Genetic identification of a non-native species introgression into wild population of the field cricket Gryllus campestris in Central Europe

Abstract

Two species of the genus Gryllus occur in Europe: G. campestris and G. bimaculatus. The first is widely distributed in the north-western Palaearctic, while the second, G. bimaculatus, occurs predominantly in the Mediterranean area. There is a visible pattern in the distribution of G. campestris, the insect being rare and threatened in the western part of its range, whereas it is still abundant in the east. Despite the fact that this species is commonly used in laboratory experiments, its natural populations are poorly characterised. In the present study, we analysed cricket populations from the lower Oder and Vistula River valleys in Poland. Based on the phylogeny of the mtDNA cytochrome b fragment, we found that 17% of the individuals studied had a G. bimaculatus-like mtDNA haplotype. Analyses of 11 autosomal microsatellite loci failed to reveal any clear genetic differentiation between individuals assigned to these two clades. This suggests, along with the spatial distribution of G. bimaculatus-like haplotypes, successful interbreeding of G. bimaculatus with native populations of G. campestris. However, both the nuclear data and additional analyses of two X-chromosome-linked microsatellite loci revealed incomplete introgression. Human-mediated introgression seems to be the most plausible explanation of the observed genotypic pattern such that caution needs to be taken in conservation efforts carried out in the western part of the species’ range.

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EUROPEAN JOURNAL OF ENTOMOLOG
Y
EUROPEAN JOURNAL OF ENTOMOLOGY
ISSN (online): 1802-8829
http://www.eje.cz
state of the fi eld cricket population seems to be visible in
Germany and Poland. Grein (2000, 2005 after Hochkirch et
al., 2007) states that only ten populations are left in Lower
Saxony and one west of the Weser River. At the same lati-
tude, but 400 km to the east in Poland, the species is still
very common and widely distributed throughout the whole
country (Bazyluk & Liana, 1990, 2000).
This contrast between populations in the west and east of
Europe may be the result of differences in land-use man-
Genetic identifi cation of a non-native species introgression
into wild population of the fi eld cricket Gryllus campestris
(Orthoptera: Gryllidae) in Central Europe
HANNA PANAGIOTOPOULOU 1, 2, *, MATEUSZ BACA 3, *, KATARZYNA BACA 4, PAWEŁ SIENKIEWICZ 5, PIOTR ŚLIPIŃSKI 1
and MICHAŁ ŻMIHORSKI 6, 7, **
1 Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warsaw, Poland;
e-mails: hpana@miiz.waw.pl, piotrs@miiz.waw.pl
2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
3 Center for Precolumbian Studies, University of Warsaw, Krakowskie Przedmieście 26/28, 00-927 Warsaw, Poland;
e-mail: bacamat@gmail.com
4 Institute of Genetics and Biotechnology, Department of Biology, University of Warsaw, Pawińskiego 5a, 02-106 Warsaw,
Poland; e-mail: katarzyna.ewa.pajak@gmail.com
5 Department of Entomology and Environmental Protection, Poznań University of Life Sciences, Dąbrowskiego 159,
60-594 Poznań, Poland; e-mail: ophonus@gmail.com
6 Institute of Nature Conservation, Polish Academy of Sciences, Mickiewicza 33, 31-120 Kraków, Poland
7 Department of Ecology, Swedish University of Agricultural Sciences, Box 7044, SE 750 07 Uppsala, Sweden
Key words. Orthoptera, Gryllidae, Gryllus campestris, Gryllus bimaculatus, conservation, mtDNA, microsatellite loci,
X-chromosome-linked markers
Abstract. Two species of the genus Gryllus occur in Europe: G. campestris and G. bimaculatus. The fi rst is widely distributed
in the north-western Palaearctic, while the second, G. bimaculatus, occurs predominantly in the Mediterranean area. There is a
visible pattern in the distribution of G. campestris, the insect being rare and threatened in the western part of its range, whereas
it is still abundant in the east. Despite the fact that this species is commonly used in laboratory experiments, its natural popula-
tions are poorly characterised. In the present study, we analysed cricket populations from the lower Oder and Vistula River valleys
in Poland. Based on the phylogeny of the mtDNA cytochrome b fragment, we found that 17% of the individuals studied had a
G. bimaculatus-like mtDNA haplotype. Analyses of 11 autosomal microsatellite loci failed to reveal any clear genetic differentia-
tion between individuals assigned to these two clades. This suggests, along with the spatial distribution of G. bimaculatus-like
haplotypes, successful interbreeding of G. bimaculatus with native populations of G. campestris. However, both the nuclear data
and additional analyses of two X-chromosome-linked microsatellite loci revealed incomplete introgression. Human-mediated in-
trogression seems to be the most plausible explanation of the observed genotypic pattern such that caution needs to be taken in
conservation efforts carried out in the western part of the species’ range.
* These authors equally contributed to the work.
** Corresponding author; e-mail: michal.zmihorski@gmail.com
INTRODUCTION
Populations of the fi eld cricket, Gryllus campestris L.
(Orthoptera: Gryllidae) vary in terms of conservation sta-
tus across the species’ European range. In the UK, Germa-
ny, Netherlands, Denmark and Switzerland, the species is
rare and included in the Red Lists (Hochkirch et al., 2007).
