ª 2004 The American Genetic Association
Journal of Heredity 2004:95(5):430–435
doi:10.1093/jhered/esh066
Eurasian Otters, Lutra lutra,
Have a Dominant mtDNA
Haplotype From the Iberian
Peninsula to Scandinavia
AINHOA FERRANDO, MONTSERRAT PONSÀ, JOSEP MARMI,
AND
XAVIER DOMINGO-ROURA
From the Departament de Biologia Cellular, de Fisiologia i d’Immunologia, Universitat Autònoma de Barcelona, 08193
Cerdanyola del Vallès, Spain (Ferrando and Ponsà), and Departament de Ciències Experimentals i de la Salut,
Universitat Pompeu Fabra, Dr. Aiguader 80, 08003 Barcelona, Spain (Ferrando, Marmi, and Domingo-Roura).
Address correspondence to A. Ferrando at the first address above, or e-mail: [email protected].
Abstract
The Eurasian otter, Lutra lutra, has a Palaearctic distribution and has suffered a severe decline throughout Europe during the
last century. Previous studies in this and other mustelids have shown reduced levels of variability in mitochondrial DNA,
although otter phylogeographic studies were restricted to central-western Europe. In this work we have sequenced 361 bp
of the mtDNA control region in 73 individuals from eight countries and added our results to eight sequences available from
GenBank and the literature. The range of distribution has been expanded in relation to previous works north towards
Scandinavia, east to Russia and Belarus, and south to the Iberian Peninsula. We found a single dominant haplotype in
91.78% of the samples, and six more haplotypes deviating a maximum of two mutations from the dominant haplotype
restricted to a single country. Variability was extremely low in western Europe but higher in eastern countries. This, together
with the lack of phylogeographical structuring, supports the postglacial recolonization of Europe from a single refugium.
The Eurasian otter mtDNA control region has a 220-bp variable minisatellite in Domain III that we sequenced in 29 otters.
We found a total of 19 minisatellite haplotypes, but they showed no phylogenetic information.
The Eurasian otter (Lutra lutra, L. 1758) has suffered an
important decline throughout much of the Western Paleartic
in the last century, a decline which has led to local extinctions
in many regions of Europe (Conroy and Chanin 2000;
Macdonald and Mason 1994; Ruiz-Olmo and Delibes 1998).
The species is protected by the Convention on International
Trade in Endangered Species of Wild Fauna and Flora
(CITES) and is the focus of important conservation efforts,
including programs to reintroduce it into its former range
(Saavedra and Sargatal 1997). It is becoming clear that there
is an urgent need to determine the genetic relationships of
otters across their distribution range to support reintroductions and management programs.
The mitochondrial (mt) DNA control region is widely
used as a genetic marker in phylogeography (Avise 1998),
and is divided into three domains: a conserved central
domain (CCD), and two hypervariable flanking regions that
can have repetitive regions known as the extended
termination associated sequences (ETAS) domain and the
conserved sequence blocks (CSB) domain. Genetic studies
430
on Eurasian otter mtDNA have been restricted to centralwestern Europe and have detected extremely low levels of
variability (Cassens et al. 2000; Effenberger and Suchentrunk
1999; Mucci et al. 1999). These results may suggest that
otters spread across Europe from a single refugium, though
its location is unknown. The main models suggested for the
postglacial recolonization of Europe are based on the
expansion of taxa from one or more southern refugia during
the Pleistocene, although more eastern refugia also may have
been involved (Hewitt 1999; Taberlet et al. 1998; but see
Stewart and Lister 2001). The importance of the Iberian
Peninsula as a glacial refugium is demonstrated by the high
levels of genetic variability and numerous endemisms
expressed as unique mtDNA haplotypes in the fauna (Bilton
et al. 1998; Hewitt 1999).
