ORIGINAL INVESTIGATION

Relative spraint density and genetic structure of otter (Lutra lutra)

along the Drava River in Hungary
J. Lanszki
a,
, A. Hidas
b
, K. Szentes
b
, T. Re vay
b
, I. Lehoczky
c
, S. Weiss
d
a
Ecological Research Group, University of Kaposvar, P.O. Box 16, 7401 Kaposvar, Hungary
b
Genetic Laboratory, Research Institute for Animal Breeding and Nutrition, Isaszegi Str. 200, 2100 Godollo, Hungary
c
Genetic Laboratory, Research Institute for Fisheries, Aquaculture and Irrigation, P.O. Box 47, 5541 Szarvas, Hungary
d
Institute of Zoology, Karl-Franzens University Graz, Universitatsplatz 2, 8010 Graz, Austria
Received 19 September 2006; accepted 23 August 2007
Abstract
In this study, we used genetic-based approaches to estimate population size and structure of Eurasian otter along the
Drava River in Hungary, and compared these results to traditional survey-based methods. The relative spraint density
of otter was estimated based on the number of fresh (D
f
) and total number (D
t
) of spraints collected on standard routes
over a 2-year period. Nine microsatellite loci were screened, generating 17 individual otter genotypes composed of 45
different alleles. The expected heterozygosity ranged from 0.53 to 0.89 and observed heterozygosity from 0.25 to 0.92.
The mean density (D
g
) estimated over six different sites was 0.17 individuals per km of shoreline. A close correlation
was found between the number of genotypes and spraint counts along a standard route (fresh spraints: r
P
0.85,
Po0.01; total spraints r
P
0.76, Po0.05). All genotypes found within the 50 km-long study area were closely related
(D
m
ranged between 0.08 and 0.21).
r 2007 Deutsche Gesellschaft fu r Sa ugetierkunde. Published by Elsevier GmbH. All rights reserved.
Keywords: Lutra lutra; Microsatellite loci; Genotype; Heterozygosity; Density
Introduction
Assessing population sizes of Eurasian otter (Lutra
lutra Linnaeus, 1758) is difcult due to its solitary,
secretive, and nocturnal behaviour (Mason and Macdo-
nald 1986; Conroy and Chanin 2002). In contrast to
some traditional approaches such as direct observation
or den coufnting (Kruuk et al. 1989), indirect methods
based on the presence of spraints or tracks are used. To
monitor population changes a number of approaches
are taken including systematic questionnaire-based
surveys (review in: Reuther et al. 2000), track counting
(Sidorovich and Pikulik 2002; Ruiz-Olmo et al. 2001b;
Wilson and Delahay 2001) or spraint survey methods
(e.g., Kruuk et al. 1986; Mason and Macdonald 1987).
There are problems with each of these methods. For
example, track counting is restricted to areas of dense
vegetation, particular soil or riverine landscapes and
snow-less periods. Spraint survey methods are the most
available techniques for studying or monitoring otters,
but their value and reliability have been widely dis-
cussed and criticised (e.g., Kruuk and Conroy 1987;
Conroy and French, 1987). Although no simple
correlations between otter numbers and spraint numbers
have been found (Kruuk et al. 1986), some authors
suggest that such relative spraint densities can
potentially be used as indicators of changes in otter
ARTICLE IN PRESS
www.elsevier.de/mambio
1616-5047/$ - see front matter r 2007 Deutsche Gesellschaft fu r Sa ugetierkunde. Published by Elsevier GmbH. All rights reserved.
doi:10.1016/j.mambio.2007.08.005 Mamm. biol. ] (]]]]) ]]]]]]

Corresponding author.
E-mail address: lanszki@mail.atk.u-kaposvar.hu (J. Lanszki).
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
population densities, between years and areas, using the
same sampling methods over time (Jefferies 1986;
Mason and Macdonald 1987; Reuther et al. 2000).
Most recently, molecular genetic methods offer a
promising alternative for population assessment. The
application of highly variable DNA markers, drawn from
non-invasive sampling of faecal material (Kohn and Wayne
1997; Taberlet and Luikart 1999) can yield estimates of
population size, genetic structure, mating patterns and sex
ratio (Dallas et al. 1999; Pertoldi et al. 2001; Dallas et al.
