Mar Biol
DOI 10.1007/s00227-012-1975-9
ORIGINAL PAPER
Recent history of the European Nassarius nitidus (Gastropoda):
phylogeographic evidence of glacial refugia
and colonization pathways
Naiara Albaina • Jeanine L. Olsen • Lucı́a Couceiro
José Miguel Ruiz • Rodolfo Barreiro
Received: 19 November 2011 / Accepted: 30 May 2012
Ó Springer-Verlag 2012
Abstract Because marine species respond differentially
to factors governing survival and gene flow, closely related
taxa may display dissimilar phylogeographic histories.
New data for the patchily distributed gastropod Nassarius
nitidus throughout its Atlantic–Mediterranean range (col-
lected during 2008 and 2009) were used to investigate its
phylogeography and recent demography. Results based on
mitochondrial COI sequences of 422 N. nitidus individuals
from 15 localities revealed contrasting phylogeographic
and demographic patterns among N. nitidus populations
from each basin. Data suggest the existence of two glacial
refugia, one in the Atlantic, around the Iberian Peninsula,
and the other in the Paleo-Mediterranean Sea (Adriatic).
Bayesian skyline reconstructions suggest that the Adriatic
population of N. nitidus remained largely unaffected by the
Last Glacial Maximum (LGM), whereas the Iberian
Atlantic region experienced dramatic exponential growth
after its conclusion. Contemporary North Sea populations
of N. nitidus are the endpoint of a leading-edge recoloni-
zation process from a southern position. Additionally, a
reanalysis of pre-existing material for the continuously
Communicated by S. Uthicke.
Electronic supplementary material The online version of this
article (doi:10.1007/s00227-012-1975-9) contains supplementary
material, which is available to authorized users.
N. Albaina (&)  L. Couceiro  J. M. Ruiz  R. Barreiro
Department of Ecology, Faculty of Sciences, Universidade da
Coruña, 15071 Coruña, Spain
e-mail: nalbaina@udc.es
J. L. Olsen
Marine Benthic Ecology and Evolution Group, Centre
for Ecological and Evolutionary Studies, University of Groningen,
Nijenborgh 7, 9747 AG Groningen, The Netherlands
•
distributed close congener N. reticulatus was used to
compare both species in the late histories. In contrast to
N. nitidus, N. reticulatus prospered during the LGM and
experienced an earlier Atlantic expansion during the pre-
vious interglacial period. Despite similar life history and
dispersal potential, the results here presented suggest that
subtle differences in microhabitat requirements between
the two species have had important consequences for their
particular distribution in response to glacial events.
Introduction
Understanding the interplay of historical, ecological, geo-
logical and climatic factors governing the distribution of
genealogical lineages within and among closely related
species is the primary objective of phylogeography (Avise
et al. 1987). Events that occurred during the Tertiary and
Quaternary periods have played an important role in spe-
cies origin and evolution towards their present status. In
particular, the last 2.5 million years (MY) have been a time
of intense climatic fluctuations in the North Atlantic marine
intertidal (Cunningham and Collins 1998; Wares and
Cunningham 2001). Following the extension of the north-
ern ice sheets during glacial periods and the drop of sea
level, the range of distribution of intertidal organisms was
generally reduced and moved southwards. Although the
fast pace of habitat alteration often prevented benthic
organisms from adapting and triggered local and regional
extinctions (Cunningham and Collins 1998; Williams et al.
1998; Thomas et al. 2004), it is also known that glacial
refugia provided survival of a fraction of individuals, while
the same type of organisms disappeared from the sur-
rounding areas (Andersen and Borns 1994; Hewitt 1996;
Maggs et al. 2008; Provan and Bennett 2008).
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The phylogeographic history of a species is the sum of
abiotic processes related to coastal topography (Wares
et al. 2001; Marko 2002; Duran et al. 2004; Waters et al.
2005), prevailing currents (Galarza et al. 2009), nutrients
and chance events (Cowen and Sponaugle 2009), as well as
to biotic interactions related to competition (Kinlan and
Gaines 2003) and species-specific traits such as larval
development (Kyle and Boulding 2000) or dispersal
capacity (Shanks et al. 2003; Gysels et al. 2004a; Roman
and Palumbi 2004). As a consequence of such complex
interaction, the fact that two species have gone through the
same paleoclimatic episodes, have a close phylogenetic
relationship or have similar larval dispersal capacities does
not necessarily guarantee that they share phylogeographic
patterns (Taberlet et al. 1998; Avise 2004).
The estuarine dog-whelk Nassarius nitidus (Jeffreys,
1867) is a benthic dweller on muddy-sandy bottoms of the
Black Sea, the Mediterranean and the NE Atlantic, the
latter extending to southern Norway (Hansson 1998).
Although mainly restricted to sheltered bays and estuaries
along the northern Atlantic coast of Spain (Fig. 1a), it is
found through a wider range of habitats in the Wadden Sea
and North Sea (Rolán and Luque 1994; Craeymeersch
and Rietveld 2005). Due to morphological similarities,
N. nitidus is still commonly misidentified as the netted dog-
whelk N. reticulatus (Linnaeus 1758). N. reticulatus,
however, has a much broader Atlantic distribution (Fig. 1a)
including some enclaves in the Spanish Mediterranean
(Rolán and Luque 1994; Couceiro et al. 2007). N. retic-
ulatus has fewer habitat requirements with respect to sed-
iment, salinity or embayment type (Fretter and Graham
1994). Although the two species display subtle differences
in habitat preferences (upper estuaries for N. nitidus; wider
range of salinity and exposure conditions for N. reticula-
tus), they are occasionally found in sympatry, particularly
along the northern coast of Spain. Both species have sim-
ilar reproductive cycles (Barroso and Moreira 1998; Valli
et al. 2003), a relatively long planktotrophic larval stage
(1–2 months), and share a scavenging-based diet (Tallmark
1980; Fretter and Graham 1994). Although our knowledge
about N. reticulatus is relatively extensive (Barreiro et al.
