Multiple interspecific hybridization and microsatellite mutations provide clonal diversity in the parthenogenetic rock lizard Darevskia armeniaca

Abstract

Background
The parthenogenetic Caucasian rock lizard Darevskia armeniaca, like most other parthenogenetic vertebrate species, originated through interspecific hybridization between the closely related sexual Darevskia mixta and Darevskia valentini. Darevskia armeniaca was shown to consist of one widespread allozyme clone and a few rare ones, but notwithstanding the origin of clonal diversity remains unclear. We conduct genomic analysis of D. armeniaca and its parental sexual species using microsatellite and SNP markers to identify the origin of parthenogenetic clonal lineages.

Results
Four microsatellite-containing loci were genotyped for 111 specimens of D. armeniaca, 17 D. valentini, and four D. mixta. For these species, a total of 47 alleles were isolated and sequenced. Analysis of the data revealed 13 genotypes or presumptive clones in parthenogenetic D. armeniaca, including one widespread clone, two apparently geographically restricted clones, and ten rare clones. Comparisons of genotype-specific markers in D. armeniaca with those of its parental species revealed three founder-events including a common and two rare clones. All other clones appeared to have originated via post-formation microsatellite mutations in the course of evolutionary history of D. armeniaca.

Conclusion
Our new approach to microsatellite genotyping reveals allele-specific microsatellite and SNP markers for each locus studied. Interspecies comparison of these markers identifies alleles inherited by parthenospecies from parental species, and provides new information on origin and evolution of clonal diversity in D. armeniaca. SNP analyses reveal at least three interspecific origins of D. armeniaca, and microsatellite mutations in these initial clones give rise to new clones. Thus, we first establish multiple origins of D. armeniaca. Our study identifies the most effective molecular markers for elucidating the origins of clonal diversity in other unisexual species that arose via interspecific hybridization.

Electronic supplementary material
The online version of this article (10.1186/s12864-018-5359-5) contains supplementary material, which is available to authorized users.

Full text

R E S E A R C H A R T I C L E Open Access
Multiple interspecific hybridization and
microsatellite mutations provide clonal
diversity in the parthenogenetic rock lizard
Darevskia armeniaca
Anastasiya E. Girnyk
1
, Andrey A. Vergun
1,2
, Seraphima K. Semyenova
1
, Andrei S. Guliaev
1
, Marine S. Arakelyan
3
,
Felix D. Danielyan
3
, Irena A. Martirosyan
1
, Robert W. Murphy
4
and Alexey P. Ryskov
1*
Abstract
Background: The parthenogenetic Caucasian rock lizard Darevskia armeniaca, like most other parthenogenetic
vertebrate species, originated through interspecific hybridization between the closely related sexual Darevskia mixta
and Darevskia valentini.Darevskia armeniaca was shown to consist of one widespread allozyme clone and a few
rare ones, but notwithstanding the origin of clonal diversity remains unclear. We conduct genomic analysis of D.
armeniaca and its parental sexual species using microsatellite and SNP markers to identify the origin of
parthenogenetic clonal lineages.
Results: Four microsatellite-containing loci were genotyped for 111 specimens of D. armeniaca,17D. valentini, and
four D. mixta. For these species, a total of 47 alleles were isolated and sequenced. Analysis of the data revealed 13
genotypes or presumptive clones in parthenogenetic D. armeniaca, including one widespread clone, two
apparently geographically restricted clones, and ten rare clones. Comparisons of genotype-specific markers in D.
armeniaca with those of its parental species revealed three founder-events including a common and two rare
clones. All other clones appeared to have originated via post-formation microsatellite mutations in the course of
evolutionary history of D. armeniaca.
Conclusion: Our new approach to microsatellite genotyping reveals allele-specific microsatellite and SNP markers
for each locus studied. Interspecies comparison of these markers identifies alleles inherited by parthenospecies from
parental species, and provides new information on origin and evolution of clonal diversity in D. armeniaca. SNP
analyses reveal at least three interspecific origins of D. armeniaca, and microsatellite mutations in these initial clones
give rise to new clones. Thus, we first establish multiple origins of D. armeniaca. Our study identifies the most
effective molecular markers for elucidating the origins of clonal diversity in other unisexual species that arose via
interspecific hybridization.
Keywords: Darevskia, Hybridization, Parthenogenesis, Microsatellites, SNP markers, Clones, Mutations, Clonal
diversity
* Correspondence: ryskov@mail.ru
1
Laboratory of Genome Organization, Institute of Gene Biology of the
Russian Academy of Sciences, Vavilova Str., 34/5, Moscow 119334, Russia
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Girnyk et al. BMC Genomics (2018) 19:979
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Background
Naturally occurring unisexual reproduction occurs in
less than 0.1% of all vertebrate species [1,2]andtrue
parthenogenesis has been described only in squamates
[1,3–5]. Recently, the origin and evolution of par-
thenogenesis, in particular in lizards, has received
considerable attention [6–13]. Unisexuality usually
arises through interspecific hybridization between
genetically related sexual species [14,15]andintro-
gressive hybridization is common among species of
lizards Jancuchova-Laskova et al. [7]. Regardless,
hybridization between the presumed ancestral species
of parthenogenetic lineages of Caucasian rock lizards
(Darevskia) does not necessary result in parthenogen-
esis [16]. Among Squamata, many parthenogenetic
species are triploid (e.g., some Aspidoscelis), however,
all unisexual species of Darevskia (Lacertidae) are
diploid. Due to their hybrid origin, these species have
the genetic diversity of their parental sexual progeni-
tors and commonly exhibit fixed heterozygosity at co-
dominant loci [17]. Sister-chromatid pairing maintains
heterozygosity, which is critical for offsetting the re-
duced fitness in parthenogenetic species [18].
