It is shown that interspecies introgressive hybrid complexes with different genetic structures were formed in Transbaikalia. Places of mass growth of D. incarnata were observed to have single plants of D. salina, the interspecies hybrids of the first and subsequent generations. Places of mass growth of D. salina were observed to contain only the hybrids that are not hybrids of the first generation. They were heterozygous not for three loci with differentiating alleles of both parents, SKDH, PGM, and IDH, but for only one of them. The degree of genetic differentiation among five populations of D. salina was on average 7.5% and that of D. incarnata was 7.1%, which in accordance with Wright’s estimation relates to mean values. The average value of FST for all studied populations of the two related species of the genus Dactylorhiza was 0.478, indicating a very high degree of genetic differentiation between D. salina and D. incarnata growing in Transbaikalia. The greatest differences between the species are for the allelic structure of loci SKDH, PGM, and IDH (FST was equal to 0.705, 0.976, and 0.762, respectively). Analysis of molecular variance (AMOVA) showed that populations of D. salina and D. incarnata in the zone where their ranges in Zabaykalsky krai and Buryatya overlap have essential differences both for the variation of alleles frequencies of eight loci (71%, d.f. = 9) and for the variability of genotypes (61%, d.f. = 9). Despite the fact that D. salina and D. incarnata explicitly share a gene flow as a result of interspecies hybridization, the genetic differentiation of populations of these related species remains at a high level.
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ISSN 1022-7954, Russian Journal of Genetics, 2017, Vol. 53, No. 3, pp. 325–337. © Pleiades Publishing, Inc., 2017.
Original Russian Text © E.G. Filippov, E.V. Andronova, 2017, published in Genetika, 2017, Vol. 53, No. 3, pp. 310–323.
Genetic Structure of Populations and Natural Hybridization
between Dactylorhiza salina and D. incarnata (Orchidaceae)
E. G. Filippova, * and E. V. Andronovab, **
aBotanical Garden, Ural Branch, Russian Academy of Sciences, Yekaterinburg, 620144 Russia
bKomarov Botanical Institute, Russian Academy of Sciences, St. Petersburg, 197376 Russia
*e-mail: filorch@mail.ru
**e-mail: elena.andronova@mail.ru
Received March 28, 2016
AbstractThe results of studying the polymorphism and genetic structure of populations of D. salina and
D. incarnata growing in Zabaykalsky krai and Buryatia are represented according to the data of allozyme anal-
ysis of eight genetic loci (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, and IDH). The specificity of the
allelic structure of loci SKDH, PGM, and IDH is established, for which D. salina and D. incarnata reliably dif-
fer from each other. It is shown that interspecies introgressive hybrid complexes with different genetic struc-
tures were formed in Transbaikalia. Places of mass growth of D. incarnata were observed to have single plants
of D. salina, the interspecies hybrids of the first and subsequent generations. Places of mass growth of
D. salina were observed to contain only the hybrids that are not hybrids of the first generation. They were het-
erozygous not for three loci with differentiating alleles of both parents, SKDH, PGM, and IDH, but for only
one of them. The degree of genetic differentiation among five populations of D. salina was on average 7.5%
and that of D. incarnata was 7.1%, which in accordance with Wright’s estimation relates to mean values. The
average value of FST for all studied populations of the two related species of the genus Dactylorhiza was 0.478,
indicating a very high degree of genetic differentiation between D. salina and D. incarnata growing in Trans-
baikalia. The greatest differences between the species are for the allelic structure of loci SKDH, PGM, and
IDH (FST was equal to 0.705, 0.976, and 0.762, respectively). Analysis of molecular variance (AMOVA)
showed that populations of D. salina and D. incarnata in the zone where their ranges in Zabaykalsky krai and
Buryatya overlap have essential differences both for the variation of alleles frequencies of eight loci (71%,
d.f. = 9) and for the variability of genotypes (61%, d.f. = 9). Despite the fact that D. salina and D. incarnata
explicitly share a gene flow as a result of interspecies hybridization, the genetic differentiation of populations
of these related species remains at a high level.
Keywords: Dactylorhiza salina, D. incarnata, allozyme analysis, interspecies introgressive hybrid complexes,
genetic polymorphism
DOI: 10.1134/S102279541703005X
INTRODUCTION
According to L.V. Aver’yanov, Dactylorhiza salina
(Turcz. ex Lindl.) Soó and D. incarnata (L.) Soó
belong to subsection Dactylorhiza of the genus Dacty-
lorhiza. This subsection includes 26 species growing
mainly in Eurasia; in Russia, there are representatives
of six taxa [1, 2].
The range of D. salina is Caucasian-Asian, cover-
ing the Caucasus, Western and Eastern Siberia, the
Far East (Zeya-Bureya region), Central Asia, North
Mongolia, and East China [3]. A typical sample of
D. salina was described from Transbaikalia.
The range of D. incarnata is Eurasian. Representa-
tives of this species grow in Europe (in the southern
part only in the mountains) from Great Britain and
Scandinavia to Central and Eastern Europe up to the
Urals. In Asia, the range covers Asia Minor, Iran, the
North Caucasus, Central Asia, Siberia, Mongolia, and
the northwestern part of China [3].
All species of orchids are rare plants. Establishment
of techniques for conservation of genetic diversity of
rare species requires information on polymorphism
and genetic structure of their populations. Quite a
major amount of work on the study of genetic charac-
teristics of representatives of the genus Dactylorhiza
was carried out mainly in Western Europe [4–11].
Samples from the territory of Russia, which accounts
for a large part of the range of D. incarnata, were
hardly used in these studies. Molecular-genetic stud-
ies carried out by foreign colleagues contain no infor-
mation about D. salina.