The aims of this study are (1) to determine the genetic
variability and principal genetic relationships among European otter populations, exploring new areas not well studied
previously, including otter samples from the Iberian
Peninsula, Scandinavia, and Eastern Europe; (2) to evaluate
Ferrando et al. Low Variability of mtDNA in European Otters
the variability and phylogenetic information of the minisatellite found in the 39 end of the control region; and (3) to
explore the possible existence of otter colonization routes,
and glacial refugia in particular, in the Iberian Peninsula.
Table 1. Geographic origin, number (n), and tissue analyzed
of individuals included in this study, according to their geographic
origin and their haplotype based on 361 bp of the control region
Materials and Methods
Country
Sampling and DNA Extraction
Portugal 10 Southeastern
Spain
17 Extremadura
Samples of 73 Eurasian otters were obtained from eight
European countries (Table 1). DNA was extracted from
blood and muscle with use of the conventional proteinase
K/phenol/chloroform method and from serum with use of
Chelex 100 chelating resin (Walsh et al. 1991). Teeth from
two historical samples of animals captured in 1919 and the
1930s in northeastern Spain were fully crushed, and DNA
was extracted with the DNeasy Tissue Kit (Qiagen),
according to Iudica et al. (2001).
Amplification and Sequencing
The whole control region of the DNA isolated from blood
and muscle was amplified with the primers L-Pro and H-Phe
(Mucci et al. 1999). Because degradation of DNA was
expected in teeth and serum samples, we amplified the 59 side
of the control region, using internal primers MelCR2,
MelCR3, MelCR4, MelCR5, and MelCR6 originally designed
from Eurasian badger, Meles meles, sequences (Marmi 2004).
Polymerase chain reaction (PCR) DNA amplification was
performed with an initial denaturation for 2 min at 948C,
then 30–40 PCR cycles, including 948C for 15 s, 55–568C for
15 s, and 728C for 15 s, and a final extension at 728C for 5 min.
The 15-s intervals were increased to 45 s when the DNA was
of poor quality. Reactions were performed in a final volume of
25 ll (except for teeth and serum samples which were 15 ll,
including 4–6 ll of extraction template), including 50–100 ng
of target DNA, 16.6 mM (NH4)2SO4, 67 mM Tris-HCl
(pH ¼ 8.8), 0.01% Tween-20, 2.5 mM MgCl2, 1.6 mM dNTPs,
4.25 pmol of each primer, and 0.85 units of EcoTaq DNA
polymerase (Ecogen). The minisatellite, located at the 39 side
of the L-strand, was also amplified in 29 individuals, with the
same conditions and primers MelCR5 and H-Phe, and with
annealing temperature increased to 648C.
PCR products were purified by Geneclean (Qbiogene),
but if they showed two bands after running in an agarose gel,
each band was cut and extracted from the gel and purified
with the GFX PCR DNA and Gel Band Purification Kit
(Amersham Biosciences). In this latter case, a new amplification was performed with the same PCR conditions,
including 1.2 ll of purified DNA elute and negative controls,
and sequenced in both directions. Purified products were
sequenced with use of the Dye Terminator Cycle Sequencing
Kit (Applied Biosystems). Sequencing products were run on
an ABI3100 automated DNA sequencer (Applied Biosystems). Sequences produced in this study and those published
by Cassens et al. (2000; GenBank accession numbers
AJ006174–AJ006178) and Mucci et al. (1999) were aligned
with ClustalW (Thompson et al. 1994) and adjusted by eye.
