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Genetic Structure and Effective Population Sizes in European Red Deer (Cervus elaphus) at a Continental Scale: Insights from Microsatellite DNA


Abstract and Figures

We analysed more than 600 red deer (Cervus elaphus) from large parts of its European distribution range at 13 microsatellite loci, presenting the first continent-wide study of this species using nuclear markers. Populations were clearly differentiated (overall FST = 0.166, Jost’s Dest = 0.385), and the BAPS clustering algorithm yielded mainly geographically limited and adjacent genetic units. When forced into only three genetic clusters our data set produced a very similar geographic pattern as previously found in mtDNA phylogeographic studies: a western group from Iberia to central and parts of Eastern Europe, an eastern group from the Balkans to Eastern Europe and a third group including the threatened relict populations from Sardinia and Mesola in Italy. This result was also confirmed by a multivariate approach to analysing our data set, a discriminant analysis of principal components (DAPC). Calculations of genetic diversity and effective population sizes (linkage-disequilibrium approach) yielded the lowest results for Italian (Sardinia, Mesola; Ne between two and eight) and Scandinavian red deer, in line with known bottlenecks in these populations. Our study is the first to present comparative nuclear genetic data in red deer across Europe and may serve as a baseline for future analyses of genetic diversity and structuring in this widespread ungulate.
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Genetic structure and effective population sizes in European red deer (Cervus elaphus) at a
continental scale: insights from microsatellite DNA
Frank E. Zachos
, Alain C. Frantz
, Ralph Kuehn
4, 5
, Sabine Bertouille
, Marc Colyn
Magdalena Niedzialkowska
, Javier Pérez-González
, Anna Skog
10, 11
, Nikica Sprĕm
, Marie-
Christine Flamand
* These authors contributed equally.
1: Natural History Museum Vienna, Burgring 7, 1010 Vienna, Austria,
2: Musée National d’Histoire Naturelle, 25, Rue Munster, L-2160 Luxembourg,,
3: Fondation faune-flore, 25, Rue Munster, L-2160 Luxembourg
4: Unit of Molecular Zoology, Chair of Zoology, Department of Animal Science, Technische Universität
München, Freising, Germany
5: Department of Fish, Wildlife and Conservation Ecology, New Mexico State University, Las Cruces,
NM 88003-8003, U.S.A.
6: partement de l’Etude du Milieu naturel et agricole, Service Public de Wallonie, 23 Avenue
Maréchal Juin, 5030 Gembloux, Belgium
7: CNRS-UMR 6553, Université de Rennes 1, Station Biologique, 35380 Paimpont, France
8: Mammal Research Institute, Polish Academy of Sciences, Białowieza, Poland,
9: Grupo de Biología y Etología, Universidad de Extremadura, 10071 Cáceres, Spain
10: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of
Oslo, 0316 Oslo, Norway
11: Cancer Registry of Norway, 0304 Oslo, Norway
12: Department of Fisheries, Beekeeping, Game Management and Special Zoology, Faculty of
Agriculture, University of Zagreb, Zagreb, Croatia
13: Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-15, 1348 Louvain-
la-Neuve, Belgium
Corresponding author: Frank E. Zachos,
Journal of Heredity Advance Access published February 24, 2016
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We analysed more than 600 red deer (Cervus elaphus) from large parts of its European
distribution range at 13 microsatellite loci, presenting the first continent-wide study of this
species using nuclear markers. Populations were clearly differentiated (overall F
= 0.166,
Jost’s D
= 0.385), and the BAPS clustering algorithm yielded mainly geographically limited
and adjacent genetic units. When forced into only three genetic clusters our data set
produced a very similar geographic pattern as previously found in mtDNA phylogeographic
studies: a western group from Iberia to central and parts of Eastern Europe, an eastern
group from the Balkans to Eastern Europe and a third group including the threatened relict
populations from Sardinia and Mesola in Italy. This result was also confirmed by a
multivariate approach to analysing our data set, a discriminant analysis of principal
components (DAPC). Calculations of genetic diversity and effective population sizes (linkage-
disequilibrium approach) yielded the lowest results for Italian (Sardinia, Mesola; N
two and eight) and Scandinavian red deer, in line with known bottlenecks in these
populations. Our study is the first to present comparative nuclear genetic data in red deer
across Europe and may serve as a baseline for future analyses of genetic diversity and
structuring in this widespread ungulate.
The present genetic structure of large mammals in Europe is mainly due to: (1) signatures of
glacial-interglacial cycles with (in temperate species) southern refugia during glacials and
subsequent recolonization of northern regions, particularly after the Last Glacial Maximum
(LGM) (Hewitt 2000); (2) anthropogenic influences over the past centuries, e. g. selective
hunting, habitat fragmentation and translocations (Hartl et al. 2003, Frantz et al. 2006).
Genetic consequences of human interference are thus grafted onto natural phylogeographic
patterns, often blurring the intraspecific structuring of pre-human eras.
The red deer (Cervus elaphus) is arguably the most important European game species and,
consequently, has been impacted by humans for centuries or even millennia (Hartl et al.
2003). A multitude of studies on its genetic structure in Europe have been carried out, both
on a local, regional and continental scale (Zachos and Hartl 2011, Carden et al. 2012,
Niedzialkowska et al. 2012, Fernández-García et al. 2014, Karaiskou et al. 2014, Krojerová-
Prokesová et al. 2015). Studies on mitochondrial DNA (for a review see Zachos and Hartl
2011) have uncovered three main phylogeographic lineages in Europe: a western
haplogroup (designated A) distributed from Iberia through France and northern Central
Europe to the British Isles, Scandinavia and parts of Eastern Europe; an eastern haplogroup
(designated C) in the Balkans and parts of Eastern and Central Europe; and an isolated
lineage B restricted to the Tyrrhenian red deer (C. e. corsicanus) on Sardinia and Corsica and
the North African Barbary red deer (C. e. barbarus). The suture zone between the lineages A
and C appears to run from Austria through Poland and Belarus to the Baltic States
(Niedzialkowska et al. 2011, Fickel et al. 2012, Krojerová-Prokesová et al. 2015).
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While the overall continental pattern does not seem to have been blurred by human
interference (there are only few geographic outliers with respect to the three lineages,
Nussey et al. 2006), the areas where geographic lineages meet may or may not show the
natural distribution patterns. In red deer, this is a general issue, even more so at regional
and local scales, where it is often not clear if and to what extent populations are “pure”, i. e.
free from artificial introductions. Translocations of farmed red deer have occurred countless
times, and what is known about them is still only the tip of the iceberg (Linnell and Zachos
2011, Apollonio et al. 2014, especially Table 3.1, and references therein). Even if, as the
mtDNA phylogeographic studies suggest, translocations have mainly been carried out within
the main lineages rather than between them, these translocations have often covered large
geographic distances (Linnell and Zachos 2011). Of course, between-lineage translocations of
stags would not leave a signature in mitochondrial patterns; however, there does not seem
to be a male bias in translocations, and available documentation confirms that most of the
times females were translocated as well (e. g. Niethammer 1963). Local or regional red deer
stocks have also been intensively studied from a population genetic point of view, often
taking into account human impacts (Kuehn et al. 2003, 2004, Zachos et al. 2007, Frantz et al.
