is one of the most abundant shorebirds using coastal habitatsin the East Atlantic migratory flyway, that links arctic breeding locations (Greenlandto Siberia) with wintering grounds (West Europe to West Africa). Differential migrationand winter segregation between populations have been indicated by morphometricsand ringing recoveries. Here, we analyse the potential of genetic markers (mitochondrialDNA – mtDNA) to validate and enhance such findings. We compared mtDNAhaplotypes frequencies at different wintering sites (from north-west Europe to WestAfrica). All birds from West Africa had western (European) haplotypes, while theeastern (Siberian) haplotypes were only present in European winter samples, reachinghigher frequencies further north in Europe. Compilation of published results frommigrating birds also confirmed these differences, with the sole presence of Europeanhaplotypes in Iberia and West Africa and increasingly higher frequencies of Siberianhaplotypes from south-west to north-west Europe. Comparison with publishedhaplotype frequencies of breeding populations shows that birds from Greenland,Iceland, and North Europe were predominant in wintering grounds in West Africa,while populations wintering in West Europe originated from more eastern breedinggrounds (e.g. North Russia). These results show that genetic markers can beused to enhance the integrative monitoring of wintering and breeding populations,by providing biogeographical evidence that validate the winter segregation ofbreeding populations.
Bird migration is a complex and adaptable life and survival strat-
egy (Berthold, 2001), used by a large variety of species, including
most of the species of shorebirds worldwide (del Hoyo
et al
.,
1996). Comparison of migratory ecology of these species reveals
a large variation in migration patterns (Pienkowski & Evans,
1984). In some species, this variability also occurs between popula-
tions, sex, or age classes that may be segregated, fully or partially,
on their wintering grounds (Nebel
et al
., 2002). Such intraspecific
differences are even more interesting when more than one popu-
lation occurs on the same migratory flyway, since populations may
mix during migration and different patterns of spatial segregation
can occur, at the intrapopulation or interpopulation level. That
may be the case of dunlin
Calidris alpina
populations that use the
‘East Atlantic flyway’. This flyway connects circumpolar breeding
areas from Greenland to Siberia with wintering areas in West
Europe and West Africa (Smit & Piersma, 1989).
The dunlin is a long distance migrant shorebird with a
circumpolar breeding distribution (del Hoyo
et al
., 1996; Clements,
2000). At least three subspecies (
C. a. arctica
,
C. a. schinzii
, and
C. a. alpina
) migrate along the East Atlantic flyway to reach their
wintering areas ranging from north-west Europe (British Isles
and South Scandinavia) to West Africa (del Hoyo
et al
., 1996).
C. a. arctica
breeds in north-east Greenland,
C. a. schinzii
in
Iceland to Baltic and South Scandinavia, and
C. a. alpina
in
1
CIBIO, Centro de Investigação em
Biodiversidade e Recursos Genéticos, Campus
Agrário de Vairão, 4485-661 Vairão, Portugal,
2
Departamento de Biología, Facultad de
Ciencias del Mar y Ambientales, Universidad de
Cádiz, Apartado 40, 11510 Puerto Real, Spain,
3
National History Museum, University of Oslo,
PO Box 1172 Blindern, N-0318 Oslo, Norway
*Correspondence: Ricardo J. Lopes, CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal. E-mail: [email protected]†Present address: Buskerud fylkeskommune, Fylkeshuset, NO-3020 Drammen, Norway
Blackwell Publishing Ltd
Geographical segregation in Dunlin
Calidris alpina
populations wintering along the East Atlantic migratory flyway – evidence from mitochondrial DNA analysis
2001a,b) and despite their relevance, integrative analyses of winter
populations have been few so far (Lopes
et al
., 2006). This is most
likely due to the logistics involved in getting DNA samples from
distant winter sites (e.g. west Africa), and because it is usually
more difficult to catch waders during winter conditions. Further-
more, the published results, which focus on single sites, need to
be integrated and interpreted on a larger geographical scale, as
shown in Wennerberg (2001a).