Two clear peaks of similar size in a single nucleotide position
Geographic
n origin
No. individuals
per haplotype
Tissue H1 H2 H3 H4 H5 H6 H7
Blood
Serum,
muscle
Asturias
Serum,
muscle
Catalunya
Tooth
Avila
Muscle
United
7 Scotland
Muscle
Kingdom
Northumbria Muscle
Wales
Muscle
Finland 23 Kälviä
Muscle
Haukipudas Muscle
Hyrynsalmi
Muscle
Kiiminki
Muscle
Kuusamo
Muscle
Oulainen
Muscle
Pelkosenniemi Muscle
Pudasjärvi
Muscle
Pulkkila
Muscle
Puolanka
Muscle
Phäjärvi
Muscle
Ruukki
Muscle
Sotkamo
Muscle
Temmes
Muscle
Tornio
Muscle
Ylitornio
Muscle
Ylivieska
Muscle
Sweden
5 Testeboån
Muscle
Steneby
Muscle
Grann
Åtvidaberg
Muscle
Umeå
Muscle
Raglunda
Muscle
Norway
4 Nordland
Muscle
Tromsö
Muscle
Belarus
3 Verhnedvinsk Muscle
Russia
4 Andreapolsky Tooth
Oleninsky
Tooth
TOTAL 73
10
11
3
2
1
5
1
1
1
2
1
1
2
2
1
1
2
1
3
1
1
1
1
1
1
1
1
1
1
1
3
1
2
1
67 1
1
1
1
1
1
2
1
1
1
within the minisatellite were considered to be the result of
heteroplasmy.
Data Analyses
Nucleotide diversity p based on the Kimura 2-parameter
model was calculated with Arlequin version 2.000 (Schneider
et al. 2000). Mean number of nucleotide differences (dA) (Nei
and Kumar 2000) was estimated with MEGA version 2.1
(Kumar et al. 2001). Mantel’s test correlating the number of
nucleotide differences and geographic distances between
countries was also performed. The DNaSP 3.51 program
(Rozas and Rozas 1999) was used to estimate overall
haplotype diversity (h). All sequences showed evidence of
431
Journal of Heredity 2004:95(5)
having recently been derived from the same ancestral
haplotype, and a minimum spanning network was obtained
by hand. We constructed a neighbor-joining tree from
minisatellite sequences with the MEGA version 2.1 program,
using the Kimura 2-parameter model and a 1,000-replicates
bootstrap. The DNA mfold program version 3.0 (SantaLucia
1998) was used to predict secondary structures in the
minisatellite array.
Results and Discussion
Mitochondrial DNA Variability in the Eurasian Otter
Sequences were obtained from the 59 end of the control
region, from 73 otters, resulting in a 361-bp final alignment.
Five variable positions (1.39%) were identified, all of them
transitions, resulting in seven haplotypes that we renamed
H1–H7. Haplotype H1 was widely distributed throughout
all countries (Table 1). None of the other haplotypes was
found in two different countries. The minimum spanning
network showed a shallow star-like feature that reflected the
extremely low level of sequence divergence and the low
frequency of rare haplotypes (Figure 1). The mean
nucleotide diversity was p ¼ 0.0006. This value was equal
to p ¼ 0.0008 in the United Kingdom, p ¼ 0.0002 in
Finland, p ¼ 0.0037 in Russia and Belarus, and p ¼ 0.0 for
the rest of the countries. The overall haplotype diversity was
h ¼ 0.16 (SD ¼ 0.06), much lower than the values obtained
in other mustelid species whose mitochondrial variability
across Europe has been explored, such as Martes martes, h ¼
0.76, data calculated from sequences of Davison et al.
(2001; GenBank accession numbers AF336949–64, 68–69)
or M. meles, h ¼ 0.90 (Marmi 2004). The low number of
mutations and the star-like feature of the network indicate
a recent genetic divergence from H1. Mantel’s test showed
no correlation between the number of differences and
geographic distances (r ¼ .093, p ¼ .555).
When we combined our sequences with those published
by Mucci et al. (1999) and Cassens et al. (2000), a total of
250–251 bp could be aligned from 236 individuals, showing
seven polymorphic sites (2.79%), six nucleotide substitutions, and one insertion, resulting in eight haplotypes.
