ORIGINAL PAPER

Phylogeography of the European stalked barnacle(Pollicipes pollicipes): identification of glacial refugia

Daniel Campo • Jose Molares • Lucia Garcia •

Pino Fernandez-Rueda • Claudia Garcia-Gonzalez •

Eva Garcia-Vazquez

Received: 3 February 2009 / Accepted: 16 September 2009 / Published online: 10 October 2009

� Springer-Verlag 2009

Abstract The alternation of glacial and interglacial

events during the Pleistocene has produced changes in

species distribution ranges leading to bottlenecks and

alterations of patterns of gene flow. The European stalked

barnacle, Pollicipes pollicipes, is a sessile pedunculate

cirripede that inhabits the rocky intertidal frame, from

Senegal to the northwestern coast of France. In this work,

we have analyzed a fragment of the mitochondrial gene

cytochrome c oxidase subunit I for 569 individuals of

P. pollicipes in order to investigate whether the shifts in

climatic conditions that occurred during the Pleistocene

influenced the current pattern of distribution of genetic

variation of P. pollicipes. A pre-last glacial maximum

pattern of demographic expansion was found, in concor-

dance with many other North Atlantic marine species. On

the other hand, three potential glacial refugia were identi-

fied: North African coasts, northwestern Iberian Peninsula

and English Channel/Brittany.

Introduction

Climate oscillations that occurred during the Pleistocene

had a dramatic effect on the geographic distribution of

terrestrial and marine species (Avise 2000), especially the

last glacial maximum (LGM), which occurred about

20,000 years ago during the Vistula glaciation. In Europe,

the extent of the marine glaciation reached as far south as

the northern limit of the Bay of Biscay, covering the British

Islands (Svendsen et al. 2004). These conditions were

especially unfavorable for sessile organisms (e.g., sea-

weeds, seagrasses and sessile invertebrates) and forced

populations into refugial areas from which they expanded

once the ice receded. The effect of such range fluctuations

left a genetic signature on the populations, which can be

measured, since we expect a higher genetic diversity within

refugial areas than in more recently colonized habitats and

high genetic differentiation between refugia (Hewitt 1996,

2004). This has allowed phylogeographers to detect glacial

refugia and infer interglacial recolonization pathways. For

marine species of the North Atlantic, these refugia were

mostly located in southern areas including Atlantic islands,

North African coast and the Mediterranean, and many

recent studies suggest the existence of Pleistocene refugial

areas at the south of the Bay of Biscay (off the coast of

northwestern Iberian Peninsula), the English Channel/

Brittany area, the southwest of Ireland and the west coast

of Iceland (reviewed in Gomez et al. 2007).

Communicated by M. I. Taylor.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00227-009-1305-z) contains supplementarymaterial, which is available to authorized users.

D. Campo � C. Garcia-Gonzalez � E. Garcia-Vazquez

Departamento de Biologia Funcional, Universidad de Oviedo,

C/Julian Claveria s/n, 33006 Oviedo, Spain

Present Address:D. Campo (&)

Molecular and Computational Biology,

University of Southern California, 1050 Childs Way,

RRI, Los Angeles, CA 90089, USA

e-mail: dcampo@usc.edu

J. Molares

Centro de Investigacions Marinas, Conselleria de Pesca

e Asuntos Maritimos, Pedras de Coron s/n,

36620 Vilanova de Arousa, Pontevedra, Spain

L. Garcia � P. Fernandez-Rueda

Centro de Experimentacion Pesquera, Consejerıa de Medio

Ambiente y Desarrollo Rural, Direccion General de Pesca,

Avda. Prıncipe de Asturias s/n, 33212 Gijon, Spain

123

Mar Biol (2010) 157:147–156

DOI 10.1007/s00227-009-1305-z

The European stalked barnacle, Pollicipes pollicipes

(Gmelin 1790), is a sessile pedunculate cirripede that

inhabits the rocky intertidal frame, in zones exposed to

strong swell. Its current distribution spans from the coast of

Senegal to the northwestern coast of France (Brittany)

(Barnes 1996). There is also an isolated population in Cape

Verde Islands, which is genetically differentiated from the

rest (Quinteiro et al. 2007). Adults are hermaphrodite and

reproduce by copulation with internal fertilization. The

reproductive period lasts 7 months, from March to Sep-

tember (Molares et al. 1994), and its larval development

takes about 1 month, with six naupliar and one cyprid

stages. All larvae are planktonic and exhibit active mobility.