2002, 2003; Randi et al. 2003; Arrendal et al. 2004).
The objective of this 2-year study was to compare
traditional methods, i.e. different spraint density indices
with genetic-based estimates and to examine the genetic
structure of the otter population along the Drava River.
Along the River Drava in Hungary, the otter shows a
continuous presence (Lanszki 2005), but at low densities
across all habitats studied. Relative density changes
were monitored continuously from 2000 until 2005 using
spraint surveys. Collection of otter DNA from fresh
spraints and anal jellies (Dallas and Piertney 1998;
Dallas et al. 1999) was initiated in 2002.
Material and methods
Study area
The study was carried out in SW Hungary, at six sites along
the River Drava, three directly on the river, and three in
backwater habitats (from D1: 46118
0
N, 16152
0
E to B3:
45157
0
N, 17130
0
E, Fig. 1a). The main stem of the Drava River
has a steep riparian region, characterised by oodplain forests.
Backwaters areas are vegetated by willows, poplar, and ash-
alder woods. More information on the habitats is given in
Juha sz and De nes (2005) and Lanszki and Sallai (2006).
Sample collection and DNA analysis
Fresh spraint and anal jelly samples (Coxon et al. 1999)
were collected monthly between June 2002 and May 2004
(between 05.00 and 10.00 h) along standard routes (Table 1).
Spraints were also collected for diet analysis, but for DNA
extraction 1 ml of fresh spraint and anal jelly were placed in
plastic vials containing 8 ml of 96% ethanol, carried in a cooler
and stored at 20 1C until analysis.
DNA was extracted using a modied hexadecyltrimethy-
lammonium bromide (CTAB)-based extraction protocol
(Coxon et al. 1999). To reduce costs and work investment an
initial control step was included in the protocol, whereby the
lysate of the spraint samples was loaded on 2% agarose gel
(stained with ethidium bromide (0.5 mg/ml) to ensure DNA
presence. In this process, samples not containing DNA were
excluded from further procedures. This step cannot exclude
DNA from other species, such as from prey, so sucessful PCR
is still not guarenteed. After diatomaceous earth binding of the
DNA, simple centrifugation was used instead of high cost
separation by ltration. The DNA was nally stored in water
(MilliQ) at 20 1C.
Nine microsatellite loci were selected Lut-435, Lut-604, Lut-
615, Lut-701, Lut-715, Lut-717, Lut-733, Lut-832 and Lut-833
described in Dallas and Piertney (1998). Annealing tempera-
ture was either 60 1C (Lut-615, Lut-833, Lut-701, Lut-715,
Lut-717, Lut-733) or 58 1C (Lut-435, Lut-604, Lut-832). The
locus Lut-SRY was used for sex identication (Dallas et al.
2000). Duplex PCR were used to identify sex from spraint
samples, where Lut-SRY was combined with Lut-701 or Lut-
615 (annealing: 60 1C). PCR reactions were performed in a
15 ml containing 50 ng of L. lutra genomic DNA, 0.01 units/ml
Dynazyme (Finnzyme) polymerase, 2.5 mM MgCl
2
, 200 mM of
each dNTP, 0.25 mM of each forward and reverse primers and
sterile ultrapure MilliQ water. The PCR programme used was:
95 1C/4 min (95 1C/15 s., annealing 30 s, 72 1C/60 s) 30 cycles,
72 1C/9 min, 20 1C/1 min. For spraint samples the number of
amplication cycles had to be increased (to 33) in order to
produce detectable PCR products (Ramekers et al. 1997). To
measure the quality and quantity of PCR products samples
were loaded on agarose gel (2% agarose gel stained with
ethidium bromide, 0.5 mg/ml). If amplication failed (no target
product detected) the PCR reaction was repeated three more
times. PCR products were genotyped (Pertoldi et al. 2001) on
an ALF Express II DNA sequencer (Amersham-Biosciences).
Internal and external molecular mass standards (100, 200,
300 bp) were used for sizing microsatellite allels and allelic
ARTICLE IN PRESS
Fig. 1. (a) Locality of the study areas in the Drava region. River sections: D1 O

rtilos, D2 Be lava r and D3 V zva r;

backwaters: B1 Be lava r, B2 Babo csa and B3 Barcs, solid lines show river and dotted lines border. (b) Neighbour-Joining
clustering of individual genotypes (Neis distance, D
m
). Brackets contain the individual sample numbers and sex, if was available, M:
male, F: female.