Haplotype Lut1 in the study of Cassens et al. (2000) matches
out haplotype H1 and is shared by all Iberian otters and by
92% of individuals included in our study. Haplotype Lut 3
from Cassens et al. (2000), dominant in some localities of
eastern Germany, presented the same nucleotide substitution
as haplotype H4 from Belarus (this study) and a haplotype
named UK from English otters (Mucci et al. 1999). Overall
haplotype diversity throughout all three studies was higher
(h ¼ 0.360) than the value obtained from our sequences,
mainly due to the high number of sequences proceeding
from otters from eastern Germany with haplotype Lut 3.
These results, expanding the distribution previously
explored by Mucci et al. (1999) and Cassens et al. (2000),
report extremely low levels of mtDNA variability in Eurasian
otters. The variability in microsatellites is higher than in
mtDNA in otters, with average, unbiased expected hetero-
432
Figure 1. Minimum spanning network of all otter samples
included in this study (n ¼ 73). Each circle represents a distinct
haplotype (H1–H7), and the area of the circle is proportional to
the number of individuals showing each haplotype. Each line
between two haplotypes represents a single substitution, and next
to the line appears the type of substitution and position.
zygosity values of He ¼ 0.62–0.71 in Scotland and He ¼
0.40–0.52 in southwestern England and Wales for 12 loci
analyzed (Dallas et al. 2002). Average expected heterozygosity values were similar (He ¼ 0.45–0.77) in otters from eight
European countries for 13 loci analyzed (Randi et al. 2003).
Haploidy, the low effective population size, reduced
recombination, and differential mutation rates of mtDNA, as
well as otter live history, such as the preferential dispersal by
males, would explain differences in the patterns of genetic
diversity between mitochondrial and microsatellite DNA. In
addition, the highly variable ETAS domain is shorter (175
bp) in otter than in other carnivores, and this fact could
contribute to the little variability observed in the otter
mtDNA control region.
Domain III Repeats
The CSB domain of Eurasian otter contains a minisatellite
composed of 22-bp repeat units. We considered that each
repeat started with nucleotide sequence ACGTACGY.
Twenty-seven individuals showed an array composed of
10 repeat units, resulting in a final array of 220 bp. The
alignment of minisatellite repeat units showed the presence
of two variable sites located at the eighth and 21st positions,
resulting in five repeat unit haplotypes (a–e; Table 2). The
first repeat unit a and the two last repeat units d were
common for all sequences. Specimens L54 and L76
presented heteroplasmy in their length, each one showing
the presence of arrays composed by eight and 10 repeat units.
The loss of two units could be a result of DNA slippage
during replication.
Nineteen different haplotypes (A1–A19) were identified
among 29 samples. Evidence of homogenization in most of
Ferrando et al. Low Variability of mtDNA in European Otters
Table 2. Description of the 19 different haplotypes (A1–A19) obtained from sequencing 220 bp of the minisatellite found in
the CSB domain of mtDNA for 29 otters of different geographic origins
Repeat array
haplotype
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
Repeat units (59fi39 of L-strand)
Country (sample code)
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
Belarus, Verhnedvinsk (L85, L88)
Finland, Hyrynsalmi (L56)
Finland, Pyhäjärvi (L60)
Finland, Pulkkila (L61)
