Molecular Ecology Notes (2007) 7, 464 466

doi: 10.1111/j.1471-8286.2006.01619.x

PRIMER NOTE
Blackwell Publishing Ltd

Isolation and characterization of 11 polymorphic

microsatellite markers for the marine gastropod Concholepas
concholepas (Brugire, 1789)
L E Y L A C R D E N A S ,* C L A I R E D A G U I N , J U A N C A R L O S C A S T I L L A * and F R D R I Q U E V I A R D
*Center for Advanced Studies in Ecology and Biodiversity (CASEB), Facultad de Ciencias Biolgicas, Pontificia Universidad Catlica
de Chile, Alameda 340, Casilla 114D, Santiago 6513677, Chile, Equipe Evolution et Gntique des Populations Marines UMR 7144
CNRS-Universit Pierre et Marie Curie, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff cedex, France, Laboratoire
International Associ Dispersal and Adaptation of Marine Species (LIA DIAMS) PUC, Chile and CNRS-UPMC, France

Abstract
Because of its long-lived planktonic stage, the marine gastropod Concholepas concholepas
is expected to exchange larvae over large distances. However, discrepancies between expected
and realized dispersal have been documented in marine invertebrates. To investigate
relationships between potential and effective (i.e. gene flow) dispersal, we developed 11
microsatellite markers and investigate their usefulness by analysing two populations
distant by c. 4000 km. The 11 loci were found to be highly polymorphic in both populations,
with 1251 alleles according to the locus. This polymorphism is strong enough to allow
fine-scale population analyses including larval studies and paternity analyses.
Keywords: Chile, Concholepas concholepas, dispersal ability, marine gastropod, microsatellites,
pelagic larvae
Received 3 July 2006; revision accepted 15 October 2006

The muricid gastropod Concholepas concholepas (Brugire,

1789), is an important component of intertidal and shallow
subtidal communities along the Chilean coast (Castilla
1999). It is also one of the main invertebrates targeted by
small-scale fisheries in the country (Leiva & Castilla 2002).
The known geographical distribution of C. concholepas
extends from central Per to Cape Horn, ranging over
7000 km of coastline in the Southeast Pacific. C. concholepas
is characterized by a bentho-pelagic life cycle with larvae
spending 3 months in the watercolumn. With such a longlived planktonic stage, dispersal of C. concholepas is expected
to be very high. Nevertheless, the occurrence of this
gastropod in areas characterized by strong and persistent
retention (Castilla et al. 2002) could reduce the dispersal of
larvae away from the parental populations with important
consequences for conservation plan and fishery management. Our purpose was to develop microsatellites to
examine the effective dispersal between populations as
well as to examine larval population diversity.
Correspondence: L.
lcardena@bio.puc.cl

Crdenas,

Fax:

56(2)6862621;

E-mail:

A genomic library was constructed of DNA isolated

from a single C. concholepas individual from central Chile
(Temblador, 2928S7118W), using a standard phenolchloroform extraction method. Two enriched libraries for
AC and TGTA repeats were constructed from extracted
DNA digested by RsaI, purified using Nucleospin columns
(Macherey-Nagel) and ligated to RsaI linkers. For the
enrichment procedure, DNA fragments were hybridized
to 5-biotinylated (AC)10 and (TGTA)5 probes separately,
and captured with Streptavidin-coated Magnesphere paramagnetic beads (Promega). The two fractions of purified
and enriched DNA were then ligated into pGEM-T Easy
vector (Promega) and transformed into Escherichia coli JM109
competent cells. Two hundred eighty-eight recombinant
clones were screened for microsatellite by standard polymerase chain reaction (PCR) using a mixture of (AC)10 or (TGTA)5
primers and the standard T7 and SP6 universal primers
consecutively. Positive clones were then sequenced with T7
and SP6 primers on an ABI 3100 sequencer (Applied Biosystems). Of a total of 56 primer pairs designed and used
in preliminary tests, 22 pairs were screened for polymorphism on a Li-Cor NEN Global IR2 DNA Analyser with
2006 The Authors
Journal compilation 2006 Blackwell Publishing Ltd