The main goal of this paper was to revise and update our
current knowledge of dunlin winter distribution along the
East Atlantic flyway using genetic markers. The presence of
geographical segregation between wintering assemblages was
investigated using samples of birds that winter in different areas
along the East Atlantic flyway and potentially mix and interact on
migration stopover sites. Our rationale is that an accurate validation
of baseline data is necessary to address biogeographical patterns
of dunlin winter distribution at the subspecies and population
level. While this is already made for some parameters, such as
species abundance and distribution (see Wetlands International,
2002), at the subspecies and population level this kind of data
will enhance our understanding of the biogeographical dynamics of
this migratory shorebird. Also, it is relevant for the conservation
and monitoring of these non-breeding populations (Piersma &
Lindström, 2004) and from site-based to flyway-based conservation
of the habitats they rely on.
METHODS
Population sampling
Dunlins were sampled at different sites along their wintering
range in the East Atlantic flyway (Sweden, UK, Portugal, South
Spain, Morocco, and Guinea-Bissau) from 1995 to 2007
(Table 1). The birds were captured using mist-nets during night
periods, with the exception of UK (collection of dead birds).
Blood samples of 20–50
μ
L were taken from all live birds by
venipuncture of the brachial vein (muscle tissue was collected
Figure 1 Statistical parsimony network of mtDNA lineages, as implemented in TCS 1.21 (Clement et al., 2000). Lineages were joined at the 95% confidence criterion. Each nucleotide substitution between haplotypes is shown as a dot, as well as the TC substitution that is used to discriminate between Eur (European) and Sib (Siberian) haplogroups using AluI restriction enzyme. Haplotypes were described in Wenink et al. (1993) and are deposited in GenBank (accession numbers L06721 to L06755).
Figure 2 The mtDNA haplotype frequencies of dunlin Calidris alpina sampled during winter and migration periods along the East Atlantic Flyway as well as in breeding regions. Each pie shows the proportion of European (black) and Siberian (white) haplotypes at each region. Where available, they represent pooled data collected from more than one site or date. The sample sizes are also presented next to each pie in brackets. Breeding data were assembled from Wenink et al. (1993, 1996), Wennerberg et al. (1999), Wennerberg (2001a) and updated with unpublished data (L.W., unpublished data). The East Atlantic Flyway indicative limits are shown in migration panels while the winter range (dark grey) is shown in the winter panel. In the inset panel we show the mtDNA European (Eur) haplotype frequencies during winter and on breeding with the 95% confidence limits. Adjacent numbers indicate sample sizes.
from dead birds). Samples were stored in 95% alcohol or in SET-
buffer (0.15
m
NaCl, 0.05
m
Tris, 0.001
m
EDTA, pH = 8.0) and
used for molecular sexing and mtDNA analysis (see below).
Additionally, previous published haplotype frequencies of
migrating and winter flocks from Wenink & Baker (1996),
Tiedemann (1999), Wennerberg (2001a), and Lopes
et al
. (2006)
were included for comparison (Table 1). They were estimated at
stopover sites in Norway, Sweden, Wadden Sea, Poland, Spain,
Portugal, and Morocco, and during winter in East Spain. We
included samples from East Spain in our analysis only for
comparison purposes, since this site may be considered to belong
to another flyway, the Mediterranean flyway. For comparison
with data from the breeding grounds, we also compiled an
updated overview of all published results from mtDNA analysis
of 280 breeding birds from 22 breeding populations (Fig. 1) from
Wenink
et al
. (1993, 1996), Wennerberg
et al
. (1999), Wennerberg
(2001a), and included unpublished additional data (L.W.,
unpublished data).