J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 2
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
ladders were run in every fth lane. Each run was repeated
following the method of Taberlet et al. (1996) to avoid the
presence of false alleles and allelic dropouts (Taberlet et al.
1999).
Statistical analysis
To compare different survey methods, relative otter spraint
density (D) indices were used (Kruuk et al. 1986; White et al.
2003). Estimated D
f
was based on the number of seasonally
collected (2 years, three collections per season) fresh (some
hours old) spraints and anal jellies, and D
t
on the number of
total (pooled fresh and older samples) otter spraints collected
per unit (100 m) length of the route section along the shoreline
(Table 1). Similarly, density based in terms of the minimum
numbers of otters alive (MNA) in each area was based on the
number of unique genotypes (D
g
). Linear regression was
applied between the relative density indices (both D
t
and D
f
)
and the MNA data using the SPSS 10 for Windows (1999)
statistical package.
Probability of identity (P
ID
/locus) was calculated by GIMLET
(ver. 1.3.3; Valie` re 2002). Testing for genotypic disequilibrium
was performed using GENEPOP (ver. 3.3; Raymond and Rousset
1995), all tests for all combinations of loci were non-
signicant, indicating independent segregation of loci.
The mean number of alleles, allele frequencies, and observed
(H
o
) and expected (H
e
) heterozygosities were calculated for
each locus using the GENEPOP software package. Genotypic
diversity and allelic richness was calculated using the FSTAT
(ver. 2.9.3.2; Goudet 1995). Departure from Hardy-Weinberg
equilibrium (HWE) and heterozygote deciency were also
tested using the Markov chain procedure implemented in
GENEPOP. Neis minimum genetic distance (Takezaki and Nei
1996) was calculated (Neighbour-Joining method) using the
POPULATIONS (ver. 1.2.28; Langella 1999) and the indivi-
dual distance tree was visualised with the TREEVIEW software
package (Page 1996). Association between genetic distance
[Fst/(1Fst)] and ln (geographic distance) for population level
data was evaluated with Mantel test (10,000 permutations) in
GENEPOP.
Results
Faecal DNA analyses and otter spraint densities
During the 2-year study 92 fresh spraints were
collected along the Drava River and 64 in backwater
habitats. (Table 1). From this sample, 22 (14.1%) DNA
extractions were successfully genotyped for nine micro-
satellite loci. Seventeen different otter DNA proles
were identied (Table 1); there was a minimum number
of 11 otters detected along the Drava River and six in
the backwater habitats. Five individuals were recorded
more than once, three in the same month (Nos. 290, 539
and 132) and two in different months (Nos. 132 and
346). Sex was determined for 10 individuals, three were
females and seven males (Fig. 1b). The mean otter
density (MNA) was equal along river and backwater
habitats (0.1770.07 and 0.1770.11 individuals/km,
7SE). Accordingly, the estimate is equivalent to at
least one otter every 5.9 km.
The mean otter spraint densities (D
t
and D
g
) were
2.171.12 total spraints per 100 m or 0.2170.144 fresh
spraints per 100 m of river, and 1.370.33 total spraints
per 100 m or 0.1170.044 fresh spraints per 100 m of
backwater habitat.
There was a close relation between the relative otter
spraint density estimates stemming from fresh spraint
(D
f
) and genetic data (D
g
) (r
P
0.85, Po0.01, Fig. 2),
as well as between total number of spraints (D
t
) and
genotypes (D
g
) (r
P
0.76, Po0.05).
Population genetic structure
All microsatellite loci were polymorphic with the
number of alleles per locus ranging from four to nine
(Table 2). The 17 distinct genotypes yielded a total of 45
ARTICLE IN PRESS
Table 1. Summarized sample data of otters (Lutra lutra) living in three river sections and three backwaters of the Drava, in
Hungary
Habitats Transect (m) Number of samples collected and analyzed
Total spraints Fresh spraints Genotypes
1st yr 2nd yr 1st yr 2nd yr 1st yr 2nd yr Total
Riverine habitats
+
Ortilos (D1)
1800 107 76 8 7 2
+1
2
+1
4
Be lava r (D2) 500 404 262 55 13 3 1 4
V zva r (D3) 1500 90 89 5 4 2 1 3
Backwaters
Be lava r (B1) 500 154 112 19 7 3 0 3
Babo csa (B2) 1600 140 248 15 15 1
+2
1
+1
2
Barcs (B3) 600 95 49 6 2 1 0 1
First year: from June 2002 to May 2003, second year: from June 2003 to May 2004,
+
same genotype.