2001; Barroso et al. 2005; Chatzinikolaou and Richardson
2008; Couceiro et al. 2009; Ruiz et al. 2010), studies on
N. nitidus are scarce and, to a great extent, have focused on
taxonomic issues (Collyer 1961; Rolán and Luque 1994;
Sanjuán et al. 1997; Carvajal-Rodrı́guez et al. 2006;
Couceiro et al. 2011). With respect to past and present
phylogeographic status of N. reticulatus, Couceiro et al.
(2007) analysed mtDNA from samples along 1,700-km of
the NE Atlantic coastline. Their results suggested the
existence of a single genetic population that experienced a
demographic expansion from a refuge in the Bay of Biscay
between 160 and 61.5 ky BP.
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Mar Biol
The aims of the present study were to assess the phy-
logeography and historical demography of the patchily
distributed N. nitidus. We used mitochondrial cytochrome
c oxidase subunit I (COI) sequences from Atlantic and
Mediterranean samples to: (1) investigate N. nitidus pop-
ulation structure along its distribution range; (2) explore
the demographic history of N. nitidus populations with a
focus on the Last Glacial Maximum (LGM; 26.5–19 years
ago) (Clark et al. 2009); and (3) ascertain whether
N. nitidus demographic processes were comparable to those
experienced by the continuously distributed N. reticulatus.
This study completes those preliminary insights of Couceiro
et al. (2011) to N. nitidus population structure and constitutes
the first approach to recent demography of both N. nitidus
and N. reticulatus species.
Materials and methods
Sampling
An extensive search of N. nitidus from Norway to Turkey
was conducted in 2008–2009. The survey covered almost
entirely the geographic range of the species (which is also
present in the Black Sea). The first author (N. A.) per-
sonally visited locations for which N. nitidus had been
reported and/or consulted with local, place-based col-
leagues (see Fig. 1a; Table given in Online Resource 1,
OR1).The species was not found along large stretches of
potentially suitable coast on the Atlantic basin. Rather,
specimens were found in only a few sheltered bays, which
were hundreds or thousands of km distant from the next
nearest area of presence. In the Mediterranean, N. nitidus
was found only in the Adriatic despite several efforts to
collect it in the western Mediterranean, Tyrrhenian and
Aegean Seas (Fig. 1a; OR1).
At each of the 15 sampling sites (Fig. 1), individuals
were attracted by mussel bait and brought into the labo-
ratory for immediate processing. When transport to the
laboratory was delayed, specimens were preserved in 96 %
ethanol.
DNA extraction and sequencing
DNA was extracted from foot tissue using Chelex-100
resin (Estoup et al. 1996) or a spin column kit (DNeasy
Blood and Tissue Kit, Qiagen International). Universal
primers were used as described by Folmer et al. (1994). A
710-bp fragment of the mitochondrial COI gene was
amplified, from which a complete 477-bp alignment was
obtained. Reactions were performed in a 25 ll solution
containing 5 lM of each primer (Bonsai Technologies
Group), 0.2 mM of each dNTP (Fermentas), 2 lM MgCl2,
Mar Biol

Fig. 1 Map of the European

coasts showing sampling sites
for N. nitidus. a Diversity plot
following Petit et al. (2003)
scheme in which red and blue
circles represent diversities
above and below the mean,
respectively. Diameter is
proportional to distance from
the average value. Beside each
the North Sea, North Iberian
Peninsula (NIP) and Adriatic
regions, the proportion of
private haplotypes with respect
to the total number of
haplotypes is indicated.
Surveyed sites lacking N.
nitidus are marked with black
stars. From N to S and from W
to E: Norway, Germany, UK (2
sites), France (Atlantic, 2),
Portugal (2), Spain (3), France
(Mediterranean, 2), Italy,
Greece, Bulgary, Turkey and
Israel. Distribution ranges for
N. reticulatus and for N. nitidus
are indicated by green and
orange lines, respectively.
b Haplotype frequencies.
Dashed lines indicate the three
clusters detected in the
parsimony network (Fig. 2).
The LGM coastline is marked
by a solid black line; the British
and Scandinavian ice sheet
extension is depicted in grey.
Redrawn and modified from
several sources (colour figure
online)

123

19 Buffer, 0.4 U AmpliTaq DNA Polymerase (Applied
Biosystems) and 1 ll of template DNA. The thermal
cycling protocol consisted of an initial denaturation of
958C for 2 min followed by 40 cycles of 95 °C for 30 s,
45 °C for 30 s and 72 °C for 1 min. PCR products were
purified with exonuclease I and shrimp alkaline phospha-
tase (Fermentas) and sequenced on ABI 3730XL (Applied
Biosystems) using BigDyeTM (Applied Biosystems) ter-
minator cycling conditions.