Mitochondrial DNA
DNA was extracted from blood by phenol/chloroform extraction
according to standard procedures (Sambrook
et al
., 1989) and
from muscle using the DNeasy Blood & Tissue Kit (Qiagen,
Venlo, The Netherlands). The mtDNA (segment of 295 bp from
the hypervariable control-region segment I) was amplified by
polymerase chain reaction (PCR), using the primers L98 and H401
(Wenink
et al
., 1993). The PCR contained 1.0
μ
L DNA (10 ng),
2.5
μ
L of each primer (10
μ
m
), 2.5
μ
L 10
×
PCR buffer, 5
μ
L
dNTP (1.25 m
m
of each nucleotide), 2
μ
L MgCl (1 m
m
), 0.1
μ
L
(1 unit) of
Taq
DNA polymerase (Boehringer Manheim,
Germany), and 9.3
μ
L dH
2
O. The PCR included 2 min at 94
°
C,
35 cycles of (30 s at 94
°
C, 30 s at 54
°
C, and 30 s at 72
°
C), followed
by 25 cycles of (30 s at 94
°
C, 30 s at 48
°
C, and 1 min at 72
°
C)
and finally 72
°
C for 10 min. PCR products were digested with
the restriction enzyme
Alu
I (Roche, Basel, Switzerland) for 3 h.
A nucleotide substitution at position 358 in the SIB lineages
creates an
Alu
I restriction site that is absent from the Eur lineages
sequences (Fig. 1) (Wenink
et al
., 1996; Wennerberg, 2001b). A
control sample with SIB lineage was always included to validate
the restriction reaction. The DNA fragments were separated by
electrophoresis in a 2% agarose gel containing ethidium bromide
and scanned using a FluoroImager (Molecular Dynamics,
USA). The length of each band was compared with reference
bands of all haplotypes, as well as with a size marker (1 kb DNA
ladder, Life Technologies, Gaithersburg, USA).
Table 1 Characterization of the sampled dunlin Calidris alpina populations along the East Atlantic flyway during migrations and on wintering grounds: sampling location, season, sample size, and mtDNA haplotype composition. The table includes data from this study, as well as from: (1) Wennerberg (2001a), (2) Wenink & Baker (1996), (3) Tiedemann (1999), (4) Lopes et al. (2006).
Location Country n Eur Sib % Eur Source
Autumn migration
Tromso Norway 33 25 8 76 (1)
Ottenby Sweden 136 95 41 70 (1)
Falsterbo Sweden 57 35 22 61 (1)
Gdansk Bay Poland 20 11 9 55 (1)
Gdansk Bay Poland 8 4 4 50 (2)
Wadden Sea Germany, Denmark 11 10 1 91 (3)
Wadden Sea Germany 13 7 6 54 (1)
Wadden Sea Germany, The Netherlands 14 11 3 79 (2)
Tarragona Spain 14 11 3 79 (1)
Mondego estuary Portugal 55 55 0 100 This study (1) (4)
Winter
Falsterbo Sweden 37 22 15 59 This study (1)
Wash, North Wales UK 16 11 5 69 This study
Tarragona Spain 5 2 3 40 (1)
Tagus estuary Portugal 80 63 17 79 This study (4)
Cadiz Bay Spain 28 25 3 89 This study
Sidi-Moussa Morocco 17 17 0 100 This study
Bijagos archipelago Guinea-Bissau 14 14 0 100 This study
Spring migration
Falsterbo Sweden 40 33 7 83 (1)
Wadden Sea Germany 15 13 2 87 (1)
Wadden Sea Germany, Denmark 24 16 8 67 (3)
Wadden Sea Germany, The Netherlands 1 0 1 0 (2)
Mondego estuary Portugal 48 48 0 100 This study (1) (4)
products were separated by gel electrophoresis in 2% agarose gels
containing ethidium bromide, using 1 kb DNA ladder, and
scanning the gel using a FluoroImager.
Statistical analysis
Differences in haplotype frequencies between samples were tested
using Fisher exact tests (Zar, 1999), while the binomial test was used
to test differences between sex ratios (Zar, 1999; Wilson & Hardy,
2002). The 95% confidence intervals of haplotype frequencies were
also estimated. All tests and estimations were calculated using the
statistics software R 2.4 (R Development Core Team, 2006).