2008, Haanes et al. 2010a, b, Niedzialkowska et al. 2012, Fernández-García et al. 2014).
In this study, we present the first microsatellite data set covering most of the European
range of red deer to infer its nuclear genetic structuring. Contrary to the roe deer and wild
boar, the other two widespread European ungulate species (Randi et al. 2004, Scandura et
al. 2008), no such analysis exists for red deer to date. The present study aims at closing this
gap, for the first time allowing for nuclear genetic comparisons across the whole continent.
In particular, our study aims are:
(1) to uncover the large-scale nuclear genetic structure of the red deer in Europe and
compare it to the known mtDNA phylogeography that is believed to bear signatures of the
Quaternary climatic cycles, especially those of refugia during the LGM and subsequent
recolonization events;
(2) to use our microsatellite data set to calculate genetic diversity and effective population
sizes (N
) at a continent-wide comparative level to get further insights into the distribution
of genetic variability in this game species and to produce important and directly comparable
diversity parameters for the endangered Tyrrhenian (Corsica, Sardinia) and Mesola (NE Italy)
Material and Methods
Sample collection and laboratory work
The present study was based on 638 red deer tissue samples from 27 locations throughout
the continent (see Table 1 and Fig. 1). We also included 30 samples from a French deer farm
(Boisgervilly) in an attempt to understand the origin of these individuals and to test the
potential to identify farmed individuals in the European data set. The samples included both
new material and individuals already analysed in previous studies (Kuehn et al. 2003, 2004,
Feulner et al. 2004, Hmwe et al. 2006a, b, Zachos et al. 2007, Skog et al. 2009, Dellicour et al.
2011, Niedzialkowska et al. 2011, rez-González et al. 2012). DNA was extracted from new
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samples using a chloroform-based extraction method (Doyle and Doyle 1990. All samples
(old and new) were genotyped at 13 microsatellite loci (BM1818, Cer14, CSPS115, CSSM14,
CSSM16, CSSM19, CSSM22, CSSM66, ETH225, Haut14, ILSTS06, INRA35 and MM12; for
references see Kuehn et al. 2003) in three multiplex polymerase chain reactions (PCR) using
the Qiagen Multiplex kit (Qiagen, Hilden, Germany). Detailed information on the PCR
composition and reaction times can be found in Dellicour et al. (2011). Reactions were
performed using a Verity thermocycler (Applied Biosystems, Warrington, UK). PCR products
were separated using an ABI 3100 automated DNA sequencer (Applied Biosystems), and the
data were analysed using GeneMapper version 3.7 (Applied Biosystems). All individuals were
genotyped at 11 loci or more, with 629 of the 668 sampled having a complete 13-locus
Data analysis
We tested for the significance of heterozygote deficiency or excess (i. e. deviation from
Hardy-Weinberg equilibrium) in the 23 European sampling locations with N≥15 (Table S1,
excluding the deer farm) with the Markov-chain method in GENEPOP 3.4 (Raymond &
Rousset 1995), with 10,000 dememorisation steps, 500 batches and 10,000 subsequent
iterations. The populations were tested for pairwise linkage disequilibrium between loci
using an exact test based on a Markov-chain method as implemented in GENEPOP 3.4. The
false discovery rate technique was used to eliminate false assignment of significance by
chance (Verhoeven et al. 2005). Mean allelic richness per locus for each pre-defined
European population was calculated with FSTAT v. 2.9.3 (Goudet 1995) to standardize
measures for a population size of ten diploid individuals. Observed (Ho) and unbiased
expected (He
) heterozygosities (Nei 1978) for the same populations were estimated using
GENETIX 4.05.2 (Belkhir 2004).
We used STRUCTURE v2.3.1 (Pritchard et al. 2000) to estimate the number of
subpopulations (K). Ten independent runs of K=110 were carried out with 10
Markov chain
Monte Carlo (MCMC) iterations after a burn-in period of 10
iterations, using the model with
correlated allele frequencies and assuming admixture. ALPHA, the Dirichlet parameter for
the degree of admixture, was allowed to vary between populations. After deciding on the
most probable number of sub-populations based on the log-likelihood values (and their
convergence) associated with each K, we calculated each individual’s percentage of
membership (q), averaging q over ten runs. Bar plots of assignments were generated using
DISTRUCT 1.1 (Rosenberg 2004). We also used BAPS v5.4 (Corander et al. 2004) to perform a
population mixture analysis based on clustering individuals. This algorithm partitions the
data into populations with non-identical allele frequencies. The program was run for K = 2 to
30 with ten replications for each K.
A discriminant analysis of principal components (DAPC) was performed using the R-package
adegenet (Jombart 2008, Jombart et al. 2010) for R v. 2.12. (R Development Core Team
2011). This method, which is not based on any assumptions regarding the population genetic
model, first extracts information by applying a principal component analysis (PCA). In a
second calculation step a discriminant analysis (DA) maximizes the between-group
component of the genetic variation. The result of the DAPC can be visualized by using RGB
colour coding; the similarity of the dot colour represents the genetic similarity of the
populations (Jombart 2008, Jombart et al. 2010). In the first step of this procedure 50
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principal components of PCA were retained in order to explain approximately 90% of the
total variation of the data set analysed in this study. We carried out the DAPC at the
population level as we did not have coordinates for a large number of single deer specimens.
To quantify overall genetic differentiation within our data set we calculated the overall F
value (indicating which portion of of the overall variance was due to differentiation among
populations) with Arlequin 3.5 (Excoffier and Lischer 2010) and an estimator of Jost’s D (D
with GenAlEx 6.502 (Peakall and Smouse 2012). These calculations were carried out over all
populations listed in Table 1 (excluding the deer farm). GenAlEx was also used to identify
private alleles and their frequencies.
We estimated effective population sizes (N
) using a bias-corrected version of the linkage
disequilibrium (LD) method by Waples and Do (2008) as implemented in the N
Estimator v2
software (Do et al. 2014). This approach is based on the rationale that in small populations
with few parent individuals genetic drift will create non-random combinations of alleles of
different loci, i.e. LD. In general this approach is reliable if effective population sizes are not
much larger than ca. 200 and the data set is based on 10 or more loci and population sample
sizes of 25 or more. These conditions are not met for all our populations, so results should
be viewed with due caution in these cases. Since rare alleles (which occur frequently in
highly polymorphic markers like microsatellites) may have a disproportionately high impact
on the linkage values, the software offers different critical threshold values (we chose the
default values of 0.05, 0.02 and 0.01) below which alleles are not considered. We were
particularly interested in the values for the endangered subspecies from Sardinia and Mesola
which have undergone serious bottlenecks and for which no values of N
derived from
genetic data have ever been published. The present data set, the largest nuclear genetic on
European red deer so far, offers a good opportunity to estimate effective population sizes of
these deer and put them into a comparative perspective. We calculated N
values for pre-
defined populations, not for the clusters retrieved by BAPS because (i) differences between
the two were often small and (ii) BAPS uses marker independence as one of the clustering
parameters, so LD values might be affected by this clustering approach. Additionally, we only
chose those populations which had a sample size of n 15 and which were not obviously part of
a much larger continuous population (which is why we did not include the red deer from the
Data availability
We have deposited the primary data underlying these analyses (ie the microsatellite
genotpyes) in Dryad (doi XXX).