Genetic studies of unisexual vertebrate species rely on
assessments of intraspecific variability and clonal diver-
sity. Their genetic diversity may owe to having the ori-
ginal clones but from different founders, post-formation
mutations (especially in hypervariable microsatellite
loci), and genetic recombination in rare cases of subse-
quent crossings [19,20]. Range-size, the age of species
and some environment factors may also affect clonal di-
versity [21–23].
Darevsky (1958) first discovered parthenogenesis in
the family Lacertidae [24]. Later, other parthenogenetic
species were described [25,26]. Darevskia has at present
24 sexual and seven parthenogenetic species. [11,14,
25]. The phylogeny of Darevskia reconstructed using
mitochondrial DNA sequences and allozyme data [27]
includes the D. caucasica,D. saxicola and D. rudis
major groups of sexual species. The formation of parthe-
nospecies is constrained phylogenetically [7,27]. They
arose from hybridization of females in the D. caucasica
group and males in the D. rudis group. Analysis of DNA
fingerprint markers has identified genetic polymorphism
in all parthenogenetic species of Darevskia [28–32].
Darevskia armeniaca has a wide distribution involving
central Armenia, southern Georgia, northeastern Anatolia
and northwestern Azerbaijan [33–35]. They live at eleva-
tions between 800 and 2700 m [33,36]. This species arose
from the hybridization of Darevskia mixta (D. caucasica
group) and Darevskia valentini (D. rudis group) [14,15,
23,27,37,38]. Darevskia mixta, which occurs in the
eastern part of the Meskheti Range, Lesser Caucasus
Mountains and on southern slops of the Greater Caucasus
between the valleys of the Rioni and Khobi rivers, is en-
demic to Georgia. The central part of the Lesser Caucasus
was the most likely area of origin for D. mixta [39].
In comparison, D. valentini occurs in eastern
Anatolia, Armenia, and adjoining Georgia [15,33]at
elevations between 1900 and 3110 m. In Armenia, this
species is locally abundant in montane habitats [33].
Triploid hybrids between D. armeniaca and sexual D.
valentini occur in some Armenian populations [34,
40,41].ThezoneofsympatryinKuchakhasanex-
tremely high number of hybrids [41]. The majority of
them are sterile triploid females, but males with fully
developed reproductive systems and presumably fertile
females also exist [41]. Putative fertile triploids prob-
ably exist in D. unisexualis,andinsomeotherpar-
thenogenetic species of reptiles [41–44]. In most
cases, spatial gaps separate the ranges of sexual and
unisexual populations of Darevskia [35]. Unisexual
forms have a superior competitive ability to their par-
ental forms in a given spatio-temporal setting [36],
expansion of the parthenogens could cause retreat of
parental species from the contact zones, preventing
further hybridization events [35].
Recently, Tarkhnishvili et al. (2017) postulated alternative
hypothesis of origin of D. armeniaca.Apparently,itarose
from backcrosses of male D. valentini with parthenogenetic
D. dahli [13]. Mitochondrial DNA analysis confirmed data
of Fu et al. (1999) that D. armeniaca and D. dahli descend
maternally from D. mixta from a limited geographic area in
central Georgia [45]. The majority of both parthenogens
shared the same genotypes at two microsatellite loci, but
they differed at the other three loci used. Therefore, they
suggested that the origin of D. armeniaca first included
hybridization between D. mixta and D. portschinskii
followed by backcrosses of these parthenogens with male
D. valentini [13]. However, new studies are needed to deter-
mine which of two or both scenarios of D. armeniaca ori-
gin have matter.
To test the alternative hypotheses, we examine the clonal
diversity and its origin in D. armeniaca using 111 samples
and microsatellite genotyping and single nucleotide poly-
morphisms (SNPs) located outside of each microsatellite
cluster [46,47]. The SNPs data yield direct information
about interspecific hybridization founder events, and
microsatellite variability provides information about pos-
sible mutations in the initial clones. Further, analyses use
partial sequences from the mitochondrial gene encoding
cytochrome b(MT-CYTB) from 14 Armenian populations
of D. armeniaca plus the parental species.
Methods
DNA samples of D. armeniaca (n= 111) from 14 popu-
lations, D. valentini (n= 17) from four populations in
Armenia, and D. mixta (n= 4) from one population in
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Georgia were analyzed (Table 1). Sample localities were
shown in Fig. 1.
All DNAs were isolated from blood samples of lizards.
Lizards were captured by Danielyan F.D. and Arakelyan
M.S. 15–20 years ago with a noose. These species are
not protected by CITES. The study was approved by the
Ethics Committee of Moscow State University (Permit
Number: 24–01) and was carried out in strict accord-
ance with their ethical principles and scientific stan-
dards. Blood samples were taken from the tail veins of
lizards under chloroform anesthesia, and then these liz-
ards were released. DNA was isolated from lizard blood
by using the standard phenol–chloroform extraction
method with proteinase K, and resuspended in TE buf-
fer, pH 8.0.
