Centre de Génétique Moléculaire (CGM), CNRS, Gif sur Yvette cedex, France;
Centro de Estudos de Ciência Animal (CECA), Campus Agrário de Vairão, Vila do Conde, Portugal;
Departamento de Zoologia e Antropologia, Faculdade de Ciências do Porto, Praça Gomes Teixeira, Porto, Portugal
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several studies based on mitochondrial DNA polymorphism within the rabbit's native range reveal two highly divergent (at least 2 Myr) maternal lineages, each with a well-defined geographical distribution: one lineage occurs in southwestern Iberia and the other in northeastern Spain, France, and in domestic breeds (Biju-Duval et al. 1991
; Monnerot et al. 1994
). Recently, Branco, Ferrand, and Monnerot (2000)
provided a more comprehensive picture of mtDNA variation within the Iberian Peninsula, reporting the existence of a relatively narrow contact zone for the two maternal lineages that bisects the peninsula along a northwest-southeast axis. Analysis of 20 polymorphic protein loci exhibiting more than 100 alleles also reveals two major groups of populations coincident with the mtDNA subdivision (Ferrand 1995
; Ferrand and Branco, unpublished data) as does the analysis of immunoglobulin polymorphism (van der Loo, Ferrand, and Soriguer 1991
; van der Loo et al. 1999
). Collectively, these data suggest that these population groups evolved separately for a significant period of time before a hybrid zone was formed following more recent secondary contact. Additionally, significant loss of genetic variability in populations north of the Pyrenees relative to those in the south, seen at both mtDNA and polymorphic protein loci, indicates a genetic bottleneck associated with the postglacial expansion of the rabbit from its pan-Iberian distribution area.
Within this phylogeographic framework, we screened a set of microsatellite markers to evaluate, on a finer scale, current hypotheses concerning both the rabbit's evolutionary past and its initial stages of geographic expansion. We are particularly interested in addressing two questions: (1) how informative are microsatellites in revealing the deep genetic divergence between groups of populations, and (2) can microsatellites reveal the pattern of a recent population expansion across France, a phenomenon that allozymes and mtDNA have failed to elucidate with any degree of explanatory resolution.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Statistical Analysis
Comparative measures of genetic diversity for each population were calculated in the form of allelic diversity (total number of alleles, mean number of alleles per locus, and private alleles), observed heterozygosity, and nonbiased expected heterozygosity (Nei 1987
) using the program GENETIX (Belkhir et al. 1996
). Hardy-Weinberg equilibrium (HWE) was evaluated for all loci across all populations, and linkage disequilibrium between pairs of loci was evaluated using GENEPOP software (Raymond and Rousset 1995
). Statistical significance was determined using Bonferroni correction (Rice 1989
). To enable large-scale inferences on the relation of major groups of rabbit populations and their expansion outside Iberia, diversity indices were averaged across all loci, and a mean value was calculated for each of the three geographical groups of populations (SWIP, NEIP, and FR). A Wilcoxon-Mann-Whitney test was used to test for significant differences in allelic diversity or heterozygosity across these three groups. All tests were conducted separately for each measure of diversity, using STATVIEW (Abacus Concepts Inc., Berkeley, Calif.).
To estimate gene flow within and among groups of populations, estimators of FST () and their 95% confidence intervals (bootstrapping over loci) were calculated using FSTAT (Goudet 1995
). Estimates of FST for microsatellite data were compared to available data on these populations based on mtDNA RFLPs and polymorphic protein loci.
To depict the genetic relationships among all populations, networks were generated using the Neighbor-Joining (NJ) algorithm with the program NEIGHBOR in the PHYLIP package. Because there is still considerable debate over the merits and drawbacks of various microsatellite-based genetic distances, we used five different matrices of genetic distances as input for the NJ algorithm. Nei's standard distance (Nei 1987
) was chosen as it assumes an infinite allele model (Kimura and Crow 1964
), whereas three microsatellite-specific measures:
µ2 (Goldstein et al. 1995
), DSW (Shriver et al. 1995
), and RST (Slatkin 1995
) all assume a stepwise mutation model (Ohta and Kimura 1973
) but may differ in how they reflect varying amounts of drift and mutation. Finally, the simple allele-sharing statistic DAS (Bowcock et al. 1994
) was used to represent a measure which makes no evolutionary assumptions. Nei's distances were calculated between all populations using GENDIST in the PHYLIP package (Felsenstein 1993
) while all other distances were calculated using MICROSAT (Minch 1996
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mean total number of alleles (a) and expected heterozygosity (He) were highest in SWIP populations (a = 83.8, He = 0.823) slightly lower in NEIP (a = 70.5, He= 0.777), and lowest in populations of France (a = 46.8, He = 0.644), conforming to our expectations of reduced genetic diversity in the area of initial geographic expansion compared to Iberian refugia (table 1 ). However, there were no significant differences in allelic diversity (P = 0.144) between SWIP and NEIP, whereas Iberian populations were significantly more diverse than populations in France (P < 0.01, table 1 ). There was a large difference in the number of private alleles (defined here as alleles found in a single population throughout the study region) between SWIP (27) and both NEIP (4) and FR (5); the similar numbers for NEIP and FR probably reflect the much higher sampling effort in FR.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