After the naupliar stages, the cyprid settles specifically to the

peduncle of adult individuals, then they move to the base and

fix permanently to the rock. As a consequence, adult indi-

viduals of P. pollicipes constitute dense aggregations.

To our knowledge, only two published studies to date deal

with genetic data of P. pollicipes. Van Syoc (1995) studied the

phylogenetic relationships of three species of the genus Pol-

licipes, and Quinteiro et al. (2007) investigated the influence of

coastal currents and mesoscale hydrographic features on the

population genetic structure of P. pollicipes. Quinteiro et al.

(2007) also suggested the existence of southern refugia (trop-

ical and subtropical latitudes) during Pleistocene glaciations.

In the present study, we investigated the mitochondrial

DNA (mtDNA) phylogeography for the entire range of dis-

tribution of the European stalked barnacle (Pollicipes pol-

licipes) using a fragment of the subunit I of the cytochrome c

oxidase (COI). The objective was to elucidate whether

extreme climatic conditions occurred during the Pleistocene

have had any influence on the current pattern of distribution of

genetic variation of P. pollicipes, specially in populations

from the northernmost boundary of its range of distribution.

Materials and methods

Samples

A total of 569 adult individuals of P. pollicipes from 11

different locations were collected, covering the entire range

of distribution of the species, from Mauritania to Brittany

(France) (Fig. 1; Table 1). The population from Cape Verde

Islands was not included in the present study due to its

unclear current taxonomic position (Quinteiro et al. 2007).

Whole individuals were collected directly from the rock and

preserved in absolute ethanol until DNA extraction.

DNA extraction, PCR amplification and sequencing

Total DNA was extracted from a small piece of muscle

(about 1 mm3) using a Chelex Resin protocol (Estoup et al.

1996) and kept at 4�C until needed. PCR amplification of a

fragment of the COI gene was initially carried out using the

set of primers K–N described by Van Syoc (1995). After-

ward, from an alignment of COI sequences obtained from

GenBank (EF032142–EF032154; Quinteiro et al. 2007),

we designed a new primer to obtain better amplification

results (primer D: 50-TGGAGGTTTTGGAAACTGAC-30).This new primer was employed in combination with the

primer N (replacing primer K). PCR were carried out in a

total volume of 40 ll containing 59 Go Taq� Flexi Buffer,

250 lM of each dNTP, 40 pmol of each primer, 0.2 ll of

Go Taq� Polymerase 5 U/ll (Promega) and 4 ll of DNA.

They were performed in a GeneAmp� PCR system 9700

(Applied Biosystems) with the following conditions: an

initial denaturing step at 95�C for 5 min, followed by 30

cycles of denaturing at 95�C for 30 s, annealing (for 30 s)

at 50�C and an extension at 72�C for 30 s, ending with a

final extension at 72�C for 10–15 min.

PCR products were visualized in 50 ml 1.5% agarose

gels with 3 ll of 10 mg/ml ethidium bromide. Stained

bands were excised from the gel, and DNA was purified

with a Wizard� SV Gel and PCR Clean-Up System (Pro-

mega) prior to sequencing. Automated fluorescence

Fig. 1 Map showing the 11 sampling sites of P. pollicipes along its

distribution range. Population codes as in Table 1

148 Mar Biol (2010) 157:147–156

123

sequencing was performed on an ABI PRISM 3100

Genetic Analyzer (Applied Biosystems) with BigDye 3.1

Terminator system, in the Unit of Genetic Analysis of the

University of Oviedo (Spain).

Population genetics

Sequences were edited using the BioEdit Sequence

Alignment Editor software (Hall 1999) and aligned with

the ClustalW application (Thompson et al. 1994) included

in BioEdit.

Since most of the neutrality tests based on nucleotide

changes are sensitive to demographic shifts, we conducted

the McDonald and Kreitman test (McDonald and Kreitman

1991) using P. polymerus as an outgroup to test whether

selection is acting on P. pollicipes COI sequences.

P. polymerus COI sequence was obtained from the com-

plete mitochondrial genome available in GenBank under

Accession number AY456188.

Haplotype diversity (Hd) and nucleotide diversity (p)

for each population were calculated with the program

ARLEQUIN version 3.01 (Excoffier et al. 2005). Due to

differences in sample size among populations, haplotype

diversity values were standardized to the smallest sample

size using the program RAREFAC (Petit et al. 1998).