Data analysis
COI sequences were unambiguously aligned and edited in
CODONCODE ALIGNER v.2.0 (CodonCode Corporation,
Dedham, MA, USA). Species identification was verified by
following Couceiro et al. (2011). Haplotype and nucleotide
diversities were determined with DNASP v.4.5 (Rozas
et al. 2003). Gene genealogies were constructed using the
statistical parsimony method described by Templeton et al.
(1992) with TCS (Clement et al. 2000). To provide sta-
tistical support to genealogical groups detected in the
parsimony network, a Bayesian phylogenetic analysis was
carried out in MRBAYES3 (Ronquist and Huelsenbeck
2003), using two N. reticulatus sequences Couceiro et al.
2007) as outgroup. Since the N. reticulatus COI fragments
were shorter than those from N. nitidus, our sequences
were trimmed and the phylogenetic analysis was performed
using only the 395-bp common segment. Searches were run
for 10 million MCMC generations and sampled every 100.
First 50,000 generations were discarded. The best-fit model
of evolution for the data was determined using hierarchical
likelihood ratio tests in MRMODELTEST v.2.3 (Nylander
2004) through PAUP v.4.0 (Swofford 2002).
Genetic structure within and between groups of samples
(based on haplotypes and diversity) was tested using
ARLEQUIN v.3.1 (Excoffier et al. 2005). Analyses of
molecular variance (AMOVA, Excoffier et al. 1992) and
also pairwise comparisons (Reynolds et al. 1983; Slatkin
1995) were performed based on F (Fst, Fct and Fsc, Wright
1951) and U (Ust, Uct and Usc, Excoffier et al. 1992)
diversity indexes. For A-statistic calculations, Tamura–
Nei ? C distance (ARLEQUIN best alternative to the
HKY ? C model followed by our data) was used. Signif-
icance was assessed by permuting data for 16,000 repli-
cates and by applying Hochberg’s correction (1988).
To test for isolation by distance (IBD) over the entire
sampling range, as well as for the Atlantic data, we used
the software ISOLDE as implemented in GENEPOP v.4.0
(GENEPOP on the Web; Raymond and Rousset 1995;
Rousset 2008), where a Mantel test (Mantel 1967) between
genetic (Ust/1 - Ust) and geographic pairwise distances
under a two-dimensional stepping stone model (Rousset
1997) was performed via 16,000 permutations.
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Mar Biol
The demographic history of each phylogeographic
group was explored in two ways. First, neutrality statistics
based on the distribution of the mutation frequencies
(Tajima’s D; Tajima 1989) and on the haplotype distribu-
tion (Fu’s Fs; Fu 1997) were estimated (H0: constant
effective population size), and the mismatch distribution
profile obtained (Rogers and Harpending 1992). Signifi-
cance was assessed by 16 000 replicates in ARLEQUIN
v.3.1 (Excoffier et al. 2005). The fit of our results to a
model of demographic expansion (H0: population expan-
sion; Rogers and Harpending 1992) was further evaluated
by considering both the sum of square deviations (SSD)
and the raggedness index (rg, Harpending 1994). When-
ever both the neutrality tests and the mismatch-based dis-
tribution tests fit an expansion model, the relationship
s = 2ut (Rogers and Harpending 1992) was used to
approach the time since expansion, where s is the empirical
estimate of the time of expansion, u is the mutation rate per
sequence per year, and t is the time to present since the
beginning of the expansion. It is important to recall that a
non-rejection of the null hypothesis of expansion should
not necessarily be treated as proof that the hypothesis is
true. In particular, the SSD test is known to be very con-
servative and rarely leads to rejection of the expansion
model (Schneider and Excoffier 1999; Ramos-Onsins and
Rozas 2002).
The posterior distribution of the effective population size
through time and the moment of haplotype coalescence
(Most Recent Common Ancestor, MRCA) was inferred
using the Bayesian Skyline Plot model (BSP; Drummond
et al. 2005) implemented in BEAST v.1.5.3 (Drummond
and Rambaut 2007). The BSP framework is a valuable tool
for tracking changes in effective population size because all
of the genealogy, demographic history and substitution-
model parameters are co-estimated in a single analysis.
Therefore, confidence intervals represent the combined
phylogenetic and coalescent uncertainty (Ho and Shapiro
2011). As the model assumes no historical gene exchange,
populations are considered to be independent. Although an
analysis of isolation with migration using IMa (Hey and
Nielsen 2007) proved impossible due to repeated crashes of
the program, the other analyses provided enough evidence
to consider the presence of two separate, isolated popula-
tions of N. nitidus, supporting the compatibility of our data
under an isolation model in BEAST. Consequently, the
Adriatic and North Iberian Peninsula (NIP) groups were
treated separately, while the SW, NE and BE samples were
excluded from the simulation. Each subset was run twice for
50 million generations and sampled every 1,000. A strict
molecular clock was assumed under an HKY ? I ? C
substitution model, starting from an UPGMA tree and
applying default priors after a burn-in period of 5 million
steps. The fit of data to a strict molecular clock rate was
Mar Biol

verified by calculating the standard deviation of the
uncorrelated lognormal relaxed clock. Log files for each run
were pooled with LOGCOMBINER to check for effective sample
size values (ESS). We assumed Wilke’s ‘‘trait-specific’’
substitution rate estimated for the COI gene of small,
aquatic, dioecious, ectothermic and one-year generation
time Protostomia (1.57 ± 0.45 % My-1, under the
HKY ? I ? C model; Wilke et al. 2009) to obtain results in
relevant time units (errors associated with the substitution
model were incorporated in data estimations). The clock
was calibrated using substitution rates calculated for 12
pairs of sister taxa from five groups of Protostomia sepa-
rated by discrete biogeographic events. As always, molec-
ular clock calculations must be interpreted with caution
because the clock rate is not corrected for ancestral poly-
morphisms and is likely to overestimate divergence times.