J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 3
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
different alleles across the nine loci analysed with a
mean of ve alleles per locus. Nine genotypes were typed
completely, three had missing data for one locus, two
for two loci, and one genotypes lacked information for
three loci. Across loci, Lut 715, 615, 717 and 701 were
ampliable in all individuals, two loci (Lut 832, 833)
failed in one individual, two loci (Lut 733, 435) in two
otters, and Lut 604 failed in three individuals. Geno-
typic diversity among populations across loci was 0.719
and allelic richness was 1.688. The expected hetero-
zygosity (H
e
) ranged from 0.53 to 0.89 (mean7SE:
0.6870.042), observed heterozygosity (H
o
) ranged from
0.25 to 0.92 (mean7SE: 0.5370.075), and there was no
signicant difference between these values (paired
samples t-test: t 2.22, P 0.057). Three loci (Lut-
615, 435 and 717) showed signicant deviations from the
HWE (Table 2) with a signicant decit of hetero-
zygotes (Po0.01).
The mean genetic distance between individuals was
0.4370.012 (7SE, range: 0.170.72), while between
sites (Table 3) it was only 0.1570.01 (range: 0.080.21).
The mean geographical distance among sites was
22.071.61 km (7SE, range: 1.748.0). There was no
association between genetic distance and ln geographic
distance (Figs 1a and b) calculated at the population
level (r
S
0.198, P 0.809).
Discussion
This study is the rst report of genetic variation in an
Hungarian otter population and further compares two
different approaches (spraint counts versus genotypes)
for calculating relative density. Overall, otter density
along the Drava River is low (one otter per 5.9 km, or
0.17 individuals/km), but similar to other densities
reported in what is considered sub-optimal habitat.
For example, Sidorovich et al. (1996) reported mean
densities based on track surveys ranging from 0.13
to 0.50 otters/km on large rivers, 0.130.19 on small
rivers, and 0.1 otter/km on very small rivers. Much
higher densities (0.81.8 otter/km) based on genotype
counts were recently reported from Taiwanese rivers
(Hung et al. 2004), and slightly higher densities
(one otter per 4.8 km shoreline) from riverine and pond
habitats of Germany (Kalz et al. 2006). The Drava
River corridor in Hungary is subject to substan-
tial uctuations in water levels and ow velocites
(Za vodszky 2005) and such disturbances may inuence
territorial marking behaviour (Jefferies 1986; Conroy
and French 1987; Kruuk and Conroy 1987; Reuther et
al. 2000). Otter density is naturally inuenced by sh
food availabilty and habitat quality (Mason and
Macdonald 1986; Kruuk et al. 1991, 1993; Ruiz-Olmo
et al. 2001a, 2002; Sidorovich and Pikulik 2002)
and sprainting behaviour is inuenced by a variety of
factors (Jefferies 1986; Ruiz-Olmo and Gosa lbez 1997;
Reuther et al. 2000; Prigioni et al. 2005), thus it is
difcult or impossible to compare the efciency of
different survey methods between studies. The relative
advantage of the traditional spraint counts in compar-
ison with the genetic method was the lower cost (0.8 vs.
25.5 EUR/sample).
Our study area, encompassing ca 50 km of the Drava
River corridor, connects a chain of otter habitats. While
there was no signicant association between genotypic
and geographic distance, and no obvious genetic
structure, genotypes within habitats were more closely
related than among habitats and no single otter was
found in two different sites. Thus, either sites did indeed
belong to distinct sub-populations that were too closely
related to detect within our limited sample size, or many
of the individuals were transients (Kalz et al. 2006),
passing through or dispersing to more distant sites.
Overnight movement of otters ranged from 7 km for a
mother with young in Sweden (Erlinge 1968), from 6.2
to 11 km in the Czech Republic (Dulfer et al. 1998),
and a maximum of 20 km in Scotland (Jenkins 1980).