The ModelTest (ver. 3.06) software (Posada and

Crandall 1998) was employed to determine the model of

sequence evolution that best fitted our dataset and to cal-

culate the proportion of invariable sites and the value of the

gamma distribution shape parameter. Population pairwise

FST values were calculated employing Tamura–Nei dis-

tances (Tamura and Nei 1993) and a value of the gamma

distribution shape parameter of 1.1698 (see ‘‘Results’’ for

the outcome of the ModelTest) with the program ARLE-

QUIN. ARLEQUIN was also employed to perform an

analysis of the molecular variance (AMOVA; Excoffier

et al. 1992) to test its hierarchical distribution at three

levels: within populations (UST), among populations (USC)

and among groups of populations (UCT). We tested several

different a priori grouping combinations, splitting popula-

tions into 2, 3 and 4 groups (Brittany vs. the rest of pop-

ulations; Brittany vs. Iberian Peninsula vs. Africa; Brittany

vs. north coast of Iberian Peninsula vs. west coast of

Iberian Peninsula vs. Africa), and that configuration with

the highest value of UCT and statistically significant was

chosen as the most likely geographic subdivision. The

statistical significance of both analyses (pairwise FST

and AMOVA) was tested through 99,224 permutations.

To test for isolation by distance, we performed a Mantel

test as implemented in ARLEQUIN, estimating the sig-

nificance level by 50,000 permutations. With this analysis,

we determined whether there is a significant correlation

between a matrix of geographic distances (represented by

the minimum coastline distance in kilometers) and a matrix

of genetic distances (FST).

Demographic history

To detect whether the populations have undergone demo-

graphic and/or range expansion, we compared the observed

frequency of pairwise sequence differences (mismatch

distribution) to the expected distribution under a sudden

expansion model with the SSD statistic (sum of squared

differences) using 1,000 bootstrap replicates, in ARLE-

QUIN. We also computed Fu’s Fs (Fu 1997) and the R2

statistic (Ramos-Onsins and Rozas 2002) for all samples

using DNASP version 4.50.3 (Rozas et al. 2003) and

estimated its significance with 1,000 coalescent simula-

tions. With this program, we can also calculate s, which

allows us to then estimate the time in generations since the

Table 1 Abbreviated codes, geographic coordinates (latitude and longitude), number of individuals (N) and haplotypes (Nh), number of private

haplotypes (Nph), percentage of Nph with respect to N (%Ph) and values of haplotype diversity (Hd) and nucleotide diversity (p) for each

location sampled

Population Code Lat/long N Nh Nph %Ph Hd p

Saint Guenole, France SG 48�19021.7000N/4�2509.9800O 35 20 5 14.3 0.950 0.0099

Quiberon, France QUIB 47�28050.9700N/3� 8031.2600O 44 15 1 2.3 0.909 0.0088

Jaizkibel, Spain JAI 43�23023.9500N/1�4806.9200O 31 14 0 0 0.938 0.0079

Monpas, Spain MON 43�2008.4400N/1�58018.6000O 35 16 0 0 0.936 0.0072

Ribadesella, Spain RIB 43�28010.1200N/5�3028.2600O 60 25 7 11.7 0.933 0.0088

Punta de la Cruz, Spain CRUZ 43�33035.7200N/7�1017.2700O 60 27 12 20 0.951 0.0082

Corme, Spain COR 43�15044.7200N/8�5805.2000O 57 25 8 14 0.945 0.0078

Vila do Conde, Portugal VC 41�21042.9500N/8�45046.5500O 30 17 1 3.3 0.940 0.0084

Berlengas, Portugal BER 39�24050.3600N/9�30025.1300O 128 30 11 8.6 0.914 0.0067

Agadir, Morocco AGA 30�24058.2100N/9�38046.1500O 53 21 5 9.4 0.923 0.0069

Mauritania MAU 18�24027.9200N/16� 5050.3200O 36 22 4 11.1 0.951 0.0068

Mar Biol (2010) 157:147–156 149

123

population expansion (t) with the formula t = s/2uk (where

u is the substitution rate in changes per nucleotide per

million years, and k is the length of the sequence).

Times of haplotypes coalescence (tmrca: time to the

most recent common ancestor) and the limits of the 95%

highest posterior density (HPD) interval, for the whole

sample and for each of the groups, were estimated with the

program BEAST ver. 1.4.8 (Drummond and Rambaut

2007). We did a run of 10 million generations sampling

every 1,000 under the strict molecular clock model

implementing the HKY substitution model, and con-

straining the priors for gamma and proportion of invariable

sites according to the values obtained in ModelTest. The

tree prior selected was exponential growth (based on

results for SSD, R2 and Fu’s Fs statistics), whereas priors

for the rest of parameters were left as default. In every case,

effective sample sizes (ESS) were all above 100, and a

good mix of the chains was observed.