The same BSP procedure in BEAST was also applied to
the close relative N. reticulatus using 156 (395-bp) COI
sequences previously published (for which only one single
but widely spread Atlantic population has been reported;
Couceiro et al. 2007). The N. reticulatus data fit an HKY
model (modeltest best-fit model of evolution), and conse-
quently, a 1.24 ± 0.22 % My-1 substitution rate (Wilke’s
clock rate under the HKY model) was used to transform
estimates in appropriate time units. To rule out that any
difference between N. reticulatus and N. nitidus could be
due to the use of different substitution rates for each spe-
cies, the demographic history of N. reticulatus was also
estimated using the same substitution model and rate
employed with N. nitidus.
Results
Genetic diversity and haplotype genealogy
The 477-bp COI fragment obtained for 422 individuals of
N. nitidus produced a total of 68 polymorphic sites
(14.3 %) of which 33 were parsimony informative (6.9 %).
Of 81 haplotypes identified, 64 (79 %) were unique to a
single location (Table 1). Haplotypes can be found in
GenBank (accession numbers: GQ219744–GQ219751,
GQ219753–GQ219765 and GU270587–GU270648).
Locations in the North Sea region of the Atlantic basin
(henceforth North Sea) exhibited the lowest haplotype
(mean: 0.16) and nucleotide (mean: 0.09 9 10-2) diversity
estimates. Diversity gradually increased southwards in NIP
sites (0.72 and 0.81 9 10-2) and reached their maxima in
the Adriatic Sea (0.976 and 0.82 9 10-2) (Table 1; Fig. 1).
The most common haplotype (h20) was present at every
locality with the exception of Venice (Fig. 1). Its relative
abundance gradually decreased from an overwhelming

Table 1 N. nitidus sampling localities, identifiers (ID), geographic coordinates, sample size (n), number of segregating sites (S), number of total
(Ht) and private (Hp) haplotypes, haplotype (h) and nucleotide (p 9 102) diversities (with SD)
Locality ID Position n S Ht Hp h p 9 102

Lysekil, Sweden SW 58°160 N, 11°250 W 41 1 2 1 0.049 (0.046) 0.01 (0.01)

Grevelingen, Netherlands NE 51°420 N, 03°590 W 30 3 2 0 0.186 (0.088) 0.12 (0.06)
Oostende, Belgium BE 51°130 N, 02°570 W 23 4 3 1 0.312 (0.115) 0.23 (0.13)
0 0
Fuenterrabı́a*, Spain SP (1) 43°22 N, 01°47 W 25 13 6 1 0.727 (0.061) 0.77 (0.13)
Santoña*, Spain SP (2) 43°260 N, 03°270 W 21 13 7 2 0.733 (0.082) 0.76 (0.32)
Neda*, Spain SP (3) 43°2900 N, 08°100 W 26 10 4 1 0.618 (0.058) 0.77 (0.08)
P. porco*, Spain SP (4) 43°200 N, 08°120 W 29 14 8 3 0.702 (0.079) 0.63 (0.12)
Sada*, Spain SP (5) 43°210 N, 08°140 W 34 13 8 4 0.783 (0.039) 0.78 (0.09)
0 0
Rianxo, Spain SP (6) 42°38 N, 08°48 W 47 15 7 3 0.698 (0.038) 0.82 (0.06)
Poio, Spain SP (7) 42°250 N, 08°410 W 31 16 7 3 0.662 (0.056) 0.80 (0.07)
S. Simón*, Spain SP (8) 42°180 N, 08°370 W 26 14 8 4 0.711 (0.079) 0.80 (0.08)
Civitanova, Italy IT (1) 43°180 N, 13°430 W 25 27 21 14 0.983 (0.017) 0.82 (0.08)
Cesenatico, Italy IT (2) 44°110 N, 12°240 W 30 33 25 14 0.982 (0.016) 0.85 (0.07)
0 0
Venice, Italy IT (3) 45°25 N, 12°20 W 20 27 17 9 0.984 (0.020) 0.96 (0.08)
Grado, Italy IT (4) 45°400 N, 13°240 W 14 12 8 4 0.890 (0.060) 0.57 (0.10)
Regional total
North Sea 94 5 4 2 0.160 (0.050) 0.09 (0.03)
NIP 239 37 27 18 0.722 (0.018) 0.81 (0.03)
Adriatic 89 48 54 47 0.976 (0.007) 0.82 (0.04)
Total 422 68 81 67 0.759 (0.019) 0.75 (0.03)
Asterisks denote sites where N. reticulatus was found in sympatry with N. nitidus

123

dominance in North Sea sites, to 23–52 % in NIP, finally
reaching minimum values in the Adriatic (7.9 % on aver-
age). Haplotypes h16 and h21 were likewise abundant, but
they occurred exclusively in the Atlantic (average fre-
quency of 12 and 23 %, respectively). H16 was found along
the entire Atlantic coastline, while h21 was only detected in
NIP sites. The predominance of just three haplotypes in the
Atlantic contrasted with the high number of low-frequency
haplotypes found in the Adriatic, where only h53, h59 and
the aforementioned h20 were locally abundant.