Transbaikalia is an area where the ranges of
D. salina and D. incarnata overlap (Fig. 1). According
to L.V. Aver’yanov, a vast area of introgression has
PLANT
GENETICS
326
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
been formed at the contact of these two closely related
species, particularly in the south of Siberia. Notewor-
thy, interspecific hybrids are fertile and owing to back-
crossing bind original parent species by a continuous
series of transitional forms [2].
Interspecies introgressive hybrid complexes [14]
produced with participation of rare species of orchids
are known, but information about their structure and
renewal remains fragmented. The study of such com-
plexes pertains to fundamental research of microevo-
lutionary processes and factors that determine the
dynamics of biodiversity. Previously, there were no
data either on the degree of genetic differentiation
between closely related species D. incarnata and
D. salina or polymorphism and genetic structure of
their local populations in the zone where their ranges
overlap. The interspecies complex in Transbaikalia
remained unstudied.
The purpose of this work is to study polymorphism
and genetic structure of local populations of D. salina
(Turcz. ex Lindl.) Soó and D. incarnata (L.) Soó
growing in the Zabaykalsky krai and Buryatia on the
basis of the data of allozyme analysis.
MATERIALS AND METHODS
The plant material was collected in natural habitats
of D. salina and D. incarnata representatives in Trans-
baikalia in 2010 (Fig. 1). Polymorphism was estimated
by the variation of allele frequencies of eight gene loci:
phosphoglucose isomerase (PGI, EC 5.3.1.9), NADH
dehydrogenase (NADHD, EC 1.6.99.5), shikimate
dehydrogenase (SKDH, EC 1.1.1.25) , glu tama te de hy-
drogenase (GDH, EC1.4 .1.2), phosph o glucomutase
(PGM, EC 5.4.2.2), diaphorase (DIA, EC 1.6.4.3),
alcohol dehydrogenase (ADH, EC 1.1.1.1 ), and i soci -
trate dehydrogenase (IDH, EC 1.1.1.4 2).
Analysis included 136 individuals of D. salina from
five local populations: (1) floodplain of the Veriya
River (Chita distinct, Zabaykalsky krai); (2) village of
Fig. 1. Location of the studied populations of Dactylorhiza salina (1, Gazimurovsky Zavod; 2, Yagye; 3, Veriya; 4, Khonkholoyka;
5, Ivolginsk) and D. incarnata (610, Petrovky Zavod and Kosurta) in Transbaikalia. Ranges of species in Eastern Siberia are
given according to the published data [12, 13] with modifications.
1
2
3
6–10
54
100°110 °120°130°140°
40°
150°
160°
50°
Dactylorhiza incarnata (L.) Soó
D. salina (Turcz. ex Lindl.) Soó
Joint growth area adjacent range
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
GENETIC STRUCTURE OF POPULATIONS 327
Gazimurovsky Zavod (Zabaykalsky krai); (3) neigh-
borhood of Ivolginsk (Buryatia); (4) floodplain of the
Yagye River (Baleysky district, Zabaykalsky krai);
(5) floodplain of the Khonkholoyka River (Mukhor-
shibirsky district, Buryatia). The data of allozyme
analysis of D. salina individuals were compared with
the data obtained in the study of specimens of five
populations of D. incarnata growing in Zabaykalsky
krai (in the floodplain of the Kosurta River and the
surrounding area of the village of Petrovsky Zavod,
points 1, 2, 3, and 5). The total sample of D. incarnata
was 118 individuals.
Local populations of D. salina were significantly
different in size. Floodplains of the Veriya and Yagye
rivers were inhabited by up to 100 flowering species,
the floodplain of the Khonkholoyka River and Gazi-
murovsky Zavod had a few hundred individuals, and
the vicinity of Ivolginsk had thousands of flowering
individuals.
Some localities showed very high polymorphism of
morphological traits (for example, leaf width and
length, plant height, spur length, and flower sizes).
Some individuals had uniform purple color of the
leaves. The color of flowers ranged from pure white to
violet-purple. The highest polymorphism was typical
of local populations in the vicinity of Ivolginsk.
None of D. salina populations contained individu-
als of another species, D. incarnata. However, it was
noted that some plants had intermediate traits between
D. salina and D. incarnata for the spur length. For
example, in D. salina individuals, the spur was not 8–
10 mm, as it should be [1], but shorter (Fig. 2). Espe-
cially many unusual specimens were found in the sur-
roundings of the Khonkholoyka River and Ivolginsk.
Samples of these populations were enlarged, account-
ing for more than 30 individuals.
In the vicinity of the station Petrovsky Zavod
(Zabaykalsky krai), a large group of D. incarnata con-
sisting of thousands of flowering individuals was dis-
covered and examined. D. incarnata plants grew on the
wet meadows for about 2 km along the railroad tracks
from the station in the direction of Chita. Very high
polymorphism of morphological traits in plants of this
species was noted. Accordingly, several samples were
taken (Petrovsky Zavod, points 1, 2, 3, and 5). The dis-
tance between the first and last points was not more
than 2 km. Another sample was taken from a local
population with a large number of individuals found
along the road to Ulan-Ude at a distance of about 6–
8 km from the Petrovsky Zavod in the f loodplain of
the Kosurta River. In addition to numerous represen-
tatives of D. incarnata, the surveyed localities in the
surroundings of Petrovsky Zavod were revealed to have
single individuals of D. salina and atypical individuals
with intermediate traits between D. incarnata and
D. salina. They were taken for analysis to determine
their genotype. Owing to the fact that, in the vicinity
of the Petrovsky Zavod, one may encounter both spe-
cies of the genus Dactylorhiza, between which hybrid-
ization is possible, samples of D. incarnata for allo-
zyme analysis were selected with caution and only
from those plants that had traits typical of representa-
tives of this species. Possible first generation hybrids
were analyzed separately. They were not included in
statistical processing of the data on D. incarnata and
D. salina.
Isoenzyme analysis was carried out using electro-
phoresis in polyacrylamide gel plates in Tris-borate-
EDTA buffer. Fresh leaves of generative shoots were
used as the material. Extraction of proteins and poly-
acrylamide gel electrophoresis and enzyme histo-
chemical staining were carried out according to stan-
dard methods [15, 16].
