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Molecular identification of marbled newts and a
justification of species status for Triturus marmoratus
and T. pygmaeus
G. Espregueira ThemudoG. Espregueira Themudo
G. Espregueira Themudo G. Espregueira Themudo
G. Espregueira Themudo1,2 1,2
1,2 1,2
1,2 & J.W. Arntzen & J.W. Arntzen
& J.W. Arntzen& J.W. Arntzen
& J.W. Arntzen2 2 2 2 2
1CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, Vairão, Portugal
2National Museum of Natural History, P.O. Box 9517, 2300 RA Leiden, The Netherlands
The marbled newts Triturus marmoratus and T. pygmaeus are common and readily diagnosed species occurring in central
Portugal, but difficult to survey in large and deep ponds. Conversely, embryos of both species are easy to locate but
morphologically indistinguishable. We studied a panel of nuclear genetic loci by starch gel electrophoresis (the enzymes
Pep-A, Pep-B and Pep-D) and isoelectric focusing techniques (the enzyme Ldh-2, post-embryonic stages only) that
together yield a species-specific signature (Cohen’s kappa = 1.00). On a locus by locus basis, the scores for correct
classification range from kappa = 0.12 to kappa = 0.97. The method allows the reliable, fast and cheap identification
of both species across life stages, with better behaviour and performance than mtDNA sequencing (i.e. bar-coding) and
nuclear DNA microsatellite profiling. The observed distribution of T. marmoratus and T. pygmaeus over 25 aquatic
breeding sites in the Caldas da Rainha area in western Portugal is parapatric, with no mixed populations and no F1
interspecific hybrids. This demonstrates that T. marmoratus and T. pygmaeus are genetically isolated, even when
populations are within the “dispersal distance per generation” range of one another. We consider the data adequate for
supporting the species status of T. marmoratus and T. pygmaeus under the Biological Species Concept.
Key words: allozymes, Amphibia, principal coordinate analysis
INTRODUCTION INTRODUCTION
INTRODUCTIONINTRODUCTION
INTRODUCTION
N
a mostly efficient and unbiased sampling technique for
adults (Arntzen, 2002a,b). This allows reliable surveying
over large areas for the purpose of, for example, the im-
proved understanding of distribution patterns and the
assessment of conservation status. In Portugal, we noted
the frequent occurrence of newts in “albercas”. These
albercas are deep (>3 m) and sizable (diameter 3–8 m),
mostly circular stone structures for agricultural and
household water supply [for typical examples of albercas,
see Malkmus (1982) and Figure 61 in Malkmus (2004)].
Albercas are exceedingly difficult to investigate. The dip-
netting approach fails, because at the first sweep the
adult newts dive beyond reach and hide in the crevices of
the stone walls. The presence of adults, however, may be
revealed by the easily spotted eggs that are individually
attached to the leaves of submerged, floating and over-
hanging vegetation (Miaud, 1993). For the eggs to be
useful in surveying, a method is required for species iden-
tification. The first aim of the present paper is thus to
develop an efficient and reliable method for identifying
the eggs of marbled newts (Triturus marmoratus and T.
pygmaeus). Since the eggs of these species are morpho-
logically indistinguishable, these will be molecular
genetic tools. On a technical note, most eggs in the field
will be fertilized and should be referred to as embryos, but
for convenience we will use the terms interchangeably.
Secondly, we apply and test the new method to qualify
the distribution of both species in central Portugal over
ewts are characterized by long annual periods (2–6
months) of pond breeding and dip-netting provides
and across a contact zone between them. Thirdly, we test
the hypothesis of Garcia-Paris et al. (2001) that T.
marmoratus and T. pygmaeus are full species under the
Biological Species Concept.