To test whether COI sequences follow a molecular clock

model, we performed a relative ratio test (Tajima 1993) in

MEGA4 (Tamura et al. 2007) with many different com-

binations of ingroup and outgroup sequences, and also

including P. polymerus COI sequence as outgroup. At least

two different substitution rates have been applied to the

mitochondrial COI of P. pollicipes. Van Syoc (1995)

employed a divergence rate of 2% per million years (pmy),

an average value commonly accepted for the whole

mtDNA, to estimate divergence times between species of

Pollicipes, whereas Wares (2001) estimated a rate of 3.1%

pmy for barnacle species of the genus Euraphia, and

applied it to the genus Chthamalus. This rate was cali-

brated using divergence values between species separated

by the Panama Isthmus, and it is in concordance with other

crustacean estimates (Wares 2001). Quinteiro et al. (2007)

employed this latter rate to calculate a little bit faster one

for mitochondrial control region in P. pollicipes. We

therefore decided to employ the rate of 3.1% pmy, as it has

been estimated from barnacles divergence data.

Phylogeographic analysis

We performed a nested clade analysis (NCA) to test for

geographic associations between related haplotypes and to

distinguish between contemporary and historical events

influence in the current distribution of genetic diversity. To

do this, a parsimony network was constructed using the

program TCS version 1.21 (Clement et al. 2000), which

implements the statistical parsimony method described by

Templeton et al. (1992). Ambiguities in the cladogram

(represented by loops) were solved following the three

criteria proposed in Pfenninger and Posada (2002). The

network was then nested by hand following the procedures

described in Templeton et al. (1987) (Fig. 2), and the NCA

was done with the program GEODIS version 2.4 (Posada

et al. 2000), through 10,000 permutations. As P. pollicipes

is a coastal species, a matrix of pairwise geographic dis-

tances between populations (the same employed for the

Mantel test) was input. The output was interpreted using

the latest available inference key (http://darwin.uvigo.

es/software/geodis.html). On the other hand, the use of

NCPA for phylogeographic inference has been lately

questioned (see respective article series in Mol Ecol, 2008),

mainly due to its inability for evaluating the relative sup-

port for the different alternative scenarios. However, we

decided to use this method as one approach more as it

can propose phylogeographic scenarios that can be sup-

ported and reinforced with other methods (Garrick et al.

2008).

Results

Population genetics

After editing and aligning all the sequences, a total dataset

of 444 base pairs (bp) length was obtained. A total of 83

haplotypes were found from the 569 P. pollicipes COI

sequences (Table S1). Only 6 out of these 83 were found in

all populations, and 54 were private haplotypes (i.e.,

present in a single location), being 53 singletons. Haplo-

type sequences are available in the GenBank database

(http://www.ncbi.nlm.nih.gov) under the accession numbers

EU998656–EU998738. The McDonald and Kreitman test

was not significant (Fisher’s exact test, P = 0.14), indi-

cating that these COI sequences are not under selection.

Total number of haplotypes (Nh), number of private

haplotypes (Nph) and percentage of Nph with respect to N

(%Ph), as well as values of standardized haplotype diver-

sity (Hd) and nucleotide diversity (p) for each sample are

listed in Table 1. Highest values of %Ph were found in

Saint Guenole, Ribadesella, Punta de la Cruz, Corme and

Mauritania. All populations showed high haplotypic

diversity, the values for Saint Guenole, Punta de la Cruz

and Mauritania being the highest ones. On the other hand,

we found low differentiation among haplotypes and groups

of haplotypes (Fig. 2), being separated by single substi-

tutions.

The model of evolution obtained with ModelTest as best

fitting our dataset was the Tamura and Nei (1993), with a

proportion of invariable sites of 0.5544 and a gamma dis-

tribution shape parameter of 1.1698. Population pairwise

FST were calculated following this model (Table 2).

Although FST values were generally low, there were some

significant genetic differences. Populations from Brittany

(Saint Guenole and Quiberon) showed significant differ-

ences with many of the other samples, except with those

150 Mar Biol (2010) 157:147–156

123

from southeastern Bay of Biscay (Jaizkibel and Monpas)

and Vila do Conde (northern Portugal).