Statistical analysis of allozyme analysis data was
carried out. Statistical indicators of polymorphism
such as allele frequencies, observed and expected het-
erozygosity, F coefficients (Wright), and the Chi-
square test to verify the Hardy–Weinberg equilibrium
and pairwise comparisons of subpopulations based on
analysis of molecular variance (AMOVA) were
obtained using the program GenAlEx version 6.4 [17].
Calculation of genetic distances between the popula-
tions according to Nei [18–20] and also dendrogram
construction using the unweighted pair-group method
with arithmetic average (UPGMA), cluster analysis,
and bootstrap value were carried out in the program
PowerMarker v. 3.25 [21] with 1000 reassembling acts.
The dendrogram with bootstrap value was obtained in
the program Phylip 3.69 [22] and visualized in the
program Mega 6.0 [23].
This paper is one of several publications on the
study of polymorphism in the genus Dactylorhiza,
which has been taking place over several years. For
ease of comparison of the data from different papers,
all alleles identified in different years received their
serial number. An allele corresponding to the fastest
protein was assigned no. 1. All subsequent slower
alleles were consecutively numbered 2, 3, etc.
RESULTS AND DISCUSSION
In D. salina, six of the eight gene loci were poly-
morphic. The highest number of alleles, three, was
noted in loci PGI, SKDH, and IDH. Two alleles each
were found in loci PGM, NADHD, and DIA. In
D. incarnata, five gene loci of the eight were polymor-
phic. The highest number of alleles, three, was noted
in loci PGI and SKDH. Two alleles each were found in
loci IDH, NADHD, and DIA. The percentage of poly-
morphic loci ranged from 25 to 62.5 (Table 1).
Polymorphism in D. incarnata for loci SKDH and
IDH was found only in samples from Zabaykalsky krai.
More than 800 individuals from other parts of the
range (not from Transbaikalia) were differentiated in
loci SKDH and IDH for allele no. 1 [24].
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RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
Fig. 2. General view of inflorescence and individual f lowers in individuals of Dactylorhiza salina (1a and 1b, large flower and long
spur), D. incarnata (2a and 2b, small flower and short spur), and atypical individuals (3a and 3b, large f lower and short spur, near
Ivolginsk; 4a and 4b, small flower and long spur, near the Kosurta River).
1a1a1a
2a
2a2a
3a
3a3a
1b
1b1b
2b
2b2b
3b
3b3b
4b
4b4b
Individuals of D. salina and D. incarnata were sig-
nificantly different for allelic composition of the three
loci SKDH, PGM, and IDH (Table 1). The major
allele in locus SKDH was no. 1 for individuals of
D. incarnata growing in Transbaikalia and nos. 2 and 7
for D. salina. Individuals of D. incarnata were differen-
tiated for allele no. 1 of locus PGM, while for D. salina
the major allele for this locus was no. 6. In locus IDH,
D. incarnata individuals were characterized by allele
no. 1, while those of D. salina were characterized by
alleles nos. 2 and 4.
The study showed that some individuals of
D. incarnata from Zabaykalsky krai had alleles charac-
teristic of D. salina, while individuals of D. salina had
alleles specific to D. incarnata (Table 1, 2–4). These
data indicate that there is hybridization between
D. incarnata and D. salina growing in Zabaykalsky krai
and Buryatia.
Two of the five subpopulations of D. salina revealed
no hybrid individuals, while in the others the number
of hybrid plants that were part of the analyzed samples
ranged from two to ten specimens. Very unusual were
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
GENETIC STRUCTURE OF POPULATIONS 329
the results of analysis of genotypes of hybrid individu-
als: no hybrids of the first generation were detected in
the samples; differentiating alleles of both parental
species were present not in the three but only one of
the loci (Table 3). Thus, in the sample of specimens
from a population of D. salina from the territory of the
village of Gazimurovsky Zavod, two individuals (out
of 26 examined) were hybrid: they were heterozygous
for differentiating alleles of D. salina and D. incarnata
in locus PGM. A sample of a D. salina population from
the vicinity of Ivolginsk was revealed to have six hybrid
individuals (out of 44), all of which were heterozygous
for differentiating alleles of both parents for one of the
loci: one individual for SKDH, two for PGM, and three
for IDH. A sample of D. salina growing in the flood-
plain of the Khonkholoyka River was revealed to have
ten hybrid plants out of 36 studied, nine of them were
heterozygous: seven for SKDH and two for IDH. One
individual was homozygous for SKDH locus allele
no. 1 nonspecific to D. salina but typical of D. incar-
nata; two other loci (IDH and the PGM) were mono-
morphic for differentiating alleles of D. salina (Table
3). All hybrid individuals identified on the basis of
allozyme analysis were not morphologically different
from individuals of D. salina.
In the sample of D. incarnata individuals from the
surroundings of the Kosurta River, one individual (out
of 26 examined) was hybrid. It was heterozygous and
had allele no. 2 in locus SKDH, which is not specific to
D. incarnata. The same location had single individuals
of D. salina. Allozyme analysis confirmed that they
belong to this taxon (Table 4). A few hybrid individuals
Table 1. Allele frequencies and parameters of genetic variation in local populations of D. salina and D. incarnata
I, Shannon’s diversity index; Ho, observed heterozygosity; He, expected heterozygosity; F, f ixation index. Values for major allele are
shown in bold. N, number of studied individuals.