After correcting for multiple tests, we observed 15 instances of a locus deviating from HWE
in one of the 26 pre-defined populations (Table S1). Four loci (BM1818, CSSM19, CSSM66,
ETH225) significantly deviated from HWE in more than one population, 16 populations
showed no deviations from HWE at all. We concluded that no locus systematically deviated
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from HWE, but that the genetic characteristics of some populations (e.g. Wahlund effect,
immigrants, non-random sampling) led to the majority of the significant deviations from
HWE. All loci were therefore retained in subsequent analyses. No pairs of loci were
characterised by systematic linkage disequilibrium. Diversity values (allelic richness,
observed and expected heterozygosities) are given in Table 1. Across all three diversity
parameters, Sardinia and Mesola showed the lowest genetic diversity (as expected), but
other populations showed similarly low values for allelic richness (Norway, Sweden) or
heterozygosity (Polish and Romanian Carpathians). As expected given our comprehensive
geographical sampling we found private alleles in several populations. Most populations only
had a single private allele, but Berchtesgaden in Germany had five (all at low frequencies of
less than 3.5%). Sardinia, arguably the evolutionarily most divergent population in our data
set, only had a single private allele which, however had a frequency of 40.6%!
Genetic structuring across Europe
The results of the STRUCTURE analysis showed that the independent runs did not converge
on an optimal solution. Log-likelihood values gradually increased, without reaching a higher
value of K where they converge reasonable well, and started to decline again after K=12 (Fig.
S1). The best convergence of log-likelihoods was obtained for K=2, K=4 and K=7. However,
even at these three values of K, assignments of individuals differed fairly widely between
runs of the same K. For example, at K=2, we obtained six different clustering solutions (Fig.
The individual-based modal population mixture analysis in BAPS inferred the presence of 26
genetic populations. The majority of the sampling locations formed distinct sub-populations
(Fig. 1). The samples from eastern Poland formed a genetic cluster with the samples from
northern and eastern Germany. Seven clusters consisted of six individuals or less. Four deer,
which had been sampled in Croatia/Slovenia, Norway, SE Germany and SE Poland,
respectively, formed single-individual partitions (not shown in Fig. 1). The SE Polish and NE
Italian (Mesola) populations, as well as the deer farm, each contained a few individuals that
formed distinct clusters (Fig. 1). The remaining deer farm individuals were grouped with the
Scottish and Eastern European clusters and it was not possible to unequivocally identify
farmed individuals in the rest of the data set.
When forced to assign all European individuals to only three clusters (K = 3), BAPS produced
a geographical pattern very much like that known from the three mtDNA lineages (Fig. S3):
there is a clear separation of western from eastern red deer with an overlap of these two
groups in eastern Central Europe and Poland. Sardinia, together with Mesola (NE Italy) and
Norway, constitutes the third cluster (at K = 4, Norway is separated from Sardinia/Mesola).
We checked for the occurrence of otherwise rare alleles in these three populations as a
possible explanation for their clustering. However, while we did find rare alleles at several
loci shared by Mesola and Sardinia, we did not find any that were shared also by Norway.
The DAPC yielded results in accordance with those from BAPS (Fig. 2). Sardinia, Mesola,
Norway and Sweden were most divergent genetically, masking differentiation among the
remaining populations. But again, when removing these four outliers from the analysis, we
found a west-east dichotomy of red deer populations (Fig. 2, right map). The Belgian red
deer were somewhat genetically differentiated from its surrounding populations. The allelic
richness values characterising the populations from Sardinia, Mesola, Norway and Sweden
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were the lowest in the data set (Fig. 3, Table 1). Furthermore, populations in France, eastern
Croatia and Iberia had low levels of allelic richness, while deer in central Europe and Poland
had the highest. The overall F
value was 0.166 (p < 0.00001), indicating that 16.6% of all
genetic variance was due to differences among populations as opposed to variability within
populations (83.4%). Jost’s D
was 0.385 (significant at p = 0.001).
Effective population sizes of European red deer
values as calculated with the LD method are given in Table 2. Values above 200 and for
samples much smaller than 25 should be viewed with due caution (see above). In line with
the diversity parameters (Table 1) values for Sardinia and Mesola were the lowest (between
2.0 and 8.2), and no other populations show similarly low effective population sizes,
although some do show values that are below 50 (e. g. Sweden, Norway and Schleswig-
Holstein in northern Germany), which is often viewed as a threshold below which inbreeding
depression is likely to occur. The comparison of different threshold values also shows that
rare alleles sometimes have a large impact on the result for a given population but do not
change the overall picture.
Our analyses of more than 600 individual multi-locus genotypes of European red deer have
uncovered substantial structuring across the continent and, as expected given the higher
mutation rates in microsatellites, the overall nuclear genetic structure was more complex
than that found in phylogeographic studies based on mtDNA. Microsatellites are generally
more appropriate for the detection of small-scale and/or more recent structuring but the
European data set once more confirmed the genetic uniqueness of both the Sardinian and
the Mesola red deer and also yielded similar patterns to those uncovered by mtDNA
European genetic structure and phylogeography
Due to convergence problems, the STRUCTURE results were inconclusive. Re-running the
analysis using the substantially longer burn-in of 10
did not solve the issue (results not
shown). To the best of our knowledge, STRUCURE does not allow a formal assessment of the
convergence of the MCMC chains, via the statistic by Gelman and Rubin (1992), for example.
While similar problems have been reported (e.g., Frantz et al. 2014), the issue is particularly
striking here. We therefore limited our inference to the results obtained by the BAPS
algorithm. Contrary to STRUCTURE, the overall picture provided by BAPS was consistent in
that Sardinian, Mesola, Norwegian and, to a lesser extent, Swedish red deer were the most
divergent of the European populations. Also, the BAPS and the DAPC analyses retrieved a
clear signal of genetic divergence between western and eastern Europe.
The BAPS analysis yielded 26 distinct genetic clusters across Europe. Even if this is an
overestimate (BAPS can overestimate the number of genetic clusters because it has a
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tendency to identify populations consisting of only a few individuals, as observed here as
well; Latch et al. 2006), it clearly shows the differentiation at a comparatively small
geographical scale in European red deer. In line with this, both F
and Jost’s D
significant differentiation at a rather high level. Our F
of 0.166 is almost exactly the same as
that found for another cervid species with a similar distribution range in Europe, the roe
deer (Capreolus capreolus; F
across Europe based on 704 specimens and 11 microsatellite
loci was found to be 0.16 by Randi et al. 2004). As expected, overall F
was much higher for
mtDNA control region sequences (because within-population diversity is lower; F
= 0.84,
Skog et al. 2009).
Most BAPS clusters comprise local populations and/or mostly geographically adjacent
sampling sites. Given the mutation rates of microsatellites, this is an expected outcome and
in line with many microsatellite studies on red deer at local or regional scales (e. g. Feulner
et al. 2004, Nielsen et al. 2008, Haanes et al. 2011, Niedzialkowska et al. 2012, Höglund et al.
