J Insect Conserv

DOI 10.1007/s10841-014-9636-6

ORIGINAL PAPER

Geographic variation in relict populations: genetics

and phenotype of bush-cricket Pholidoptera frivaldskyi
(Orthoptera) in Carpathians
Peter Kanuch Benjamn Jarcuska
Elena Iulia Iorgu Ionut S tefan Iorgu
Anton Kristn

Received: 4 October 2013 / Accepted: 2 April 2014

Springer International Publishing Switzerland 2014

Abstract A decreasing population size is often causing

species extinction, however, relict species persisting in
small-sized populations counter this. We analysed spatial
genetic variation and past changes in population size at the
maternally-inherited mitochondrial DNA level to clarify
the origin of all recently known isolated populations of
Pholidoptera frivaldskyi occurring in the range of Carpathian Mountains. Along with that we analysed also morphological variation as some phenotypic traits can retain
useful information on population genetic structure. We
found a relatively low genetic diversity within isolated
populations as 778 bp COI gene sequences revealed only
13 unique haplotypes (n = 173 individuals from 10 populations). The spatial analysis of molecular variance identified three geographically homogenous genetic clusters
(one in Slovakia and two in Romania) with a high level of
differentiation among them, suggesting restricted gene
flow, whilst Bayesian skyline simulation reconstructed a
negative demographic change through evolutionary time.
Inferred genetic pattern clearly coincides with differences
in males colour phenotype as the extent of pigmentation
on the lateral pronotum varied significantly among genetic
lineages. We suggest that geographical variation in the
species populations has relict-like character and their isolated occurrence is not a result of recent introduction
events. Identification of evolutionary units may help in

P. Kanuch (&)  B. Jarcuska  A. Kristn

Institute of Forest Ecology, Slovak Academy of Sciences,
L. Stura 2, 960 53 Zvolen, Slovakia
e-mail: kanuch@netopiere.sk
E. I. Iorgu  I. S . Iorgu
Grigore Antipa National Museum of Natural History,
S os. Kiseleff 1, 011341 Bucharest, Romania

the conservation and management of this rare insect

species.
Keywords Phylogeography  Genetic drift  Population
decline  Mountain habitats  Tettigoniidae

Introduction
Climate change, fragmentation or loss of habitats and human
exploitation are the main reasons why many species survive
in small and isolated populations today (Hochkirch et al.
2007; Polus et al. 2007; Griebeler and Gottschalk 2010;
Holusa 2012). A decreasing number of reproducing individuals in a previously widespread population is often
causing local extinction due to inbreeding depression and
reduced fitness (Keller and Waller 2002; Willi et al. 2006).
However, the long-term persistence of some relict or endemic species in small-sized populations counter this extinction destiny and evolutionary processes such as speciation or
differentiation of population genetic structure take effect in a
restricted gene flow regime (e.g. Barascud et al. 1999;
Ritchie et al. 2001; Ciplak 2004; Krzysztofiak et al. 2010).
Gene flow among geographically isolated populations is
highly restricted in species that have a limited ability to
disperse. Good examples of these are found in flightless
insect species (e.g. Colembola, Cicconardi et al. 2010;
Orthoptera, Ritchie et al. 2001, Brouwers and Newton 2009;
Hemiptera, Phillipsen and Lytle 2013; Coleoptera, Garnier
et al. 2004, Keller et al. 2004). On the other hand, the disruption of gene flow among populations may also result from
an introduction mediated via passive transport. It was
observed that some insects with limited dispersal ability may
establish isolated populations far beyond their primary area
of distribution (Wilson et al. 2009; Kanuch et al. 2013).