Fig. 2 Statistical parsimony network for 81 N. nitidus COI haplo-
types. Each circle represents a haplotype with its name (outside) and
sampling sites (inside). Circle size is proportional to strain frequency.
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Mar Biol
The haplotype network (Fig. 2) revealed three secondary
lineages, each one derived from the common haplotypes h16,
h21, and h59 and interconnected through the central, highly
abundant h20. For these secondary lineages, posterior
probabilities were C 0.92 (see the Bayesian phylogenetic
tree OR2). Clade 1 (around haplotype h21) was endemic to
the NIP (posterior probability value 1.00) and considerably
divergent (six mutational steps) from the ancestral h20.
Clade 2 (posterior probability value 0.92), although mostly
found at the NIP sites, also had representatives in the North
Coloured dashed lines delineate the three main lineage clusters. Lines
between haplotypes are single mutational steps; small black dots
indicate unrecorded haplotypes (colour figure online)
Mar Biol
Sea. Interestingly, both clades 1 and 2 emerged from h21
through haplotypes found only in the Adriatic (for clade 1,
this observation was supported by a 0.98 posterior proba-
bility of the phylogenetic analysis). Mean sequence diver-
gence between clades 1 and 2 was 2.22 % (Tamura–Nei
distance) and included 19 fixed mutational positions. Finally,
clade 3 (posterior probability value 0.99) consisted of 19
haplotypes recently derived from h59 and was confined to the
Adriatic Sea (phylogenetic posterior probability value 0.99;
this analysis included in clade 3 two single types sampled
elsewhere, probably as a result of the shortening of sequen-
ces necessary for tree construction). The remainder of the
network (81 %) consisted of predominantly single-mutation
haplotypes always \ 3 mutations away from the dominant
h16, h21, h59 and h20. This star-like pattern is often regarded
as a signature of a recent population expansion. It is worth
noting that the haplotypes found on each side of the Strait of
Gibraltar were mutually exclusive, with the exception of the
ancestral, highly abundant in the Atlantic h20 and the phy-
logenetically close h42, for which nine individuals (seven
and two, respectively) were collected in the Mediterranean.
At least two of the terminal haplotypes collected in the NIP
(h29 and h40, and probably h41) appear as direct descen-
dants of a series of Adriatic types (2–3 mutational steps not
sampled in the NIP). In addition, the h41 position was sup-
ported by the Bayesian phylogenetic inference (see OR2).
Population structure
Estimates of population differentiation based on haplotype
and nucleotide distances were nearly identical; therefore,
only the results for nucleotide distances are presented. The
AMOVA revealed a highly significant structure (Uct =
0.196, P \ 0.001) among the three sets of samples (North
Sea, NIP and Adriatic). Differences within groups were not
significant and accounted for only 3.4 % of the total vari-
ance. Except for a few cases involving sites NE, BE and IT
(3), most inter-regional pairwise comparisons were signif-
icant after Hochberg’s correction. Pairwise Ust values
ranged from 0.067 to 0.563 for the North Sea–NIP con-
trasts, from 0.073 to 0.435 for the NIP–Adriatic combina-
tions, and from 0.063 to 0.491 for the Adriatic–North Sea
ones (see Table in OR3).
Genetic divergence estimates were positively correlated
with geographic distance for both the whole set of samples,
as well as for the more restricted Atlantic set (P \ 0.001,
r = 0.494 and P \ 0.001, r = 0.518, respectively; not
shown).
Historical demography
Under the assumption of selective neutrality, Tajima’s
D and Fu’s Fs regional estimates for the North Sea and NIP
failed to detect any significant departure from the null
hypothesis of equilibrium (Table 2). For the Adriatic,
significant D and Fs estimations indicated an excess of
low-frequency haplotypes. Subsequent mismatch distribution
analyses revealed different patterns among regions (Fig. 3).
In the North Sea, a strongly skewed, unimodal profile was
obtained, and neither the SSD nor the rg index rejected the
null hypothesis of demographic growth. The NIP graph
showed a multimodal distribution. In this case, the statis-
tical tests for population expansion produced conflicting
results. Whereas the rg statistic rejected an event of pop-
ulation growth, the SSD did not. Finally, the Adriatic was
characterized by a smooth unimodal curve with high sta-
tistical support. As for this region both the neutrality and
mismatch distribution tests were consistent with an episode
of population proliferation, we used the ARLEQUIN
inference of s to estimate the time since expansion.
According to a s = 4.084 estimation, expansion would
have started some 273 ky BP (95 % confidence interval:
182–315 ky BP). It must be noted that the molecular clock
rate employed in this study resulted in wide posterior
probability intervals for date estimations. For this reason,
we should be circumspect when interpreting results. Nev-
ertheless, even taking the previous fact into account,
BEAST coalescent reconstruction of past demographic
events again showed contrasting patterns between the NIP
and the Adriatic (Fig. 4). According to the BEAST inference,
the Adriatic N. nitidus population underwent a slow
exponential growth approximately from 220 (150–320) ky
BP and then reached a plateau after the LGM (0–15 ky BP;
Fig. 4). Effective population size increased by 60-fold in
the course of this 200-ky expansion. The MRCA is esti-
mated to have occurred 347 (156–584) ky BP. For the NIP
haplotypes, the MRCA was estimated to have arisen some
522 ky BP (95 % highest posterior density interval:
203–899 ky BP), due to an early split between the lineages
leading to clades 1 and 2. Only after the end of the LGM
(15–30 ky BP) did a demographic burst take place of
approximately 40-fold.