Two grouping configurations yielded significant values

of UCT in the AMOVA. In one of them, populations from

Brittany (Saint Guenole and Quiberon) were on one group,

and all the other populations on another group. The other

significant configuration was that splitting samples in three

groups: Brittany, Iberian Peninsula and Africa. The first

one (two groups of samples) obtained the highest among

group variation (UCT), indicating that the significance of

the second one (three groups) could be due to the among

group variation of the first partition. Thus, we took as the

most likely regional configuration the first one (i.e., Brit-

tany vs. the rest of samples), and the AMOVA revealed

that 2.96% of the variance could be explained by differ-

ences between these two groups (UCT = 0.02962,

P \ 0.05; Table 3). In addition, variation among popula-

tions within groups (0.15%) was not significant

Fig. 2 Statistical parsimony

cladogram showing the nestedclade design for the 83

haplotypes of P. pollicipesfound in the present study. Each

branch represents a mutational

step and the black dots denote

missing haplotypes. Biological

inferences are detailed in

Table 5

Table 2 Matrix of pairwise FST genetic distances between P. pollicipes populations

Population 1 2 3 4 5 6 7 8 9 10

1: SG

2: QUIB 0

3: JAI 0.01344 0.01094

4: MON 0.01864 0.01823 0

5: RIB 0.02746* 0.00603 0 0

6: CRUZ 0.03110* 0.02238* 0 0 0

7: COR 0.02788* 0.02354* 0 0 0.00594 0

8: VC 0 0 0.00748 0.00549 0 0.01153 0.00919

9: BER 0.05656* 0.03886* 0 0 0.00190 0 0.00817 0.02603*

10: AGA 0.03865* 0.00895 0.00985 0.00240 0 0.00520 0.00656 0 0.00517

11: MAU 0.06806* 0.06028* 0.00607 0.00223 0.00794 0 0.00845 0.04641* 0.00393 0.02002

They were calculated employing the Tamura–Nei correction and a value of the gamma distribution shape parameter of 1.1698. Significant values

after 99,224 permutations are indicated with an asterisk

Mar Biol (2010) 157:147–156 151

123

(USC = 0.00150) supporting that the main differentiation

was among groups. In addition, most molecular variation

was explained by within population variability (96.89%),

which agrees with the high levels of haplotype diversity

found in all samples.

A positive correlation was found between genetic and

geographic distances, which yielded a significant value

of the correlation coefficient in the Mantel test

(r = 0.441911, P \ 0.05), indicating a scenario of isola-

tion by distance. However, there was a wide dispersion of

the data, explained by some low FST values between distant

populations. Moreover, when we removed the two Brittany

localities (SG and QUIB), Mantel test became non-signif-

icant (r = 0.326241, P = 0.14).

Demographic history

Sudden demographic expansion was supported by non-

significant SSD values for Saint Guenole, Ribadesella,

Punta de la Cruz and Agadir populations (Table 4). For

other four samples (Jaizkibel, Monpas, Corme and Mau-

ritania), the least square procedure did not converge after

1,800 steps, not allowing to achieve a value of the SSD

statistic. The statistic R2 yielded significant values indi-

cating demographic expansion in Ribadesella, Punta de la

Cruz, Vila do Conde, Berlengas, Agadir and Mauritania.

Moreover, the Fs statistic, which is more sensitive to

population growth in large samples (Ramos-Onsins and

Rozas 2002), was significant in all samples except Quib-

eron. On the other hand, SSD values for the fitting to the

range expansion model were non-significant (therefore

supporting range expansion) for Saint Guenole, Quiberon,

Ribadesella and Agadir (data not shown).

Relative ratio test yielded non-significant values for all

tested combinations of ingroup and outgroup sequences. This

means that nucleotide substitutions accumulate in a clocklike

manner in the COI sequences here analyzed, and therefore we

can apply a fixed substitution rate. Values of s ranged from

2.242 for Berlengas to 3.613 for Quiberon. The overall s value

for Brittany samples was 3.243 whereas for the rest of

localities was 2.388. Generation time of P. pollicipes has been

observed to be of 1 year, at least in populations of the Iberian

Peninsula (Cruz 1993; Molares 1993). Thus, assuming this

value as a mean for all populations along its distribution range

and using the substitution rate of 3.1% pmy (estimated in

barnacles), the time since demographic expansion is calcu-

lated to be 117,807 years ago for Brittany populations and

86,748 years for Iberian and African samples, both falling

within the Eem interglacial. On the other hand, times of

haplotype coalescence (tmrca) were 198,258 (106,903–

297,387) years (Holstein interglacial) for Brittany samples,

and 167,645 (114,774–226,387) years (Warthe/Saale glacia-

tion) in the case of Iberian and African samples.