Four loci, Du215, Du281, Du323, and Du47G were
PCR-amplified using previously described primer pairs [46,
48,49]. Data on genetic variation of these loci in D. arme-
niaca and on allelic variation of the homologous loci in D.
valentini and D. mixta [49] were shown in Additional file 1:
Table S1. PCR was performed on 50 ng of DNA in a total
volume of 20 μl using a GenePak PCR Core Kit (Isogene)
and 1 μM of each primer. The reaction conditions were as
follows: one cycle of 3 min at 94 °C; 30 cycles of 1 min at 94 °
C; 40s at the annealing temperature (58 °C for Du215, 50 °C
for Du281, 52 °C for Du323, and 54 °C for Du47G); and 40s
at 72 °C followed by one cycle of 5 min at 72 °C. PCR prod-
ucts (7 μl) were loaded onto an 8% non-denatured polyacryl-
amide gel (to separate allelic variants for each locus) and run
for12hat60V.A100bpladder(Fermentas)wasusedasa
size marker. The amplified products were visualized by stain-
ing DNA in the gel with ethidium bromide. Well-resolved
individual PCR products, which corresponded to the two in-
dividual alleles of the locus, were excised from the gel, puri-
fied by ethanol precipitation, and sequenced directly in both
directions using a chain termination reaction with an ABI
PRISM BigDye Terminator v.3.1 on an Applied Biosystems
3730 DNA analyzer. Allelic identity was checked and con-
firmed via the comparison of sequences obtained independ-
ently. All unique de novo sequences were deposited in
GenBank (GU972533-GU972535; HM070259-HM070264;
HM013992-HM013994; KT070998-KT071004; GU972551;
GU972553; HM013997; KM573717-KM573727; MH18798
8-MH187999). Sometimes for genotypes 9–13, which were
represented by only one individual, the PCR products of the
Du47G locus were excised from the polyacrylamide gel, puri-
fied and cloned into pMos blue vectors according to stand-
ard procedures (pMos blueBlunt ended Cloning kit RPN
5110, Amersham Biosciences). The clones were amplified in
MOSBlue competent cells grown at 37 °C, and sequenced as
described previously [50].
Diversity parameters for each locus were estimated only
for D. armeniaca, because sample sizes of the parental
sexual species were insufficient. The number of alleles, al-
lelic richness (Rs), as a measure of allele counts adjusted
for sample size, and expected heterozygosity, as a measure
of gene diversity, were calculated per locus and per popu-
lation by using R package Poppr v.2.5.0 [51], GenePop
v.4.2, and Web-version of POPTREEW [52].
A statistical parsimony haplotype network was used to
visualize variation. It was calculated using TCS software
v.1.21, with gaps being considered as a second state [53].
This approach has been used at the population-level for
comparing mitochondrial SNPs, which have linear ar-
rangements [54]. Clonality in parthenogenetic lizards re-
sulted in homologous alleles having linear arrangements
with little or no recombination. This served to link the
parthenogenetic genotypes with mutations (repeat num-
ber changes). Tarkhnishvili et al. (2017) used a similar
approach for linking microsatellite genotypes of D. dahli
and D. armeniaca from Georgia, Armenia and Turkey,
but with Network v.5.0 [13,55]. The method of coding
genotypes was described previously [46].
A 320 bp fragment of MT-CYTB was amplified and se-
quenced for 30 specimens of D. armeniaca (2–3 individ-
uals from each population) including those with a distinct
microsatellite genotype, and four specimens of D. mixta
from one population.The primers used were L14841:
5′-CCATCCAACATCTCAGCATGATGAAA-3′and H1
5149: 5′-GCCCCTCAGAATGATATTTGTCCTCA-3′
Table 1 Species and population samples used in this study
Species Populations Number of
individuals in
populations
Total number
of species
individuals
D.
armeniaca
Dsegh 3 111
Harich 18
Kuchak 7
Sevan 1
Lchashen 1
Meghradzor 9
Medved-gora (vicinity of
Stepanavan)
12
Dilijan (Papanino) 4
Pushkin Pass 7
Dilijan-Semyonovka Pass 8
Sotk 3
Stepanavan 9
Artavaz (Hankavan) 21
Tezh (Pambak Ridge) 8
D.
valentini
Hatis (Geghama Mountains) 4 17
Kuchak 2
Lchashen 5
Tezh (Pambak Ridge) 6
D. mixta Akhaldaba (Georgia) 4 4
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[56], as described for these taxa in [27]. PCR analysis was
performed as described in [13]. The amplicons were se-
quenced on an automated sequencer (Applied Biosystems
3730 DNA analyzer). Sequence alignment was performed
with BioEdit v.7.0 [57].
Results
All 111 specimens of D. armeniaca, including 10 indi-
viduals with coloration difference [37] from Artavaz
(Hankavan), 17 specimens of D. valentini, and four D.
mixta were analyzed successfully using locus-specific
PCR and DNA sequencing of PCR amplificants. The
320 bp fragment of MT-CYTB did not vary among D.
armeniaca and D. mixta; all sequences assigned to
haplotype A of D. mixta [45].
All but four individuals of D. armeniaca (from Dsegh,
Dilijan-Semyonovka Pass and Stepanavan) were hetero-
zygous at microsatellite loci used; the alleles differed
from each other in length and structure, and in single
nucleotide variations (SNVs) in fixed positions of the
flanking allelic regions (Additional file 1: Table S1),
which was similar to previous reports for D. dahli,D.
unisexualis, and D. rostombekowi [32,46–48]. Locus
Du323, also had a unique (AC)
n
microsatellite cluster
that differentiated parental alleles.
In D. armeniaca, Du215 and Du323 had three alleles,
Du281 had four, and Du47G had seven (Additional file 1:
Table S1). Allelic variants formed distinct groups accord-
ing to the fixed SNVs, which in hybrid genomes resulted
from the different parental genomes. They correspond
ed to parent-specific markers such that unique clonal
SNVs likely reflected independent founder events. SNVs
in alleles 1 and 2 of Du215 formed the set TGC and al-
lele 3 of Du215 formed the set ACT. Similarly, SNVs in
alleles of Du281 had T and C variants, and SNVs in al-
leles of Du323 had CT and AC variants. The seven al-
leles of Du47G formed three sets of SNVs: TAGT,
TTCA, and AAGA. Each sets associated with specific-
ally organized microsatellite clusters. Thus, together
the sets of SNVs and microsatellite clusters differenti-
ated distinct genotypes inherited by parthenospecies
from parental species.