In contrast, populations from central-eastern Europe are
abundant, and appear to show stable dynamics over time
(Bazyluk & Liana, 2000). The most distinct contrast in the
Eur. J. Entomol. 113: 446–455, 2016
doi: 10.14411/eje.2016.058
ORIGINAL ARTICLE

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Panagiotopoulou et al., Eur. J. Entomol. 113 : 446–455, 2016 doi: 10.14411/eje.2016.058
et al., 2011, 2013; Tyler et al., 2013). Post-zygotic barri-
ers between these two species seem to be much weaker,
though hybrid offspring are negatively sexually selected
and have lower fi tness. This includes reduced hatching
success, which is probably a result of higher mortality in
the very early life stages (Veen et al., 2013). Hybrids do
however live longer (Veen et al., 2013), which makes their
life-time reproductive success higher (Rodríguez-Muñoz
et al., 2010). This incomplete reproductive isolation sug-
gests the possibility of successful introgression in the wild
in areas where both species coexist naturally or come into
secondary contact due to human introductions.
In the present study, we have attempted to characterize
the genetic polymorphism and structure of wild G. camp-
estris populations in the eastern part of the species’ distri-
bution range. More specifi cally, we studied both mitochon-
drial as well as nuclear genetic markers of 488 crickets
captured in the wild. During the research we discovered
introgression of G. bimaculatus-like haplotypes into the G.
campestris gene pool. Possible explanations for the occur-
rence of G. bimaculatus’ far outside its natural distribution
and the possible consequences for conservation of natural
populations of G. campestris are discussed.
MATERIALS AND METHODS
Sampling
In total 414 individuals were collected in the lower Oder River
valley of north-western Poland (52.89°N, 14.32°E) in May–June,
2010 and 2011 in 21 different localities within this region. These
particular sampling sites, covering different types of habitats uti-
lized by the species − xerothermic grasslands, fi elds, meadows
and clear-cut areas in the forest complex − were selected in order
to characterize genetic structure of the populations living under
different environmental conditions. Additionally, we collected
74 individuals from the lower Vistula River valley (53.05°N,
18.40°E) at three localities on the basis of visual and aural de-
tection in appropriate habitats. The trapped animals were kept in
agement, landscape history and agriculture intensifi cation
(see Table 2 in Tryjanowski et al., 2011). Studies conduct-
ed on various Orthoptera species in the Czech Republic
suggest habitat loss, due to changes in land management
and forestations, as a predominant factor that has resulted
in considerable fragmentation and isolation of many inver-
tebrate populations (Holusa, 2012; Holusa et al., 2012).
The decline of G. campestris in Western Europe is likely
to be due to isolation of small populations accelerating in-
breeding and the associated increased extinction risk (Wit-
zenberger & Hochkirch, 2008). Nevertheless, knowledge
concerning the status and temporal trends in population dy-
namics of the fi eld cricket in different locations across its
European distribution range is still poor. While the species
is commonly used in laboratory studies, its natural popula-
tions are rarely investigated (but see e.g. Ritz & Kohler,
2007; Witzenberger & Hochkirch, 2008; Rodríguez-Mu-
ñoz et al., 2010; Bretman et al., 2011). Even basic ecologi-
cal parameters are unknown, and there is no reliable data
on the genetic diversity of the species, especially in the
eastern part of its range.
The distribution of G. campestris includes all of Europe
and overlaps with the distribution of its sister species, G.
bimaculatus De Geer towards the south (Fig. 1). The latter
species’ range in Europe is mainly restricted to the Medi-
terranean coastal regions. Both species share common life
histories, utilizing similar habitats and have recently been
studied for patterns of reproductive barrier development
that may have caused their speciation and prevent hybridi-
zation (Tyler et al., 2013; Veen et al., 2011, 2013). Con-
tact zones between these two fi eld cricket species exist in
south-eastern Spain and probably also in Italy and further
east, although geographical barriers like the Cantabrian
Mountains probably prevent secondary contact (Bazyluk
& Liana, 2000; Veen et al., 2013 and references therein).
In contact zones of the two species, the possibility of in-
trogression exists, and it has indeed been experimentally
demonstrated that they can hybridize (Veen et al., 2011,
2013; Tyler et al., 2013). The intensity of inter-specifi c
crossing in the natural environment remains unknown,
though earlier studies suggest that gene fl ow between these
two species ought to be highly restricted via multiple re-
productive barriers (Veen et al., 2013).
The natural ability of fi eld crickets to hybridize has re-
ceived considerable attention as a very good model to in-
vestigate evolutionary forces that promote and maintain
speciation. The topic has also been recently intensively
studied using molecular (DNA) markers (Harrison, 1983;
1986; Harrison et al., 1987; Maroja et al., 2009, 2014; Veen
et al., 2011, 2013; Larson et al., 2012; Andrés et al., 2013;
Tyler et al., 2013). These studies aimed to discover repro-
ductive barriers acting at pre- and post-mating stages. It
was proven that prezygotic barriers between G. campes-
tris and G. bimaculatus are strong and include pre-mating
isolation via sexual signals and selection (based on calling
song preferences and probably species-specifi c cuticular
hydrocarbons) as well as post-mating prezygotic conspe-
cifi c sperm precedence and cryptic female choice (Veen
Fig. 1. Schematic distribution of the two cricket species in Central-
Western Europe: Gryllus campestris (blue) and G. bimaculatus
(pink).