2013, Karaiskou et al. 2014, Krojerová-Prokesová et al. 2015). However, if low K values of the
BAPS analysis are considered, the geographical distribution of the clusters are very
interesting. Indeed, if all European red deer are clustered into only three groups, the
geographical pattern bears a striking resemblance to the phylogeographic pattern derived
from mtDNA: two main groups (west and east, respectively) that show an overlap in Central
Europe and Poland, and a minor group containing the red deer from Sardinia. These groups
correspond to the mtDNA lineages A (west), C (east) and B (Sardinia and North Africa).
Instead of the North-African Barbary deer (of which unfortunately no samples were available
for the present study), the microsatellite analysis particularly groups the Sardinian red deer
with Mesola whose mtDNA affinities are somewhat intermediate between the western and
eastern clade (Skog et al. 2009, Niedzialkowska et al. 2011), with the most recent study
favouring closer relationships to the eastern group (Lorenzini and Garofalo 2015). Norway
becomes separated from this group at the next higher level of K = 4. The DAPC confirmed
these results in that after the removal of the outlier populations there was a clear
differentiation between Western, Central and Central-Eastern Europe on the one hand and
South-Eastern and southern Central Europe on the other. In red deer, concordance between
mtDNA phylogroup distribution and microsatellite structuring has been found before, albeit
at a smaller geographical scale, in the Czech Republic (Krojerová-Prokesová et al. 2015) and
in Greece (Karaiskou et al. 2014).
The most convincing explanation for the biogeographic pattern observed in the present
study is that the microsatellite structure of red deer across Europe still carries a signature of
the postglacial recolonization process from two main glacial refugia (Iberia/southern France
in the west, the Balkans and possibly the Carpathian region in the east, Sommer et al. 2008).
In line with this, values of allelic richness are highest where the two main BAPS clusters and
DAPC groups (west and east) meet in Central Europe and Poland, which is also where the
western and eastern mtDNA lineages co-occur (Niedzialkowska et al. 2011, Fickel et al. 2012,
Krojerová-Prokesová et al. 2015). To what extent this zone of overlap is natural, however,
cannot be definitively answered due to the high number of translocations that are known to
have occurred. It would be interesting to see whether phylogeographic data from one or
more nuclear markers with lower mutation rates than microsatellites also confirm the large-
scale pattern of three groups found in mtDNA and microsatellites.
Within the West-Palaearctic red deer, the Tyrrhenian red deer from Corsica and Sardinia (C.
e. corsicanus) and the North-African Barbary red deer (C. e. barbarus) comprise a distinct
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mtDNA lineage (B), and their phylogeographic history is not entirely clear, e. g. it is still being
debated whether the Tyrrhenian deer are derived from introduced Barbary deer or vice
versa (see Zachos and Hartl 2011 and references therein). If the Tyrrhenian deer are
descendants of Italian mainland deer, then they should be closely related to the red deer
from Mesola (recently described as C. e. italicus, Zachos et al. 2014) which are the last
surviving native Italian red deer. While this is not supported by mtDNA studies, close
affinities between the two have been found based on microsatellites (Hajji et al. 2008). It is
interesting to note that the present data set of 13 microsatellites none of which is identical
to the eight loci used by Hajji et al. (2008) yielded the same result of a close relationship
between C. e. corsicanus and C. e. italicus. It seems therefore unlikely that these results are
simply an artefact due to drift effects in two recently severely bottlenecked populations for
this to be true the bottlenecks would have to have resulted in similar and unique allele
frequencies in two completely non-overlapping sets of altogether 21 loci. Rather, it seems
more likely that the result is indicative of a true signal of phylogeographic relationships
between mainland Italian and Sardinian/Corsican red deer, a question that ultimately only
ancient DNA studies will be able to answer.
Genetic diversity and effective population sizes
Genetic diversity values were in the range known for microsatellite loci in red deer (see
Table 1 in Zachos and Hartl 2011 for a compilation of values from all over Europe). The
lowest values when considering both allelic diversity/richness and heterozygosities were
expectedly found in the severely bottlenecked populations from Sardinia and Mesola in Italy,
and known bottlenecks can also account for the low diversity (at least in terms of allelic
richness) found in Scandinavian red deer from Sweden and Norway which is in accordance
with findings from other studies (Haanes et al. 2010a, b, 2011).
We present here also the first estimation of effective population sizes in red deer across a
large area of their distribution. Overall, N
values were in the range of, although with lower
maximum values than, those previously calculated for German and Spanish red deer based
on genetic and demographic data (Martinez et al. 2002, Kuehn et al. 2003). The calculations
have also confirmed the genetic depletion of the red deer from Sardinia and Mesola as a
consequence of past bottlenecks and near-extinction. Values between two and eight are the
lowest in Europe and even considerably lower than the N
= 20 calculated with the same
approach (LDNe) for the endangered Kashmir red deer or hangul (C. e. hanglu) whose total
census population size is estimated at just above 200 (Mukesh et al. 2015). While both the
Sardinian and Mesola red deer have increased in numbers recently and are not threatened
with immediate extinction anymore, the long-term consequences of low genetic diversity
and inbreeding remain unclear. While overall the LD approach is viewed as a reliable
method, there are many unknowns in any calculation of effective population size (Luikart et
al. 2010). The values therefore might best be viewed in a comparative context rather than as
absolute values for each of the populations separately.
Although a common and locally abundant game animal today, the red deer faced extirpation
in many parts of its range during past centuries. Documentation on recolonization whether
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natural or human-mediated is usually scarce, and what is known from the literature (e. g.
Niethammer 1963, Apollonio et al. 2014) is almost certainly only the tip of the iceberg. In
fact, it is believed that the present gene pool of many if not most free-living populations of
red deer in Europe contains at least some genetic material that goes back to introductions
(Hartl et al. 2003). Evidence for purely autochthonous populations is rare (and usually not
conclusive), with some possible examples being red deer in Mesola (Zachos et al. 2014),
Skåne in southern Sweden (Höglund et al. 2013) and the Scottish Highlands (Pérez-Espona et
al. 2009). Although genetic analyses are a powerful means to elucidate the status of
populations with respect to their natural or anthropogenic origin (see Kuehn et al. 2004,
Frantz et al. 2006), such analyses have not been carried out for most of the distribution
range. Many of the populations analysed in the present study will therefore not be
completely natural units (it is known, for example, that the Châteauroux red deer have
partly been introduced from the Domaine National de Chambord). However, a “purist”
approach allowing only completely native populations in a species as deeply impacted
anthropogenically as the red deer in Europe is neither feasible nor would it, in our view, be
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Fig. 1. Location of the genetic populations inferred using the individual-based BAPS
algorithm. The size of the pie charts indicates the number of samples collected from a
locality, while the pattern of the pie chart indicates the identity of the genetic clusters. The
four deer that had been sampled in Croatia/Slovenia, Norway, SE Germany and SE Poland
and that formed single-individual partitions were omitted from the plot. For the pattern
based on only three genetic clusters, see Fig. S3 in the Appendix.
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Fig. 2. DAPC of European red deer populations. Similar RGB colour codes signify genetic similarity. Sardinia, Mesola, Norway and Sweden are genetically very
different from the rest of Europe, effectively veiling differentiation among the latter (left). When running the analysis without these four outliers (right), the
European pattern largely shows a dichotomy between a western group (red-purple colours) that ranges from Iberia through western Europe and the British
Isles to eastern central Europe, and an eastern group (green-blue colours) in the Balkans and southern central Europe. In Central and Eastern Europe these
groups admix (brownish colours). This is in accordance with both mtDNA phylogeography and the BAPS results for K = 3. The Belgian red deer are the only
outliers in this pattern (yellow dots).
