A P P L I C AT I O N N O T E

Arlequin (version 3.0): An integrated software package

for population genetics data analysis
Laurent Excoffier, Guillaume Laval, Stefan Schneider
Computational and Molecular Population Genetics Lab, Zoological Institute, University of Berne, Baltzerstrasse 6, 3012 Berne, Switzerland
Abstract: Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics
data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies,
tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation
or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA
framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of
input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation
of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several
data types like DNA sequences, microsatellite data, or standard multi-locus genotypes. A Windows version of the software
is freely available on http://cmpg.unibe.ch/software/arlequin3.
Keywords: Computer package, population genetics, genetic data analysis, AMOVA, EM algorithm, gametic phase estimation,
spatial expansion.

Introduction

Most genetic studies on non-model organisms require a description of the pattern of diversity within and
between populations, based on a variety of markers often including mitochondrial DNA (mtDNA)
sequences and microsatellites. The genetic data are processed to extract information on the mating
system, the extent of population subdivision, the past demography of the population, or on departure
from selective neutrality at some loci. A series of computer packages have been developed in the last
10 years to assist researchers in performing basic population genetics analyses like Arlequin2 (Schneider
et al. 2000), DNASP (Rozas et al. 2003), FSTAT (Goudet 1995), GENEPOP (Raymond and Rousset
1995b), or GENETIX (Belkhir et al. 2004). These programs have been widely used in the molecular
ecology and conservation genetics community (Labate 2000; Luikart and England 1999; Schnabel et al.
1998). Among these, Arlequin is a very versatile (though not universal) program, and complements
the other programs listed above. It can handle several data types like RFLPs, DNA sequences,
microsatellite data, allele frequencies, or standard multi-locus genotypes, while allowing the user to
carry out the same types of analyses irrespective of the data types.
We present here the version 3 of Arlequin with additional methods extending its capacities for the
handling of unphased multi-locus genotypes and for the estimation of parameters of a spatial expansion.
Note that these new developments are mainly implementations of new methodologies developed in our
lab. We believe these methods will be useful to the research community, but we do not claim that
alternative methods implemented by other groups in other programs are inadequate. A new graphical
interface has been developed to provide a better integration of the different analyses into a common
framework, and an easier exploration of the data by performing a wide variety of analyses with different
settings. The tight coupling of Arlequin with the simulation programs SIMCOAL2 (Laval and Excoffier
2004) and SPLATCHE (Currat et al. 2004) should also make it useful to describe patterns of genetic
diversity under complex evolutionary scenarios.

Methods implemented in Arlequin

Arlequin provides methods to analyse patterns of genetic diversity within and between population samples.

Correspondence: Laurent Excoffier, Tel: +41 31 631 30 31, Fax: +41 31 631 48 88 Email: laurent.excoffier@
zoo.unibe.ch
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Excoffier et al

Intra-population methods

Computation of different standard genetic

indices, like the number of segregating
sites, the number of different alleles, the
heterozygosity, the base composition of DNA
sequences, gene diversity, or the population
effective size Ne scaled by the mutation rate
as =4Neu.

Maximum-likelihood estimation of allele

and haplotype frequencies via the EM
algorithm (Excoffier and Slatkin 1995).

Estimation of the gametic phase from

multi-locus genotypes via the ExcoffierLaval-Balding (ELB) algorithm (Excoffier
et al. 2003).

Estimation of the parameters of a demographic

(Rogers and Harpending 1992; Schneider
and Excoffier 1999) or a spatial (Excoffier
2004; Ray et al. 2003) expansion, from the
mismatch distribution computed on DNA
sequences.
Calculation of several measures of linkage
disequilibrium (LD) like D, D, or r2 (Hedrick
1987), and test of non-random association of
alleles at different loci when the gametic
phase is known (Weir 1996) or unknown
(Slatkin and Excoffier 1996).

Exact test of departure from Hardy-Weinberg

equilibrium (Guo and Thompson 1992).

Computation of Tajimas D (Tajima 1989)

and Fus FS (Fu 1997) statistics, and test of
their significance by coalescent simulations
(Hudson 1990; Nordborg 2003) under the
infinite-site model.

Tests of selective neutrality under the

infinite-alleles model, like the EwensWatterson test (Slatkin 1996; Watterson
1978), and Chakrabortys amalgamation test
(Chakraborty 1990).

Inter-population methods

Search for shared haplotypes between

populations

Analysis of population subdivision under

the AMOVA framework (Excoffier 2003;
Excoffier et al. 1992), with three hierarchical
levels: genes within individuals, individuals
within demes, demes within groups of

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demes. Computation of F-statistics like the

local inbreeding coefficient FIS or the index
of population differentiation FST.

Computation of genetic distances between

populations related to the pairwise FST index
(Gaggiotti and Excoffier 2000; Reynolds
et al. 1983; Slatkin 1995).

Exact test of population differentiation

(Goudet et al. 1996; Raymond and Rousset
1995a).

A simple assignment test of individual

genotypes to populations according to their
likelihood (Paetkau et al. 1997).