448
Panagiotopoulou et al., Eur. J. Entomol. 113 : 446–455, 2016 doi: 10.14411/eje.2016.058
captivity until their natural death, which took place in August–
September, and afterwards they were preserved in 75% ethanol.
DNA extraction, amplifi cation and sequencing
DNA was isolated from one rear leg of each specimen using
the Wizard SV 96 Genomic DNA Purifi cation System (Promega,
Madison, WI, USA) according to the manufacturer’s recommen-
dations. In order to determine species affi liation of Gryllus speci-
mens, a 846 bp region of the cytochrome b sequence was am-
plifi ed with primers 846For 5’-AGA GTA AGT CAC ATT TGC
CGA G-3’ and 846Rev 5’-GGT TCT TCA ACT GGT CGG GCA
CC-3’ designed with Primer 3 v.0.4.0 software (Untergasser et
al., 2012). PCR reactions were performed in 20 μl of reaction mix
containing 0.2 μM of each primer, 1 × Biomix (Bioline, London,
UK), and 1 or 2 μl of extracted DNA. Initial denaturation of 5
min at 95°C was followed by 31 cycles of 30 s at 95°C, 30 s at
58°C, 1 min at 72°C followed by a fi nal extension of 7 min at
72°C. PCR products were purifi ed using PEG precipitation and
sequenced. DNA chromatograms were analyzed using FinchTV
v.1.4.0 (Geospiza, Seattle, WA, USA), and aligned with BioEdit
software v.7.0.5.3 (Hall, 1999).
In addition to the mitochondrial marker, 14 microsatellite loci,
12 autosomal and two sex-linked i.e. Gbim59 and Gbim71 (Daw-
son et al., 2003; Bretman et al., 2008) were amplifi ed, divided into
4 multiplex reactions (Panagiotopoulou et al., 2015). Capillary
electrophoresis of PCR products, divided into two panels, was
conducted in Oligo.pl service (IBB, Warsaw, Poland) on an ABI
PRISM 3730 Genetic Analyzer (Applied Biosystems, Carlsbad,
CA, USA). The Peak Scanner program v.1.0 (Applied Biosys-
tems) was used to bin, score and output the microsatellite alleles.
Phylogenetic analyses
A phylogenetic tree was reconstructed to confi rm species affi li-
ation of the studied individual crickets. Cytochrome b sequences
of two Gryllus species that are common in Europe, G. campes-
tris and its sister species G. bimaculatus, were obtained from
the literature (Huang et al., 2000; Fereira & Ferguson, 2010).
Phylogenetic analyses were based on 575 bp long sequences.
Phylogenetic trees were constructed using three approaches: (1)
Maximum likelihood (ML) and (2) Neighbour Joining (NJ) trees
were constructed in MEGA v.5 software (Tamura et al., 2011)
with 1,000 bootstrap replicates used for each analysis. Tamura-
Nei with a proportion of invariable sites (TN93 + I) was chosen
as a best fi tting substitution model with MEGA v.5 software and
used for NJ and ML analyses. In addition, a Bayesian tree (3)
was reconstructed in MrBayes v.3.2.3 (Ronquist & Huelsenbeck,
2003). We used a nst=mixed option to allow MrBayes to sam-
ple over all possible reversible substitution models. We applied
gamma distributed rate variation across sites and allowed for pro-
portion of invariable sites. Different substitution rates for each
codon position were allowed. Two independent runs with four
Markov chains each were run for 10,000,000 generation with pa-
rameters sampled every 1,000 generations. Runs were checked
for stationarity and convergence using the program Tracer v. 1.6
(ESS above 200 and the standard deviation of split frequencies
lower than 0.01).
Spatial distribution of individuals carrying the two
cytochrome b haplotypes
To check, whether the G. bimaculatus-like cytochrome b hap-
lotype was randomly distributed in the study area, multiple logis-
tic regression was used. The share of G. bimaculatus-like haplo-
type among all crickets sampled in a given location was modeled
as a function of latitude and longitude and presented on the map
in form of pie plots. The analysis was performed in R (R Devel-
opment Core Team, 2015).
Genetic polymorphism and structure estimations
In order to verify the amount of gene fl ow – introgression or
reproductive separation between the two detected mitochondrial
lineages of crickets, we applied a microsatellite screening ap-
proach. For this, only individuals whose cytochrome b haplotype
was identifi ed were used. Micro-Checker v.2.2.3 software (van
Oosterhout et al., 2004), was applied, using 1,000 iterations and
95% CI, to search for genotyping errors caused by false alleles,
stuttering, null alleles (assuming the Brookfi eld 1 equation) and
allelic dropouts in cricket groups from each sampling location
treated separately. Locus Gbim58 was discarded from further
analyzes, because it possessed a high and signifi cant frequency of
null alleles (> 0.19) in nearly all of the analyzed populations. As
loci Gbim59 and Gbim71 were shown to be linked to the chromo-
some X (Bretman et al., 2011), they were analyzed separately.
The observed allelic range, number of alleles per locus (NA) and
effective number of alleles (Ne) were calculated using GeneAlEx
v.6.4 software (Paekal & Smouse, 2006). Observed heterozygos-
ity (HO) and expected heterozygosity (HE) were obtained with
Arlequin v.3.5.1.2 (Excoffi er & Lischer, 2010). Allelic richness
(R) and inbreeding coeffi cients (FIS) were estimated in FSTAT
v.2.9.3.2 (Goudet, 2002). Deviation from Hardy-Weinberg equi-
librium (HWE) was tested using Genepop v.4.0.10 (Raymond &
Rousset, 1995).