Locus Allele no.
D. salina D. incarnata
Veriya Gazim.
Zavod Ivolginsk Yagye Khon-
kholoyka Kosurta Petrovsky Zavod
N = 14 N = 26 N = 44 N = 16 N = 36 N = 26 N = 25 N = 27 N = 16 N = 24
PGI 6 0.036 0.038 0.057 0.000 0.194 0.212 0.000 0.296 0.094 0.396
7 0.071 0.192 0.023 0.375 0.264 0.000 0.000 0.019 0.000 0.021
80.893 0.769 0.920 0.625 0.542 0.788 1.000 0.685 0.906 0.583
SKDH 1 0.000 0.000 0.012 0.000 0.125 0.978 0.979 0.980 1.000 0.979
20.929 0.818 0.558 0.964 0.611 0.022 0.021 0.000 0.000 0.021
70.0710.1820.430 0.036 0.264 0.000 0.000 0.020 0.000 0.000
PGM 1 0.000 0.040 0.023 0.000 0.000 1.000 1.000 1.000 1.000 1.000
61.000 0.960 0.977 1.000 1.000 0.000 0.000 0.000 0.000 0.000
IDH 1 0.000 0.000 0.034 0.000 0.028 1.000 0.980 0.981 1.000 0.979
2 0.286 0.058 0.341 0.000 0.097 0.000 0.000 0.000 0.000 0.000
40.714 0.942 0.625 1.000 0.875 0.000 0.020 0.019 0.000 0.021
ADH 21.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
DIA 11.000 0.962 1.000 1.000 1.000 1.000 1.000 0.963 1.000 1.000
2 0.000 0.038 0.000 0.000 0.000 0.000 0.000 0.037 0.000 0.000
NADHD 31.000 1.000 0.977 1.000 1.000 0.519 0.960 0.889 0.844 0.938
4 0.000 0.000 0.023 0.000 0.000 0.481 0.040 0.111 0.156 0.063
GDH 21.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
Polymorphic loci, %37.562.562.525 37.537.537.562.525 50
I0.158 0.209 0.257 0.102 0.295 0.164 0.046 0.174 0.093 0.150
Ho0.098 0.107 0.143 0.071 0.139 0.073 0.010 0.051 0.031 0.068
He0.092 0.116 0.154 0.067 0.171 0.109 0.020 0.098 0.054 0.088
F0.170 0.022 0.059 –0.052 0.121 0.261 0.319 0.358 0.461 0.057
330
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
Table 2 . Observed (Obs) and expected (Exp) number of genotypes for loci with taxon-specific alleles in local populations of D. salina and D. incarnata
m, all individuals are monomorphic.
Locus Genotype D. salina D. incarnata
Veriya Gazimur. Zavod Ivolginsk Khonkholoyka Yagye Kosurta P. Zavod-1 P. Zavod-2 P. Zavod-3 P. Zavod-5
SKDH 11 Obs 0 1 22 23 24 m 23
Exp 0.006 0.563 22.011 23.010 24.010 23.010
12 Obs 0 4 1 1 1
Exp 0.558 5.500 0.978 0.979 0.979
22 Obs 13 15 15 15 13 0 0 0
Exp 12.071 14.727 13.395 13.444 13.018 0.011 0.010 0.010
17 Obs 1 3 1
Exp 0.430 2.375 0.980
27 Obs 0 6 18 10 1
Exp 1.857 6.545 20.651 11.611 0.964
77 Obs 1 1 9 3 0 0
Exp 0.071 0.727 7.959 2.507 0.018 0.010
IDH 11 Obs 0 0 m 24 26 m 23
Exp 0.051 0.028 24.010 26.009 23.010
12 Obs 1 0
Exp 1.023 0.194
22 Obs 0 0 5 0
Exp 1.143 0.087 5.114 0.340
14 Obs 2 2 1 1 1
Exp 1.875 1.750 0.980 0.981 0.979
24 Obs 8 3 19 7
Exp 5.714 2.827 18.750 6.125
44 Obs 6 23 17 27 m 0 0 0
Exp 7.143 23.087 17.188 27.563 0.010 0.009 0.010
PGM 11 Obs 0 0 mmmmm
Exp 0.040 0.023
16 Obs 2 2
Exp 1.920 1.955
66 Obs m 23 42 m m
Exp 23.040 42.023
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
GENETIC STRUCTURE OF POPULATIONS 331
Table 3. Genotypes of hybrid individuals (for loci with taxon-specific alleles) found in places of mass growth of D. salina
Alleles not specific to D. salina are shaded.
Population
Genotype by different loci
SKDH PGM IDH
Gazimurovsky Zavod 221644
221644
Near Ivolginsk
176644
276614
226614
276612
221644
771624
Near Khonkholoyka
176644
126644
176644
176644
116624
126624
126644
126644
276614
226614
Table 4. Genotypes of hybrids and individuals of D. salina (for loci with taxon-specific alleles) found in places of mass
growth of D. incarnata
Alleles not specific to D. incarnata are shaded.
Population, sample size
Genotype for different loci
Note
SKDH PGM IDH
Floodplain of the Kosurta River
121111
121614Hybrid F1
2 2 6 6 4 4 D. salina
121614Hybrid F1
7 7 6 6 2 4 D. salina
Near Petrovsky Zavod, point 1 1 21114
Near Petrovsky Zavod, point 2 1 71114
Near Petrovsky Zavod,
point 3
2 2 6 6 4 4 D. salina
121614Hybrid F1
Near Petrovsky Zavod, point 5 1 21114
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RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
with intermediate morphological traits (between
D. incarnata and D. salina) were found here. Allozyme
analysis showed that they are the first generation
hybrids since they were heterozygous for differentiat-
ing alleles of both the parental species for all three loci
(SKDH, PGM, and IDH).