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Fig. 3. Microsatellite-based allelic richness measures for 28 pre-defined European red deer
populations (incl. the deer farm). The estimate of allelic richness is based on a sample of 10 diploid
individuals and 13 microsatellite markers. Sardinia, Mesola, Norway and Sweden show the lowest
values (little black squares).
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Table 1: Geographic distribution of the European samples analysed in this study and summary of
genetic diversity measures. A
: allelic richness; Ho: observed heterozygosity; He
: unbiased expected
Microsatellite diversity
Wallonia NE
Wallonia central
Wallonia West
E Croatia
NW Croatia / S Slovenia
Central France (Châteauroux)
E France (Meurthe)
NW France (Hardouinais, Brittany)
E Germany (Saxony)
N Germany (Schleswig-Holstein)
NE Bavaria (Fichtelberg/Goldkronach)
NE Germany (Mecklenburg)
SE Germany (Berchtesgaden)
N Italy (Southern Tyrol/Vinschgau)
NE Italy (Mesola)
W Norway (Sogn og Fjordane)
E Poland (Białowieża)
NE Poland (Warmia-Masuria Province)
SE Poland (N Carpathians)
SE Romania (Carpathians)
NE Serbia (Bachka)
SE Spain (Andalucía)
W Spain (Extremadura)
S Sweden (Skåne)
Deer Farm
France, Brittany (Boisgervilly)
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Table 2. Effective population sizes (N
) as calculated with NeEstimator based on the linkage disequilibrium approach. For each population, N
values are
given for three different thresholds for the lowest allele frequency used. The values in parentheses are the 95% confidence intervals based on jackknifing on
loci. n: sample size. “infinite” values of N
refer to cases where there is no evidence of variation of the genetic characteristic due to finite numbers of
parental individuals, i. e. all can be explained by sampling error (Do et al. 2014). Only those populations are included for which evidence was present that
they were not just artificially designated sample sites and for which n was ≥ 15. The two Spanish populations, after mostly yielding infinite values separately,
were pooled.
Population n Effective population size (N
Frequency threshold: 0.05 0.02 0.01
Sardinia 16 4.3 (2.3 10.4) 8.2 (3.3 - 17.1) 8.2 (3.3 17.1)
Mesola 22 2.0 (1.4 3.0) 2.6 (1.8 5.3) 2.6 (1.8 5.3)
Sweden 16 infinite (9.8 infinite) 20.4 (7.8 549.8) 20.4 (7.8 549.8)
Norway 31 40.6 (16.9 532.1) 30.2 (15.5 87.8) 10.3 (3.9 21.6)
Schleswig-Holstein 19 19.2 (13.7 29.0) 26.2 (18.6 40.5) 26.2 (18.6 40.5)
Saxony 15 infinite (122.2 infinite) 283.5 (69.8 infinite) 283.5 (69.8 infinite)
Serbia 19 303.7 (41.1 infinite) 131.3 (37.1 infinite) 131.3 (37.1 infinite)
E Croatia 53 480.6 (128.7 infinite) 1384.1 (206,6 infinite) 1127.7 (217.6 infinite)
NW Croatia/S Slovenia 49 84.5 (52.5 177.2) 155.4 (93.1 394.9) 139.5 (80.6 397.4)
Berchtesgaden 29 46.2 (32.1 75.4) 51.7 (35.2 89.1) 41.4 (28.0 71.2)
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Fichtelgebirge 31 28.8 (20.2 44.7) 38.3 (27.7 57.7) 41.1 (30.4 59.9)
Liechtenstein 29 139.6 (60.3 infinite) 166.0 (75.3 infinite) 156.3 (74.0 infinite)
Vinschgau (Italy) 26 101.0 (45.6 infinite) 119.5 (54.9 infinite) 149.5 (62.3 infinite)
E France (Meurthe) 27 42.0 (22.1 144.8) 57.6 (30.3 229.9) 79.2 (40.8 424.2)
NW France (Hardouinais) 22 77.7 (26.6 infinite) 176.4 (45.1 infinite) 176.4 (45.1 infinite)
C France (Châteauroux) 23 62.1 (27.0 infinite) 85.1 (40.6 1797.9) 85.1 (40.6 1797.9)
Spain (pooled) 30 38.0 (24.9 68.8) 42.1 (28.8 70.4) 68.0 (45.3 124.8)
NE Wallonia 20 infinite (118 infinite) infinite (121.4 infinite) infinite (121.4 infinite)
C Wallonia 20 82.8 (35.9 infinite) 114.3 (45.6 infinite) 114.3 (45.6 infinite)
W Wallonia 20 126.2 (44.2 infinite) 167.5 (57.4 infinite) 167.5 (57.4 infinite)
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... The genetic structure of the red deer in Europe has been widely investigated (Ludt et al. 2004;Skog et al. 2009;Perez-Espona et al. 2009;Niedziałkowska et al. 2011;Fernández-García et al. 2014;Zachos et al. 2016;Rey-Iglesia et al. 2017;Schnitzler et al. 2018;Doan et al. 2022) even though a small percentage of data involved peninsular Italian populations (Lorenzini et al. 2005;Hmwe et al. 2006A, BA;Zachos et al. 2016;Doan et al. 2017Doan et al. , 2022. Doan et al. (2022) also highlighted Italy as one of the countries with the lowest genetic amount of data as to red deer populations. ...
... The genetic structure of the red deer in Europe has been widely investigated (Ludt et al. 2004;Skog et al. 2009;Perez-Espona et al. 2009;Niedziałkowska et al. 2011;Fernández-García et al. 2014;Zachos et al. 2016;Rey-Iglesia et al. 2017;Schnitzler et al. 2018;Doan et al. 2022) even though a small percentage of data involved peninsular Italian populations (Lorenzini et al. 2005;Hmwe et al. 2006A, BA;Zachos et al. 2016;Doan et al. 2017Doan et al. , 2022. Doan et al. (2022) also highlighted Italy as one of the countries with the lowest genetic amount of data as to red deer populations. ...
... Therefore, although the original population in ACQUERINO derived from free-ranging individuals captured in North-Eastern Italy (Mazzarone and Mattioli 1996), several unofficial releases from local caged areas may have occurred in the last 50 years, thus contributing to this genetic diversity. Genetic studies on the red deer have involved mitochondrial DNA sequences (Ludt et al. 2004;Skog et al. 2009;Perez-Espona et al. 2009;Niedziałkowska et al. 2011;Fernández-García et al. 2014;Borowski et al. 2016;Schnitzler et al. 2018;Queiròs et al. 2019;Doan et al. 2022) andmicrosatellites (Hwme et al. 2006B;Zachos et al. 2016). These authors have shown that the large-scale genetic structure of European red deer has been shaped by the Late Pleistocene and Holocene glacial-interglacial cycles. ...