Finland, Oulainen (L63)
Finland, Puolanka (L44)
Finland, Kuusamo (L59)
United Kingdom, Walsh (L92)
Finland, Ruukki (L54)
Finland, Ylivieska (L55)
Sweden, Testeboan (L71)
Norway, Tromsö (L77)
United Kingdom, Scotland (L80)
United Kingdom, Scotland (L84)
Spain, Asturias (L26)
Spain, Asturias (L33)
Spain, Asturias (L89)
Spain, Extremadura (L37)
Spain, Extremadura (L35, L38)
Spain, Extremadura (L24, L34, L39, L90)
Spain, Avila (L69)
Portugal, Alentejo (L18)
Portugal, Alentejo (L17)
Norway, Nordland (L76)
a
a
y
b
b
c
b
b
b
c
b
b
c
c
d
d
d
d
d
d
a
a
b
b
c
c
b
c
c
b
b
b
z
c
d
d
d
d
d
d
a
a
a
a
a
a
a
a
a
a
a
a
c
c
b
c
b
c
b
b
b
c
c
c
c
c
z
c
b
c
c
c
c
c
c
c
c
c
c
c
b
b
b
b
b
c
c
c
c
c
c
z
c
b
b
b
b
b
b
c
c/e
d
c
z
c
c
b
b
b
z
b
c
c/d
c
c
c
c
b
b
z
c
c
c
c
e/d
d
d
d
d
d
d
d
d
e
e
d
d/d
d
d
d
d
d
d
d
d
d
d
d/d
d
d
d
d
d
d
d
d
d
d
a
a
a
c
c
c
c
c
c
c
c
c/b
b
c
c/c
b
b
b/d
c
c
c/d
b
d
d/d
d
d
d/-
d
d
d/-
Each letter represents a repeat unit with the following sequences: a ¼ ACGTACGTATACACGCACACTC, b ¼ ACGTACGTATACACGCACACCC, c ¼
ACGTACGCATACACGCACACCC, d ¼ ACGTACGCATACACGCACACGC, e ¼ ACGTACGTATACACGCACACGC, y ¼ ACGTACGTATACACGCACACYC, z ¼ ACGTACGYATACACGCACACCC. Y represents C/T and is found in y–z heteroplasmic sequences.
the array (e.g., L85 and L88 for b repeat, or L24, L34, L39,
and L90 for c repeat) is important. These homogenizations
and the constant size (220 bp) of the complete repeat region
across Europe suggest the prevalence of cross-talk mechanisms leading to the generation of variability in these
sequences. Using the mfold program, we found that the
minisatellite region can form secondary structures (Figure 2).
The graphic representation of this folding shows stable stem
structures and that the variable positions eighth and 21st of
the minisatellite repeat units fall outside these structures in
locations more prone to mutation. The accumulation of
mutations in two variable positions and the regular pattern
observed in many of the sequences is against the role of
random point mutations.
The neighbor-joining tree failed in resolving phylogenetic
relationships among repeat array haplotypes, with no
branches showing bootstrap values above 50%, and no
further arrays were sequenced. Thus, we could not extract
any relevant phylogeographic information from the comparison of minisatellite sequences across countries. Only
similarities are evident in populations from Extremadura
(L24, L34, L39, and L90) or Asturias (L33 and L89). In otters
the analysis of the mini satellite variability might be
informative for individual identification as seen in other
species (Matsuhashi et al. 1999), for instance in combination
with genetic markers such as microsatellites.
Pleistocene Refugia
Climatic fluctuations during the Quaternary are responsible
for local extinctions and reduced genetic variability in
European fauna (Hewitt 1999). The widespread monomorphism and lack of phylogeographical structuring in our
study supports a single genetic lineage colonizing Europe
from a single refugium after the last glaciation, as has been
suggested for Martes martes and Mustela putorius (Davison et al.
2001). The lack of structuring in mtDNA and haplotypes
shared over long distances is not an exception in wideranging
carnivores (Vilà et al. 1999). This phenomenon could be the
result of leptokurtic dispersal, where there is a rapid and
long-distance dispersal of individuals to set up colonies far
ahead of the main dispersing population. For the otter, this
dispersal would be favored in wet habitats and periods.