 2006 The Authors

Journal compilation 2006 Blackwell Publishing Ltd

Table 1 Characteristics of 11 microsatellite loci isolated from Concholepas concholepas (GenBank Accession nos DQ379460DQ379470). NTall, and HT, number of alleles and total
heterozygosity over the whole sample (N = 54); Nall and HE, number of alleles and expected heterozygosity at the population level; R, allelic richness over the whole sample (RT) and at
the population level (RS); R estimation were based on a minimum sample size of 21 diploid individuals using the software fstat version 2.9.3 (Goudet 1995). FIS, fixation index and test
for deviation of HardyWeinberg expectations (*P < 0.05; ***P < 0.005)
Puerto Aguirre
(4515S 7240W)
N = 24
Repeat array

Primer sequences (53)

Cc1A2

(GT)5(TG)8

Cc2A11-1

(GT)13

Cc2A5

(TGTGTC)2
(TG)6
(CA)13

F: GTGTTCTTGCTTGAGCTGGTGTGC
R: CGCTTCTGAAAACACATGTCTCTCC
F: CCACTTGGTTCAGGATGGTCAC
R: CATTGTTTTGGTGTTGTGGGAA
F: GCACCAGCGAAACAAGTCCC
R: CTGTGCTGATGGGAGCCTTG
F: AGGACATTGTAAGAGTAACGGTGGC
R: ACCAAGTGGTGGTGATTTCTGTTG
F: TTGGCGAAGACACTAAAAAATAATG
R: TAGGCAGGACATGTTTGTTGATG
F: TCATCTGTTGTTTTGTCATTCC
R: GCTGTCCCCTTATTCATTGTTC
F: GTAGATATGCAGTGTAATGCAGTAG
R: ACACTTATGCCAATTATCAGTAATG
F: AACTCACCTCATTCTTCTACTAAAC
R: AAGCGGTGGTCCTATAACTGGC
F: GATGGTGCAAGTCACAGTGAATG
R: GCCCCTCACTGTCTGTGTTCC
F: GCGAAGAAGAAAATGACGACTAC
R: ACTTCAAGATTAGAAACACCAATAC
F: TCTGACAGAAACCCAATGCTGAG
R: CAACGCCGATAACAGCAGAATGG

Cc1B6
Cc2F5
Cc1E5
Cc1D8
Cc2A11-2
Cc2B5
Cc1H2
Cc1H1
Mean over loci
SD

(GAAT)2N3(TGAA)3N16(TGAA)3
N6(AAGG)8N4(AAGG)2
(TG)7N5(TG)8
N4(TG)10
(CA)3N4(CA)2N4
(CA)27N(CA)5
(TC)29(TG)3
N6(TC)5N4(TC)4
(CA)8N2(CA)2(GA)10
N2(GA)2N2(GA)25
(CTA)20N6(ACC)7a
(CTA)5N3(CTA)5N20(CTA)16
(TGTC)6N4(TGTC)5N2
(TGTC)3

Allele size
range (bp)

NTall

HT

132149

12

0.65

127149

12

262286

RT

Nall

HE

RS

8.8

10

0.73

9.6

0.82

9.8

0.82

12

0.74

8.6

187238

18

0.86

11.4

215333

18

0.90

299347

21

209266

FIS

Nall

HE

RS

FIS

0.14

0.57

7.4

0.06

8.7

0.02

11

0.83

10.4

0.13

0.75

8.7

0.05

0.74

7.4

0.15

10

0.81

9.5

0.33***

14

0.88

12.4

0.07

13.2

13

0.89

12.4

0.02

16

0.90

14.1

0.11

0.90

14.5

18

0.93

17

0.06

14

0.88

12.1

0.02

27

0.90

18

15

0.87

15

0.01

22

0.93

18.7

0.10

228315

34

0.97

23.7

25

0.96

23.4

0.05*

29

0.97

24.5

0.04

194348

48

0.98

29.3

23

0.97

23

0.47***

34

0.98

28

0.30***

268361

48

0.98

29.7

26

0.97

26

0.07

34

0.98

27.7

0.01

280395

51

0.99

30.9

27

0.97

25.7

0.24***

33

0.98

28.4

0.42***

0.88
0.11

17.9
8.8

16.8
7.3

0.88
0.09

16.3
7.1

0.13***
(P = 0.000)

20.4
10.4

0.88
0.13

17.4
8.4

0.10***
(P = 0.000)