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J Insect Conserv
Fig. 1 a Isolated populations of
P. frivaldskyi occurring in the
range of Carpathian Mountains
with a geographic distribution
of 13 haplotypes (coded as
pf113, GenBank accession
numbers KF706416
KF706428) based on a 778 bp
fragment of the mtDNA COI
gene (colours are congruent
with haplotype network). The
size of the circle is the number
of haplotypes in a population,
rarefied to represent 15
sequences per site (the real
numbers of haplotypes are
shown for populations STR,
SUC, and GOV due to a low
sampling success). b A
minimum spanning haplotype
network where each node
represents one mutational step
and haplotype frequencies are
represented by the size of the
circle. (Color figure online)

Thanks to increasing human activity and traffic, such events

are more frequent over the last century (di Castri 1989).
If there is restricted gene flow among populations, natural selection or genetic drift can also drive a rapid phenotypic divergence (Whitney and Gabler 2008). Natural
selection is likely to occur when a population is introduced
into a new environment where individuals are adapting
something that happens often during range expansion
(Lande 2009; Fauvergue et al. 2012). Contrary to that,
genetic drift may cause local morphological variation if
isolated populations suffer from a negative demographic
event and persist as a relict in the long term (Barascud et al.
1999; Ritchie et al. 2001; Krzysztofiak et al. 2010).
Knowledge about population origin, its colonisation history
and demographic changes over evolutionary time are prerequisites towards understanding the importance of local
genetic and/or phenotypic variations. Determination of
such significant evolutionary units may help in the conservation and management of a species distributed in an
isolated or scattered pattern (Palsbll et al. 2007; Ortego
et al. 2009; Habel et al. 2010; Kajtoch et al. 2014). For our
study, we chose Pholidoptera frivaldskyi (Herman, 1871),
an insect species which recent populations would potentially have various genetic and phenotypic characteristics.
It is an example of a very locally distributed bush-cricket
(Orthoptera, Tettigoniidae); with isolated populations
whose origins we could only hypothesise as either a historical relict or as a result of recent introduction.
Frivaldskys bush-cricket, originally described as
Thamnotrizon Friwaldszkyi from Romania (type locality

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Gyergyo, today Gheorgheni in Harghita), has been classified as a species with a Paleo-European origin (Warchalowska-Sliwa and Michailova 1993). Although
distributional and habitat data are scarce (Nagy 2005), P.
frivaldskyi was reported mostly from the Carpathian
Mountains (or Carpathians) region (Romania, Slovakia,
Ukraine), but also from the Balkans (e.g. Harz 1969;
Warchalowska-Sliwa et al. 2005; Fabriciusova et al. 2008;
Kristn et al. 2013). Currently, in the mountains of
Romania we can probably find about dozen of species
populations (Iorgu et al. 2008; Kristn et al. 2013). In
Slovakia, four other long-term known populations exist
(Ebner 1914; Kristn 2000; Fabriciusova et al. 2008) while
data from Ukraine is more than 130 years old and the
existence of some recent populations is doubtful (cf.
Lomnicki 1875, 1879; Storozhenko and Gorochov 1992).
Even so, it was reported from several mountain areas in
Bulgaria (Warchaowska-Sliwa and Michailova 1993) and
one male collected in Greece indicates the southernmost
known area of the species distribution (WarchaowskaSliwa et al. 2005). Other historical data from Serbia,
Bosnia and Macedonia would need updating (Chobanov
and Mihajlova 2010). All known populations are geographically isolated from each other and the distance
between neighbouring populations ranges from 33 to
183 km in the Carpathian region.
The species is an obligatory short-winged (body length
2027 mm), thus active dispersal is very limited. The
dominant body colour is solid light green with an individually specific pattern of black spots on the lateral

J Insect Conserv
Table 1 Sample size,
geographic coordinates, altitude
and characteristic type of the
habitat of P. frivaldskyi
populations in the Carpathian
Mountains

Code

Site (Countrya)

nM/nbF

Lon, Lat ()

Altitude (m a.s.l.)

Habitatc

HRB

Hrochotska Bukovina (SK)

Velke Biele Vody (SK)

9/10

19.43, 48.66

880

10/10

20.34, 48.94

597

Puste Pole (SK)

Strbske Pleso (SK)

10/10

21.44, 48.94

630

2/3

19.99, 49.12

1,170

C, M

9/12

23.64, 46.68

546

VBV
PUP
STR

SK Slovakia, RO Romania

VAN

Valcele (RO)
Vanatori Neamt (RO)

5/13

26.22, 47.23

470

nM males, nF females

SUC

Suceava-Bosanci (RO)

2/2

26.26, 47.58

357

BEC

10/14

26.71, 45.36

337

CAM

Beciu (RO)
Candes tiGiuvala (RO)