Phylogeography

NCA yielded significant results for six clades (Table 5), at

different nesting levels. A biological pattern could be

Table 3 Analyses of molecular variance (AMOVA) of the Pollicipes pollicipes COI sequences

Source of variation Variance

components

Percentage

of variation

U statistic P value

Brittany vs. the rest of samples Among groups 0.05281 2.96 0.02962 0.019

Among populations within groups 0.00260 0.15 0.00150 0.292

Within populations 1.72757 96.89 0.03108 0.008

Brittany vs. Iberian Peninsula vs. Africa Among groups 0.02646 1.51 0.01506 0.023

Among populations within groups 0.00313 0.18 0.00181 0.287

Within populations 1.72757 98.32 0.01684 0.009

Regional configurations with significant value of among groups variation (UCT) are shown. The first one has a higher UCT value, and therefore it

has been taken as the most likely configuration

Table 4 SSD statistic and neutrality tests values for the Pollicipespollicipes populations analyzed in this study

Population SSD R2 Fs s

SG 0.00433 0.0841 -9.5880* 3.249

QUIB 0.03707 0.0906 -3.2085 3.613

JAI – 0.0855 -4.4198* 2.521

MON – 0.0687 -6.7820* 2.592

RIB 0.01431 0.0570* -12.5158* 2.770

CRUZ 0.00413 0.0457* -16.6692* 2.587

COR – 0.0567 -14.9515* 2.396

VC 0.02721 0.0625* -8.0651* 2.686

BER 0.01650 0.0389* -16.9178* 2.242

AGA 0.00668 0.0551* -10.9764* 2.341

MAU – 0.0510* -17.0997* 2.455

Abbreviation codes for population names are given in Table 1.

Numbers in bold letter for SSD values indicate non-significant values

after 1,000 bootstrap replicates, whereas asterisks for R2 and Fs

values indicate significant values (P \ 0.05) through 1,000 coalescent

simulations. Values of s (mutational parameter to estimate time since

demographic expansion) are also shown

152 Mar Biol (2010) 157:147–156

123

inferred for four of them. For Clade 1-20, which includes

haplotypes from Saint Guenole, Punta de la Cruz, Vila do

Conde, Berlengas, Agadir and Mauritania, the inference

was restricted gene flow with isolation by distance. For

Clade 1-25, including haplotypes from all the populations

but Agadir, the outcome was a contiguous range expansion.

For Clade 2-1, the estimated pattern was again restricted

gene flow with isolation by distance. Finally, for Clade 3-2,

the inference was past fragmentation followed by a range

expansion.

Discussion

According to our results, at least two groups of P. pollic-

ipes populations can be identified, one comprises the

French Brittany and the other one includes the rest of the

distribution range. This result agrees with Quinteiro et al.

(2007), who also found significant differences between

Brittany and Iberian Peninsula samples. In addition, our

results also point out isolation by distance as an important

biological factor in shaping the current pattern of popula-

tion structure for P. pollicipes. When excluding Brittany

localities from the analysis, it became non-significant,

suggesting that those more divergent populations were

responsible for the initial significance. However, NCPA

also suggested such a pattern to be present, and Quinteiro

et al. (2007) detected a weak, although significant, pattern

of isolation by distance with mitochondrial D-loop

sequences analysis and suggested that this may reflect the

divergent oceanographic features between European and

African regions.

With the exception of pairwise comparisons involving

Brittany populations, very low levels of genetic divergence

have been found in the present study. This could be due to

either high levels of ongoing gene flow, recent colonization

after a period of isolation, or a combination of both

(Stamatis et al. 2004). Quinteiro et al. (2007) found high

Table 5 Results of the nested

clade analysis (NCA)

Only clades with significant

values of clade (Dc), nested

clade (Dn) and/or interior-tip

distances (I-T) are shown.