P R I M E R N O T E 465

Locus

Matarani
(1700S7218W)
N = 30

466 P R I M E R N O T E
forward primers labelled with infrared fluorescent dye
IRD700 or IRD800. Genomic DNA was extracted using the
Nucleospin 96 Tissue kit (Macherey-Nagel). All loci were
amplified as follows: touchdown PCR procedure using a
PTC-200 thermocycler (MJ Research): 15 L final volume,
2 L of DNA template diluted to 1:10, 1 buffer (Euroclone),
0.25 mm of each dNTP, 1.5 mm MgCl2, 2 m of bovine
serum, 0.32 U of Eurotaq polymerase (Euroclone), 0.10 m
of fluorescent labelled forward primer, 0.27 m of unlabelled
forward primer and 0.33 m of unlabelled reverse primer.
Touchdown PCR program included 4 min at 95 C, followed by 10 cycles with 45 s at 95 C, 45 s at 60 C and then
decreasing by 1 C per cycle to 50 C, 45 s at 72 C, and then
20 cycles at 95 C for 45 s, 50 C for 45 s, 72 C for 45 s, and
7 min at 72 C. Of the 22 primer pairs, 11 were discarded
because of unclear banding patterns or monomorphism.
For the 11 remaining loci, allele sizes were determined
using a known DNA sequence and the polymorphism was
assessed using samples from two populations distant by
c. 4000 km, Matarani and Puerto Aguirre (Table 1). These
two populations were chosen to test for artefacts (e.g. null
alleles) that might arise from constructing a library using
individuals sampled in a population strongly divergent
from others in the species range. Exact test for genotypic
disequilibrium and deviations from HardyWeinberg
equilibrium (HWE) as well as number of alleles and
expected heterozygosity (Table 1) were computed using
the program genepop version 3.4 (Raymond & Rousset
1995). No linkage disequilibria across loci were detected.
The total number of alleles varied across loci, ranging from
12 to 51. With one exception (Cc1A2 in Matarani), the
expected heterozygosity (HE) was high and similar across
populations and loci. In most cases, no significant deviations from HWE were found. The only exceptions were in
Puerto Aguirre at loci Cc1B6 and Cc2A11-2 and in both
populations at loci Cc2B5 and Cc1H1 (Table 1). For the two
latter, this result could be due to null alleles. The number
of null amplifications was nevertheless low (less than 5%)
whatever the population and of the same order than for
other loci that fit HWE. An alternative explanation is sampling drift effects given that are Cc2B5 and Cc1H1 highly
polymorphic with numerous rare alleles. Given the large
size difference between alleles at these two loci as well as
for Cc2F5, Cc2A11-2 and Cc1H2, nonhomology of alleles
could also be suspected. However, computations made
for each of the 11 loci with the software micro-checker
(Van Oosterhout et al. 2004) did not reveal experimental
artefacts (e.g. no drop-out effects). Banding patterns (e.g.
unambiguous amplification pattern and regular distribution of allele sizes between the two extreme allele sizes)

also supported homology of alleles. Moreover, population

analyses carried out with the software bottleneck showed
that HE values fall into the range or were even below those
expected under a stepwise mutation model or an infinite
allele model (Cornuet & Luikart 1996). Altogether, the two
study populations were found to be highly polymorphic
(mean allelic richness of 16.3 7.1 and 17.4 8.4 in Puerto
Aguirre and Matarani, respectively) with same level of
expected heterozygosity. Besides, the lack of genetic
differences between the two populations suggested either
a recent common origin or homoplasy effects often documented for microsatellite markers with high mutation rate
(Estoup et al. 2002). This does not preclude their usefulness
for population studies over a fine-spatial scale including
larval diversity studies with the most polymorphic markers
being most useful for paternity analyses.

Acknowledgements
This study was funded by grants awarded by ECOS Program n
C03B04, by FONDAP-FONDECYT 1501-0001 Program 6 to cases,
and by the French Embassy in Santiago. Molecular work was
also supported by program 2 Metapopulation and Dispersal of
LIA DIAMS. L. Crdenas acknowledges a doctoral support from
CONICYT (AT 24050101) and a grant from CNRS and UPMC for
travel and stay in France as part of her co-supervised thesis.

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2006 The Authors

Journal compilation 2006 Blackwell Publishing Ltd