Genetic distances between pairs of individuals were calculat-
ed using the DA measure (Nei, 1983) with MSA software v.4.05
(Dieringer & Schlötterer, 2003). The obtained values matrix of
DA values was visualized using principal coordinate analysis
(PCoA) implemented in GeneAlEx. The locations of individuals
belonging to the two haplotypes were visualized with the kernel
density estimator plotted along the fi rst two axes of the PCoA. It
was checked, moreover, whether or not the two haplotypes (G.
campestris and G. bimaculatus-like) were separable along the
PCoA axes. More specifi cally, we compared the mean values of
all PCoA axes scores (n = 123 axes) for G. bimaculatus-like and
G. campestris individuals with t-tests for independent samples.
Genetic structure was analyzed with the Bayesian approach
implemented in STRUCTURE v.2.3.4 (Pritchard et al., 2000).
Simulations assumed admixture, no a priori population informa-
tion, and the correlated allele frequency model. Ten clusters (K)
from 1 to 10 were inferred. In each case, the program was run
with ten repetitions of 500,000 MCMC iterations following a
burn-in period of 50,000 iterations. The most probable number
of clusters (K) was determined in the Structure Harvester v.0.6.8
program (Earl & von Holdt, 2012) by calculating ΔK as described
by Evanno et al. (2005). The average coeffi cients of membership
(Q) of individuals to each of the pre-defi ned clusters across the
ten repetitions were computed using CLUMPP v.1.1.2 (Jako-
bbson & Rosenberg, 2007) and displayed with the DISTRUCT
v.1.1 (Rosenberg, 2004) software. Next, the relationship between
the genetic structure of the whole population and assignment of
the crickets to the two mitochondrial groups (G. bimaculatus-like
vs. G. campestris) was investigated. Here, we used the chi-square
test (2 × 2 contingency table) in order to check whether assign-
ment of the crickets into one of the two mitochondrial groups
depended on its classifi cation to a particular cluster according to
the results obtained using STRUCTURE.
RESULTS
Phylogenetic analyses
Three distinct haplotypes were discovered in the Gryllus
dataset. Phylogenetic analyses showed that two of them (H1
and H2) grouped with G. campestris haplotypes. The third

449
Panagiotopoulou et al., Eur. J. Entomol. 113 : 446–455, 2016 doi: 10.14411/eje.2016.058
haplotype (H3) was located on the G. bimaculatus branch,
although it was highly divergent from all G. bimaculatus
specimens (Fig. 2). The position of this haplotype was
consistent across all tree reconstruction methods used with
high bootstrap and posterior probability values. Specimens
carrying haplotype H3 could not be easily assigned to G.
bimaculatus; however, since available cytochrome b se-
quence data of G. bimaculatus is limited to South African
and three western Mediterranean populations, we cannot
therefore exclude the possibility that this haplotype came
from a divergent near Eastern or Asiatic subpopulation of
this species. The recorded haplotypes were deposited in
GenBank under accession no. KF182315-17, along with
the computed trees that were uploaded to TreeBase data-
base (accession no. S19499).
Haplotype H3 was present in 72 of 391 individuals from
the lower Oder River and absent in the lower Vistula River
valley sampling localities (i.e. of 23 individuals tested) and
the difference in the observed frequencies (i.e. 72 vs. 319
and 0 vs. 23) was signifi cant (2 × 2 contingency table chi-
square test with Yates correction, χ2
1 = 3.92; P = 0.048).
At a lower spatial scale, a clear and statistically signifi cant
east-west frequency gradient of G. bimaculatus-like haplo-
types was observed in the lower Oder River valley region
(multiple logistic regression, estimate = 3.19, t = 3.61, P =
0.0019). This haplotype had a frequency of ≤ 75% of in-
dividuals in populations from the east and their frequency
decreased towards the western part of the sampling area
(Fig. 3). The effect of latitude was not signifi cant (estimate
= 1.77, t = 0.82, P = 0.4216).
Populations’ genetic polymorphism
Analyses of the 11 autosomal microsatellite markers
showed a very similar level of polymorphism and nearly
identical allelic size range across the 414 individuals tested,
when the two groups of crickets representing G. campes-
tris (n = 342) and G. bimaculatus-like (n = 72) haplotypes
were compared (Table 1). The mean number of alleles (NA)
and allelic richness (R) were however slightly lower for
the group of individuals with the G. bimaculatus-like hap-
lotype, which indicates intermixing of a small group into
a bigger gene pool. In the case of the NA values, this dif-
ference results mainly from the larger sample size of the
G. campestris group. Despite this, all other differences in
diversity indices between the two crickets groups were not
Fig. 2. Phylogenetic tree based on a 575 bp of cytochrome b se-
quences. The position of the haplotypes obtained in this study
(H1–H3) is as shown. Bootstrap support and posterior probabilities
of main nodes are indicated. The outgroup Acheta domestica (L.)
(Orthoptera: Gryllidae) was removed for display purposes.
Fig. 3. Share of G. campestris and G. bimaculatus-like specimens within the sampled populations from the Oder River valley region. Visu-
alization of the partial effects of longitude and latitude is given in the inner subplot – the longitude is a signifi cant predictor for the sharing
of G. bimaculatus-like haplotypes. The pale grey polygon indicates forested areas, dark grey – water bodies.