Computation of correlations or partial

correlations between a set of 2 or 3 distance
matrices (Mantel test: Smouse et al. 1986)

New features in Arlequin 3

Version 3 of Arlequin integrates the core

computational routines and the interface in
a single program written in C++ for the
Windows environment. The interface has
been entirely redesigned to provide better
usability.

Incorporation of two new methods to

estimate gametic phase and haplotype
frequencies:

The ELB algorithm (Excoffier et al.

2003) is a pseudo-Bayesian approach
aiming at reconstructing the gametic
phase of multi-locus genotypes, and
the estimation of the haplotype
frequencies are a by-product of this
process. Phase updates are made on the
basis of a window of neighbouring loci,
and the window size varies according to the local level of linkage
disequilibrium.

The EM zipper algorithm, which is an

extension of the EM algorithm for estimating haplotype frequencies (Excoffier and Slatkin 1995), aims at
estimating the haplotype frequencies in
unphased multi-locus genotypes. The
estimation of the gametic phases are a
by-product of this process. It proceeds
by adding loci one at a time and
progressively extending the length of
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Arlequin 3.0

the reconstructed haplo-types. With this

method, Arlequin does not need to build
all possible genotypes for each
individual like in the conventional EM
algorithm, but it only considers the
genotypes whose sub-haplotypes have
non-null estimated frequencies. It can
thus handle a much larger number of
polymorphic sites than the strict EM
algorithm. It also gives final haplotype
frequencies that often have a higher
likelihood than those estimated under
the strict EM algorithm, due to the
difficulty in exploring the space of all
possible genotypes when the number
of polymorphic loci in the sample is
large. Note that this version of the
EM algorithm is equivalent to that
implemented in the SNPHAP program
by David Clayton fully described
on http://www-gene.cimr.cam.ac.uk/
clayton/software/snphap.txt, and whose
efficiency for inferring gametic phase
has been favorably evaluated (Adkins
2004).

Estimation of the parameters of a spatial

expansion (age of the expansion and deme
size scaled by the mutation rate, as well as
the number of migrants exchanged between
neighbouring demes) from the patterns of
polymorphism in a sample of DNA sequences.
The estimation is based on a simple model
of instantaneous and infinite range expansion,
where some time ago, a single deme
instantaneously colonized an infinite number
of demes subsequently interconnected by
migration (as under an infinite-island model)
(Excoffier 2004). The parameters are
obtained by a least-square approach
maximizing the fit between the observed and
expected distribution of pair-wise differences
(the mismatch distribution) computed on
DNA sequences. Confidence intervals of the
estimates are obtained under a parametric
bootstrap approach involving the simulation
of an instantaneous expansion under a
coalescent framework.
Estimation of confidence intervals for
F-statistics estimated under the AMOVA
framework by bootstrapping over loci for
multi-locus data. A minimum of 8 loci are

Evolutionary Bioinformatics Online 2005:1

necessary for the computation of these

confidence intervals.

A completely rewritten and more robust

input file parsing procedure, giving more
precise information on the location of
potential syntax and format errors in input
files.

Use of the ELB algorithm described above

to generate samples of phased multi-locus
genotypes, which allows one to analyse
unphased multi-locus genotype data as if
the phase was known. The phased data sets
are output in Arlequin projects that can
be analysed in a batch mode to obtain
the distribution of statistics taking phase
uncertainty into account.

New output files fully compatible with

modern web browsers.

Availability

A Windows executable version Arlequin ver 3 can

be freely downloaded on http://cmpg.unibe.ch/
software/arlequin3, together with an up-to-date
user manual in Adobe Acrobat PDF format
incorporating more technical details on the methods
used in Arlequin 3, as well as several example
files.

Acknowledgements

This work was partially made possible thanks to a

Swiss NSF grant No 31-56755.99 to LE.

References

Adkins RM. 2004. Comparison of the accuracy of methods of computational

haplotype inference using a large empirical dataset. BMC Genet. 5:22.
Belkhir K, Borsa P, Chikhi L et al. 2004. GENETIX 4.05, logiciel sous
Windows pour la gntique des populations. Laboratoire Gnome,
Populations, Interactions, CNRS UMR 5000, Universit de
Montpellier II, Montpellier.
Chakraborty R. 1990. Mitochondrial DNA polymorphism reveals hidden
heterogeneity within some Asian populations. Am J Hum Genet. 47:
87-94.
Currat M, Ray N and Excoffier L. 2004. SPLATCHE: a program to simulate
genetic diversity taking into account environmental heterogeneity.
Mol Ecol. 4: 139-142.
Excoffier L. 2003. Analysis of Population Subdivision. In Balding D Bishop
M, and Cannings C, eds. Handbook of Statistical Genetics, 2nd
Edition. New York: John Wiley and Sons, Ltd. p 713-750.
Excoffier L. 2004. Patterns of DNA sequence diversity and genetic structure
after a range expansion: lessons from the infinite-island model. Mol
Ecol. 13: 853-864.
Excoffier L, Laval G and Balding D. 2003. Gametic phase estimation over
large genomic regions using an adaptive window approach. Mol Ecol.
1: 7-19.