Home range of otters on the basis of reading tracks
and signs in freshwater habitats of southern Sweden
ranged from 0.7 to 1.0 km
2
(Erlinge 1968) or by radio-
telemetry between 1.2 and 2.6 km
2
in the Czech Republic
(Dulfer et al. 1998). Thus, our study area should have
been much larger than any individual home range, but
not necessarily large enough to encompass more than
one population unit, when considering gene ow or
dispersal.
For our microsatellite analysis, global parameters
such as H
e
(0.68), H
o
(0.53), and the mean number of
ARTICLE IN PRESS
0.0 0.2 0.4 0.6 0.8 1.0
Df (number of fresh spraints/100 m)
0.00
0.02
0.04
0.06
M
N
A

(
n
u
m
b
e
r

o
f

o
t
t
e
r
s

g
e
n
o
t
y
p
e
d
/
1
0
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m
)
Fig. 2. Linear regression between fresh otter (Lutra lutra)
spraint density and MNA. Genetic survey based on the
minimum number of otters alive (MNA); and the relative
density based on the number of fresh spraints and anal jellies
(D
f
). MNA 0.01+0.05D
f
, R
2
0.72, n 10, P 0.002.
J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 4
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
ARTICLE IN PRESS
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J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 5
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
alleles (5.0) were well within the ranges of those reported
in both European and Asian studies (e.g., Randi et al.
2003; Hung et al. 2004). However, our result of nine
alleles at the Lut-717 locus was higher than that
reported across studies screening 1400 indviduals
(summarising data can be found in Table 4).
In conclusion, the spraint survey method yielded
results in close relation with abundance of otters, as
revealed by non-invasive molecular techniques. These
results may be universal for the monitoring in otter
populations in primarily natural freshwater habitats. In
this rst study of otters in Hungary, based on non-
invasive sampling, high genetic diversity, no migration
of individuals and only low densities were found.
Further investigations, with increased DNA extraction
efciency, carried out on a larger catchment area needed
to better estimate patterns of metapopulation structure
and dynamics.
Acknowledgements
We express sincere thanks to Drs. Jim Conroy and
Rob Marrs for advice and comments on early draft of
the manuscript. This work was supported by the
Hungarian Fund for Scientic Research (OTKA F
037557 and K 62216), the Directorship of the Danube-
Drava National Park and the Bolyai Scholarship (JL).
Zusammenfassung
Relative Losungsdichte und genetische Struktur von
Fischottern (Lutra lutra) entlang der Drau in Ungarn
In dieser Untersuchung verwendeten wir genetische
Methoden, um die Populationsgro e und -struktur des
eurasischen Fischotters am Flu Drau in Ungarn
abzuscha tzen. Die Ergebnisse wurden mit jenen aus
traditionellen Untersuchungen verglichen. Die relative
Losungsdichte wurde berechnet auf Grund der Anzahl
von frischem (D
f
) und der Anzahl von gesamter Losung
(D
t
), die entlang von Standardrouten in einem zwei-
ja hrigen Zeitraum gesammelt wurden. Neun Mikrosa-
telliten wurden verwendet, die insgesamt 45 Allele
aufwiesen. Dadurch konnten 17 individuelle Genotypen
unterschieden werden. Die erwartete Heterozygotie
reichte von 0.53 bis 0.89, und die beobachtete Hetero-
zygotie reichte von 0.25 bis 0.92. Die mittlere Dichte
(D
g
) u ber sechs Stellen war 0.17 Individuen/km. Es
gab eine signikante Korrelation zwischen der Anzahl
von Genotypen und der Anzahl gefundener Losung
ARTICLE IN PRESS
Table 4. Summary data of molecular genetic analyses of Eurasian otter (Lutra lutra) populations
Location Sample
type
Number of H
e
/H
o
Mean A Source
samples loci
Great Britain, Ireland,
Germany
T 32 13 n/0.55 6.69 Dallas and Piertney
(1998)
Denmark T, M 124 9 0.46/0.43 3.89 Pertoldi et al. (2001)
England, Wales,
Scotland, N. Isles
T 618 12 0.55/n 7.08 Dallas et al. (2002)
England T, F 122 7-9 0.54/n 5.11 Dallas et al. (2003)
Great Britain, Ireland,
Spain, Lithuania,
Denmark, Germany,
Sweden, France
T, O 102 11 0.74/0.55 8.09 Randi et al. (2003)
Germany F, T 59 6 0.65/0.63 5.30 Kalz et al. (2006)
Norway, Sweden T, M 114 6 0.65/0.65 8.50 Arrendal et al. (2004)
Taiwan F 38 7 0.61/0.76 3.86 Hung et al. (2004)
Czech Republic T, F 132 10 0.53/0.51 4.50 Ha jkova et al. (2007)
Slovak Republic T, F 65 10 0.59/0.55 4.70 Ha jkova et al. (2007)
Hungary F 17 9 0.68/0.53 5.00 Present study
Sample type: T: tissue, M: museum tissue (e.g., skull), F: faecal sample, O: other (e.g., hair, blood); loci: number of examined microsatellite loci; H
e
/
H
o
: expected and observed heterozygosity, n: no calculation available; mean A: mean number of alleles.