A similar pattern was observed in the vicinity of
Petrovsky Zavod (point 3). This location also, in addi-
tion to D. incarnata, was inhabited by single individu-
als of D. salina and first generation hybrids (Table 4).
D. salina and first generation hybrids were not
encountered in other places of collecting D. incarnata
in the surroundings of Petrovsky Zavod (points 1, 2,
and 5). However, according to the allozyme analysis
data, each of the samples contained one hybrid indi-
vidual. They were heterozygous and had alleles for loci
SKDH and IDH not specific to D. incarnata.
In total, the samples collected for the study of
D. incarnata contained four hybrids: three of them
were heterozygous and had differentiating alleles for
D. incarnata and D. salina in only two loci, and one
hybrid, in only one locus (Table 4). Morphologically,
they were not different from D. incarnata.
According to the data, two local populations of
D. salina are in Hardy–Weinberg equilibrium for all
polymorphic loci. In others, although from two to five
loci were polymorphic, the disequilibrium was
observed in only one locus: either for PGI or for
SKDH. In all cases, the equilibrium was shifted toward
an excess of homozygotes (Table 5). One of the studied
populations of D. incarnata for all the polymorphic
loci is in Hardy–Weinberg equilibrium; in other pop-
ulations, the equilibrium was shifted toward an excess
of homozygotes for some loci: in two for locus PGI, in
one for both PGI and DIA, and in another one only for
NADHD.
Despite the fact that features of the allelic compo-
sition of loci SKDH, PGM, and IDH indicate hybrid-
ization between D. incarnata and D. salina in the zone
of adjacent range in Transbaikalia, almost all popula-
tions are in equilibrium for these loci, although there
is an insignificant excess of heterozygotes (Table 5).
The exception is one population of D. salina near the
Veriya River, which lacks heterozygotes for locus
SKDH and shows a significant divergence from
Hardy–Weinberg equilibrium. However, this is proba-
bly an error of the calculations, since the studied sam-
ple was very small; in this case, the calculated value of
expected heterozygosity may be overstated [25].
Subdivision of D. incarnata and D. salina popula-
tions was estimated using Wright’s F-coefficients [25].
In the case of D. salina, the individual inbreeding
coefficient (I) with respect to the subpopulation (S)
FIS varied from –0.165 for locus IDH to 0.185 for locus
SKDH; its mean value was 0.011 (the deficiency of het-
erozygotes was only 1%). The individual inbreeding
coefficient (I) with respect to the species as a whole
(T) FIT varied from –0.013 for locus PGM to 0.298 for
locus SKDH; its mean value was 0.083 (the deficiency
of heterozygotes was 8%). The subpopulation inbreed-
ing coefficient (S) with respect to the species as a
whole (T) FST had a minimum value of 0.018 for locus
NADHD and a maximum value of 0.138 for locus
SKDH (average of 0.075). It was also high for some
other loci: for PGI, it was equal to 0.107, and for IDH,
it was equal to 0.133.
In the case of D. incarnata, the individual inbreed-
ing coefficient (I) with respect to the subpopulation
(S) FIS ranged from 0.021 for locus SKDH to 0.519 for
locus PGI; its mean value was 0.342 (the deficiency of
heterozygotes was 34.2%). The individual inbreeding
coefficient (I) with respect to the species as a whole
(T) FIT ranged from –0.014 for locus SKDH to 0.580
for locus PGI; its mean value was 0.386 (the deficiency
of heterozygotes was 38.6%). The subpopulation
inbreeding coefficient (S) with respect to the species as
a whole (T) FST had a minimum value of 0.007 for
locus SKDH and a maximum value of 0.182 for locus
NADHD (average of 0.071). FST for another locus PGI
was also high (0.127).
Thus, the degree of genetic differentiation among
the five populations of D. salina was on average of
7.5%, and for D. incarnata, it was 7.1%, which in
accordance with Wright’s estimation [25] pertains to
mean values.
The mean value of FST for all the studied popula-
tions of the two closely related species of the genus
Dactylorhiza was 0.478, indicating a very high degree
of genetic differentiation between populations of
D. salina and D. incarnata growing in Transbaikalia.
The species show the highest differences for the allelic
structure of loci SKDH, PGM, and IDH (FST was
0.705, 0.976, and 0.762, respectively). For loci PGI
and NADHD, FST also had high values (0.146 and
0.248, respectively).
Analysis of variation of allele frequencies
(AMOVA) for the five populations of D. salina showed
that the most significant differences occur at the indi-
vidual level (73%, d.f. = 136) compared to the inter-
population one (11%, d.f. = 4). Analysis of variance of
genotype frequencies indicates that all differences are
at the intrapopulation level (83%, d.f. = 131).
Analysis of variation of allele frequencies
(AMOVA) for the five populations of D. incarnata
showed that differences are largely at the individual
(46%, d.f. = 118) and intrapopulation (41%, d.f. = 113)
levels. Analysis of variance of genotype frequencies
indicates that the greatest diversity occurs within pop-
ulations (83%, d.f. = 113).
Analysis of variance (AMOVA) of the data showed
that the populations of D. salina and D. incarnata in
the area where their ranges overlap in Zabaykalsky krai
and Buryatia have substantially significant differences
in the variation of allele frequencies of the eight loci
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
GENETIC STRUCTURE OF POPULATIONS 333
Table 5 . Parameters of genetic variation and compliance with Hardy–Weinberg equilibrium for polymorphic loci in local populations of D. salina (1–5) and D. incar-
nata (6–10)
See Table 1. Na, number of differentiating alleles; Ne, number of effective alleles; d.f., number of degrees of freedom; ChiSq, value of chi-square; Prob, critical probability of the
observed value; Signif, significance: ns, nonsignificant. * P < 0.05, ** P < 0.01, *** P < 0.001.