MATERIALS AND METHODSMATERIALS AND METHODS
MATERIALS AND METHODS MATERIALS AND METHODS
MATERIALS AND METHODS
Research was carried out in an area of approximately 4000
km2 around Caldas da Rainha to the north of Lisbon, Por-
tugal, where T. marmoratus and T. pygmaeus have both
been observed in a pilot study (J.W.A. & E. Froufe,
unpubl. data). Ponds and other potential newt breeding
sites were located by motorized field searches, assisted
by military topographical maps and with help and infor-
mation from local inhabitants. At each spot we checked
the submerged, floating and marginal vegetation for the
presence of marbled newt eggs, which are clearly distin-
guishable from those of other amphibians by size,
structure and the way they are deposited. The eggs of the
sympatric small bodied newt T. boscai (placed in the ge-
nus Lissotriton by Garcia-Paris et al., 2004 and in the
genus Lophinus by Litvinchuk et al., 2005) are readily dis-
tinguished from marbled newt eggs on account of their
small size, bipolar pigmentation and the round (as op-
posed to ovoid) shape of the jelly capsule around them.
Eggs were collected from the vegetation all over the ac-
cessible parts of the water body and placed in Eppendorf
vials. Occasionally, adult and larval marbled newts were
caught by dip-netting. The adults were identified as T.
marmoratus or T. pygmaeus on the basis of size, colour
and colour-pattern, whereas larvae remained unidentified.
Tail tips were removed and placed under buffer in indi-
Correspondence: G. Espregueira Themudo, CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus
Agrário de Vairão, 4485-661 Vairão, Portugal. E-mail: themudo@mail.icav.up.pt
HERPETOLOGICAL JOURNAL 17: 24-30, 2007HERPETOLOGICAL JOURNAL 17: 24-30, 2007
HERPETOLOGICAL JOURNAL 17: 24-30, 2007HERPETOLOGICAL JOURNAL 17: 24-30, 2007
HERPETOLOGICAL JOURNAL 17: 24-30, 2007
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25
vidual Eppendorf vials. All vials were placed in liquid ni-
trogen for transportation to the laboratory and then
stored in a freezer at –80 ºCfor future electrophoresis.
In the laboratory, the tail tips and entire embryos were
homogenized in an aliquot amount of ice cold buffer (100
mM Tris, 1 mM EDTA, 0.05 mM NADP, adjusted to
pH=7.0 with HCl) and centrifuged for 15 minutes at 13,000
rpm at 4 ºC. The supernatant was treated with dithiotreitol
(120 µ M DTT) for 1 hour at 37 ºC prior to electrophoresis
and staining on starch gels for three peptidases (Pep-A,
Pep-B and Pep-D) and on acrylamide gels with isoelectric
focussing for the enzyme lactate dehydrogenase (Ldh-2),
following standard protocols (e.g. Pinho et al., 2003).
Electromorphs were interpreted as alleles at the corre-
sponding genetic locus. We used the program Genepop
(Raymond & Rousset, 1995) to test for population genetic
differentiation by Fisher’s exact test, to calculate expected
heterozygosity (He) and to test for departure from Hardy-
Weinberg equilibrium (HW) under standard Bonferroni
correction.
Principal Coordinate Analysis (PCA) was performed
on a binary data set with alleles as characters and pres-
ence (1) or absence (0) of alleles as character states.
Character states were assumed to be independent, al-
though in reality limited to a maximum of two scores of 1
per locus. Homozygotes were not distinguished from
heterozygotes (i.e. they were represented by a single
score of 1). The subroutine SIMQUAL of the program
NTSYS 1.7 (Rohlf, 1992) was used to compare the enzyme
profiles and to calculate a matrix with pairwise similarity
coefficients. We chose the Jaccard similarity coefficient
because it ignores joint absences. The subroutine
DCENTER was used to transform the similarity matrix into
scalar product form, after which it was factored using the
subroutine EIGEN.
RESULTS RESULTS
RESULTSRESULTS
RESULTS
The presence of marbled newts was confirmed in 25
aquatic sites, from which we sampled 101 eggs and em-
bryos, 41 larvae and 84 adults. In 14 sites we failed to
catch any adults, either because of timing (adults had left
the water) or because the site had inaccessible parts. Tis-
sue samples from adults and larvae were scored for four
loci (with few exceptions) and embryos were scored for
the three peptidase loci.
Analysis of the results indicates the existence of two
separate genetic units, corresponding to T. pygmaeus (17
populations) and T. marmoratus (eight populations) re-
spectively . The observed number of alleles was four at
Pep-A, three at Pep-B, eight at Pep-D and three at the Ldh-
2 locus. The allele frequencies are presented in Table I.