Table 3. Matrix of Nei minimum genetic distances (D
m
)
among ve otter sub-populations (on the basis of n 15
individuals) along the Drava River, Hungary
Habitats D2 D3 B1 B2
D1 0.107 0.122 0.177 0.155
D2 0.133 0.181 0.081
D3 0.212 0.117
B1 0.187
In the B3 habitat only one individual was genotyped, and only three
microsatellites amplied (for abbreviations see Table 1).
J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 6
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
entlang von Standardrouten (frische Losung: r
P
0.85,
Po0.01; gesamte Losung r
P
0.76, Po0.05). Alle
Genotypen, die innerhalb des 50 km langen Untersu-
chungsgebietes gefunden wurden, waren nah mit einan-
der verwandt (D
m
reichte von 0.08 bis 0.21).
References
Arrendal, J., Walker, C.W., Sundqvist, A.K., Hellborg, L.,
Vila, C., 2004. Genetic evaluation of an otter translocation
program. Conserv. Genet. 5, 7988.
Conroy, J.W.H., Chanin, P.R.F., 2002. The status of
the Eurasian otter (Lutra lutra). IUCN OSG Bull. 19A,
2448.
Conroy, J.W.H., French, D.D., 1987. The use of spraints to
monitor populations of otters (Lutra lutra L). Symp. Zool.
Soc. London 58, 247262.
Coxon, K., Chanin, P., Dallas, J., Sykes, T., 1999. The use of
DNA ngerprinting to study the population dinamics of
otters (Lutra lutra) in Southern Britain: a feasibility study.
Research and Development Technical Report W202.
Dallas, J.F., Piertney, S.B., 1998. Microsatellite primers for the
Eurasian otter. Mol. Ecol. 7, 12481251.
Dallas, J.F., Bacon, P.J., Carss, D.N., Conroy, J.W.H., Green,
R., Jefferies, D.J., Kruuk, H., Marshall, F., Piertney, S.B.,
Racey, P.A., 1999. Genetic diversity in the Eurasian otter,
Lutra lutra, in Scotland. Evidence from microsatellite
polymorphism. Biol. J. Linn. Soc. 68, 7386.
Dallas, J.F., Carss, D.N., Marshall, F., Koepi, K.P., Kruuk,
H., Bacon, P.J., Piertney, S.B., 2000. Sex identication of
the Eurasian otter Lutra lutra by PCR typing of spraints.
Conserv. Genet. 1, 181183.
Dallas, J.F., Marshall, F., Piertney, S.B., Bacon, P.J., Racey,
P.A., 2002. Spatially restricted gene ow and reduced
microsatellite polymorphism in the Eurasian otter Lutra
lutra in Britain. Conserv. Genet. 3, 1529.
Dallas, J.F., Coxon, K.E., Sykes, T., Chanin, P.R.F.,
Marshall, F., Carss, D.N., Bacon, P.J., Piertney, S.B.,
Racey, P.A., 2003. Similar estimates of population
genetic composition and sex ratio derived from carcasses
and faeces of European otter Lutra lutra. Mol. Ecol. 12,
275282.
Dulfer, R., Foerster, K., Roche, K., 1998. Habitat use, home
range and behaviour. In: Dulfer, R., Roche, K. (Eds.), First
Phase Report of the Trebon Otter Project. Scientic
Background and Recommendations for Conservation and
Management Planning, vol. 93. Council of Europe Publish-
ing, Strasbourg, pp. 3146.