Table 2 N. nitidus. Analyses of population stability (Tajima’s D and
Fu’s Fs tests) and population expansion (sum of squared deviations
SSD and raggedness rg mismatch distribution tests)
Region Tajima’s D Fu’s Fs Mismatch SSD, rg
North Sea -1.260 -0.285 0.020, 0.670
(0.088) (0.285) (0.101, 0.700)
Northern Iberian -1.040 -5.538 0.072, 0.175
Peninsula (0.143) (0.087) (0.076, 0.017)
Adriatic Sea -1.887 -25.951 0.001, 0.019
(0.007) (<0.001) (0.421, 0.548)
Parameters and probabilities (in brackets) are shown for each regional
group. Significant P values are indicated in bold and italics
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Fig. 3 Mismatch distribution for the three regional groups of
N. nitidus populations, North Sea, North Iberian Peninsula (NIP)
and Adriatic. Bars represent the observed distribution of pairwise
differences, while solid lines show the expected distribution under a
pure demographic expansion model (colour figure online)
According to the genealogical trees generated for the
BSP reconstructions of N. nitidus (figures not shown), the
point estimate for the coalescence time of clade 3 hap-
lotypes (220; 95 % HPD: 110–350 ky BP) is older than
the upper confidence interval for those in both clades
1 (76; 95 % HPD: 18–164 ky BP) and 2 (62; 95 % HPD:
16–127 ky BP) (posterior probabilities C0.93). These
estimates suggest an earlier persistence of the Adriatic
clade 3 and a more recent origin of both the Atlantic
clades 1 and 2, consistent with events surrounding the
LGM.
The historical demographic analysis of the congener
N. reticulatus (from Belgium to southern Portugal) indicated
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Fig. 4 Bayesian skyline plot (BSP) for N. nitidus in Atlantic (NIP) and
Mediterranean (Adriatic) refugia and for N. reticulatus in the Atlantic.
Curves represent population sizes (effective population size per
generation time; Ne.s) progress to their most recent common ancestor
(MRCA). Dashed vertical lines indicate the lower end of the 95 %
highest posterior density (HPD) interval for the MRCA estimate, and
grey fill represents the 95 % HPD of the BSP. The LGM extent
according to Clark et al. (2009) is shown on each chart. Above and below
the graphs, two horizontal diagrams indicate warm (white) and cold
(grey) Marine Isotopic Stages (MIS; Gibbard) (colour figure online)
that the MRCA may have existed at least 353 (75–847) ky
BP. This species seems to have increased its effective
population size around 75-fold in an exponential expansion
that started at 100 ky BP (88–113 ky BP) and ended at the
time of the LGM (Fig. 4). According to these models,
Mar Biol
contemporary population sizes for both the NIP and
Adriatic N. nitidus groups would be similar but smaller as
compared to those of N. reticulatus.
Discussion
The impact of a major historical event is expected to be
manifested by phylogeographic concordances across
unrelated taxa (Avise 2004). While there is agreement
about the homogenizing effects that glaciations had in the
NW Atlantic biota (Wares and Cunningham 2001; Addi-
son and Hart 2005), current evidence for the NE Atlantic
is more difficult to interpret, and a single generalized
pattern across taxa cannot be inferred (Maggs et al. 2008).
Reasons for this are related to sea surface temperature
isotherms, the complexity of the coastline and the much
greater variation in suitable habitat types along the
European coasts. Nevertheless, some of the large-scale
patterns are fairly consistent among taxa, and N. nitidus is
no exception.
The Mediterranean–Atlantic split
The Strait of Gibraltar, together with the Almeria-Oran
oceanographic front (Tintore et al. 1988), is a well-docu-
mented barrier to gene flow between the Atlantic and
Mediterranean, as illustrated for seaweeds (Andreakis et al.
2004), seagrasses (Coyer et al. 2004; Olsen et al. 2004),
invertebrates (Roman and Palumbi 2004) and fishes
(Gysels et al. 2004a, b) (revised by Patarnello et al. 2007).
Our analyses of N. nitidus also support this phylogeo-
graphic break, with each region comprising distinct clades
and radically different diversities, that is, a few abundant
haplotypes in the Atlantic and many low frequency ones in
the Adriatic. Indeed, only two haplotypes were shared
between the two basins.
Within the Atlantic, two groups were identified
(Table 2). Although the ancestral h20 and the more recent
h16 occurred along the entire Atlantic coastline, their
dominance reached maximum values only in the North Sea.
In addition, the mismatch distribution indicates different
demographic histories for each of these subregions,
admixture at the NIP (see below) and a very recent
northward expansion into the North Sea. A division
between the North Sea and the NE Atlantic has been
repeatedly observed for other species (Luttikhuizen et al.
2003; Jolly et al. 2006; Spalding et al. 2007; Strasser and
Barber 2009), and it is believed that a transition area
between the Northern European and the Lusitanian marine
provinces is situated in the Hurd Deep within the present-
day English Channel (Provan et al. 2005; Maggs et al.
2008).