Observed genetic heterozygosity averaged 0.20±0.11. A
significant departure from Hardy-Weinberg equilibrium
was observed for Pep-D in the sample from Valado dos
Frades (population 4, P<0.05). No significant genetic dif-
ferentiation was found between cohorts in any
population. Pep-A showed significant population differ-
entiation within T. marmoratus and Pep-B and Pep-D
showed significant population differentiation within T.
pygmaeus (P<0.001 in all three cases).
The PCA scores fell in two non-overlapping groups
with values <-0.22 and >0.11, that we term the “M”-group
and the “P”-group, respectively. Seven adult T.
marmoratus had a genetic make-up that placed them in
the M-group and 77 adult T. pygmaeus had a genetic
make-up that placed them in the P-group. Common alleles
with diagnostic properties are Pep-Dd and Pep-Df and, to
a lesser extent, Pep-Aa and Pep-Ad. Individuals hetero-
zygous for the Pep-D diagnostic alleles were observed in
the populations from Alqueidão (population 3), São
Bartolomeu dos Galegos (5), Genrinhas (15), Santa
Susana (16) and Fonte da Pena da Couvinha (23) (n=1 in
all five cases). Less common and rare alleles associated
with either group are Pep-Ab, Pep-Ae, Pep-Da, Pep-Db and
Ldh-2f in the M-group and Pep-De, Pep-Dg, Pep-Dh and
Ldh-2c in the P-group. Alleles shared between the groups
are Pep-Bbde, Pep-Dc and Ldh-2b (Table I). Correct classifi-
cation on a locus-by-locus basis is very good for Pep-D
(k=0.97) and Pep-A (k=0.92), moderate for Ldh-2 (k=0.56)
and poor for Pep-B (k=0.12), in the terminology of Altman
(1991). The distribution of the two groups was spatially
structured to the extent that, first, all ponds yielded either
M- or P-group individuals and not both, and second,
ponds in the centre of the study area had M-group indi-
viduals whereas ponds at the fringe had P-group
individuals. The average distance to the nadir point of the
study area was 12.8±4.3 km for M-ponds and 14.0±7.5 km
for P-ponds. Note that aspects of the spatial distribution
of T. marmoratus and T. pygmaeus in western Portugal
will be dealt with separately (Espregueira Themudo &
Arntzen, in press).
DISCUSSION DISCUSSION
DISCUSSION DISCUSSION
DISCUSSION
We are interested in the distribution and ecology of am-
phibians from the Iberian peninsula, with the particular
aim of elucidating those environmental correlates that
help to reconstruct, explain, predict and understand spe-
cies ranges (e.g. Teixeira et al., 2001; Arntzen, 2006). This
requires extensive surveying based upon reliable species
identification. In Portugal and Spain, as in most other tem-
perate regions, amphibians gather in ponds and streams
for breeding; this, if the time and place of the fieldwork are
chosen advantageously, facilitates the gathering of data.
In practice, most surveys concentrate on offspring, be-
cause adult pond presence may be short, as in “explosive
breeders” (e.g. Rana temporaria), species that mate on
land and only come to the water for offspring deposition
(e.g. Salamandra salamandra), or species that are espe-
cially secretive (e.g. Pelodytes punctatus). As a rule,
however, the earlier the life stage, the more problematic
identification in the field proves to be. Classical keys for
identification of eggs, spawn and larvae (Heron-Royer &
Bambeke, 1889; Boulenger, 1891) have recently been up-
graded (Miaud & Muratet, 2004), and most modern field
guides will include identification keys for adults, larvae
and eggs (e.g. Ferrand de Almeida et al., 2001; Duguet &
Melki, 2003).
We set out to develop a molecular marker technique for
the unambiguous species identification of marbled newt
eggs that would allow all ponds to be investigated, in-
Molecular identification of marbled newtsMolecular identification of marbled newts
Molecular identification of marbled newtsMolecular identification of marbled newts
Molecular identification of marbled newts
Page 3
26
Table I.
Table I.
Table I.
Table I.