Significantly small (S) or large

(L) values are indicated. We

also indicate the steps followed

in the inference key for each

clade and the biological inferred

pattern. The nested cladogram is

shown in Fig. 2

Clade Subclades Dc Dn Steps in the key Inferred pattern

1-13 3 1-2-11-17-NO Inconclusive outcome

14

17

32

47 S

61

76

I-T

1-20 9 1-2-3-4-NO Restricted gene flow with

isolation by distance42

63 S S

I-T

1-25 10 S S 1-19-20-2-11-YES-12-NO Contiguous range expansion

81 L

I-T S S

2-1 1-25 S S 1-2-3-4-NO Restricted gene flow

with isolation by distance1-26 L

I-T L

3-1 2-1 L 1-2-11-17-NO Inconclusive outcome

2-2

2-3

2-4

2-5

I-T

3-2 2-6 1-2-3-5-6-13-YES Past fragmentation followed

by range expansion2-7 S

2-8 L

2-9 L L

2-10

I-T

Mar Biol (2010) 157:147–156 153

123

levels of gene flow for P. pollicipes, which must be med-

iated by larval drift and likely favored by oceanographic

features. A gene flow of just a few individuals per gener-

ation is sufficient to prevent the rise of genetic differenti-

ation between populations (Hartl and Clark 1997). In

contrast, the high number of private haplotypes found

could indicate restricted gene flow (Allendorf and Luikart

2007) or past fragmentation of populations. The results

here obtained suggest that the most likely scenario for the

P. pollicipes samples here analyzed is a past population

fragmentation and moderate to high levels of ongoing gene

flow. NCPA supported this scenario and suggested sub-

sequent range expansion of the fragmented populations. In

addition, a clear pattern of both demographic and range

expansions has been observed in all the localities sampled,

as derived from the mismatch distributions and neutrality

tests results. The starting point of such expansions and the

time of coalescence of the haplotypes seems to be older in

the case of Brittany populations. Estimated dates for these

events were associated with glacial and/or interglacial

periods within the Pleistocene. We estimated a time since

demographic expansion of about 118,000 years for Brit-

tany and 87,000 for Iberian and African populations, both

coinciding with the Eem interglacial period, whereas the

times to the most recent common ancestor were 198,000

(Holstein interglacial) and 168,000 (Warthe/Saale glacia-

tion) years, respectively. Therefore, according to all the

results obtained in this study, we can conclude that the

extreme climatic conditions occurred during the Pleisto-

cene, with alternation of glaciations and interglacial peri-

ods, had a strong influence in the contemporary distribution

of the genetic variation of the European stalked barnacle,

P. pollicipes.

Glacial refugia and contact zones

Genetic signatures of areas that served as refugia for bio-

diversity during Pleistocene glacial stages typically consist

of a higher genetic (haplotypic) diversity with respect to

the adjacent, more recently recolonized areas, and a high

number of private haplotypes; we also expect a high

genetic differentiation between refugia, as genealogical

lineages evolve separately in each one (Hewitt 1996, 2004;

Provan and Bennett 2008). On the other hand, secondary

contact zones, where individuals from different refugia

meet during recolonization at interglacial periods, are also

characterized by high genetic diversity; however, we do not

expect the presence of locally restricted haplotypes (i.e.,

private haplotypes) at these zones. In addition, contact

zones between previously fragmented populations can be

also inferred from the NCA if haplotypes belonging to

different fragmented groups are now present in the same

location (Templeton 2001). In the present study, the

highest haplotypic diversity values have been found in

samples from Saint Guenole, a population located in north

Brittany near the English Channel; in Punta de la Cruz,

located in the northwest of the Iberian Peninsula and in

Mauritania, in North Africa. In addition, Saint Guenole, the

three northwestern Iberian localities (Ribadesella, Punta de

la Cruz and Corme) and the two African samples exhibited

high percentages of private haplotypes (Table 1). All these

data together suggest the existence of a Pleistocene refugial

area off the North African coasts and two northern addi-

tional glacial refugia for P. pollicipes, in the English

Channel/Brittany region and in the northwestern Iberian

Peninsula. These two northern areas have also been sug-

gested to act as refugia for other marine sessile species

(Gomez et al. 2007 for a review; Hoarau et al. 2007;

Provan and Bennett 2008). The high levels of gene flow

encountered for this species (Quinteiro et al. 2007; present

study) could have rapidly homogenized the expanding

populations from North Africa and Iberian Peninsula,

impeding the detection of genetic differences. On the other

hand, the existence in southwestern France of a region

spanning along the coasts of Aquitaine, Poitou-Charentes

and Pays de la Loire, which is characterized by long sandy

beaches and lack of rocky environments, can constitute a

partial barrier for P. pollicipes larval dispersal from the

Iberian Peninsula to Brittany and vice versa, making

homogenization of those populations much slower, even

though gene flow in this area is favored by the Iberian

Poleward Current.