49

Excoffier et al
Excoffier L and Slatkin M. 1995. Maximum-likelihood estimation of
molecular haplotype frequencies in a diploid population. Mol Biol
Evol. 12: 921-927.
Excoffier L, Smouse P and Quattro J. 1992. Analysis of molecular variance
inferred from metric distances among DNA haplotypes: Application
to human mitochondrial DNA restriction data. Genetics. 131:
479-491.
Fu Y-X. 1997. Statistical tests of neutrality of mutations against population
growth, hitchhiking and backgroud selection. Genetics. 147:
915-925.
Gaggiotti O and Excoffier L. 2000. A simple method of removing the effect
of a bottleneck and unequal population sizes on pairwise genetic
distances. Proceedings of the Royal Society London B. 267: 81-87.
Goudet J. 1995. Fstat version 1.2: a computer program to calculate
F-statistics. J Heredity. 86: 485-486.
Goudet J, Raymond M, de Mees T et al. 1996. Testing differentiation in
diploid populations. Genetics. 144: 1933-1940.
Guo S and Thompson E. 1992. Performing the exact test of Hardy-Weinberg
proportion for multiple alleles. Biometrics. 48: 361-372.
Hedrick P. 1987. Gametic disequilibrium measures: proceed with caution.
Genetics. 117: 331-3412.
Hudson RR. 1990. Gene genealogies and the coalescent process. In Futuyma
DJ and Antonovics JD, eds. Oxford Surveys in Evolutionary Biology.
New York: Oxford University Press. p 1-44.
Labate JA. 2000. Software for Population Genetic Analyses of Molecular
Marker Data. Crop Sci. 40: 1521-1528.
Laval G and Excoffier L. 2004. SIMCOAL 2.0: a program to simulate genomic
diversity over large recombining regions in a subdivided population
with a complex history. Bioinformatics. 20: 2485-2487.
Luikart G and England PR. 1999. Statistical analysis of microsatellite DNA
data. Trends Ecol Evol. 14: 253-256.
Nordborg M. 2003. Coalescent Theory. In Balding D Bishop M, and Cannings C, eds. Handbook of Statistical Genetics, 2nd edition. New
York: John Wiley and Sons Ltd. p 602-635.
Paetkau D, Waits LP, Clarkson PL et al. 1997. An empirical evaluation of
genetic distance statistics using microsatellite data from bear
(Ursidae) populations. Genetics. 147: 1943-1957.
Ray N, Currat M and Excoffier L. 2003. Intra-Deme Molecular Diversity
in Spatially Expanding Populations. Mol. Biol. Evol. 20: 76-86.

50

Raymond M and Rousset F. 1995a. An exact test for population

differentiation. Evolution. 49: 1280-1283.
Raymond M and Rousset F. 1995b. GENEPOP Version 1.2: Population genetics
software for exat tests and ecumenicism. J Heredity. 248-249.
Reynolds J, Weir BS and Cockerham CC. 1983. Estimation for the coancestry
coefficient: basis for a short-term genetic distance. Genetics. 105:
767-779.
Rogers AR and Harpending H. 1992. Population growth makes waves in
the distribution of pairwise genetic differences. Mol Biol Evol. 9:
552-569.
Rozas J, Sanchez-DelBarrio JC, Messeguer X et al. 2003. DnaSP, DNA
polymorphism analyses by the coalescent and other methods.
Bioinformatics. 19: 2496-2497.
Schnabel A, Beerli P, Estoup A et al. 1998. A guide to software packages
for data analysis in molecular ecology. In Carvalho G, eds. Advances
in Molecular Ecology. Amsterdam: IOS Press. pp 291-303.
Schneider S and Excoffier L. 1999. Estimation of demographic parameters
from the distribution of pairwise differences when the mutation rates
vary among sites: Application to human mitochondrial DNA.
Genetics. 152: 1079-1089.
Schneider S, Roessli D and Excoffier L. 2000. Arlequin: a software for
population genetics data analysis. User manual ver 2.000. Genetics
and Biometry Lab, Dept. of Anthropology, University of Geneva,
Geneva.
Slatkin M. 1995. A measure of population subdivision based on microsatellite
allele frequencies. Genetics. 139: 457-462.
Slatkin M. 1996. A correction to the exact test based on the Ewens sampling
distribution. Genet Res. 68: 259-260.
Slatkin M and Excoffier L. 1996. Testing for linkage disequilibrium in
genotypic data using the EM algorithm. Heredity. 76: 377-383.
Smouse PE, Long JC and Sokal RR. 1986. Multiple regression and
correlation extensions of the Mantel Test of matrix correspondence.
Syst Zool. 35: 627-632.
Tajima F. 1989. Statistical method for testing the neutral mutation hypothesis
by DNA polymorphism. Genetics. 123: 585-595.
Watterson G. 1978. The homozygosity test of neutrality. Genetics. 88:
405-417.
Weir BS. 1996. Genetic Data Analysis II: Methods for Discrete Population
Genetic Data. Sinauer Assoc., Inc.: Sunderland, MA, USA.

Evolutionary Bioinformatics Online 2005:1