Populati
on no.
Population,
sample size Locus NaNeIHoHeFChi-square test to check Hardy–Weinberg equilibrium
d.f. ChiSq Prob Signif
1Veriya
N = 14
PGI 3 1.244 0.409 0.214 0.196 –0.091 3 0.202 0.977 ns
SKDH 21.153 0.257 0.000 0.133 1.000 114.000 0.000 ***
IDH 2 1.690 0.598 0.571 0.408 –0.400 1 2.240 0.134 ns
2
Gazimur.
Zavod
N = 26
PGI 31.587 0.644 0.308 0.370 0.168 38.905 0.031 *
SKDH 2 1.424 0.474 0.273 0.298 0.083 1 0.153 0.696 ns
PGM 2 1.083 0.168 0.080 0.077 –0.042 1 0.043 0.835 ns
IDH 2 1.122 0.221 0.115 0.109 –0.061 1 0.097 0.755 ns
DIA-1 2 1.080 0.163 0.077 0.074 –0.040 1 0.042 0.838 ns
3Ivolginsk
N = 44
PGI 3 1.175 0.325 0.114 0.149 0.237 3 5.904 0.116 ns
SKDH 3 2.013 0.740 0.442 0.503 0.122 3 1.987 0.575 ns
PGM 2 1.046 0.108 0.045 0.044 –0.023 1 0.024 0.877 ns
IDH 3 1.968 0.776 0.500 0.492 –0.016 3 0.068 0.995 ns
NADHD 2 1.046 0.108 0.045 0.044 –0.023 1 0.024 0.877 ns
4Yagye
N = 16
PGI 2 1.882 0.662 0.500 0.469 –0.067 1 0.071 0.790 ns
SKDH 2 1.074 0.154 0.071 0.069 –0.037 1 0.019 0.890 ns
5
Khonk-
holoyka
N = 36
PGI 32.495 1.002 0.389 0.599 0.351 315.332 0.002 **
SKDH 3 2.180 0.912 0.472 0.541 0.128 3 1.414 0.702 ns
IDH 3 1.289 0.443 0.250 0.224 –0.115 3 0.735 0.865 ns
6Kosurta
N = 26
PGI 21.501 0.516 0.115 0.334 0.654 111.12 4 0.001 ***
SKDH 2 1.044 0.105 0.043 0.043 –0.022 1 0.011 0.915 ns
NADHD 2 1.997 0.692 0.423 0.499 0.153 1 0.605 0.437 ns
7P. Zavod -1
N = 25
SKDH 2 1.043 0.101 0.042 0.041 –0.021 1 0.011 0.917 ns
IDH 2 1.041 0.098 0.040 0.039 –0.020 1 0.010 0.919 ns
NADHD 21.083 0.168 0.000 0.077 1.000 125.000 0.000 ***
8P. Zavod -2
N = 27
PGI 31.793 0.693 0.185 0.442 0.581 311.302 0.010 *
SKDH 2 1.041 0.098 0.0 40 0.039 –0.020 1 0.010 0.919 ns
IDH 2 1.038 0.092 0.037 0.036 –0.019 1 0.010 0.922 ns
DIA-1 21.077 0.158 0.000 0.071 1.000 127.000 0.000 ***
NADHD 2 1.246 0.349 0.148 0.198 0.250 1 1.688 0.194 ns
9P. Zavod -3
N = 16
PGI 21.205 0.311 0.063 0.170 0.632 16.395 0.011 *
NADHD 2 1.358 0.433 0.188 0.264 0.289 1 1.335 0.248 ns
10 P. Zavod -5
N = 24
PGI 3 2.010 0.762 0.333 0.503 0.337 3 3.954 0.267 ns
SKDH 2 1.043 0.101 0.042 0.041 –0.021 1 0.011 0.917 ns
IDH 2 1.043 0.101 0.042 0.041 –0.021 1 0.011 0.917 ns
NADHD 2 1.133 0.234 0.125 0.117 –0.067 1 0.107 0.744 ns
334
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
(71%, d.f. = 9) and in the variability of genotypes
(61%, d.f. = 9).
The data of allozyme analysis showed that there is
a clear exchange of genetic material among individuals
of D. salina and D. incarnata as a result of interspecific
hybridization; however, populations of these species
have well-defined genetic differentiation.
Data on allele frequencies of D. salina and D. incar-
nata were used to construct phylogenetic trees. As is
known, the correctness of topology and lengths of
branches of a phylogenetic tree depend on the algo-
rithm of its construction and the genetic distance
measuring method [20]. Genetic distances represent
the degree of genetic differentiation (genomic differ-
ences) between populations [26]. There are different
methods for evaluating genetic distances. Most often,
genetic differentiation between populations is com-
pared using standard genetic distances according to
Nei, D [18]. However, when the sample size is very
small, standard genetic distances in the case of two
genetically identical populations may be greater than
zero. Nei [19] called them spurious distances. Because
of this fact, in cases where there is a sampling error, he
introduced an unbiased estimate of genetic distances,
where all negative values in the construction of phylo-
genetic trees are changed to zero [19].
Using the computer modeling of Nei et al. [20], it
was shown that, for the restoration of the true evolu-
tionary tree topology, the most effective formula for
calculating the genetic distances DA is the one in which
the estimate of differences between populations does
not take into account low-frequency alleles. As is
known, the number of low-frequency alleles signifi-
cantly increases with increasing sample size and
thereby genetic distances decrease with increasing
samples even when comparing the same populations.