Recolonization pathways

Recolonization pathways from refugial areas should not be

difficult to infer for a species that inhabits the high inter-

tidal zone of continental coasts. In the case of the Brittany

refugium, habitat expansion at interglacial periods was

likely southward, since there is no occurrence of P. pol-

licipes northern than Brittany and genetic diversity

decreased from north to south. From the Iberian refugium,

diversity decreased southward but not to the east; in this

case, although the haplotype diversity was lower for all

samples eastern to Punta de la Cruz, it increased from

Ribadesella to Jaizkibel. In addition, all the haplotypes

found in Monpas and Jaizkibel were also present in both

Brittany and the rest of Iberian samples, and no private

haplotypes were found. This could be explained by a sec-

ondary contact in that area between individuals from the

Brittany and Iberian Peninsula groups during the intergla-

cial expansions since Jaizkibel is located at the eastern

boundary of the Iberian range of distribution. Finally,

expansion from African populations should be toward the

north, since this is the southernmost limit of the species.

Although Quinteiro et al. (2007) only recognized one

154 Mar Biol (2010) 157:147–156

123

refugium for P. pollicipes at tropical and subtropical lati-

tudes, the values of haplotypic diversity found by them at

the AT-rich region of the mitochondrial control region and

a flanking fragment are in agreement with the data here

presented, since they decreased from western Iberian

Peninsula to the south and to the east, and from Canary

Islands to Morocco.

Comparative phylogeography

For marine sessile organisms, climatic conditions during

glacial periods were likely even harder than for non-sessile

species, due to their low capacity of displacement to more

suitable areas, the exposure to cold atmosphere during low-

tide periods (Marko 2004) and the change in sea level

(down to 120 m at LGM). Most marine species are sug-

gested to have followed a pre-LGM expansion model

(Duran et al. 2004; Stamatis et al. 2004; Chevolot et al.

2006; Hoarau et al. 2007), with the exception of some

North Atlantic taxa (Wares and Cunningham 2001) and

two species of the genus Zoostera in Europe (Coyer et al.

2004; Olsen et al. 2004). This could be due to the fact that

climatic conditions during Pleistocene glaciations were less

severe in Europe than in North America, where LGM

conditions should have been especially difficult for rocky

intertidal species (Wares and Cunningham 2001). On the

other hand, the Warthe/Saale glaciation (180,000–

128,000 years ago), which preceded the Eem interglacial

period, probably had a broader effect than the LGM

(Kellaway et al. 1975). Thus, the pattern of demographic

expansion during the Eem interglacial found for P. pol-

licipes is in concordance with that found in many other

marine invertebrates and sessile species of the North

Atlantic, like the bivalve Macoma balthica (Luttikhuizen

et al. 2003), the polychaete tubeworms Pectinaria koreni,

P. auricoma and Owenia fusiformis (Jolly et al. 2006), the

red alga Palmaria palmata (Provan et al. 2005) and the

brown seaweed Fucus serratus (Hoarau et al. 2007). In

addition, Van Syoc (1994) also found a congeneric species,

the eastern Pacific goose barnacle, P. elegans, to have been

affected by periods of cooling and warming during the

Pleistocene, leading to the separation of two differentiated

amphitropical subpopulations with alternating periods of

genetic exchange and isolation.

Conclusions

The current pattern of distribution of the genetic diversity

in the European stalked barnacle, P. pollicipes, can be at

least partially explained by climatic conditions that

occurred during the Pleistocene. During cold glaciation

periods, populations could survive in southern areas off the

North African coast and in two small northern refugial

areas, in the English Channel (Brittany region) and in

northwestern Iberian Peninsula. During warmer interglacial

stages, populations expanded from these areas and sec-

ondary contacts between individuals from those groups

could take place, impeding a total genetic differentiation of

such lineages, and so preventing differential evolution and

speciation events. High levels of gene flow due to high

capacity of larval dispersal also allowed a rapid homoge-

nization of populations, and thus most of the genetic dif-

ferentiation that could arise during population contractions

in refugial areas has been currently minimized.

The identification of cryptic refugia has important

implications in current and future periods of global climate

change (Provan and Bennett 2008) in order to predict and

model the ecological behavior of the species. On the other

hand, barnacles are very plastic species (Barnes and Barnes

1962; Crisp and Bourget 1985) and this, together with the

high levels of gene flow found for P. pollicipes, could

likely guarantee its survival in future climatic changes.

Acknowledgments Samples were collected with the help of Ramon

Riveiro, Jorge L. Alcazar and other anonymous collaborators. We

want to thank Dr. Galice Hoarau for precious help in focusing the

paper. Dr. David Posada helped us with the NCA. Prof. Francis

Juanes revised the article and corrected the English. We also want

to thank two anonymous reviewers for helpful comments. This study

has been financially supported by the Xunta de Galicia (Spain), Grant

SV-05-XUNTA-1.

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