20/21

25.14, 45.38d

6751,182

P, M

GOV

Govora (RO)

1/0

24.22, 45.09

240

VAL

M well preserved meadow,

C forest clear-cut, R ruderal
plot, P regularly grazed pasture

Approximate midpoint

pronotum (shield) and on the dorsal part of the head. This

is the most variable morphological trait, while it is more
distinct in males (Fabriciusova et al. 2008). It was found as
a grassland-dwelling species but details about its biology
and ecology are almost unknown (Kristn 2000). The first
aim of this paper was to understand the population history
of P. frivaldskyi and to clarify the origin of isolated populations occurring in the Carpathian Mountainsthe primary species distribution area. To do this, we studied
phylogeographic patterns where we analysed spatial
genetic variation at the maternally-inherited mitochondrial
DNA level. Along with that, we analysed past changes in
population size using the same molecular marker. Secondly, we also aimed to analyse morphological variation
according to inferred population genetic structure. For this
purpose, we used the striking colour pattern of the shield as
a quantitative trait that is most characteristic of the species
phenotype.

Materials and methods

Sampling of populations
Using hand-nets, we collected in total 173 adult individuals
of P. frivaldskyi (78 males and 95 females) from ten sites
along the range of the Carpathian Mountains in central
(Slovakia) and in south-eastern (Romania) Europe
(N455.20 497.30 , E1925.80 2642.80 ), between 13 and
30 July 2012 (Fig. 1). We sampled often around 20 individuals at all recently known populations within this
region. However, at three sites, the sampling success was
low and only 15 individuals were collected (Table 1). For
all these populations except for one near Campulung
(CAM), samples were collected at a single site (meadow or
pasture of area 12 ha) where the species occurs in isolation. In CAM an abundant population extends continuously
over an exceptionally large area of mountain meadows and
pastures. Thus, two sampled sites were located at its

margins, ca. 28 km apart (Giuvala mountain pass and

Candes ti village; Table 1). Despite large altitudinal range
in this population, distribution of the species was continuous and non-interrupted by some geographical barriers.
As this spatial attribute contrasts significantly to other
populations, we designated it as a stronghold population
in our study.
Sequencing of mtDNA
In order to acquire enough genetic material and to save the
life of a rare species (i.e. let some chance for possible
reproduction), we only amputated one hind leg from each
individual and stored it in 96 % ethanol. After the procedure, all individuals were released at the site of capture.
DNA from muscle tissue was obtained using the DNeasy
Blood & Tissue Kit (Qiagen, Inc.) following the manufacturers instructions. Maternally-inherited mtDNA is
relatively conserved comparing to some nuclear genes, thus
it is suitable when searching for historical processes and
mostly used in phylogeography (Avise 2000). A fragment
of the mitochondrial cytochrome oxidase subunit I (COI)
gene was amplified, using the general insect primer pair
C1-J-2183
(50 -CAACATTTATTTTGATTTTTTGG-30 )
and TL2-N-3014 (50 -TCCAATGCACTAATCTGCCATATTA-30 , Simon et al. 1994), in 20 lL reaction mixtures
containing: 0.2 lM of each primer, 300 lm of each dNTP,
2.5 mm MgCl2, 19 Taq DNA buffer (Qiagen, Inc.),
0.5 lg/lL BSA, 19 Q-solution, 1 U of HotStar Taq DNA
polymerase (Qiagen, Inc.) and 1 lL of genomic mtDNA
(*100 ng lL-1). The polymerase chain reaction (PCR)
conditions consisted of an initial denaturation (95 C,
5 min) followed by 45 cycles of strand denaturation
(94 C, 30 s), primer annealing (47 C, 30 s) and extension
(72 C, 90 s), then a final extension step at 72 C for
10 min. The PCR products were purified using ExoSAP-IT
(Affymetrix, Inc.), then sequenced using a BigDye Terminator Sequencing Kit with both the forward and reverse
primers and analysed in an ABI3130XL Genetic Analyzer