Analysis of the parental species showed homozygosity
for Du215 and Du323 in D. mixta (n= 4), and Du281 and
Du47G had two alleles each. In D. valentini (n=17),
Du215 was homozygous, Du281 had five, Du323 had six,
and Du47G had 10 alleles (Additional file 1: Table S1). All
parental alleles contained microsatellite clusters and vari-
able nucleotides (SNVs) at fixed positions in neighboring
regions which differentiated D. mixta and D. valentini.
Microsatellite motifs and/or the sets of SNVs discerned
parental alleles.Most alleles of Du215, Du281, Du323, and
Du47G in D. armeniaca occurred in the parents. Alleles
Du215(arm)1 and 2 coincided with allele Du215(mix)1, and
Fig. 1 Collection localities of parthenogenetic lizards Darevskia armeniaca and their parental species D. valentini and D. mixta. Sampling localities
are indicated by the following colors: D. armeniaca –yellow; D. valentini –blue; D. mixta –red. Numbers indicate populations: 1 –Dsegh (41°04′
50.8″N 44°39′27.1″E); 2 - Harich (40°38′25.9″N 43°54′14.4″E); 3 - Kuchak (40°31′49.81″N 44°17′3.43″E); 4 - Sevan (40°28′02.4″N 45°03′43.5″E); 5 -
Lchashen (40°30′45.92″N 44°54′3.22″E); 6 - Meghradzor (40°36′45.1″N 44°36′23.5″E); 7 - Medved-gora (vicinity of Stepanavan) (40°58′45.8″N 44°24′
32.7″E); 8 - Dilijan (Papanino) (40°42′27.76″N 44°45′43.89″E); 9 - Pushkin Pass (40°54′42.1″N 44°25′55.6″E); 10 - Dilijan-Semyonovka Pass (40°39′52.6″N
44°53′24.4″E); 11 - Sotk (40°12′43.8″N 45°52′42.6″E); 12 - Stepanavan (41°03′21.8″N 44°21′33.5″E); 13 - Artavaz (Hankavan) (40°37′20.2″N 44°34′51.4″E);
14 - Tezh (Pambak Ridge) (40°42′8.08″N 44°36′30.80″E); 15 - Hatis (Geghama Mountains) (40°18′14.91″N 44°43′40.71″E); 16 - Akhaldaba (Georgia)
(41°41′3.840″N 44°39′29.880″E). The map was created in the licensed version ArcGIS Desktop 10.4.1 by the authors (http://desktop.arcgis.com)
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allele Du215(arm)3 coincided with Du215(val)1. Further, al-
leles Du281(arm)1 and 2 coincided with Du281(mix)1 and
2, and alleles Du281(arm)3 and 4 coincided with
Du281(val)1–5. Alleles Du323(arm)1 and 2 with the spe-
cific (AC)
6
microsatellite cluster occurred among Du323 al-
leles from D. valentini, and allele Du323(arm)3 with
specific (AC)
5
microsatellite cluster coincided with
Du323(mix)1. Finally alleles, Du47G(arm)3–6 corre-
sponded to alleles of Du47G in D. valentini,and
Du47G(arm)7 coincided with Du47G(mix)2. Due to the
high mutation rate of microsatellite DNAs [50], in some
cases we did not find direct correlation between distinct al-
leles of D. armeniaca and its parents. Parental alleles with
the SNV TAGT, which was specific for Du47G(arm)1 and
2, was not found probably due to either genome divergence
through genetic recombination or mutation in D. arme-
niaca, or insufficient parental sampling. Allelic combina-
tions of four loci identified genotypic diversity in D.
armeniaca (Fig. 2).
Analyses resolved 13 genotypes that differed in their
frequencies and distribution (Table 2). Widespread geno-
type 1 occurred in 61 individuals (54.9% of the total co-
hort). Genotype 4 (n= 10; 9%) and genotype 6 (n=5;
4.5%) were found in four and three populations, respect-
ively. All other genotypes (2, 3, 5, 7–13) occurred in one
or two populations (n= 35; 32.5%). Although relatively
uncommon, genotypes 2–5 made up each the majority
of individuals in some populations: genotype 2 at Harich
(12 of 18 specimens), genotype 3 at Kuchak (5 of 8),
genotype 4 at Pushkin Pass (5 of 7), and genotype 5 at
Lchashen (7 of 9) (Table 2). All 10 color-variant individ-
uals from Artavaz (Hankavan) had genotype 1.
Genotypic diversity of D. armeniaca varied from 0 to
66.7% (Table 2). The highest level of genotypic diversity
was observed at Dsegh and Sotk, which had two geno-
types in three individuals. Five genotypes, one common
and four rare, occurred in the nine individuals from Ste-
panavan. The lowest levels of genotypic diversity were
observed at Sevan, Lchashen, Artavaz (Hankavan), and
Tezh (Pambak Ridge), which had genotype 1 only.
Population genetic indices for four loci and genotypes
1–9 were given in Table 3. Five individuals with unique
genotypes 10–13, and populations at Sevan and Lchashen,
which were represented by one individual each, were ex-
cluded from the analysis. The estimates of expected het-
erozygosity varied from 0.51 to 0.67 (average 0.55–0.63
depending on locus) whereas observed heterozygosity did
not vary among loci and populations (Table 3). The num-
ber of alleles varied from 2 to 5 (average, 2.00–2.83 de-
pending on locus). Values of allelic richness ranged from
1.89 to 3.20 (average, 1.94–2.24 depending on locus). The
highest values of heterozygosity occurred at Dsegh (loci
Du215, Du281, Du323) and Sotk (locus Du47G). The
highest values of allelic richness occurred at Dsegh (loci
Du215 and Du281), Sotk (loci Du215, Du281, and
Du47G) and Medved-gora (vicinity of Stepanavan) (locus
Du323), while the highest values of allelic number varied
from 2 to 5 depending on the locus in the populations
studied.