Refugia
The high haplotype diversity, deep divergence and abun-
dance of private haplotypes in the Adriatic and the NIP are
consistent with at least two glacial refugia (Hoarau et al.
2007; Maggs et al. 2008). One refugium, located in the
low-stand, Paleo-Mediterranean Sea (Bianchi and Morri
2000) would account for the current Adriatic haplotypes,
while the other, situated on the Atlantic coast of the Iberian
Peninsula, would account for the Atlantic lineages.
The high haplotype diversity and phylogenetic depth of
the Adriatic branch are consistent with long residency in a
Mediterranean refugium. Population growth and expansion
are evident, and no sign of admixture was detected. Our
estimate places the start of this steady proliferation in the
Saalian glaciation, probably during the interstadials
180–240 ky BP (Marine Isotopic Stages, MIS, 7a, c, e).
The slight slowdown detected during the LGM indicates
that this population was only weakly affected by cooling
events.
The Atlantic Iberian coast was the second refugium
based on the dominance of three distant lineages, one of
them the ancestral lineage. Although the results obtained
for the tests based on the multimodal mismatch distribution
for the NIP may be indicative of a large stable population
(Rogers and Harpending 1992), they are also consistent
with the admixture of at least two different expanding units
(Alvarado Bremer et al. 2005). Indeed, we identified two
highly divergent Atlantic clades in the Iberian samples,
each of them with a star-like topology (Figs. 2, OR2).
Moreover, the Bayesian reconstruction showed a mini-
mum-sized population that underwent rapid expansion
after the LGM. The North Sea region comprises a sub-
sample of clade 1 haplotypes that undertook a process of
sudden population expansion. These patterns are consistent
with Pleistocene marine refugia found in several other
marine intertidal organisms including a seaweed (Hoarau
et al. 2007), a crustacean (Campo et al. 2010) and a ver-
tebrate (Chevolot et al. 2006).
Mediterranean origin
Lineages surviving in the Atlantic refugium (clade 1 at
76 ky BP and clade 2 at 62 ky BP) had a Mediterranean
origin given the MRCA estimated for the Adriatic clade 3
at 220 ky BP. Previous studies of the benthic vertebrate,
Pomatoschistus microps (Gysels et al. 2004b) and the
gastropod Hydrobia acuta (Wilke and Pfenninger 2002)
also support a Mediterranean origin for lineages surviving
in two different Mediterranean and Atlantic refugia during
the Pleistocene. Sea level fluctuations between 76 and 220
ky BP (Gibbard and Cohen 2008) are believed to have
facilitated the movement of organisms through the Strait of
123

Gibraltar (Kooistra et al. 1992). The early Mediterranean
N. nitidus population expansion (220 ky BP) and the very
recent NIP expansion (19 ky BP) estimates are in good
agreement with this hypothesis.
A scenario in which clades 1 and 2 arose in the Mediter-
ranean and migrated into the Atlantic after the LGM is
unlikely. First, each Atlantic clade occupies a peripheral
position in the genealogical network and only shares
one haplotype with the Mediterranean cluster. Second, the
star-like pattern of each Atlantic clade is typical of a pre-
established population undergoing expansion rather than
experiencing a leading-edge colonization process (Hewitt
1996). Third, if a glacial episode had removed the less fre-
quent haplotypes (including ancestors to modern clades
derived from h20) after migration of the clade to the Atlantic
basin, the ancestral genetic signature for the Atlantic popu-
lations (but not the Mediterranean) would be expected to
have disappeared (as it is in this basin where climatic pres-
sure reached extreme conditions). In contrast, a high number
of Adriatic haplotypes in the branch leading to the Atlantic
clade 1 are present. In this light, the ancestral h20 would be
absent, or at least scarce, in the Atlantic coast; yet it is
abundant. Thus, even taking into account the limitations
inherent to the interpretation of haplotype networks, the
requirement that all of these events would have co-occurred
makes support for this scenario weak.
Pre- and post-LGM expansions
Although the LGM affected both the Atlantic and Mediter-
ranean, it did so in different ways. In the Atlantic basin, the
Scandinavian and English ice sheets covered the Baltic Sea
(totally) and the North Sea (partially). Coastal areas such as
the Hurd Deep and the English Channel were left exposed, as
the sea level reached a low stand of -100 m (Lambeck 1997;
Clark and Mix 2002). In the Mediterranean, conditions were
milder, with sea level changes shifting benthic taxa to the
deeper central basins (Bianchi and Morri 2000; Patarnello
et al. 2007). Despite the southern and eastern displacement of
the Adriatic population of N. nitidus, demographic expansion
continued, whereas in the Atlantic, this snail survived in the
NIP refugium but did not expand until the end of the LGM
20 ky BP. Subsequent colonization of the North Sea area
from the NIP then proceeded. Our results match the post-
LGM patterns obtained for other marine invertebrates
(Gysels et al. 2004a; Maggs et al. 2008) and seaweeds (Coyer
et al. 2003; Provan et al. 2005; Hoarau et al. 2007; Olsen et al.
2010) inhabiting the Atlanto-Mediterranean territories and
are also comparable to those obtained for seaweeds that
experienced a post-LGM expansion in distant, temperate-
sub-Antartic waters (Fraser et al. 2009).
Although survival in glacial refugia during the LGM
was widespread, the demographic impact that we can infer
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Mar Biol
depends upon whether the refugia were large or small.