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(Applied Biosystems, Inc.). Prior to generating a consensus

sequence for each individual, forward and reverse
sequences were assembled into contigs using SeqTrace 0.8
(Stucky 2012) and chromatograms were visually inspected
for accuracy of base calls. If some unreliable or double
peaks occurred (26 % of individual sequences had such
base calls but in very low frequency of 14 bp in a
sequence), nucleotide identity was left as missing at such
sites. In order to control for possible NUMTs (nuclear
copies of mtDNA), we translated sequences into amino
acids and checked for unexpected stop codons. After a
removal of low quality end sections of the sequenced
fragments and after trimming them to the same reading
length, we aligned sequences by the Clustal W algorithm in
BioEdit 5.0.9 (Hall 1999).
Spatial analysis of genetic diversity
The identities and frequencies of haplotypes were determined using DnaSP 5.0 (Librado and Rozas 2009). Sites with
missing nucleotide identities were not considered when
generating the haplotype list. In order to assess the relationship between haplotypes, we constructed a minimum
spanning haplotype network in HapStar 0.7 (Teacher and
Griffiths 2011). The geographic distribution of haplotypes
was analysed with respect to the sample size, thus the number
of haplotypes was rarefied with a rarefaction calculator
(available at http://www.biology.ualberta.ca/jbrzusto/rar
efact.php) to represent 15 sequences per site. In order to infer
geographically homogeneous genetic clusters that are maximally differentiated from other clusters we applied a spatial
analysis of molecular variance with SAMOVA 1.0 (Dupanloup et al. 2002). A simulated spatial annealing procedure (calculated with geographic distances) was performed
for a number of clusters (K = 25) to identify the optimal
number of population groups. We selected the optimal value
of K from the point where UCT values reached their maximum (plateau) and tested the strengths of the associations
within and between the identified clusters through an analysis of molecular variance (AMOVA) using Arlequin 3.5
software (Excoffier and Lischer 2010).
Historic population demography
Population demography in the past was estimated by two
different methods. Possible population expansion or contraction was tested by Fus FS and Tajimas D in Arlequin
3.5 (Excoffier and Lischer 2010). These frequency based
neutrality tests were calculated with 10,000 simulated
samples to detect population departure from mutation-drift
equilibrium (Wright-Fisher model). In order to model past
changes in effective population size we used a coalescentbased Bayesian skyline plot method in the BEAST 1.7.5

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software (Drummond and Rambaut 2007). This method

implements a Markov chain Monte Carlo (MCMC) sampling for intervals which are determined from coalescent
events and generates a posterior distribution of effective
population size through time (Drummond et al. 2005).
Before this simulation we applied an AICc selection criterion to choose the best-fit substitution site model using
jModelTest 2 (Darriba et al. 2012). All sequences were
sampled at the same time, thus, time was set relatively in
mutational units (substitution/site). A determination of
population size resulted from total effective population size
times mutation rate (Nel). MCMC chains were run for 10
million iterations and sampled every 10,000 iterations
while discarding the first 50,000 as burn-in. The result of
the simulation was examined in Tracer 1.5 (available at
http://beast.bio.ed.ac.uk/Tracer). An accurate timing of
demographic events by mutation units is limited for
changes during the past 10,000 years (cf. Ho et al. 2011,
Grant et al. 2012). Therefore, instead of a molecular clock
calibration, we used an interspecific comparison in our
demographic reconstruction (cf. OBrien et al. 2013). Here
we employed data of Metrioptera roeselii (raw data from
Kanuch et al. 2013), i.e. the species which occurs in recent
sites of P. frivaldskyi and which exhibits a rapid range
expansion (for details see Kanuch et al. 2013).
Morphological versus genetic variability
In the field, a side-portrait picture of each individual was
taken using a hand-held camera with a fixed zoom and flash
light. Subsequently, a digital-imaging analysis was applied
in order to estimate the ratio of a black spotted area on the
right side of the shield (lateral pronotum) in the software
ImageJ 1.46r (Schneider et al. 2012). The ratio of the black
area was calculated from the whole area of the lateral
pronotum measured along its distinct edge. The morphological data of males (females were omitted for these
purposes due to a low variability in colouration) was
combined with a Bayesian inference for phylogenetic
reconstruction. In MrBayes 3.2 software (Ronquist et al.
2012) we set the same best-fit evolutionary substitution site
model as for a demographic reconstruction. Using a
MCMC method, we twice ran four independent chains of
one million replicates and sampled every 1,000 generations. The first 5,000 generations were discarded as a burnin. The remaining trees were then used to calculate a 50 %
majority-rule consensus tree topology. Finally, a Kruskal
Wallis test was applied in order to compare the median
values of a measured phenotypic trait according to geographically homogeneous genetic clusters identified by
SAMOVA (see above). Unless otherwise noted, computation and plot drawings were performed in R 2.14.1 (R
Development Core Team 2011).