Population genetic indices for four loci and four popula-
tions (17 individuals) were given in the Additional file 2:
Table S2. The estimates of expected heterozygosity varied
from 0.50 to 0.89 (average 0.52–0.84 depending on locus),
whereas observed heterozygosity from 1 for locus Du215
(all populations) to 0.33–0.94 for other loci. The number
of alleles varied from 2 to 6 (average, 2–5dependingon
locus). Values of allelic richness ranged from 1.79 to 3.43
(average, 1.96–3.36 depending on locus). The highest
Fig. 2 Schematic representation of thirteen genotypes formed by allelic combinations of microsatellite loci Du215, Du281, Du323, and Du47G in
111 individuals of D. armeniaca. Parent-specific SNV markers are shown in yellow squares. Variable microsatellite clusters are shown in each of
two alleles
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value of allelic richness occurred in the population at
Lchashen (loci Du47G, Du323, Du215). The highest
values of expected heterozygosity occurred in Adis (locus
Du47G). These data demonstrated that higher genetic
variability in populations of D. valentini rather than popu-
lations of D. armeniaca. Linkage disequilibrium analysis
(Additional file 3: Table S3) suggested that all populations
except Lshachen had no association between loci. The
population at Lshachen appeared to have linked loci prob-
ably because of either a recent bottleneck or association of
microsatellite markers with unknown loci under selection.
Combinations of parent-specific SNVs and those of D.
armeniaca differentiated between single or multiple in-
terspecies hybridization event(s). The structural compos-
ition of the 13 genotypes (Additional file 1: Table S1)
were shown schematically in Fig. 2. Genotypes 1–9
matched all parent-specific SNV combinations TAGT/
TTCA (Du47G), TGC/ACT (Du215), T/C (Du281), and
CT/AC (Du323). This did not reject the hypothesis of a
common origin from a single hybridization event. How-
ever, the analysis did not rule out the possibility of inde-
pendent crossings of the parental individuals because
they differed from each other by microsatellite se-
quences only at loci Du47G, Du281, and Du323. Some
of the rarer genotypes may have arisen via
post-formation microsatellite mutation.
Genotypes 10–12, which occurred in four individuals
from three populations, matched parent-specific SNV
combinations at all loci, but they differed from geno-
types 1–9 by the parent-specific SNV combination
TTCA/TTCA for locus Du47G. These four specimens
were uniquely homozygous at Du47G and differed from
each other only by microsatellite sequences at Du215
and Du281. Therefore, this rejects the hypothesis of a
single origin of D. armeniaca, and their variation likely
arose through microsatellite mutations. Further, rare
Table 2 Sample size, genotype composition, diversity and distribution in the populations of D. armeniaca
Genotype
number
Genotype composition
(see, Fig. 2)
Population
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Number of
individuals with
definite genotype
(genotype
frequencies)
1 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(2 + 5)
6112103211521861(0,549)
2 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(2 + 4)
12 1 13 (0,117)
3 Du215(2 + 3) + Du281(2 + 3) +
Du323(2 + 3) + Du47G(2 + 5)
5 5 (0,045)
4 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(1 + 5)
2 1 2 5 10 (0,090)
5 Du215(2 + 3) + Du281(2 + 4) +
Du323(1 + 3) + Du47G(2 + 5)
7 7 (0,063)
6 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(1 + 4)
2 2 1 5 (0,045)
7 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(2 + 3)
2 1 3 (0,027)
8 Du215(2 + 3) + Du281(1 + 4) +
Du323(2 + 3) + Du47G(2 + 5)
1 1 (0,009)
9 Du215(2 + 3) + Du281(1 + 4) +
Du323(2 + 3) + Du47G(1 + 5)
1 1 (0,009)
10 Du215(2 + 3) + Du281(2 + 3) +
Du323(2 + 3) + Du47G(5 + 5)
2 2 (0,018)
11 Du215(1 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(5 + 5)
1 1 (0,009)
12 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(5 + 5)
1 1 (0,009)
13 Du215(2 + 3) + Du281(2 + 4) +
Du323(2 + 3) + Du47G(6 + 7)
1 1 (0,009)
Total number of individuals 3 18 7 1 1 9 12 4 7 839218111
Genotype diversity (%) 2
(66,7)
5
(27,8)
2
(28,6)
1
(0)
1
(0)
2
(22,2)
2
(16,7)
2
(50)
2
(28,6)
3
(37,5)
2
(66,7)
5
(55,5)
1
(0)
1
(0)
13
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Table 3 The population indices of gene diversity for four studied loci in twelve sampled populations of D. armeniaca
Locus Population Alelle (N) R
S
H
E
H
O
Du215 Dsegh 2 2.00 0.67 1
Harich 2 1.89 0.52 1
Kuchak 2 1.93 0.54 1
Meghradzor 2 1.90 0.53 1
Medved-gora (vicinity of Stepanavan) 2 1.92 0.52 1
Dilijan (Papanino) 2 1.97 0.57 1
Pushkin Pass 2 1.93 0.54 1
Dilijan-Semyonovka Pass 2 1.93 0.54 1
Sotk 2 2.00 0.60 1
Stepanavan 2 1.92 0.53 1
Artavaz (Hankavan) 2 1.89 0.51 1
Tezh (Pambak Ridge) 2 1.92 0.53 1
Total 2 2.00 0.50 1
Mean ± SE 2 ± 0.00 1.94 ± 0.01 0.55 ± 0.01 1 ± 0.00
Du281 Dsegh 2 2.00 0.67 1
Harich 2 1.89 0.52 1
Kuchak 3 2.18 0.60 1
Meghradzor 2 1.91 0.53 1
Medved-gora (vicinity of Stepanavan) 2 1.92 0.52 1
Dilijan (Papanino) 2 1.97 0.57 1
Pushkin Pass 2 1.93 0.54 1
Dilijan-Semyonovka Pass 3 2.18 0.60 1
Sotk 2 2.00 0.60 1
Stepanavan 3 2.14 0.59 1
Artavaz (Hankavan) 2 1.89 0.51 1
Tezh (Pambak Ridge) 2 1.92 0.53 1
Total 4 4.00 0.54 1