Table I. Allele frequencies over four loci in marbled newts from the Caldas da Rainha area. Values in parentheses are the electrophoretic mobilities relative to the most common
allele, shown as 100. Six alleles present elsewhere in Portugal were not encountered in the Caldas da Rainha area. Panels at the bottom present heterozygosity on the assumption
of Hardy-Weinberg equilibrium and average score at the first PCA axis, with and without the locus Ldh-2.
G. Espregueira ThemudoG. Espregueira Themudo
G. Espregueira ThemudoG. Espregueira Themudo
G. Espregueira Themudo & J.W. Arntzen& J.W. Arntzen
& J.W. Arntzen& J.W. Arntzen
& J.W. Arntzen
Triturus pygmaeus
T. marmoratus
Population
Total
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Sample size
226
13
19
12
22
6
8
8
9
8
1
7
10
8
2
7
3
4
9
8
10
21
10
7
4
10
adults
84
0
19
12
22
0
5
0
0
1
1
7
10
0
0
0
0
0
4
0
0
1
0
2
0
0
larvae
41
5
0
0
0
6
0
0
0
0
0
0
0
0
0
0
3
0
5
0
1
20
0
1
0
0
embryos
101
8
0
0
0
0
3
8
9
7
0
0
0
8
2
7
0
4
0
8
9
0
10
4
4
10
Locus and allelePep-A
a
(122)
0.56
0.83 0.85 0.91 0.95 1.00 1.00 1.00
b
(107)
0.05
d
(100)
1.00
1.00
1.00
1.00 1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.44
0.17 0.15 0.05
e
(78)
0.05
Pep-B
b
(113)
0.13
0.29
0.07 0.08 0.19
0.15
0.17
0.11
0.06
0.07
d
(100)
0.38
0.76
0.46
0.66 0.92 0.75 0.81 1.00 0.94
1.00 0.93 0.60 1.00 1.00 0.79 0.83 1.00
0.89
0.94 0.95 0.88 1.00 0.93 0.88 0.75
e
(88)
0.62
0.11
0.25
0.27
0.06 0.19
0.06
0.07 0.25
0.21
0.05 0.05
0.07 0.13 0.25
Pep-D
a
(125)
0.06
0.14
0.07
b
(121)
0.06
c
(117)
0.50
0.06
d
(112)
0.04
0.08
0.07 0.17
0.78
1.00 1.00 0.86 1.00 0.93 1.00 1.00
e
(106)
0.11
0.04
0.18
0.25
0.50
f
(100)
0.96
0.84
0.71
0.71 0.92 0.56 0.94 0.50 0.50
1.00 1.00 1.00 0.94 1.00 0.79 0.83 1.00
0.06
g
(95)
0.04
0.05
0.11
0.06
0.06
0.14
h
(88)
0.21
0.19
Ldh-2
b
(100)
1.00
1.00
0.96
1.00 1.00 1.00
1.00
1.00 1.00
0.83
0.56
1.00 0.45
1.00
c
(72)
0.04
f
(36)
0.17
0.44
0.55
Three loci
He
0.19
0.23
0.38
0.32 0.11 0.35 0.15 0.18 0.22
0.00 0.05 0.20 0.04 0.00 0.25 0.22 0.00
0.38
0.14 0.12 0.22 0.03 0.10 0.08 0.13
SE on He
0.15
0.12
0.20
0.16 0.06 0.18 0.10 0.18 0.16
0.00 0.05 0.20 0.04 0.00 0.13 0.11 0.00
0.09
0.09 0.08 0.02 0.03 0.05 0.08 0.13
Weighted average
0.214±0.132
0.163±0.063
Four loci
He
0.05
0.09
0.25
0.19 0.08 0.25
0.00 0.04 0.15
0.25
0.39
0.26
0.13
SE on He
0.05
0.06
0.15
0.13 0.05 0.15
0.00 0.04 0.15
0.08
0.11
0.07
0.13
Weighted average
0.150±0.100
0.284±0.084
Average score along first PCA axis
Three loci
0.34
0.32
0.28
0.29 0.28 0.28 0.32 0.25 0.25
0.33 0.32 0.33 0.33 0.33 0.28 0.23 0.33
-0.44 -0.60 -0.66 -0.68 -0.73 -0.72 -0.72 -0.69
Four loci
0.41
0.37
0.36
0.35 0.33 0.37
0.37 0.36 0.39
0.23
-0.36
-0.57
-0.51
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27
cluding technically problematic ones such as albercas
and lakes. In ponds with aquatic vegetation absent, rare
or out of reach, collecting may be facilitated by the intro-
duction of strings of thin plastic liner available for
egg-deposition, cut from, for example, garbage bags. With
the phenotypic identification of adult T. marmoratus and
T. pygmaeus as a reference and acknowledging the
equivalent allelic expression among embryos, larvae and
adults, the observed “M” and “P” enzyme profiles can be
equated with T. marmoratus and T. pygmaeus, respec-
tively. The single case of departure from Hardy-Weinberg
equilibrium, with a lower than expected number of
heterozygotes, may well be attributed to a less than opti-
mal resolution of Pep-D zymogram, perhaps caused by
interference of the enzyme and the mucous components
in larval tissue.