However, our study does not support the role of the Iberian
Peninsula as a refugium for otters. We detected most
mutations in eastern Europe, that is, in otters from Russia
and Belarus. This result could suggest an otter colonization
of Europe from the Balkans or western Asia as has been
suggested for other species (Bilton et al. 1998; Desmesure et
al. 1996). The reduced number of mutations and eastern
samples involved in this study prevent any firm conclusion
on this issue but prompt further research with additional
samples from the Balkan Peninsula and eastern Europe, and
433
Journal of Heredity 2004:95(5)
Figure 2. Example of folded secondary structure of the complete minisatellite array for individual L92. Triangles indicate
variable sites in the 8th and 21st positions of each repeated unit.
with other molecular markers in addition to mtDNA. The
low genetic variability observed indicates that this colonization must be quite recent, maybe even subsequent to the
main glacial period of the Pleistocene, with otter populations
recovering only after two more recent episodes of drought,
13,000 and 10,000 years ago (Starkel 1991), which may have
been critical for otter survival.
also thank O. Lao and L. Pérez-Jurado for their valuable help in data analyses
and interpretation, N. Sastre for her assistance in obtaining samples, and C.
Simmons for editorial support. A. Ferrando and J. Marmi are supported by
scholarships from the DURSI, Generalitat de Catalunya (Ref. 2002FI-00280
and 2000FI-00698, respectively). This project is financed by the Fundació
Territori i Paisatge, Caixa de Catalunya, Barcelona, Spain. A. Ferrando and
X. Domingo-Roura are now at the Department de Genètica de la
Conservació, Institut de Recerca i Technologica Agroalimentàries, Centre
de Cabrils, Carretera de Cabrils s/n, 08348 Cabrils, Spain.
Acknowledgments
References
Samples used in this study were kindly provided by A. Bradshaw, University
of Wales Lampeter; S. Filella and J. Fernández-Morán, Parc Zoològic de
Barcelona; E. Garcia-Franquesa, Museu de Zoologia de Barcelona; A.
Kitchener, National Museums of Scotland; J. Pastor, Museo Anatómico de la
Universidad de Valladolid; A. Ross, Swedish Museum of Natural History;
D. Saavedra, Fundació Territori i Paisatge, Barcelona; P. Korabljov, Central
Forest Reserve of Zapovednik; V. Sidorovich, National Academy Science of
Belarus; and R. Tornberg, Zoological Museum of the University of Oulu. We
are very grateful to A. Pérez-Lezaun, O. Andrés, G. Fernández, A. B. Ibarz,
and J. F. López-Giráldez for their advice and support in the laboratory. We
Avise JC, 1998. The history and purview of phylogeography: a personal
reflection. Mol Ecol 7:371–379.
434
Bilton DT, Mirol PM, Mascheretti S, Fredga K, Zima J, and Searle JB, 1998.
Mediterranean Europe as an area of endemism for small mammals rather
than a source for northwards postglacial colonization. Proc R Soc Lond B
Biol Sci 265:1219–1226.
Cassens I, Tiedemann R, Suchentrunk F, and Hartl GB, 2000.
Mitochondrial variation in the European otter (Lutra lutra) and the use of
spacial autocorrelation analysis in conservation. J Hered 90:31–35.
Ferrando et al. Low Variability of mtDNA in European Otters
Conroy JWH and Chanin PRF, 2000. The status of the Eurasian otter
(Lutra lutra) in Europe—a review. J Int Otter Surviv Fund 1:7–28.
Dallas JF, Marshall F, Piertney SB, Bacon PJ, and Racey PA, 2002. Spatially
restricted gene flow and reduced microsatellite polymorphism in the
Eurasian otter Lutra lutra in Britain. Conserv Genet 3:15–29.
Davison A, Birks JDS, Brooks RC, Messenger JE, and Griffiths HI, 2001.
Mitochondrial phylogeography and population history of pine martens
Martes martes compared with polecats Mustela putorius. Mol Ecol 10:
2479–2488.
Desmesure B, Comps B, and Petit RJ, 1996. Chloroplast DNA
phylogeography of the common beech (Fagus sylvatica L.) in Europe.
Evolution 50:2515–2520.
Rozas J and Rozas R, 1999. DnaSP version 3: an integrated program for
molecular population genetics and molecular evolution analysis. Bioinformatics 15:174–175.