RESULTS
Population segregation on wintering grounds
The mtDNA haplotype frequencies of wintering dunlin populations
(Fig. 2) differed between locations along the East Atlantic flyway
(Table 2). A clear distinction was observed between West Africa,
where only European haplotypes were present, and West Europe,
which included both European and Siberian haplotypes
(increasing higher frequencies of Siberian haplotypes with latitude).
Birds wintering in Sweden and the UK, which are among the
northernmost wintering population in this flyway, had haplo-
type compositions that differed significantly from all wintering
sites further south, with the exception of South Spain (Table 2), with
a considerably higher proportion of Siberian haplotypes (Fig. 2)
and they were not significantly different from each other (Table 2).
Sex segregation on wintering grounds
The haplotype composition in the wintering sites did not differ
significantly between males and females (Table 3). With the
exception of Guinea-Bissau (Table 3), uneven sex ratios (higher
proportions of females) seemed to be present in the other winter-
ing sites (proportion of males: Sweden = 32%; UK = 37%;
Portugal = 43%; South Spain = 36%; Morocco = 35%). However,
these sex ratios were not significantly higher than 1 : 1 ratio, with
the exception of Sweden (one tailed binomial tests: Sweden
P
= 0.02; UK
P
= 0.23; Portugal
P
= 0.11; South Spain
P
= 0.09;
Morocco
P
= 0.17; Guinea-Bissau
P
= 0.60).
Genetic composition during migration
Migrating flocks also showed clear differences in mtDNA haplo-
type frequencies (Fig. 2). West Iberian and West African migrants
only had European haplotypes, while Siberian haplotypes
occurred from Iberia, Wadden Sea, Baltic Sea, to Scandinavia.
While frequencies of European haplotypes were similar between
the Wadden Sea and Scandinavia, the highest proportion of
Siberian haplotypes occurred in populations from the Gdansk
Gulf. Differences between spring and autumn migration could
be tested in the locations that were sampled in both migration
periods but no significant differences between migratory periods
were found at these sites, with the exception of Sweden (Table 4).
Assignment of breeding origin
Wintering flocks could be assigned to different breeding ranges
by comparing their haplotype frequencies with those from
breeding populations (Fig. 2). The frequency of haplotypes from
the West African winter assemblages corresponded to frequencies
found in breeding areas in the western part of the breeding range
(Greenland, Iceland, Baltic Sea) and was significantly different
from eastern (Siberia) populations (Table 5). Bird assemblages
wintering in South Spain corresponded to frequencies found in
Table 2 Probabilities obtained using Fisher exact tests, comparing mitochondrial haplotypes of wintering dunlin Calidris alpina from different locations along the winter range.
North Scandinavia. In contrast to birds using Portugal during
migration, which mainly seem to originate from Icelandic breeding
areas, the Portuguese winter assemblage was similar to North
Scandinavian and North Russian breeding populations (Fig. 2).
The mtDNA haplotype frequencies from wintering birds in
Sweden and UK corresponded to breeding populations in north
Russian since their haplotype frequencies matched the ones
from North Russia and Taimyr (Fig. 2, Table 5). Probably due to
the lack of samples from important breeding areas in the east of
Yamal (Gydan Peninsula, west Taimyr), the Swedish winter
sample differed significantly from all breeding samples included
in Table 5.
DISCUSSION
To interpret the results, we must be aware that each winter flock
may not always correspond to a single breeding population.
Despite the several types of evidence of winter and migrating
Table 3 Mitochondrial DNA haplotype frequencies of wintering dunlin Calidris alpina discriminated by sex. Fisher exact tests were used to compare haplotype ratios between seasons.
Table 4 Mitochondrial DNA haplotype frequencies of migrating dunlin Calidris alpina sampled in autumn and spring migration at the same stopover site. Fisher exact tests were used to compare haplotype ratios between seasons.
Table 5 Fisher exact tests comparing mitochondrial haplotypes of breeding populations with wintering dunlin Calidris alpina from various regions along the East Atlantic Flyway (see Figs 1 and 2).