J Insect Conserv

frequency polymorphisms resulting from a population

contraction or balancing selection. Using the selected bestfit evolutionary HKY ? I substitution model, the Bayesian
skyline simulation reconstructed a negative change in
population size through evolutionary time. Considering the
mean mutation rate per nucleotide site, which is about 10-8
bp per generation in invertebrates (Lynch 2010), the start
of this demographic decline may date back to the Boreal
period (*42 ka before present, Fig. 2). Given the
uncertainty in the mutation rate of a specific mtDNA region
we cannot accurately date this event. However, using this
relative comparison with expanding M. roeselii, we can
clearly assume that the population size of P. frivaldskyi has
rapidly decreased (Fig. 2).
Spatial genetic structure

Fig. 2 Demographic reconstruction of estimated effective population

size of P. frivaldskyi resulting from the Bayesian skyline plot using
the mtDNA COI gene (black lines). Demographic reconstruction for
Metrioptera roeselii (raw data from Kanuch et al. 2013), the species
which occurs in recent sites of P. frivaldskyi and which exhibits a
rapid range expansion, is shown for comparison (grey lines). The zero
value of the relative time indicates the present, the solid lines are the
median population size and dashed lines are the upper and lower
95 % credible intervals

Results
Haplotype diversity and population demography
We found a relatively low mitochondrial diversity within
isolated populations of P. frivaldskyi. Analysis of the
778 bp COI gene sequences revealed only 21 (2.7 %)
variable sites (without missing nucleotide identities) with
13 unique haplotypes (GenBank accession numbers
KF706416KF706428) in 173 individuals from ten populations. The average haplotype diversity per site was also
low, i.e. only 1.03.7 haplotypes if rarefied to represent 15
sequences per site (Fig. 1). Surprisingly, the designated
stronghold population (CAM) in Romania did not have the
maximum diversity, though we had the highest sampling
effort there (Table 1). The minimum spanning haplotype
network showed a dumbbell pattern of two haplogroups
with two dominating haplotypes: pf1 which shared 33 %
and pf5 which shared 26 % of the individuals, respectively
(Fig. 1), with remarkably distant position of pf13 (nine
nucleotide substitutions from pf5) suggesting possibility
for separate phylogenetic lineage. Fus FS and Tajimas
D had positive, but non significant (p [ 0.05), values in all
populations (mean SD: Fus FS = 0.76 1.21, Tajimas D = 0.40 0.80). This may indicate a low level of

The geographical distribution of 13 haplotypes suggests

two distinct population units of P. frivaldskyi that became
separated in the Carpathian Mountains, while single specimen from GOV may indicate another population unit
located in southern Carpathians. The genetic profiles
clearly show a difference between Slovak and Romanian
populations (Fig. 1). However, the spatial analysis of
molecular
variance
identified
three
(maximum
UCT = 0.734, p \ 0.01) geographically homogenous
genetic clusters with a high level of differentiation among
them (Table 2); whereas significant pairwise FST values
(p \ 0.001) for all three clusters combinations were found
(Table 3). These clusters included the following populations (Table 3): Slovak sites (cluster 1), all Romanian sites
except Beciu (cluster 2) and site Beciu (BEC) in Romania
(cluster 3). The AMOVA confirmed the association of
clusters that were identified with the highest variation
found among clusters (73.4 %) and the lowest among
individuals within sites (11.7 %, Table 2). Thus, we considered the BEC as a genetically different population
compared to other Romanian sites.
Colour phenotypes of genetic lineages
A phylogenetic tree which plotted all sampled males identified three well supported ([92 %) main genetic lineages
(HKY ? I substitution model), and the extent of their pigmentation on the lateral pronotum varied significantly
among these lineages (Fig. 3). The ratio of black spotted
area ranged from 0.0 to 37.8 % on the males lateral
pronotum. Lineages inferred by this Bayesian procedure
clearly supported geographically homogeneous genetic
clusters identified by SAMOVA, which considered both
males and females in studied populations. The darkest males
were found in Slovak sites whilst the lightest were found in
the BEC. These represented the two most distant lineages