The red deer Cervus elephus has been a common species in Italy until the Middle Ages and the Renaissance, when its distribution range started to considerably decrease, due to gradual deforestation and hunting pressure. Afterwards, the red deer has been reintroduced to many regions of the world, including Italy. In the Italian Apennines, the Acquerino-Cantagallo Natural Reserve (ACQUERINO) hosts one of the largest peninsular red deer populations, originated from a series of successful reintroductions. In this study, we meant to detect the level of genetic variability of Acquerino-Cantagallo Natural Reserve deer population and to investigate the genetic relationships with the other Italian and European populations. We identified five mitochondrial DNA control region (D-loop) haplotypes, four falling in lineage A and one falling in lineage C, derived from at least two maternal lineages, confirming that ACQUERINO population should be the result of multiple reintroductions. Haplotype diversity (H = 0.50) and nucleotide (π = 0.004) diversity were low, but included into the deer range values. ACQUERINO population showed low levels of genetic diversity when compared to other European and Mediterranean populations, confirming that this expanding population may have been generated from a low number of founders.
... Red deer Cervus elaphus stags produce their rutting calls for attracting potential mates and deterring competitive males (Clutton-Brock and Albon 1979). Studies of acoustic variation of stag rutting calls (Frey et al. 2012;Passilongo et al. 2013;Della Libera et al. 2015;Volodin et al. 2015aVolodin et al. ,b, 2019Golosova et al. 2017) are in agreement with the subdivision of red deer to phylogenetic lineages (Mahmut et al. 2002;Ludt et al. 2004;Skog et al. 2009;Zachos and Hartl 2011;Zachos et al. 2016). The acoustics of stag rutting calls proved to be helpful population markers in red deer (Frey et al. 2012;Passilongo et al. 2013;Volodin et al. 2019) in addition to the genetic markers, such as mtDNA and microsatellites (Feulner et al. 2004;Niedziałkowska et al. 2012;Krojerova-Prokešova et al. 2015;Carranza et al. 2016;Zachos et al. 2016). ...
... Studies of acoustic variation of stag rutting calls (Frey et al. 2012;Passilongo et al. 2013;Della Libera et al. 2015;Volodin et al. 2015aVolodin et al. ,b, 2019Golosova et al. 2017) are in agreement with the subdivision of red deer to phylogenetic lineages (Mahmut et al. 2002;Ludt et al. 2004;Skog et al. 2009;Zachos and Hartl 2011;Zachos et al. 2016). The acoustics of stag rutting calls proved to be helpful population markers in red deer (Frey et al. 2012;Passilongo et al. 2013;Volodin et al. 2019) in addition to the genetic markers, such as mtDNA and microsatellites (Feulner et al. 2004;Niedziałkowska et al. 2012;Krojerova-Prokešova et al. 2015;Carranza et al. 2016;Zachos et al. 2016). ...
... The results of the 8-loci microsatellite analysis also confirmed the low genetic diversity in Voronezh red deer. Six of these 8 loci were previously used in the largest-scale microsatellite analysis of European red deer (Zachos et al. 2016;Frantz et al. 2017), so the obtained data are well comparable with those on other red deer populations. It was expected that all indices of genetic diversity would be the lowest for Voronezh red deer compared to Pannonian and Iberian red deer (Table 4). ...
Full-text available
This study investigates a population of red deer Cervus elaphus, founded by 10 individuals introduced in the nineteenth century from Germany to the Voronezh region of the European part of Southern Russia and then developed without further introductions. We characterize for the first time the vocal phenotype of the Voronezh red deer male rutting calls in comparison with similar data on the Pannonian (native Central European) and Iberian (native West European) red deer obtained by the authors during preceding studies. In addition, we provide for the first time the genetic data on Pannonian red deer. In Voronezh stags, the number of roars per bout (2.85 ± 1.79) was lower than in Pannonian (3.18 ± 2.17) but higher than in Iberian (2.11 ± 1.71) stags. In Voronezh stags, the duration of main (the longest within bouts) roars was longer (2.46 ± 1.14 s) than in Pannonian (1.13 ± 0.50 s) or Iberian (1.90 ± 0.50 s) stags. The maximum fundamental frequency of main roars was similar between Voronezh (175 ± 60 Hz) and Pannonian (168 ± 61 Hz) but higher in Iberian stags (223 ± 35 Hz). Mitochondrial cytochrome b gene analysis of red deer from the three study populations partially supports the bioacoustical data, of closer similarity between Voronezh and Pannonian populations. In contrast, microsatellite DNA analysis delineates Voronezh red deer from either Pannonian or Iberian red deer. We discuss that population bottlenecking might affect the acoustics of the rutting roars, in addition to genotype.
... The genus Cervus is widely distributed in the Holarctics. Its European member, the red deer (Cervus elaphus L. 1758) inhabits a wide range of environments [23][24][25]. Deer have cultural, ecological and an increasing economic importance. Being among the most important game animals for trophies, their populations have been managed, translocated and selectively hunted throughout their history and distribution area [26][27][28][29][30]. ...
... Genetic identification of red deer has become very important in forensic and population genetics as well as for wildlife conservation and parentage testing in animal breeding [25,31,34,35]. Although microsatellite markers have been used in red deer, genetic studies mostly rely on adopted STR markers originally developed in other cervids [31,35,36]. ...
... We believe that the newly developed X-and Y-chromosome markers have the potential to provide a useful tool for recording male-specific gene flows, thus are feasible for use in population and evolutionary studies, especially when combined with mitochondrial and nuclear markers. Combinations of our newly developed markers with nuclear marker sets developed recently so far for genetic studies of red deer populations [25,31,35] will provide a complex toolkit for investigating their genetic structure at fine resolution. A good example, as demonstrated in our study here (Figs 6 and 7), is that with ten DeerPlex STRs supplemented with the five Y-and ten X-chromosomal STRs, the Principal Component Analyses (PCoAs) were improved, and we moved closer to our primary aim, i.e. studying correlations between geographical origins and genetic diversity of red deer in the Carpathian Basin, shown best in the case of Gemenc (SWG) vs. Zemplén (NE). ...
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Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.
... Studies of non-human species that utilized a hierBAPSbased phylogeny vary with respect to the description of the relationships between subclades and the genetic material being analyzed, for example, mtDNA [63] or chloroplast DNA and genomic markers [64][65][66]. These studies are typically supplemented by additional analyses, such as admixture and estimates of genetic diversity, or the addition of other biomarkers in the population, to draw inferences about their geographical dispersal [64][65][66]. ...
... Studies of non-human species that utilized a hierBAPSbased phylogeny vary with respect to the description of the relationships between subclades and the genetic material being analyzed, for example, mtDNA [63] or chloroplast DNA and genomic markers [64][65][66]. These studies are typically supplemented by additional analyses, such as admixture and estimates of genetic diversity, or the addition of other biomarkers in the population, to draw inferences about their geographical dispersal [64][65][66]. ...