Mean ± SE 2.25 ± 0.13 2.00 ± 0.03 0.56 ± 0.01 1 ± 0.00
Du323 Dsegh 2 2.00 0.67 1
Harich 2 1.89 0.52 1
Kuchak 2 1.93 0.54 1
Meghradzor 3 1.91 0.62 1
Medved-gora (vicinity of Stepanavan) 2 2.26 0.52 1
Dilijan (Papanino) 2 1.97 0.57 1
Pushkin Pass 2 1.93 0.54 1
Dilijan-Semyonovka Pass 2 1.93 0.54 1
Sotk 2 2.00 0.60 1
Stepanavan 2 1.92 0.53 1
Artavaz (Hankavan) 2 1.89 0.51 1
Tezh (Pambak Ridge) 2 1.92 0.53 1
Total 3 3.00 0.53 1
Mean ± SE 2.08 ± 0.08 1.96 ± 0.03 0.56 ± 0.01 1 ± 0.00
Du47G Dsegh 2 2.00 0.67 1
Harich 5 2.45 0.67 1
Girnyk et al. BMC Genomics (2018) 19:979 Page 7 of 12
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genotype 13 (n= 1) differed from all other genotypes by
the SNV combination TTCA/AAGA at locus Du47G.
This also rejected the null hypothesis. Consequently,
analyses pointed to at least three independent
hybridization events in the genesis of the 13 genotypes
of D. armeniaca.
The TCS network displayed the geographical distribu-
tion of genotypes 1–9 (Additional file 4: Figure S1). The
network displayed a star-like appearance that was typical
of mutations deriving from a central, ancestral genotype.
Discussion
In their review of allozyme variation in parthenogenetic
lizards, Parker et al. (1989) proposed that species having
a single origin will usually have a widespread numerous
clone along with a few rare clones, where species with
multiple hybrid origins are highly variable with random
allele combinations [22]. In some cases, high diversity in
allozymes but low variation in mitochondrial DNA sug-
gests multiple origins from a geographically restricted
sample of females [22,58,59]. Add to this, low diversity
in allozymes and mitochondrial DNA suggests restricted
origins, both geographically and numerically [60,61]. In
a laboratory creation of hybridogenetic Poeciliopsis line-
ages, relatively low clonal diversity resulted from selec-
tion of the most-fit clones from a broad spectrum of
genotypes [2,16].
The clonal diversity of parthenogenetic species was de-
tailed using allozyme and mitochondrial DNA analysis
[16,23,27,38,45,62–65]. Allozyme analyses resolved
several clones in all species except for D. rostombekowi),
and all consist in one major widespread clone and a few
rare clones. Some peculiarities in allozyme clone pat-
terns were found for D. armeniaca [38,66].
Darevskia armeniaca has a karyotype of 2n = 38 [67]
characterized by fixed heterozygosity of allozyme loci
[38,66] and exhibits no [45] or low variability of mito-
chondrial DNA inherited from D. mixta [27]. Using five
loci, Uzzell and Darevsky (1975) found one clone in 11
specimens of D. armeniaca from two populations from
Armenia [14]. MacCulloch et al. (1995) examined 35 loci
and 75 specimens from seven Armenian populations
and found one widespread clone with two rare clones
[38]. One of the rare clones occurred in 19 out of 27 in-
dividuals at Dilijan (Papanino) and the other one the
only clone in Kuchak (n= 2). Fu et al. (2000) used 35
allozyme loci and 117 specimens, including some lizards
with the color variation described by Darevsky (1992)
and Danielyan (1999) as being four clones, one being
common [37,66,68]. One rare allozyme clone domi-
nated in two populations and a rare color-variant clone
at Ankavan (n = 2) differed at two loci [66]. This is un-
like D. dahli, whose morphological difference did not
correspond with either allozyme or microsatellite
markers [16,46].
MacCulloch et al. (1995) attributed the clonal variation
that associated with morphological data in D. armeniaca
to mutations [37,38]. They argued that (1) some rare
clones had alleles not detected in the parental species;
(2) the pattern of clonal variation in D. armeniaca was
typical of parthenogenetic lizards of single hybridization
origin such as in teiid Aspidocelis [22]; and (3) overall
variation in D. armeniaca was less than that found in
species of multiple hybrid origin, such as parthenogen-
etic gecko Heteronatia binoei [19]. Alternatively, Fu et
al. (2000) suggested that although mutation was a pos-
sible explanation of the origin of the clonal variation in
D. armeniaca, but multiple origins was equally likely
[66]. The rare clones in the Kuchak and Dilijan (Papa-
nino) may have owed to an independent origins because
of dominance of rare alleles and (2) the peripheral distri-
bution to the common clone suggested multiple origin
Table 3 The population indices of gene diversity for four studied loci in twelve sampled populations of D. armeniaca (Continued)
Locus Population Alelle (N) R
S
H
E
H
O
Kuchak 2 1.93 0.54 1
Meghradzor 2 2.17 0.53 1
Medved-gora (vicinity of Stepanavan) 3 1.92 0.59 1
Dilijan (Papanino) 3 2.41 0.68 1
Pushkin Pass 3 2.34 0.65 1
Dilijan-Semyonovka Pass 2 1.92 0.53 0.86
Sotk 4 3.20 0.87 1
Stepanavan 4 2.67 0.73 1
Artavaz (Hankavan) 2 1.89 0.51 1
Tezh (Pambak Ridge) 2 1.92 0.53 1
Total 5 5.00 0.65 1
Mean ± SE 2.83 ± 0.30 2.24 ± 0.12 0.63 ± 0.03 0.93 ± 0.07
NNumber of alleles, R
S
Allelic richness, H
E
Expected heterozygosity, H
O
Observed heterozygosity
Girnyk et al. BMC Genomics (2018) 19:979 Page 8 of 12
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[22]. Further, the young age of D. armeniaca implied
from low mtDNA variation and substantial allozyme
variation favored the multiple-origin scenario over a
rapid accumulation of mutations [66].