450
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statistically signifi cant (Table 1). The allelic size ranges of
single loci of the two crickets group were almost identical
and in most cases in the range observed for G. campestris
(Dawson et al., 2003; Bretman et al., 2008). In general,
both groups exhibited very similar pattern across all calcu-
lated diversity indices. Estimated FIS values were positive
and signifi cant, indicating internal genetic structure (Wah-
lund effect).
Genetic polymorphism levels of the 13 cricket collec-
tions (n = 262 individuals), for which the sampled indi-
viduals number exceeded 15, were generally higher than
observed in the cricket populations in northern Germany
(Witzenberger & Hochkirch, 2008). The calculated values
ranged for NA from 4.18 to 6.64 (in comparison to 3.75–
5.75), for Ne from 2.48 to 4.06 (compared to 2.07–4.22)
and for R from 4.08 to 5.77 (in comparison to 3.58–5.67).
The observed differences in the genetic variability be-
tween the Polish and German populations are rather small,
although the eastern populations seem to be more poly-
morphic, which is consistent with the abundances trends
and population dynamic in both countries. The fi eld cricket
populations in north Germany have been greatly reduced
in number and isolated, contrary to the situation in Poland,
where populations remain stable and abundant (Bazyluk &
Liana, 1990, 2000; Hochkirch et al., 2007; Witzenberger &
Hochkirch, 2008). In our present study, the mean number of
alleles in the 13 cricket populations sampled was, however,
signifi cantly lower compared to the G. campestris popula-
tion from Asturias in Northern Spain (n = 541) where NA
was ~ 11.4 across 11 loci (Bretman et al., 2011). Interest-
ingly, we obtained similar NA value when we pooled all of
the studied crickets (n = 414) sampled over sites occupying
several dozens of square km, while the population from
North Spain inhabited only a 800 m2 meadow.
Introgression assessment
Admixture analyzes showed no clear subdivision of in-
dividuals possessing the two different haplotypes. Both G.
campestris as well as G. bimaculatus-like individuals were
recorded in the two clusters in STRUCTURE (Fig. 4); even
so, the distribution of G. campestris and G. bimaculatus-
like individuals in the two clusters was not random and the
proportion of individuals assigned to cluster 1 represented
16% and 60% in case of G. bimaculatus-like and G. camp-
estris individuals, respectively, the difference being highly
signifi cant (Chi-square test, χ2
1 = 59.5, P < 0.001). Simi-
larly, some genetic divergence was observed when pair-
wise genetic distances (DA) were calculated and expressed
via PCoA (Fig. 5). Mean values of the scores of particular
individuals along the fi rst PCoA axis did not differ between
the two species (t-test, t131 = 0.48, P = 0.631). However, the
two species were separable along the second PCoA axis:
TABLE 1. Summary of the allelic variation and gene diversity indices at 11 autosomal microsatellite markers in the two fi eld crickets groups representing G.
campestris (n = 342) and G. bimaculatus-like (n = 72) haplotype lineages. NA – number of alleles; Ne – effective number of alleles; R – allelic richness; HO –
observed heterozygosity; HE – expected heterozygosity; FIS – inbreeding coeffi cient; HWE – deviation from Hardy-Weinberg equilibrium estimated from FIS
values: * – signifi cant FIS values at a probability level equivalent to P < 0.05 and NS – not signifi cant FIS values. Signifi cance obtained from paired samples
t-test are given in the last column.
Measure Cricket group Gbim15 Gbim35 Gbim21 Gbim40 Gbim72 Gbim29 Gbim57 Gbim33 Gbim49 Gbim04 Gbim66 Mean P-value
NAG. camprestris 9 8 27 14 7 12 7 18 4 6 10 11.09 0.014
G. bimaculatus-like 9 3 16 12 5 11 5 15 3 6 6 8.27
NeG. camprestris 4.5 1.7 10.6 2.9 3.2 7.6 1.5 5.5 3.0 4.1 3.1 4.33 0.741
G. bimaculatus-like 4.0 1.4 7.4 4.5 2.3 6.9 1.4 7.9 2.9 3.9 3.5 4.21
R G. camprestris 8.6 4.8 21.8 11.4 5.4 10.8 5.5 14.6 3.9 6.0 6.7 9.05 0.133
G. bimaculatus-like 8.9 3.0 16.0 11.7 4.9 10.9 5.0 14.8 3.0 5.8 6.0 8.18
HOG. camprestris 0.68 0.06 0.57 0.58 0.64 0.73 0.31 0.71 0.34 0.64 0.59 0.53 0.240
G. bimaculatus-like 0.74 0.12 0.54 0.70 0.60 0.79 0.29 0.79 0.45 0.54 0.58 0.56
HEG. camprestris 0.78 0.41 0.91 0.65 0.69 0.87 0.34 0.82 0.67 0.76 0.68 0.69 0.623
G. bimaculatus-like 0.76 0.31 0.87 0.78 0.56 0.86 0.31 0.88 0.66 0.75 0.72 0.68
FIS G. camprestris 0.12 0.85 0.37 0.12 0.07 0.16 0.07 0.13 0.49 0.15 0.13 0.23 0.146
G. bimaculatus-like 0.03 0.61 0.38 0.10 –0.06 0.09 0.06 0.11 0.32 0.28 0.19 0.18
HWE G. camprestris ******NS*****
G. bimaculatus-like NS * * NS NS NS NS * * * NS *
Range
(bp)1
G. camprestris 151–177
(167–197)
202–220
(215–219)
256–324
(262–317)
132–162
(142–166)
179–211
(180–192)
250–284
(270–281)
155–169
(163–178)
194–262
(203–276)
185–195
(187–197)
201–215
(211–257)
289–323
(303–320)
G. bimaculatus-like 151–177
(150–220)
202–214
(217–265)
256–332
(246–387)
132–162
(125–183)
181–211
(180–241)
258–286
(270–299)
155–167
(157–181)
194–262
(265–347)
185–189
(206–240)
207–219
(205–239)
305–319
(310–437)
1 In parentheses: allele sizes observed for wild G. campestris and laboratory G. bimaculatus populations originating from Spain (Dawson et al., 2003; Bretman
et al., 2008). Both populations were large and represented by 15–30 individuals. For locus Gbim58 that was excluded from statistical analysis, no differences
in allele sizes ranges between these two cricket groups were observed in this study (G. campestris: 83–113 bp vs. G. bimaculatus-like: 89–111 bp), in contrary
to the populations from Spain (G. campestris: 95–99 bp vs. G. bimaculatus: 114–149 bp).