The program PowerMarker 3.25 [21] makes it pos-
sible to construct UPGMA (unweighted pair-group
method with arithmetic average) and NJ (neighbor
joining) dendrograms on the basis of 16 different ways
to measure genetic distances. To construct dendro-
grams, we used three matrices of genetic distances:
standard genetic distances D according to Nei [18],
genetic distances (unbiased) DM [19], and genetic dis-
tances DA [20]. Dendrograms based on the UPGMA
algorithm were the most plausible as compared to
those constructed on the basis of the NJ method. As it
turned out, the D. salina and D. incarnata populations
in all UPGMA dendrograms constructed on the basis
of different estimates of genetic distances were divided
into two different groups (Fig. 3).
Dendrograms constructed on the basis of genetic
distances D and DM were similar in topology but dif-
fered in branch lengths. These dendrograms were dif-
ferent from those constructed on the basis of genetic
distances DA in different grouping of D. salina popula-
tions, while clustering in the group of D. incarnata
populations was similar.
UPGMA dendrograms with bootstrap value of
nodes on the basis of genetic distances DM and DA of
populations of different species were divided into two
groups with different probabilities: 74% for the den-
drogram constructed on the basis of genetic distances
DM and 100% for the dendrogram based on DA.
In the dendrogram constructed on the basis of
genetic distances DM, five populations of D. salina
were divided with a probability of 97% into two groups
of two or three populations. Clustering within sub-
groups had low support (less than 45%). In the group
of D. incarnata populations, a population from the
vicinity of the Kosurta River had a probability of 97%;
the remaining four populations were divided into two
and then further between each other with a probability
of 61–67%.
With a high probability (98%), the dendrogram
constructed on the basis of genetic distances DA
revealed a population of D. salina from the surround-
ing area of the Yagye River and a population from the
group of D. incarnata from the surrounding area of the
Kosurta River. Division of the remaining four popula-
tions of D. salina into clusters did not have high sup-
port (less than 53%). Four populations of D. incarnata
were divided into two subgroups with a probability of
53%; in one of them, the populations were divided
with a probability of 61%.
The bootstrap consensus tree is some average of
bootstrap replications. For nodes with a low bootstrap
value, bootstrap consensus trees can differ from the
original trees [26, p. 126]. This was evident in cluster
analysis of the studied populations of D. salina. When
using different methods of estimating the genetic dis-
tances DM and DA, the topologies of the original trees
and bootstrap consensus trees did not coincide in the
group of D. salina populations, where nodes were
characterized by very low bootstrap values.
According to Nei and Kumar [26], it is necessary to
use genetic distances DA for accurate topology of the
phylogenetic tree and standard genetic distances D to
estimate branch lengths. However, in the case of
closely related populations, DA increases almost lin-
early; therefore, for estimation of branch lengths and
correct topology for loci of proteins, one should use
only DA [26]. We agree with the above point of view,
because the trees constructed on the basis of genetic
distances DA seem more plausible than dendrograms
based on DM.
The study made it possible to establish the specific-
ity of the allelic composition of loci SKDH, PGM, and
IDH for D. salina and D. incarnata, for which these
species significantly differ from each other. The data of
allozyme analysis support the view of L.V. Aver’yanov
[2] expressed on the basis of the morphological study
that the contact zone between the ranges of D. incar-
nata and D. salina has a vast area of introgression and
that owing to backcrossing the hybrid individuals bind
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
GENETIC STRUCTURE OF POPULATIONS 335
the original parent species by a continuous series of
transitional forms. High polymorphism of D. salina
and D. incarnata in Zabaykalsky krai and Buryatia can
be explained by the presence of introgressive hybrid-
ization.
These findings are consistent with the view that, in
the interspecific introgressive hybrid complexes, the
evolution proceeds in the direction of one of the par-
ents [14, 27–31]. The hybrid individuals in the sam-
ples of the studied hybrid complexes of D. salina and
D. incarnata had heterozygous genotypes only for one
or two of the three loci with differentiating alleles for
different taxa. Thus, with the change of hybrid gener-
ations, genotypes for loci with taxon-specific alleles
changed toward an excess of homozygotes. This could
have occurred as a result of backcrossing. Further-
more, in some cases, the fitness of heterozygotes may
be lower than that of homozygotes. For example, this
occurs during renewal of interspecific and intraspe-
cific hybrids [25]. It is possible that the studied hybrid
complexes may have selection against heterozygotes.
Despite the apparent gene flow resulting from intro-
gressive hybridization and the presence of hybrids in the
samples, almost all subpopulations of D. incarnata and
D. salina were in Hardy–Weinberg equilibrium for loci
SKDH, PGM, and IDH, whose polymorphism is deter-
mined by the presence of introgressive hybridization.
No Hardy–Weinberg equilibrium was observed for
some populations of D. salina for loci PGI and SKDH
and populations of D. incarnata for loci PGI, NADHD,
and DIA (Table 5); almost all of these loci are not
related to introgressive hybridization. Absence of
equilibrium was observed not for all but only for some
of them; it was shifted toward an excess of homozy-
gotes.
As known, the equilibrium may be disrupted owing
to the selective nature of crossings and other import-
ant evolutionary factors, such as selection and gene
flow [25, 32, 33]. Predominance of homozygotes in
subpopulations of D. incarnata in Europe is associated
with inbreeding [5, 7]. It is quite difficult to imagine
inbreeding in the case of the studied local populations
in Zabaykalsky krai and Buryatia. The populations
have greater abundance and individuals are character-
ized by high polymorphism of morphological charac-
ters. The populations are characterized by interspe-
Fig. 3. UPGMA dendrograms based on genetic distances DM (1) and DA (2) according to allele frequencies of eight gene loci for
populations of Dactylorhiza salina and D. incarnata growing in Zabaykalsky krai and Buryatia (genetic distances and files for con-
struction of original trees (a) and trees with bootstrap value for 1000 replications (b) were obtained in the program PowerMarker
v. 3.25, consensus trees were constructed in the program Phylip 3.69, and visualization of the trees was carried out in the program
Mega 6.06). Data for D. fuchsii from Irkutsk oblast are given as an outgroup.