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Table 2 Partitioning of mtDNA variation at different hierarchical levels in samples of P. frivaldskyi in Carpathian Mountains based on an
analysis of molecular variance including three clusters identified in a SAMOVA
Source of variation

df

Among clusters

Variance components

Percentage of variation (%)

Fixation indices

293.7

2.574 Va

73.4

UCT = 0.734*

55.9

0.522 Vb

14.9

USC = 0.560**

163

66.7

0.409 Vc

11.7

UST = 0.883**

Among sites within clusters

Among individuals within sites

Sum of squares

Populations included in each cluster are shown in Table 3

* p \ 0.01, ** p \ 0.001, according to significance tests with 1,023 permutations
Table 3 Average number of pairwise differences between genetic
clusters (upper diagonal) and within cluster (diagonal elements in bold)
and FST values (lower diagonal) between clusters defined by SAMOVA
Cluster

1 (SlovakiaHRB, VBV, PUP, STR)

0.61

7.46

8.88

2 (RomaniaVAL, VAN, SUC, CAM, GOV)

0.783

2.42

4.16

3 (RomaniaBEC)

0.937

0.576

0.43

All FST values significantly differ from zero at p \ 0.001, according

to tests with 1,000 permutations

with an intermediate phenotype occurring among the rest of

the Romanian sites (see typical colour patterns in Fig. 3). A
male of intermediate phenotype (black spots 9.4 %) found in
GOV, having distinct haplotype (pf13), was clustered into
Romanian lineage by this Bayesian inference. The variability of a measured phenotypic trait differs highly among
three geographical clusters (v2 = 33.1, df = 2, p \ 0.0001)
and a post hoc comparison found all pairwise combinations
significantly different at p \ 0.01 (Fig. 3).

Discussion
Analysis of mtDNA sequences inferred a clear geographical pattern in the genetic variation of P. frivaldskyi populations in the Carpathian Mountains (Fig. 1). Combination
of single genetic marker with distinct individuals colour
phenotype strongly suggests separate genetic clusters with
restricted gene flow among populations at selected geographical scale (Fig. 3). Genetic isolation of populations in
long term also supports differences found in a species
karyological profile at a similar geographic scale (Warchaowska-Sliwa and Michailova 1993). In light of the fact
that this species experienced a recent population decline,
we will discuss the likely mechanism behind the genetic
and morphological variations we found.
Dispersal limits of P. frivaldskyi are unknown, however,
data from similar flightless congeneric bush-crickets suggests its low ability to cross geographical barriers or larger
areas of hostile habitat (e.g. Diekotter et al. 2005). Comparing the large inhabited area of abundant population near

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Campulung (CAM) in the southern Carpathians with other

populations, which are much smaller in size, one could
expect that some recent passive introductions would play a
role in population establishment. Historical movements of
livestock herders were found to be such a vector as they
introduce a range of insects with their fodder and stored
food or dunnage, while some introduced insects successfully colonised new areas (Vickers and Sveinbjarnardottir
2013). Similarly, the transport of hay and other agricultural
commodities mediated the large-distance translocation of
propagules of grassland-dwelling bush-crickets (Wagner
2004, Kanuch et al. 2013). As northern parts of the Carpathian Mountains (i.e. Slovakia) were colonised by Wallachian shepherds coming from the southern Carpathians in
Romania (Turnock 2003), the possibility for the unintentional transport of P. frivaldskyi existed. However, CAM as
a hypothetical source (founding) population, expected due
to its abundant population extending continuously over an
exceptionally large area, neither maintains a full spectrum
of haplotype diversity of putative progeny populations, nor
has a higher diversity compared to others (three observed
haplotypes differed in 12 substitutions only). In addition
to that, we also did not observe a starburst pattern in the
haplotype network structure, which would arise in the case
of a recent population expansion from a small number of
founders (Avise 2000). Thus, we do reject the hypothesis
about the current geographical distribution of P. frivaldskyi
populations as a result of introduction events.
On the other hand, we consider another historical scenario more likely. Firstly, a dumbbell pattern that was
observed in the haplotype network of P. frivaldskyi is
typical for allopatric geographic distributions of populations (Avise 2000). Taking into account the assumed low
dispersal abilities of the species, its present spatial distribution may be explained as the result of past gene flow
followed by the extinction of intermediate populations
(Avise 2000; Habel et al. 2010). However, restricted gene
flow is probably also within closely located populations in
southern Carpathians (BEC, GOV). Our data indicates that
a low level of local genetic diversity and restricted gene
flow between Romanian and Slovak populations resulted in
genetic drift, which also caused a significant amount of