Fig. 4. Admixture analyses of 414 fi eld crickets performed using STRUCTURE with K = 2. Each individual is represented by a vertical bar.
The vertical black line separates individuals belonging to the two distinct haplotype lineages. K – number of clusters.

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Panagiotopoulou et al., Eur. J. Entomol. 113 : 446–455, 2016 doi: 10.14411/eje.2016.058
G. campestris individuals were associated with the higher
score values compared to G. bimaculatus-like individuals
(unequal variance t-test, t100 = 8.06, P < 0.0001). Among
all the PCoA axes (n = 123), there were 12 cases show-
ing signifi cant differences (P < 0.05, with no correction
for the multiple comparisons) in the location between G.
campestris and G. bimaculatus-like individuals. As here
demonstrated, the G. campestris group is more genetical-
ly complex than hitherto assumed, forming at least three
concentrations visible on the kernel density plot, whereas
the G. bimaculatus-like individuals were contrastingly re-
stricted to one.
Separate calculations of the two X-chromosome-linked
loci, Gbim59 and Gbim71, showed a similar pattern as the
autosomal loci, revealing an almost complete introgression
of individuals carrying the G. bimaculatus-like haplotype
into the G. campestris gene pool. Analyzes of males (n =
261), for which these loci were treated as one unit in the
haploid-STR way (males are X0), revealed the existence
of seven different genotypes (Table 2). We followed the
distributions of these genotypes across six populations rep-
resenting “pure” G. campestris collections and compared
them with 11 collections, where both mitochondrial haplo-
types were recorded (separately for G. campestris and G.
bimaculatus-like individuals). Most of the individuals bore
the same genotype, regardless of the mitochondrial haplo-
type contribution. Differences in the proportions of geno-
types in each group were statistically signifi cant for two
genotypes: thus genotype 134/88 was recorded in > 12%
males in the G. campestris group, regardless of whether the
populations were pure or mixed, whereas in the G. bimac-
ulatus-like individuals, this genotype was absent, as was
genotype 136/82, which was more common in G. bimacu-
latus-like individuals compared to G. campestris (Table 2).
DISCUSSION
In this study, we investigated the variability of cyto-
chrome b gene fragment (mtDNA) and microsatellite loci
(autosomal and sex-linked) of fi eld crickets representing 24
different sub-populations from the Oder and Vistula River
valleys of north-western Poland. The genetic diversity in-
dices for the separate populations were rather low when
compared to the old and large population of G. campestris
in North Spain (Bretman et al., 2011), though higher than
described for the depleted populations in northern Germa-
ny (Hochkirch et al., 2007). Mitochondrial sequence analy-
ses revealed the occurrence of a G. bimaculatus-like hap-
lotype, which was unexpected in this region. It seems that
this introduced species has successfully introgressed into
the gene-pool of local G. campestris populations, which
is worrying, considering its endangered status in Western
Europe.
Fig. 5. Principal coordinate analyses (PCoA) of the 414 fi eld crick-
ets based on the matrix of DA distances of Nei et al. (1983). The
two axes of PCoA explain 12% of the total variance of the distance.
The bottom subplot presents the projections of particular individu-
als on the plot delimited by fi rst and second axis of PCoA, middle
and upper subplots present kernel density estimators for the G.
campestris and G. bimaculatus-like specimens.
TABLE 2. Genotype distribution of the two chromosome X-linked loci in males, belonging to the two fi eld crickets groups of G. campestris and G. bimaculatus-
like mitochondrial haplotypes. n total = 230 individuals without missing data consisting of 11 populations with mixed origin (n = 123 G. campestris and n =
43 G. bimaculatus-like crickets) and 6 populations of pure G. campestris individuals (n = 64). Results of the Chi-square test (test statistics and P values)
comparing genotype frequencies between groups of crickets are given in the last two columns: “y” denotes Yates continuity correction applied, signifi cant
differences are bolded.