Our analyses indicate that at least three interspecific
hybridizations were involved in the genesis of D. arme-
niaca and microsatellite mutations followed these. A
previous allozyme analysis reported four clones with the
rare ones occurring in restricted populations [65]. This
allozyme pattern generally followed Parker et al.’s (1989)
model [22], which is consistent with single hybridization
origin. Nevertheless, Fu et al. (2000) noted that multiple
origins could also explain the clonal diversity [66].
Our modified microsatellite genotyping [46] detects
both microsatellite and SNVs variability. The method re-
veals 13 genotypes in 111 individuals using four micro-
satellite loci. Previously, the approach revealed a high
level of clonal diversity (11 clones) in D. dahli [46] and
rejected the hypothesis of monoclonality of D. rostombe-
kowi [47], and our analyses also detect greater variation
in D. armeniaca than did allozymes. Thus, this method
is a more precise measure of assessing genetic variability
compared with allozymes.
Previously, the two color-variant individuals of D.
armeniaca from Ankavan were investigated using allo-
zymes [65]. These specimens differed from the common
allozyme clone forming separate clones. However, our
data failed to distinguish 10 specimens of D. armeniaca
with differences in coloration; they all have the most
abundant and widespread genotype 1. The color-variants
consist of about half of the samples from Artavaz
(Hankavan). Similarly, our microsatellite genotyping data
[46], as well as allozyme [16] and mtDNA [23] studies,
failed to distinguish the color-varieties of parthenogen-
etic D. dahli.
The pattern of distribution of the clones rejects the hy-
pothesis of a single origin for D. armeniaca. All individ-
uals with genotypes 1–9 exhibit an identical combination
of parent-specific SNVs, suggesting their common origin
via one hybridization event. Given that D. mixta and D.
valentini are the parental species for D. armeniaca [14,23,
37,38,66], the identical SNVs but different microsatellite
markers indicates that the hybridization event involved
one population. The five individuals with genotypes 10–
12 also exhibit an identical combination of parent-specific
SNVs, suggesting their common origin, but one of that
differs from individuals with genotypes 1–9. Clonal diver-
sity in both groups owes to microsatellite unstable
(GATA)
n
mutations only. Such mutations in parthenogen-
etic D. unisexualis occur in one generation via deletion or
insertion of a single repeat at one or at both alleles of the
locus [50]. Finally, the individual from Harich has geno-
type 13 and a unique combination of parent-specific SNVs
at locus Du47G. Allele Du47G(arm)7 was inherited by D.
armeniaca from D. mixta (Additional file 1:Table S1).
Therefore, this individual may represent a third
hybridization event.
The rather high clonal diversity in Darmeniacalikely
owes to its multiple hybrid origin and subsequent microsat-
ellite mutations. Similarly, most of the observed clonal diver-
sity within parthenogenetic D. dahli in 9 out of 11 detected
genotypes owe to microsatellite mutations within the com-
mon clone, and two out of 111 individuals were suggested to
be members of independent hybridization events [46].
Analyses of parthenogenetic D. rostombekowi [47] revealed
one common and four rare clones, each represented by one
or several individuals from one or two populations. The re-
sults were consistent with single hybridization origin of D.
rostombekowi, with clonal diversity arising via post-formation
microsatellite mutations [47].
Tarkhnishvili et al. (2017) hypothesized that D. arme-
niaca originated from a series of crosses between D.
valentini and parthenogenetic D. dahli, rather than be-
tween D. valentini and D. mixta [13]. Their microsatel-
lite genotyping of Du215, Du281, Du481, Du323, and
Du47G revealed identical genotypes for D. dahli and D.
armeniaca at Du323 and Du47, but not at the other
three loci. They suggested that backcrosses and muta-
tions best explained the genetic and clonal diversity than
a multiclonal origin. However, species of Darevskia ex-
hibit rather high levels of genomic similarity and very
similar microsatellite alleles. They assumed alleles of
equal length had identical nucleotide sequences. Given
our discoveries, more detailed molecular data on the
structure of the alleles and genotypes is needed to sup-
port their hypothesis. Our discovery of multiple origins
provides a more parsimonious hypothesis than the com-
plex hypothesis of backcrossing. Further, projections on
the historical distribution of sexual and parthenogenetic
species during glacial waves is necessary to document
the possibility of hybrids between D. dahli and D. valen-
tini, as well as possible hybridizations between D. mixta
and D. portschinskii that might be according to [13] the
first stage in origin of D. armeniaca.
Parker et al. (1989) proposed that unisexual species ori-
ginating through a single hybridization event will exhibit a
common clone with a few rare clones [22]. Accordingly,
among clones 1–9, genotype 1 might be ancestral (Table 2).
The TCS network (Additional file 4: Figure S1) has a
star-like structure with common genotype 1 occupying the
central location and the others differing by from one to two
mutational events. This star-like structure is consistent with
a recent origin and diversification of clones.The same in-
ference is not possible for genotypes 10–12 because none
of them is numerous and widespread. Dsegh and Harich do
not appear to have genotype 1. However, genotype 11 at
Dsegh and genotype 13 at Harich arose independently from
genotype 1. Genotype 4 at Dsegh and Harich, and
Girnyk et al. BMC Genomics (2018) 19:979 Page 9 of 12
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genotypes 2, 6 and 7 at Harich appear to be variants of
genotype 1. If so, then their occurrence in these localities
must owe to the dispersal of lizards from other regions.