The method of identifying T. marmoratus versus T.
pygmaeus through expressed protein loci is lethal when
applied to embryos. However, considering the high fe-
cundity of marbled newts (Arntzen & Hedlund, 1990) we
presume that the effect will be negligible at the population
level. Tissue sampling by clipping tail-tips has been
shown to have no ill effects in adult big-bodied newts
(Arntzen et al., 1999). The effect of tail-tip sampling of lar-
vae has not yet been studied. For applying our method of
species identification in other parts of the Iberian penin-
sula a note of warning is in place, since we observed
geographic variation in the level of discrimination
achieved by Pep-B and Pep-D (unpublished data). This
reservation is in line with the observed population genetic
differentiation in the Caldas da Rainha area for both spe-
cies. An alternative technique for species identification
would be the DNA sequencing of a mitochondrial gene
such as COI, currently known as “bar-coding” (Hebert et
al., 2003; Vences et al., 2005). An important shortcoming
of this marker is that, in a phylogenetic sense, it may not
represent the species from which it is isolated correctly,
due to hybridizsation or incomplete lineage sorting, in
Fig. 1. Fig. 1.
Fig. 1. Fig. 1.
Fig. 1. Histograms representing the scores along the
first principal coordinate axis for enzyme genetic
markers in marbled newts from the Caldas da Rainha
area in western Portugal, with four enzyme loci studied
in 39 larvae and 82 adults (A) and three enzyme loci
studied for an additional 101 embryos, two larvae and
two adults (B). Individuals in B also figuring in A are
marked by grey shading. Adults identified from
morphology are shown by horizontal hatching (Triturus
marmoratus, n=6) and diagonal hatching (T. pygmaeus,
n=76). Note that these fall into different, non-
overlapping M- and P- groups, respectively.
Fig. 2.Fig. 2.
Fig. 2. Fig. 2.
Fig. 2. Histograms representing the scores along the
first principal coordinate axis derived from 87 alleles in
a panel of 30 variable enzyme genetic markers in the
newts Triturus helveticus and T. vulgaris in Mayenne,
western France (A: after Arntzen et al., 1998) and hybrid
index based on nine diagnostic enzyme genetic markers
for the newts T. cristatus and T. marmoratus in the same
area (B: after Arntzen & Wallis, 1991).
Molecular identification of marbled newtsMolecular identification of marbled newts
Molecular identification of marbled newts Molecular identification of marbled newts
Molecular identification of marbled newts
Page 5
28
combination with maternal inheritance and low effective
population size compared to nuclear genetic markers.