Erlinge, S., 1968. Territoriality of the otter Lutra lutra L.
Oikos 19, 8198.
Goudet, J., 1995. FSTAT (ver. 1.2): a computer program to
calculate F-statistics. J. Hered. 86, 485486.
Ha jkova , P., Pertoldi, C., Zemanova , B., Roche, K., Ha jek, B.,
Bryja, J., Zima, J., 2007. Genetic structure and evidence for
recent population decline in Eurasian otter populations in
the Czech and Slovak Republics: implications for con-
servation. J. Zool. (London) 272, 19.
Hung, C.M., Li, S.H., Lee, L.L., 2004. Faecal DNA typing to
determine the abundance and spatial organization of otters
(Lutra lutra) along two streams in Kinmen. Anim. Conserv.
7, 301311.
Jefferies, D.J., 1986. The value of otter Lutra lutra surveying
using spraints: an analysis of its successes and problems in
Britain. Otters, J. Otter Trust 1, 2532.
Jenkins, D., 1980. Ecology of otters in northern Scotland: I.
otter (Lutra lutra) breeding and dispersion in mid-Deeside,
Aberdeenshire, in 197479. J. Anim. Ecol. 49, 713735.
Juha sz, M., De nes, A., 2005. Biomonitoring of alluvial willow
forests. Nat. Somogy. 7, 1118.
Kalz, B., Jewgenow, K., Fickel, J., 2006. Structure of an otter
(Lutra lutra) population in Germany results of DNA and
hormone analyses from faecal samples. Mamm. Biol. 71,
321335.
Kohn, M.H., Wayne, R.K., 1997. Facts from feces revisited.
Tree 12, 223227.
Kruuk, H., Conroy, J.W.H., 1987. Surveying otter Lutra lutra
populations: a discussion of problems with spraints. Biol.
Conserv. 41, 179183.
Kruuk, H., Conroy, J.W.H., Glimmerween, U., Ouwerkerk,
E.J., 1986. The use of spraints to survey populations of
otters Lutra lutra. Biol. Conserv. 35, 187194.
Kruuk, H., Moorhouse, A., Conroy, J.W.H., Durbin, L.,
Frear, S., 1989. An estimate of numbers and habitat
preference of otters Lutra lutra in Shetland, UK. Biol.
Conserv. 49, 241254.
Kruuk, H., Conroy, J.W.H., Moorhouse, A., 1991. Recruit-
ment to a population of otters (Lutra lutra) in Shetland, in
relation to sh abundance. J. Appl. Ecol. 28, 95101.
Kruuk, H., Carss, D.N., Conroy, J.W.H., Durbin, L., 1993.
Otter (Lutra lutra) numbers and sh productivity in rivers
in N.E. Scotland. Symp. Zool. Soc. London 65, 171191.
Langella, O., 1999. POPULATIONS 1.2.28 software /http://
www.prs-gif.frS.
Lanszki, J., 2005. Otter monitoring between 2000 and 2004 in
the Drava region (Hungary). Nat. Somogy. 7, 169178.
Lanszki, J., Sallai, Z., 2006. Comparison of the feeding habits
of Eurasian otters on a fast owing river and its backwater
habitats. Mamm. Biol. 71, 336346.
Mason, C.F., Macdonald, S.M., 1986. Otters: Ecology
and Conservation. Cambridge University Press, Camridge.
Mason, C.F., Macdonald, S.M., 1987. The use of spraints for
surveying otter Lutra lutra populations: an evaluation. Biol.
Conserv. 41, 167177.
Page, R.D.M., 1996. TREEVIEW: an application to display
phylogenetic trees on personal computers. Comput. Appl.
Biosci. 12, 357358.
Pertoldi, C., Hansen, M.H., Loeschcke, V., Madsen, A.B.,
Jacobsen, L., Baagoe, H., 2001. Genetic consequences of
population decline in the European otter (Lutra lutra): an
assessment of microsatellite DNA variation in Danish
otters from 1883 to 1993. Proc. R. Soc. London, Ser. B.
268, 17751781.
Prigioni, C., Remonti, L., Balestrieri, A., Sgrosso, S., Priore,
G., Misin, C., Viapiana, M., Spada, S., Anania, R., 2005.