MacCulloch et al. (1995) and Fu et al. (2000) observed
some peculiarities in allozyme-clone variation [62,65].
Among their five populations of D. armeniaca, rare
clones made up the majority of the individuals in
Kuchak and Dilijan (Papanino), although the common
clone covered most of the species’distribution. This
contributed to the suggestion that numerous rare clones
might indicate independent hybrid origins [65]. How-
ever, our parent-specific SNVs for these clones are iden-
tical to those of genotype 1.
The distribution of microsatellite clones in D. arme-
niaca is not exceptional. Among 11 detected clones in the
parthenospecies D. dahli [46], two were numerous and
widespread geographically and all others were rare, al-
though one rare clone in “Dendropark”(near Stepanavan)
occurred in 6 out of 9 individuals. Similar analysis re-
vealed one common and four rare clones in D. rostombe-
kowi, and one of the rare clones occurred in all 8
individuals at Tsovak [47]. Thus, in all three parthenogen-
etic species investigated, the numerous rare clones most
likely arose via post-formation microsatellite mutations of
the common clone, and not through independent inter-
specific hybridizations. Ecological differences among the
habitats of various populations, and the relative fitness of
clonal lineages of D. armeniaca and other parthenogenetic
Darevskia may explain the differences in the distribution
and existence of successful rare clones.
In summary, the analyses suggest that the clonal diver-
sity in D. armeniaca derives from three interspecific hy-
bridizations and subsequent microsatellite mutations.
Future studies may identify the role mutations play in al-
tering of the initial clones. The methodological approach,
which is based on the detection of parent-specific micro-
satellite and SNV markers, can elucidate the origin of gen-
etic and clonal diversity in other unisexual species that
arose via interspecific hybridization.
Conclusions
Our interspecific genomic analysis used microsatellites and
single nucleotide polymorphisms (SNP). A comparison of
these markers between parthenogenetic D. armeniaca and
its parental sexual species D. valentini and D. mixta reveals
13 genotypes or presumptive clones. Clonal diversity in D.
armeniaca appears to result from at least three independ-
ent interspecific hybridization events. All other clones of D.
armeniaca appear to have derived from subsequent
microsatellite mutations. This methodological approach,
which is based on the detection of parent-specific microsat-
ellite and SNP markers, can be applied for study clonal
diversity in other unisexual species that arose via interspe-
cific hybridization.
Additional files
Additional file 1: Table S1. Allelic variations of microsatellite containing
loci in the lizard species D. armeniaca, D. valentini, and D. mixta.(PDF132kb)
Additional file 2: Table S2. The population indices of gene diversity for
four studied loci in twelve sampled populations of D. valentini. (PDF 99 kb)
Additional file 3: Table S3. Indices of association in four populations of
D. valentini. (PDF 347 kb)
Additional file 4: Figure S1. Schematic representation of the TCS
network that reflects distribution of genotypes 1–9inD. armeniaca.
Concatenated sequences of D. armeniaca genotypes were analyzed using
TCS software v.1.21. Genotypes 10–13 are plotted separately. Population
distribution of the genotypes is shown by different colors. Numbers
indicate the number of individuals in populations. The black circles show,
unsampled, but computer-predicted genotypes. (TIF 802 kb)
Acknowledgments
Authors thank to Fedor Osipov for help in preparing the manuscript.
Funding
This research was partly funded by RFBR according to the research projects
№17–00-00430 (17–00-00426), №17–04-00396 and by the RAS Program
«Molecular and Cell Biology». The work was conducted on the base of the
Center for Collective Use, Institute of Gene Biology, Russian Academy of
Sciences (GK-02.451.11.7060). These funding bodies had roles in the
experimental work, analysis of the data, and in preparation and writing the
manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article and its supplementary information files. All unique de novo
sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/).
Authors’contributions
APR: Conceptualization. AAV, and APR: Methodology. SKS, and ASG: Software.
AAV, FDD, and IAM: Validation. SKS, and ASG: Formal Analysis. AAV, and AEG:
Investigation. APR: Resources. AAV, and AEG: Data Curation. APR: Writing
original. APR, RWM, and MSA: Writing, review & editing. APR, AEG, and AAV:
Visualization. APR, RWM, and FDD: Supervision. APR: Project administration.
APR: Funding acquisition. All authors read and approved the final
manuscript.
Ethics approval
Studied lizards species are not protected by CITES. Lizards were captured by
Danielyan F.D. and Arakelyan M.S. 15–20 years ago with a noose. Blood
samples were taken from the tail veins of lizards under chloroform
anesthesia, and then these lizards were released. The study was approved by
the Ethics Committee of Moscow State University (Permit Number: 24–01)
and was carried out in strict accordance with their ethical principles and
scientific standards.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Laboratory of Genome Organization, Institute of Gene Biology of the
Russian Academy of Sciences, Vavilova Str., 34/5, Moscow 119334, Russia.
2
Department of Biochemistry, Molecular biology and Genetics, Institute of
biology and chemistry, Moscow State Pedagogical University, M.
Pirogovskaya Str., 1/1, Moscow 119991, Russia.
3
Faculty of Biology, Yerevan
State University, 1 Alex Manoogian, 0025 Yerevan, Armenia.
4
Department of
Girnyk et al. BMC Genomics (2018) 19:979 Page 10 of 12
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Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, ON M5S
2C6, Canada.
Received: 23 October 2018 Accepted: 9 December 2018
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