This phenomenon, that ultimately may involve the com-
plete “mtDNA-capture” by one species from the other,
appears fairly frequent in salamanders. Discordance be-
tween the signature of mitochondrial and nuclear genetic
markers has been found in various genera of plethodontid
(e.g. Batrachoseps, Wake & Jockusch, 2000) and
salamandrid salamanders (e.g. Chioglossa, Sequeira et
al., 2005; Salamandra, Garcia-Paris et al., 2003; Taricha,
Kuchta & Tan, 2005). Well-documented cases in big-bod-
ied newts (genus Triturus) include 1) the near-complete
bias for T. cristatus mothered hybrids in T. marmoratus x
T. cristatus interspecies hybrids in western France
(Arntzen & Wallis, 1991) and 2 ) the presence of mtDNA
typical of T. karelinii in T. carnifex and T. dobrogicus
over a large area of northern Serbia (Wallis & Arntzen,
1989; Arntzen & Wallis, 1999). An example among small-
bodied newts, genus Triturus (or Lissotriton or
Lophinus) is the replacement across the entire Carpathian
mountain range of the original T. montandoni mtDNA by
that of T. vulgaris (Babik et al., 2005). Under the notion
that flawed inferences from mtDNA may not be infre-
quent, its choice as a species marker was in this study
rejected a priori. Nuclear microsatellite DNA markers
have been successfully used to uncover genetic variation
in T. marmoratus (Jehle et al., 2001, 2005; Krupa et al.,
2002), but in our experience, it is not easy to isolate and
amplify nuclear DNA from freshly deposited embryos.
This technique may require a larger number of copies of
nuclear DNA than is available in this life stage, the one
most frequently observed in the field, and its application
would involve raising the embryos in the laboratory.
The contact zone between T. marmoratus and T.
pygmaeus runs over approximately 600 km across central
to western Iberia, from approximately Madrid in central
Spain to north of Lisbon in Portugal. None of the 25
ponds around Caldas da Rainha had a mixed population
and individuals with intermediate enzyme profiles were
not found (Fig. 1). This suggests the absence in our sam-
ple of F1 interspecies hybrids. On the other hand, the
pattern of allozyme discrimination here revealed is flat U-
shaped (Fig. 1), rather than more sharply -shaped as in
the T. helveticus–T. vulgaris and T. cristatus–T.
marmoratus situations (Fig. 2). The extent to which the
shape of the curve represents incomplete diagnosticity of
the enzyme genetic markers (as in T. marmoratus and T.
pygmaeus and T. helveticus–T. vulgaris) versus gene
flow has yet to be determined.
The minimum observed distance between T.
marmoratus and T. pygmaeus populations in this study
was 3.3 km. This contrasts to observations in Spain where
the recorded minimum distance between the species was
about 26 km between Cilleros and Zarza la Mayor in west-
ern Spain and about 6 km between Hoyo de Manzanares
and Villalba in central Spain (García-París et al., 2001).
With just three localities of T. marmoratus and eight lo-
calities of T. pygmaeus recorded in the province of
Madrid (Garcia-Paris et al., 1993), both species are locally
rare and the contact zone between them has presumably
deteriorated, effectively forming a residual contact zone,
sensu Szymura (1993).
In the section of the T. marmoratus–T. pygmaeus dis-
tribution considered here, the contact zone between the
species is firmly parapatric. Moreover, the data point to
the complete or near-complete genetic isolation of the
taxa, therewith supporting their specific status under the
Biological Species Concept. The taxonomic change was
first put forward by García-París et al. (2001) and imple-
mented by, for example, Frost (2004) and Montori et al.
(2005). Our study differs from theirs in the following re-
spects: 1) a fully diagnostic panel of nuclear genetic
markers; 2) utilization of the mitochondrial genetic marker
rejected; 3) larger number of populations (25 versus six);
4) small minimum inter-pond distances (3.3 km versus 6.0
km); and 5) not part of a residual contact zone. All too fre-
quently, taxonomic and nomenclatorial change is
proposed on the basis of a single type of data, including
cases that rely on mtDNA data exclusively (e.g.
Salamandrina, Mattoccia et al., 2005; Plethodon, Mead
et al., 2005; Carlia, Couper et al., 2005). This contrasts
with studies that integrate evidence from two or more
sources, such as morphology, allozymes, mtDNA and
nuclear DNA (e.g. Calotriton, Carranza & Amat, 2005;
Scaphiophryne, Glos et al., 2005; Salamandrina, Nascetti
et al., 2005; Hyla, Salducci et al., 2005).
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
We thank N. Ferrand for support and discussion. The
work was carried out under licence from the ICN (Instituto
de Conservação da Natureza) in accordance with national
law for capturing wild fauna, and was financed by FCT
(Fundação para a Ciência e Tecnologia) research project
POCTI/34110/99.
REFERENCESREFERENCES
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Accepted: 21 June 2006
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