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Background We combined an unsupervised learning methodology for analyzing mitogenome sequences with maximum likelihood (ML) phylogenetics to make detailed inferences about the evolution and diversification of mitochondrial DNA (mtDNA) haplogroup U5, which appears at high frequencies in northern Europe. Methods Haplogroup U5 mitogenome sequences were gathered from GenBank. The hierarchal Bayesian Analysis of Population Structure (hierBAPS) method was used to generate groups of sequences that were then projected onto a rooted maximum likelihood (ML) phylogenetic tree to visualize the pattern of clustering. The haplogroup statuses of the individual sequences were assessed using Haplogrep2. Results A total of 23 hierBAPS groups were identified, all of which corresponded to subclades defined in Phylotree, v.17. The hierBAPS groups projected onto the ML phylogeny accurately clustered all haplotypes belonging to a specific haplogroup in accordance with Haplogrep2. By incorporating the geographic source of each sequence and subclade age estimates into this framework, inferences about the diversification of U5 mtDNAs were made. Haplogroup U5 has been present in northern Europe since the Mesolithic, and spread in both eastern and western directions, undergoing significant diversification within Scandinavia. A review of historical and archeological evidence attests to some of the population interactions contributing to this pattern. Conclusions The hierBAPS algorithm accurately grouped mitogenome sequences into subclades in a phylogenetically robust manner. This analysis provided new insights into the phylogeographic structure of haplogroup U5 diversity in northern Europe, revealing a detailed perspective on the diversity of subclades in this region and their distribution in Scandinavian populations.
... Assuming that red deer populations in a geographically narrow region such as the study area should naturally be in a lively genetic exchange, the anthropogenic fracturing of the landscape in this region appears to have a considerable impact. These findings are in line with previous studies that identified motorways as obstacles to gene flow in red deer (Kinser and Herzog 2008;Frantz et al. 2012;Zachos et al. 2016). ...
... In absolute terms however, the data of the Hessian population show rather favourable heterozygosity and lower Fis values in comparison with other national (Poetsch et al. 2001;Kuehn et al. 2003;Zachos et al. 2007;Edelhoff et al. 2020) and international studies (Hmwe et al. 2006a, b;Nussey et al. 2007;Nielsen et al. 2008;Sanchez-Fernandez et al. 2008;Zsolnai et al. 2009). The lowest heterozygosity and the highest F values are typically found in small islet populations and populations with longer history of isolation and low population sizes (Hmwe et al. 2006a, b;Hajji et al. 2008;Zachos and Hartl 2011;Zachos et al. 2016;Edelhoff et al. 2020). However, it must be taken into account that both measures are decisively influenced by the markers used, their number, and the sample size (Reiner et al. 2019). ...
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Nineteen red deer areas in a densely populated region with a huge network of fenced motorways and the division into administrative management units (AMUs) with restricted ecological connectivity were investigated. In the season 2018/2019, a total of 1291 red deer samples (on average 68 per area) were collected and genotyped using 16 microsatellite markers. The results show a clear genetic differentiation between most of the AMUs. Fourteen AMUs may be combined into four regions with a considerable internal genetic exchange. Five areas were largely isolated or showed only a limited gene flow with neighbouring areas. Ten of the 19 AMUs had an effective population size below 100. Effective population sizes greater than 500–1000, required to maintain the evolutionary potential and a long-term adaptation potential, were not achieved by any of the studied AMUs, even when AMUs with an appreciable genetic exchange were aggregated. Substantial genetic differentiation between areas can be associated with the presence of landscape barriers hindering gene flow, but also with the maintenance of ‘red deer–free’ areas. Efforts to sustainably preserve the genetic diversity of the entire region should therefore focus on measures ensuring genetic connectivity. Opportunities for this goal arise from the establishment of game bridges over motorways and from the protection of young male stags migrating through the statutory ‘red deer–free’ areas.
... Microsatellite DNA studies showed that in most study sites, the hetorozygosity (expected heterozygosity He) of European moose was relatively high (range He ¼ 0.57-0.75, Haanes et al. 2011, Kangas et al. 2013, Niedziałkowska et al. 2016a in comparison with other common European ungulate species as red deer Cervus elaphus (e.g., Niedziałkowska et al. 2012;Zachos et al. 2016) or roe deer Capreolus capreolus (Lorenzini and Lovari 2006;Olano-Marin et al. 2014) and slightly higher than in moose populations in North America (He ¼ 0.45-0.64, Hundertmark 2009, Schmidt et al. 2009). ...
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This comprehensive species-specific chapter covers all aspects of the mammalian biology, including palaeontology, physiology, genetics, reproduction and development, ecology, habitat, diet, mortality, and behavior. The economic significance and management of mammals and future challenges for research and conservation are addressed as well. The chapter includes a distribution map, a photograph of the animal, and a list of key literature.
... Most European populations of C. elaphus belong to three haplogroups: A, B and C (Skog et al., 2009;Niedziałkowska et al., 2011). A signal concordant with the mitochondrial DNA has recently been identified in the nuclear genome (Zachos et al., 2016). Haplogroup A is most widely distributed from Iberia in the south-west of the continent through western Europe and from the British Isles to Scandinavia and central/eastern Europe, where it co-occurs with haplogroup C that is otherwise distributed in southeastern Europe, including the Balkans and the Carpathians. ...
The present phylogeographic pattern of red deer in Eurasia is not only a result of the contraction of their distribution range into glacial refugia and postglacial expansion, but probably also an effect of replacement of some red deer s.l. mtDNA lineages by others during the last 50 000 years. To better recognize this process, we analysed 501 sequences of mtDNA cytochrome b, including 194 ancient and 75 contemporary samples newly obtained for this study. The inclusion of 161 radiocarbon-dated samples enabled us to study the phylogeny in a temporal context and conduct divergence-time estimation and molecular dating. Depending on methodology, our estimate of divergence between Cervus elaphus and Cervus canadensis varied considerably (370 000 or 1.37 million years BP, respectively). The divergence times of genetic lineages and haplogroups corresponded to large environmental changes associated with stadials and interstadials of the Late Pleistocene. Due to the climatic oscillations, the distribution of C. elaphus and C. canadensis fluctuated in north–south and east–west directions. Some haplotypes dated to pre-Last Glacial Maximum periods were not detected afterwards, representing possibly extinct populations. We indicated with a high probability the presence of red deer sensu lato in south-eastern Europe and western Asia during the Last Glacial Maximum.
... Сматра се да је анализа >600 јединки обичног јелена из великих делова његовог европског ареала помоћу 13 микросателитних локуса (Zachos et al., 2016), прва студија ове врсте употребом нуклеарних маркера, која је утврђено значајно структурирање широм европског континента и, како се очекивало узимајући у обзир веће стопе мутације у микросателитима, укупна нуклеарна генетичка структура је сложенија него што је пронађена у филогеографским студијама заснованим на mtDNA. На основу анализе mDNK издвојене су три велике филогеографксе групе обичног јелена у Европи, и то: ...
... All of the above-mentioned mechanisms, which have a strong influence on genetic diversity and large-scale genetic structure of the red deer could be investigated based on polymorphic microsatellite loci specific to cattle (Bos taurus taurus), sheep (Ovis aries), elk (Cervus canadensis) or reindeer (Rangifer tarandus) (Niedziałkowska et al. 2012a;Hoffmann et al. 2016;Zachos et al. 2016). Additionally, the recently deposited de novo entire red deer genome CerEla1.0 (Acc. ...