Distribution and sprainting activity of the otter (Lutra
lutra) in the Pollino National Park (southern Italy). Ethol.
Ecol. Evol. 17, 171180.
Ramekers, J., Hummel, S., Hermann, B., 1997. How many
cycles does a PCR need? Determination of cycle numbers
ARTICLE IN PRESS
J. Lanszki et al. / Mamm. biol. ] (]]]]) ]]]]]] 7
Please cite this article as: Lanszki, J., et al., Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in....
Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005
depending of the number of targets and the reaction
efciency factor. Naturwissenschaften 84, 259262.
Randi, E., Davoli, F., Pierpaoli, M., Pertoldi, C., Madsen,
A.B., Loeschcke, V., 2003. Genetic structure in otter (Lutra
lutra) populations in Europe: implications for conservation.
Anim. Conserv. 6, 93100.
Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2):
population genetics software for exact tests and ecumeni-
cism. J. Hered. 86, 248249.
Reuther, C., Dolch, D., Green, R., Jahrl, J., Jefferies, D.,
Krekemeyer, A., Kucerova, M., Madsen, A.B., Roma-
nowski, J., Roche, K., Ruiz-Olmo, J., Teubner, J.,
Trindade, A., 2000. Surveying and monitoring distribution
and population trends of the Eurasian otter (Lutra lutra).
Habitat 12, Hankensbu ttel, Germany.
Ruiz-Olmo, J., Gosa lbez, J., 1997. Observations on the
sprainting behaviour of the otter Lutra lutra in the NE
Spain. Acta Theriol. 42, 259270.
Ruiz-Olmo, J., Lopez-Martin, J.M., Palazon, S., 2001a. The
inuence of sh abundance on the otter (Lutra lutra)
populations in Iberian Mediterranean habitats. J. Zool.
(London) 254, 325336.
Ruiz-Olmo, J., Saavedra, D., Jime nez, J., 2001b. Testing the
surveys and visual and track censuses of European otter
(Lutra lutra). J. Zool. (London) 253, 359369.
Ruiz-Olmo, J., Olmo-Vidal, J.M., Manas, S., Batet, A., 2002.
The inuence of resource seasonality on the breeding
patterns of the Eurasian otter (Lutra lutra) in Mediterra-
nean habitats. Can. J. Zool. 80, 21782189.
Sidorovich, V.E., Jedrzejewska, B., Jedrzejewski, W., 1996.
Winter distribution and abundance of predatory mustelids
and beavers in the river valleys of Bialowieza Primeval
Forest. Acta Theriol. 41, 155170.
Sidorovich, V.E., Pikulik, M.M., 2002. Factors allowing high
density of otters in Eastern Europe. IUCN OSG Bull. 19A,
326333.
SPSS 10 for Windows, 1999. SPSS Inc., Chicago.
Taberlet, P., Luikart, G., 1999. Non-invasive genetic sampling
and individual identication. Biol. J. Linn. Soc. 68,
4155.
Taberlet, P., Grifn, S., Goossens, B., Questiau, S., Manceau,
V., Escaravage, N., Waits, L.P., Bouvet, J., 1996. Reliable
genotyping of samples with a very low DNA quantities
using PCR. Nucleid Acids Res. 24, 31893194.
Taberlet, P., Waits, L.P., Luikart, G., 1999. Noninvasive
genetic sampling: look before you leap. Trends Ecol. Evol.
14, 323327.
Takezaki, N., Nei, M., 1996. Genetic distances and reconstruc-
tion of Phylogenetic trees from Microsatellite DNA.
Genetics 144, 189399.
Valie` re, N., 2002. Gimlet: a computer program for analysing
genetic individual identication data. Mol. Ecol. Notes 2,
377379.
White, P.C.L., McClean, C.J., Woodroffe, G.L., 2003. Factors
affecting the success of an otter (Lutra lutra) reinforcement
programme, as identied by post-translocation monitoring.
Biol. Conserv. 112, 363371.
Wilson, G.J., Delahay, R.J., 2001. A review of methods to
estimate the abundance of terrestrial carnivores using eld
signs and observations. Wildl. Res. 28, 151164.
Za vodszky, S., 2005. Hydroelectricity or national park? Nat.
Somogy. 7, 59.
ARTICLE IN PRESS
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Mamm. Biol. (2007), doi:10.1016/j.mambio.2007.08.005