D. incarnata,D. fuchsii,
Kosurta
D. incarnata,
Kosurta
D. incarnata,
Petrovsky Zavod-2
D. incarnata,
Petrovsky Zavod-2
D. incarnata,
Petrovsky Zavod-5
D. incarnata,
Petrovsky Zavod-5
D. incarnata,
Petrovsky Zavod-1
D. incarnata,
Petrovsky Zavod-1
D. incarnata,
Petrovsky Zavod-3
D. incarnata,
Petrovsky Zavod-3
D. salina,
Khonkholoyka
D. salina,
Khonkholoyka
D. salina,
Gazimur. Zavod
D. salina,
Gazimur. Zavod
D. salina,
Yagye
D. salina,
Yagye
D. salina,
Ivolginsk
D. salina,
Ivolginsk
D. salina,
Veriya
D. salina,
Veriya
D. incarnata,
Kosurta
D. incarnata,
Petrovsky Zavod-2
D. incarnata,
Petrovsky Zavod-5
D. incarnata,
Petrovsky Zavod-1
D. incarnata,
Petrovsky Zavod-3
D. salina,
Yagye
D. salina,
Khonkholoyka
D. salina,
Ivolginsk
D. salina,
Gazimur. Zavod
D. salina,
Veriya
0.05
0.05
(1a)
(2a)
(1b)
(2b)
100
98
98
53
48
61
23
53
37
67
65
45
28
61
40
97
97
74
Irkutsk oblast
D. fuchsii,
D. incarnata,
Kosurta
D. incarnata,
Petrovsky Zavod-2
D. incarnata,
Petrovsky Zavod-5
D. incarnata,
Petrovsky Zavod-1
D. incarnata,
Petrovsky Zavod-3
D. salina,
Khonkholoyka
D. salina,
Gazimur. Zavod
D. salina,
Yagye
D. salina,
Ivolginsk
D. salina,
Veriya
Irkutsk oblast
336
RUSSIAN JOURNAL OF GENETICS Vol. 53 No. 3 2017
FILIPPOV, ANDRONOVA
cific hybridization, which is only possible in the case
of cross-pollination.
The excess of homozygotes may be due to a phe-
nomenon such as the Wahlund effect [25, 32, 33]. This
effect manifests itself when studied samples of individ-
uals are taken from the structured population that
contains heterogeneous subsamples with different
allele frequencies. It is possible that an excess of
homozygotes indicates a high degree of structuring of
populations in Transbaikalia. However, the question
about the causes of the displacement of equilibrium in
the direction of excess of homozygotes in subpopula-
tions of D. salina and D. incarnata remains open and
requires further special studies.
ACKNOWLEDGMENTS
We express our gratitude to Candidates of Biology
Alla Vasil’evna Verkhozina and Ol’ga Dmitrievna
Chernova, as well as to the administration of the
Zabaykalsky Botanical Garden (Chita) for their assis-
tance in conducting field research in Zabaykalsky krai.
This work was carried out according to government
task nos. 01201255606 and 012001459509 with finan-
cial support of the Russian Foundation for Basic
Research (project no. 12-04-01560) and the Program
of the Presidium of the Russian Academy of Sciences
“Living Nature: Contemporary State and Problems of
Development” (Subprogram “Dynamics and Reten-
tion of Gene Pools”), the Complex Program of the
Ural Branch of the Russian Academy of Sciences
(project no. 15-12-4-35), and a grant of the President
of the Russian Federation for Support of Leading Sci-
entific Schools of Russia (NSh-5282.2014.4).
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We carried out an allozyme analysis to investigate polymorphism and genetic structure of the populations of D. incarnata and D. ochroleuca in regions of their joint growth in Russia and Belarus. We found that D. ochroleuca individuals in the populations of the Urals and Siberia, which are distant fragments from the main range of the species, do not differ significantly from individuals within the main part of the area (Belarus) on the basis of the allelic composition of eight gene loci. We revealed that D. ochroleuca and D. incarnata are differentiated by different alleles of the GDH locus. Thus, we established a genetic marker suitable to distinguish these closely related taxa. In addition to the GDH locus, D. ochroleuca and D. incarnata in the places of their joint growth, differ in the allelic structure of the PGI and NADHD loci. D. incarnata from the Urals and Siberia were polymorphic for both loci, and individuals from Belarus were polymorphic for one locus (PGI). In contrast, all D. ochroleuca individuals growing in sympatric populations with polymorphic D. incarnata were homozygous for the same alleles. Thus, comparison of the genetic structure of D. ochroleuca and D. incarnata points to the existence of a genetic isolation and a functioning isolation mechanism even under conditions of their joint growth. We found that the GDH locus in D. incarnata is polymorphic only in populations which grow together with D. ochroleuca, with exception a few examples. Thus, we conclude that variability of the GDH locus in D. incarnata is associated with hybridization with D. ochroleuca.
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Аверьянов Л.В. 1988. Конспект рода Dactylorhiza Neck. ex Nevski (Orchidaceae), 1. Новости Сист. Высш. раст. т. 25: 48-67. Averyanov L.V. 1988. Conspectus generis Dactylorhiza Neck. Ex Nevski (Orchidaceae), 1. Nov. Syst. Pl. Vasc. (Leningrad) 25: 48-67 (in Russian).
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