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The conservation of biodiversity and rational use of biological resources should be conducted with a view to population genetics. Thus, the purpose of this study was to compare the genetic diversity of highly distributed red deer groups from Słowiński National Park (NP) and hunting areas (HA) in northern Poland using 10 dinucleotide microsatellites. In 254 animals, 112 alleles were observed with a high allelic number (NA = 7–15). The NP and HA groups showed high mean heterozygosity (Ho = 0.548 ± 0.063–0.613 ± 0.061) compared to other deer populations in Europe, although a slight decrease in heterozygosity and an increase in inbreeding was identified in NP, compared to HA. A high level of intra-group and a low level of inter-group genetic differentiation was found, which was supported by a high level of gene flow between NP and HA. Structure analysis revealed two genetic clusters (NP and HA) within the population. The HA cluster was distinguished by numerous private alleles, although the NP cluster was distinguished by a high frequency of two private alleles. These study results show that unhunted conservation areas may affect red deer density without increasing genetic diversity whereas hunting does not seem to have a negative impact on deer genetic diversity. This study affirms the anthropogenic impact on the red deer groups in both NP and HA. Ongoing monitoring of the red deer population with an effective sampling strategy is recommended.
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Australian arboreal mammals are experiencing significant population declines, particularly due to land clearing and resulting habitat fragmentation. The squirrel glider, Petaurus norfolcensis , is a threatened species in New South Wales, with a stronghold population in the Lake Macquarie Local Government Area (LGA) where fragmentation due to urbanization is an ongoing problem for the species conservation. Here we report on the use of squirrel glider mitochondrial (385 bp cytochrome b gene, 70 individuals) and nuclear DNA (6,834 SNPs, 87 individuals) markers to assess their population genetic structure and connectivity across 14 locations sampled in the Lake Macquarie LGA. The mitochondrial DNA sequences detected evidence of a historical genetic bottleneck, while the genome-wide SNPs detected significant population structure in the Lake Macquarie squirrel glider populations at scales as fine as one kilometer. There was no evidence of inbreeding within patches, however there were clear effects of habitat fragmentation and biogeographical barriers on gene flow. A least cost path analysis identified thin linear corridors that have high priority for conservation. These areas should be protected to avoid further isolation of squirrel glider populations and the loss of genetic diversity through genetic drift.
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Molecular forensic methods are being increasingly used to help enforce wildlife conservation laws. Using multilocus genotyping, illegal translocation of an animal can be demonstrated by excluding all potential source populations as an individual's population of origin. Here, we illustrate how this approach can be applied to a large continuous population by defining the population genetic structure and excluding suspect animals from each identified cluster. We aimed to test the hypothesis that recreational hunters had illegally introduced a group of red deer into a hunting area in Luxembourg. Reference samples were collected over a large area in order to test the possibility that the suspect individuals might be recent immigrants. Due to isolation-by-distance relationships in the data set, inferring the number of genetic clusters using Bayesian methods was not straightforward. Biologically meaningful clusters were only obtained by simultaneously analysing spatial and genetic information using the program baps 4.1. We inferred the presence of three genetic clusters in the study region. Using partial Mantel tests, we detected barriers to gene flow other than distance, probably created by a combination of urban areas, motorways and a river valley used for viticulture. The four focal animals could be excluded with a high certainty from the three genetic subpopulations and it was therefore likely that they had been released illegally.
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Red deer populations in the Iberian glacial refugium were the main source for postglacial recolonization and subspecific radiation in north-western Europe. However, the phylogenetic history of Iberian red deer (Cervus elaphus hispanicus) and its relationships with northern European populations remain uncertain. Here, we study DNA sequences at the mitochondrial control region along with STR markers for over 680 specimens from all the main red deer populations in Spain and other west European areas. Our results from mitochondrial and genomic DNA show contrasting patterns, likely related to the nature of these types of DNA markers and their specific processes of change over time. The results, taken together, bring support to two distinct, cryptic maternal lineages for Iberian red deer that predated the last glacial maximum and that have maintained geographically well differentiated until present. Haplotype relationships show that only one of them contributed to the northern postglacial recolonization. However, allele frequencies of nuclear markers evidenced one main differentiation between Iberian and northern European subspecies although also supported the structure of both matrilines within Iberia. Thus, our findings reveal a paraphyletic nature for Iberian red deer but also its genetic identity and differentiation with respect to northern subspecies. Finally, we suggest that maintaining the singularity of Iberian red deer requires preventing not only restocking practices with red deer specimens belonging to other European populations but also translocations between both Iberian lineages.
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Due to a restriction of the distributional range of European red deer (Cervus elaphus L.) during the Quaternary and subsequent recolonization of Europe from different refugia, a clear phylogeographical pattern in genetic structure has been revealed using mitochondrial DNA markers. In Central Europe, 2 distinct, eastern and western, lineages of European red deer are present; however, admixture between them has not yet been studied in detail. We used mitochondrial DNA (control region and cytochrome b gene) sequences and 22 microsatellite loci from 522 individuals to investigate the genetic diversity of red deer in what might be expected to be an intermediate zone. We discovered a high number of unique mtDNA haplotypes belonging to each lineage and high levels of genetic diversity (cyt b H = 0.867, D-loop H = 0.914). The same structuring of red deer populations was also revealed by microsatellite analysis, with results from both analyses thus suggesting a suture zone between the 2 lineages. Despite the fact that postglacial recolonization of Central Europe by red deer occurred more than 10000 years ago, the degree of admixture between the 2 lineages is relatively small, with only 10.8% admixed individuals detected. Direct translocations of animals by humans have slightly blurred the pattern in this region; however, this blurring was more apparent when using maternally inherited markers than nuclear markers. © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail:
The package adegenet for the R software is dedicated to the multivariate analysis of genetic markers. It extends the ade4 package of multivariate methods by implementing formal classes and functions to manipulate and analyse genetic markers. Data can be imported from common population genetics software and exported to other software and R packages. adegenet also implements standard population genetics tools along with more original approaches for spatial genetics and hybridization. Availability: Stable version is available from CRAN: Development version is available from adegenet website: Both versions can be installed directly from R. adegenet is distributed under the GNU General Public Licence (v.2). Supplementary information:Supplementary data are available at Bioinformatics online.
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from
Molecular phylogeny and evolutionary history of Cervus, the most successful and widespread cervid genus, have been extensively addressed in Europe, fairly in eastern Asia, but scarcely in central Asia, where some populations have never been phylogenetically investigated with DNA-based methods. Here, we applied a coalescent Bayesian approach to most Cervus taxa using complete mitochondrial cytochrome b gene and control region to provide a temporal framework for species differentiation and dispersal, with special emphasis on the central Asian populations from the Tarim Basin (C. elaphus bactrianus, C. elaphus yarkandensis) and Indian Kashmir (C. elaphus hanglu) aiming at assessing their phylogenetic and phylogeographic patterns. Red deer (C. elaphus), wapiti (C. canadensis) and sika deer (C. nippon) are confirmed as highly differentiated taxa, with genetic distances, divergence times and phylogenetic positions compatible with the rank of species. Similarly, the red deer of the Tarim group, hitherto considered as subspecies of C. elaphus, showed a comparable pattern of genetic distinction in the phylogeny and, according to our results, are thus worthy of being raised to the species level. The systematic position of the endangered red deer from Indian Kashmir is assessed here for the first time, and implications for its conservation are also outlined. Based on phylogeny and divergence time estimates, we propose a novel evolutionary pattern for the genus Cervus during the Mio/Pliocene, in the light of palaeo-climatological information.