Genotype Eleven “mixed” populations (%) Six “pure” populations (%) Differences – chi-square test
Gbim711Gbim59 [A]
G. campestris
[B]
G. bimaculatus-like
[C]
G. campestris [A] vs. [B] [A] vs. [C]
1 134 82 81.30 76.74 79.69 χ2 = 0.41; P = 0.519 χ 2 = 0.07; P = 0.790
2 134 88 12.20 0.00 12.50 χ 2 = 4.38; P = 0.036y χ 2 = 0.01; P = 0.952
3 140 82 0.81 0.00 3.13 χ 2 = 0.30; P = 0.581y χ 2 = 0.34; P = 0.561y
4 140 88 0.00 0.00 3.13 — χ 2 = 1.49; P = 0.222y
5 136 88 0.00 4.65 1.56 χ 2 = 2.54; P = 0.111y χ 2 = 0.11; P = 0.734y
6 132 82 3.25 6.98 0.00 χ 2 = 0.34; P = 0.545y χ 2 = 0.86; P = 0.354y
7 136 82 2.44 11.63 0.00 χ 2 = 4.03; P = 0.045y χ 2 = 0.42; P = 0.518y
1 Allele sizes for both loci observed in wild G. campestris and laboratory G. bimaculatus populations (Dawson et al., 2003; Bretman et al., 2008) were similar
for both species and in this study.

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Gryllus bimaculatus occurs in Europe predominantly in
the Mediterranean areas, in Africa and Asia in tropical and
subtropical regions (Heller et al., 1998; Bazyluk & Liana,
2000; Ferreira & Ferguson, 2010). Ferreira & Ferguson
(2010) suggest that the distribution and abundance of this
species in Europe is shaped by migration from warmer re-
gions (mainly from Africa) and limited toward the north by
the winter temperature isotherm around 16°C. Until now,
no natural G. bimaculatus population has been described
in Poland or other regions of northern Europe. The occur-
rence of G. bimaculatus-like crickets in north-western Po-
land (lower Odra River valley), whilst unexpected, must
nevertheless be viewed in the light of the fact that G. bi-
maculatus was recently recorded in a few localities both in
Germany and France (following the Fauna Europea web-
site: http://www.faunaeur.org/distribution_table.php). It is
thus possible that the presence of xerothermic vegetation in
the region facilitates the persistence of this thermophilous
cricket’s population, but habitat preferences of the two spe-
cies needs further investigations. Moreover, winter tem-
peratures are much lower than the G. bimaculatus survival
threshold proposed by Ferreira & Ferguson (2010).
Several scenarios may explain the presence of the G.
bimaculatus-like haplotype in G. campestris populations.
It could result from incomplete lineage sorting during
speciation or the maintenance of an ancestral polymor-
phism. However, in such a scenario, one might expect ran-
dom distribution of the mtDNA haplotypes in the studied
populations. The observed eastern-western gradient of G.
bimaculatus-like haplotype frequencies, given the limited
mobility of crickets, is therefore consistent with the scenar-
io of a recent introduction of a small number of individuals
into the natural population. It is possible that an artifi cially-
introduced species has successfully introgressed into the
natural G. campestris populations. This explains the high
frequency of the mtDNA haplotype G. bimaculatus which,
due to climatic conditions, could not survive winters in
that part of Europe. In light of this, it seems reasonable
to conclude that a group of G. bimaculatus-like individu-
als was accidentally or intentionally (probably the former)
introduced into the wild and thanks to propitious circum-
stances intermixed into local populations of G. campestris.
This view is supported by the fact that G. bimaculatus is
commonly kept as food for spiders and reptiles, as well
as being a model species used in scientifi c research (Sim-
mons, 1986, 1991).
The analysis of microsatellite loci provides additional
support for these assumptions, as individuals carrying the
G. bimaculatus-like haplotype did not form a separate clus-
ter, but were grouped together with G. campestris crickets.
This means that the observed intermixing likely lasted for
at least several generations. The genetic diversity indices
and heterozygosity levels between the two cricket groups
differed only slightly. In the study of Bretman et al. (2008),
where individuals representing pure G. bimaculatus or G.
campestris species were analysed, allelic ranges of several
microsatellite loci (i.e. Gbim49, 58, 72, 33 and 66) were
different and only partially overlapping for the two species.
In contrast, in our study, the observed allelic size ranges
were almost identical, indicating advanced intermixing.
However, the spatial genetic structure seems to be still af-
fected by the observed introgression, as the probability of
successful prediction of a randomly chosen individual car-
rying G. bimaculatus-like haplotype to the second cluster
(at the uppermost level of substructure) was high.
It was proven that gene-fl ow between the two cricket-
species is asymmetrical with G. campestris females almost
never hybridizing. In contrast, G. bimaculatus females
hybridize more readily, which is explained by their higher
degree of polyandry (Veen et al., 2011, 2013; Tyler et al.,
2013). Hybrid females prefer mating with G. bimaculatus
males, while hybrid males have reduced attractiveness
in general, as fact that should restrict further gene fl ow
(Veen et al., 2013). These particular studies showed that
in the case of hybridization, the interspecifi c introgression
should occur mainly through G. bimaculatus females and
G. campestris males (Veen et al., 2011, 2013). In such cir-
cumstances, the mtDNA haplotype of a relatively small
group of introduced individuals could spread within the
natural population due to genetic drift. Similar conclusions
were made by Hochkirch & Lemke (2011) for asymmetri-
cal hybridization in the case of other Orthoptera species,
i.e. Chorthippus montanus and C. parallelus (Orthoptera:
Acrididae).