J Insect Conserv
Fig. 3 Bayesian phylogenetic
unrooted tree of 78 males of P.
frivaldskyi sampled in the
Carpathian Mountains based on
mtDNA COI sequences inferred
by MrBayes software. Posterior
probabilities for nodes with
values higher than 50 % are
shown. Tip labels show a ratio
of black spots on the lateral
pronotum (three different colour
patterns are presented) with
reference to three
geographically homogeneous
genetic clusters defined by
SAMOVA. Box-plot represents
clusters medians, 2575 %
percentiles, non-outlier ranges
and outliers; p value
corresponds to non-parametric
KruskalWallis test. (Color
figure online)

morphological variation (cf. C

iplak 2004; Whitney and
Gabler 2008; Krzysztofiak et al. 2010). These results
confirmed that morphological traits can retain useful
information on population genetic structure (Ortego et al.
2012). Population differentiation using other morphological traits is also possible; however, more comprehensive
data from the species range is needed (Fabriciusova et al.
2008). A reconstructed negative demographic event suggests a post-glacial origin of the observed genetic and

morphological variation (cf. Ritchie et al. 2001) and it

remains a question which demographic, environmental or
climatic factor induced such a change (Hampe and Jump
2011). Similarly as in some other cold-adapted insects,
especially tundra-alpine species, it appears that the range of
P. frivaldskyi was much larger in the past cold periods and
recently isolated distributions are only relicts in warmstage refugia of former range (Grimaldi and Engel 2005;
Ashcroft 2010; Stewart et al. 2010). Different genetic

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lineages of P. frivaldskyi in the north-western and the

southern parts of the Carpathians suggest long lasting
separation of these two refugia (Schmitt 2009).
While the species was found in various grasslands, wellpreserved mountain habitats predominate there (Kristn
2000; Fabriciusova et al. 2008; Kristn et al. 2013). For
example, all four of the Slovak populations occur in protected mountain regions that have historically conserved
agriculture (National Parks, Biosphere and Nature
Reserves) and could indicate a beneficial effect of such a
traditional land-use management on species persistence.
Mountain regions that have diverse grassland habitat areas,
sustainably managed over the centuries as mown meadows
or sheep and cattle pastures are todays refuges for P. frivaldskyi and other rare species (Holusa 2012; Albrecht &
Haider 2013). Essentially, we are unsure of which environmental factors are causing a relict-like character of this
species occurrence (e.g. Coope 1994; Kajtoch et al. 2009;
Krzysztofiak et al. 2010). Regardless of missing ecological
data, due to a small inhabited area of the majority of these
isolated populations, the species is subjected to a high risk
of local decline (Kristn 2000). Based on current knowledge, we can not clearly predict whether some speciation is
evolving in these refuges (Ciplak 2004) or if species
extinction is pending (Britten et al. 1994). However, legal
protection (now in Slovakia only) along with a suitable
habitat management of recognised so-called management
units (i.e. discrete genetic populations by Palsbll et al.
2007) could be a means for conserving/prolonging the
current status.
Acknowledgments We would like to thank P. Tucek and M. Mikus
for helping us out in the field and for taking colour measurements of
the studied individuals. Two anonymous reviewers are greatly
acknowledged for valuable and insightful comments on a previous
version of this manuscript. The study was financed by the Slovak
Research and Development Agency (APVV-0497-10).

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