Ruiz-Olmo J and Delibes M, 1998. La Nutria en España ante el Horizonte
del Año 2000. Málaga: Sociedad Española para la Conservación y Estudio
de los Mamı́feros (SECEM).
Saavedra D and Sargatal J, 1997. Reintroduction of the otter (Lutra lutra) in
northeast Spain (Girona province). Galemys 10:191–199.
SantaLucia J Jr, 1998. A unified view of polymer, dumbbell, and
oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad
Sci USA 95:1460–1465.
Effenberger S and Suchentrunk F, 1999. RFLP analysis of the
mitochondrial DNA of otters (Lutra lutra) from Europe—implications for
conservation of a flagship species. Biol Conserv 90:229–234.
Schneider S, Roessli D, and Excoffier L, 2000. Arlequin ver.2000:
A software for populations genetics data analysis. Geneva: Genetics and
Biometry Laboratory, University of Geneva.
Hewitt GM, 1999. Post-glacial re-colonization of European biota. Biol J
Linn Soc Lond 68:87–112.
Starkel L, 1991. Environmental changes at the Younger Dryas-Preboreal
Transition and during the early Holocene: some distinctive aspects in
central Europe. Holocene 1:234–242.
Iudica CA, Whitten WM, and Williams NH, 2001. Small bones from dried
mammal museum specimens as a reliable source of DNA. Biotechniques
30:732–736.
Kumar S, Tamura K, Jakobsen IB, and Nei M, 2001. MEGA2: Molecular
Evolutionary Genetics Analysis software. Bioinformatics 17:1244–1245.
Macdonald SM and Mason CF, 1994. Status and conservation needs of the
otter (Lutra lutra) in the western Palaearctic. In: Nature and Environment
no. 67. Strasbourg: Council of Europe Press.
Marmi J., 2004. Sistemàtica molecular, filogeografia i genètica de la
conservació de mustèlids i de macacs (PhD thesis). Barcelona: Universitat
Pompeu Fabra.
Stewart JR and Lister AM, 2001. Cryptic northern refugia and the origins of
the modern biota. Trends Ecol Evol 16:608–613.
Taberlet P, Fumagalli L, Wust-Saucy AG, and Cossons JF, 1998.
Comparative phylogeography and postglacial colonization routes in Europe.
Mol Ecol 7:453–464.
Thompson JD, Higgins DG, and Gibson TJ, 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting position-specific gap penalties and weight
matrix choice. Nucleic Acids Res 22:4673–4680.
Matsuhashi T, Masuda R, Mano T, and Yoshida MC, 1999. Microevolution
of the mitochondrial DNA control region in the Japanese brown bear
(Ursus arctos) population. Mol Biol Evol 16:676–684.
Vilà C, Amorim IR, Leonard JA, Posada D, Castroviejo J, Petrucci-Fonseca F,
Crandall KA, Ellegren H, and Wayne RK, 1999. Mitochondrial DNA
phylogeography and population history of the grey wolf Canis lupus. Mol Ecol
8:2089–2103.
Mucci N, Pertoldi C, Madsen AB, Loeschcke V, and Randi E, 1999.
Extremely low mitochondrial DNA control-region sequence variation in
the otter Lutra lutra population of Denmark. Hereditas 130:331–336.
Walsh PS, Metzger DA, and Higuchi R, 1991. Chelex 100 as a medium for
simple extraction of DNA for PCR-based typing from forensic material.
Biotechniques 10:506–513.
Nei M and Kumar S, 2000. Molecular evolution and phylogenetics: New
York: Oxford University Press.
Randi E, Davoli F, Pierpaoli M, Pertoldi C, Madsen AB, and Loeschcke V,
2003. Genetic structure in otter (Lutra lutra) populations in Europe:
implications for conservation. Anim Conserv 6:93–100.
Received October 15, 2003
Accepted June 1, 2004
Corresponding Editor: Rob DeSalle
435