Knowing that introgression under laboratory conditions
is asymmetric and that hybrid males are much less attractive
to females than paternal species (Veen et al., 2011, 2013),
means that different genotypes distributions on the X-chro-
mosome-linked markers between individuals carrying the
two mitochondrial haplotypes are expected. We wished to
test if any signs of reduced gene fl ow could be observed,
especially as it is generally accepted the X chromosome is
very important in the evolution of reproductive isolation
(reviewed by Turelli & Moyle, 2007; Bolnick et al., 2008;
Good et al., 2008). We compared these patterns between
males, as they possess only one X chromosome, inherited
from their mothers. Indeed, the G. campestris males had
one genotype present both in pure and intermixed popula-
tions with similar, quite high frequency (> 10%), that was
absent in the G. bimaculatus-like individuals. The observed
differences of the X-chromosome-linked loci between the
G. campestris and G. bimaculatus-like groups were, how-
ever, weak (although signifi cant) and masked probably by
several generations of interbreeding. Such differences may
though serve as a signal that the observed intermixing is an
ongoing process in the studied populations of fi eld cricket.
When able to choose, both species (and both sexes)
strongly prefer conspecifi cs as mates (Tyler et al., 2013;
Veen et al., 2013). Interbreeding avoidance could be even
stronger in natural contact zones, where both species have
co-existed in sympatry for a long time and the species may
develop additional behavioral mechanisms which reinforce
ecological-evolutionary divergence. For example, G. ful-
toni (Alexander) differs in calling songs depending on if it
occurs in sympatry or allopatry with G. vernalis Blatchley
(Jang & Gerhard, 2006). However, in artifi cially created

453
Panagiotopoulou et al., Eur. J. Entomol. 113 : 446–455, 2016 doi: 10.14411/eje.2016.058
contact zones, the native species usually outnumbers the
introduced one, facilitating hybridization. As shown in this
study, costs of hybridization are suffi ciently low to enable
gene-fl ow at least through the females of G. bimaculatus.
Backcrossing with parental G. campestris must be unavoid-
able and seems not to affect the F2 fi tness in preventing fur-
ther reproduction. This will probably result in persistence
of the hetero-specifi c mitochondrial haplotype in the next
generations. The important issue that needs to be quantifi ed
is the autosomal contribution of G. bimaculatus to the G.
campestris gene pool, as it have been shown that this may
be negligible even when introgression of mitochondrial
DNA has occurred due to sex-biased asymmetries or/and
adaptive forces causing mito-nuclear discordances (Toews
& Brelsford, 2012; Good et al., 2015).
The phenomenon observed in this study emphasizes
the threat for both the numerous and stable populations of
fi eld crickets in Poland, as well as the declining and en-
dangered populations in Germany, essentially because of
the continuing diffusion of G. bimaculatus genes, which
is highly probable. The genetic integrity of G. campestris
as a species has been locally disrupted and the surround-
ing, currently unaffected populations are at the same risk.
What is more, occasional reports of G. bimaculatus in
Germany and France indicate that the problem of non-in-
tentional introductions of G. bimaculatus and the species’
introgression into native populations of G. campestris may
also have taken place in other regions of western Europe.
In theory, G. campestris and G. bimaculatus can be eas-
ily distinguished morphologically (i.e. width of the head
in relation to body, length of wings, size of yellow patch-
es and general body size; Bazyluk, 1956). There are also
some phenological differences in the activity of the two
species during the year − G. campestris is in general active
earlier than G. bimaculatus (Bellman, 2006). Having said
that, three randomly selected individuals from the groups
of crickets carrying G. bimaculatus-like haplotypes were
morphologically determined by an Orthoptera expert to be
G. campestris (A. Liana, pers. comm.), showing that ge-
netic evaluation is necessary in this case.
The introgression of genes from non-native species is
a well-known phenomenon (cf. Crispo et al., 2011 and
references therein), yet the effects are poorly understood.
It can decrease adaptation and affect fi tness of the local
populations. The impact of the observed introgression of
G. bimaculatus-like individuals into natural populations of
G. campestris, should therefore be characterized, especial-
ly by quantifying the extent of nuclear gene fl ow between
these species through collection of genome-wide data. Our
fi ndings seem to be especially important in the light of res-
titution efforts, including translocations of G. campestris
(Witzenberger & Hochkirch, 2008), more especially as in-
troductions of G. bimaculatus individuals may negatively
infl uence the restitution process. Populations scheduled
for use as a source for reintroductions should be fi rst ge-
netically characterized to exclude possible introgression.
Populations proven to be already introgressed with G. bi-
maculatus-like haplotypes should not be used for translo-
cations even if their genetic parameters like heterozygosity
or effective population sizes are high.
ACKNOWLEDGEMENTS. We are grateful to the anonymous re-
viewers for their valuable comments. This study was supported by
National Science Center grant no. NN303322234 directed by M.
Żmihorski and the project fi nancing agreements POIG.02.02.00-
14-024/08-00. We are grateful to K. Barańska and M. Molak for
fi eld work assistance. English proofreading of the manuscript
was kindly performed by B. Przybylska and A. Baca, whilst H.D.
Loxdale also made valuable editorial suggestions for the im-
provement of the manuscript.
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Received November 9, 2015; revised and accepted May 25, 2016
Published online August 29, 2016