Interestingly, the Atlantic congener N. reticulatus (scarcely
present in the Mediterranean; Fig. 4) appears to have not
been significantly affected by the LGM. Its population
expansion started during the penultimate interglacial period
and has remained relatively stable since the LGM. Our
results are consistent with the estimation of Couceiro et al.
(2007) of a population growth starting 62–160 ky BP.
These authors also found little evidence for any population
structure in N. reticulatus and concluded that the high
genetic diversity in the mid-Atlantic was consistent with
long-term presence of the species in the Bay of Biscay.
Similarly, examples of other taxa whose status was little
affected by the LGM include the brown seaweed Asco-
phyllum nodosum (Olsen et al. 2010) and the barnacle
Pollicipes pollicipes (Campo et al. 2010).
Patchy versus continuous distributions
Our intertidal and subtidal surveys of potential habitat for
N. nitidus suggest a naturally patchy distribution. While we
cannot claim to have searched every kilometre of coastline,
a major effort was made to visit sites recorded in the lit-
erature, those reported by colleagues with local knowledge
and many new ones through our own newly initiated
efforts. Dependency on soft, sheltered, brackish habitats
appears to be more important than traditionally thought,
especially recalling the fact that the congener N. reticulatus
is comfortable in a broader range of muddy-sandy habitats
that overlaps with those of N. nitidus. While acknowledg-
ing that further field explorations should be accomplished
to confirm the evidences presented here, the observed
differences between N. nitidus and N. reticulatus phylog-
eographies are comparable to those reported in previous
studies of marine gastropods where small differences in the
life history of congenerics have resulted in contrasting
distributions (Wilke and Pfenninger 2002; Crandall et al.
2008; Marko 2004). Likewise, and despite the fact that
N. nitidus and N. reticulatus are closely related and share
similar life histories and dispersal capacities, differences in
microhabitat requirements may have shaped their specific
response to climatic shifts and thus their respective post-
LGM distributions—one patchy and one continuous.
Genetic drift versus artificial population connectivity
The presence of three Atlantic haplotypes at the tips of the
Adriatic clade, one of them supported by the phylogenetic
analysis, remains difficult to explain in evolutionary terms.
Two hypotheses may account for our observations. First,
both regions have a long tradition of shellfish culture and
commercial exchange. The Adriatic is a typical source for
clam transfers to the Iberian Peninsula, and it has been
Mar Biol
identified as the source of several nonindigenous gastro-
pods (Gibbula adansonii, Gibbula albida, Rapana venosa
and Hexaplex trunculus) in sites close to our locations SP
(6) and SP (7) (Rolán 1992; Rolán and Bañón 2007). In the
case this theory was true, gene flow and genetic diversity
estimations here presented would be hardly reliable.
However, our results are in proper agreement with geo-
climatic history and with studies on other coastal benthic
organisms. A second possibility would be strong genetic
drift acting on the Atlantic populations. As mentioned
before, it is assumed the NE Atlantic was a hostile envi-
ronment for intertidal species during glacial intervals,
especially for brackish habitat species. Brackish biota is
characterized by a high degree of population differentiation
resulting from the discrete nature of habitat and their rel-
ative confinement. Thus, for both Nassarius species, the
effects of genetic drift acting on local settlements are
expected (reviewed by Bilton et al. 2002; Johannesson and
André 2006). To our opinion, in order to avoid stochastic
events acting on small size mitochondrial populations,
further investigations based on non-mitochondrial genetic
information are required to resolve this issue.
Conclusion
Pleistocene climatic shifts affected population structure
and recent demography of the Atlanto-Mediterranean
estuarine dog-whelk N. nitidus. A sharp division between
Adriatic and Atlantic populations was found, with the
Atlantic populations originally derived from the Adriatic.
Two refugia were identified: the Adriatic experienced a
steady population growth over 200 ky, while the NIP
experienced rapid expansion only during the last 20 ky.
Our results are consistent with numerous other studies,
most of which are also exclusively based on mtDNA.
However, it is increasingly recognized that the addition of
nuclear loci (especially those under selection) may provide
a more complete and more nuanced view of the phyloge-
ographic history of a species and the superimposed eco-
logical pressures that have further shaped distribution. In
the present study, for example, the Atlantic congener
N. reticulatus seems to have been scarcely affected by the
LGM despite similar morphology and life history features.
Such results highlight the fact that seemingly insignificant
intertidal habitat differences may have allowed some spe-
cies to avoid the ice by going slightly lower and deeper
down the shore.
Acknowledgments We thank C. Bernárdez, J. Craeymeersch, L.
Grassia, L. Kellner, F. Kerchov, B. Lundve and C. Mazziotti for
providing samples, R. Bao for information and advice about Qua-
ternary chronostatigraphy and paleoceanography and J. Coyer and
G. Hoarau for discussions about data analysis and interpretation while
N. A. hosting in the Olsen lab. Financial support for this work was
provided by the Spanish Ministerio de Educación y Ciencia (MEC)
grant CTM2004-04496/MAR (partially co-funded by FEDER, Fondo
Europeo de Desarrollo Regional) and the Xunta de Galicia grant
PGIDT05PXIC10302PN. N. A. acknowledges her postgraduate
fellowship from the Ministerio de Educacion (FPU-MEC, AP2006-
03231).
Conflict of interests The authors declare that we have no conflict of
interests.
Ethical standards The experiments performed in this study comply
with the current laws in Spain.
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