High and/or differential rates of drift should be reflected in at least some microsatellite-specific distance measures such as Goldstein's µ2 where the mean variance across loci is expected to remain equal in magnitude but undergo a modal shift between two groups of populations over time. However, given allele size range overlap, we must conclude that the mutation spectrum has become saturated during 2 million years of divergence, through a combination of mutation constraints and back mutations that have homogenized allele size distributions as predicted by Nauta and Weissing (1996)
. This form of homoplasy, which we refer to as size homoplasy, simply means that alleles are identical in state but have different mutational histories that have led to their present state. This definition neither necessitates nor excludes the possibility that alleles also differ at the sequence level. A mechanistic model for such allele size homogenization for tetranucleotide repeats in humans has recently been shown in Xu et al. (2000)
. The only alternative scenario that could explain our observations would be a pattern of long-term, sex-biased dispersal in which females remain in their breeding groups and gene flow is essentially male mediated. However, Ferrand (1995)
and Ferrand and Branco (unpublished data) were able to describe a relatively strong phylogenetic signal (compatible with mtDNA) between southwestern and northeastern Iberian rabbits as well as strong population substructure within regions using protein polymorphisms. Likewise, in our study, the large numbers of region-specific private alleles (as opposed to private for the study area) for both NEIP (18) and SWIP (38) suggest fine-scaled population structure in Iberian rabbits. Further evidence for the lack of gene flow over time is seen at the locus sat13 where intermediately sized private alleles (sizes 113, 115, and 127) in NEIP suggest that there has been a sufficient period of isolation for a combination of point mutations and/or a point mutation and subsequent slippage to occur without spreading to SWIP. Such population structure is incompatible with strong gene flow across the Iberian Peninsula. Thus, to our knowledge, we provide the first example of empirically stationary allele distributions across a set of microsatellite loci applied to intraspecific populations in a known phylogeographic context.
Despite stationary allele distributions, it is illuminating to evaluate the effectiveness of different genetic distance measures in assessing the genetic relation among populations. Goldstein's µ2 and RST failed to reveal the three main geographic regions as they depend heavily on detecting differences in allele size variance, a parameter which can become wholly obscured by homoplasy. However, both Nei's distance and DAS are not affected by allele size variances, being more weighted toward demonstrating differences in allele frequencies and the presence or absence of alleles. These measures clearly identified the three main geographic areas, and additionally were concordant with subregion division within France. DSW, which incorporates allelic variance to some extent, also supported differentiation of NEIP, SWIP, and FR, but was discordant with Nei's distance and DAS-based trees in depicting the pattern of differentiation within FR regions.
Colonization of France and Expansion to the North
Our microsatellite data on FR populations clearly reflects depleted levels of genetic diversity when compared with Iberian populations (table 1
). The disjunct allelic size distributions in contrast with those displayed by SWIP and NEIP are especially informative in supporting a founder effect resulting from colonization from Iberia. Furthermore, as allelic diversity in FR populations has not been restored, the lack of new mutations supports a very recent (i.e., postglacial) founding event. The derivation of French populations from NEIP is strongly suggested by several microsatellite loci, and especially by the occurrence of alleles 195 at sat4 (totally absent in SWIP), 247 at sat2, 140 at sat8 and 184 at sat7. Within France, the overall results show significant differences in allele frequencies (data not shown) allowing the definition of three population assemblages: southwest, southeast, and the north of France (fig. 3a
). This pattern suggests a two-step colonization of France by the rabbit. In the first step, rabbits may have expanded following two main geographical routes, colonizing the southwest and the southeast (geographically separated by the mountains of Massif Central) from the Mediterranean region immediately adjacent to the eastern Pyrenees, after a single colonization event. In a second step, the recent colonization of the north of France may have resulted from an expansion of the southwestern group, as indicated by the phylogenetic reconstruction depicted in figure 3a
and illustrated in the map of figure 4 .
|
For example, Allendorf and Seeb (2000)
compared gene flow estimates among microsatellites, mtDNA, allozymes, and RAPD markers and concluded that there was little difference in FST-type estimates provided that they are corrected for differing numbers of alleles and heterozygosity. This study was conducted on a set of geographically proximate populations with a shallow evolutionary history, a situation thought to be most appropriate for the application of microsatellites (Takezaki and Nei 1996
; Angers and Bernatchez 1998
). However, several studies have successfully applied microsatellite markers in a deeper phylogeographic context as well as across species boundaries. Estoup et al. (1995)
found basic concordance between microsatellites and mtDNA for honeybee subspecies, but suggested that allele size homoplasy resulted in underestimation of divergence among major lineages. Similarly, Harr et al. (1998)
reported an unambiguous phenetic relation of four closely related species of Drosophila based on 39 microsatellite loci, but these same data provided divergence estimates an order of magnitude or more lower than those based on DNA sequence data. Thus, despite claims that some microsatellite distance measures can be linear with time (Takezaki and Nei 1996
), it is clear that empirical studies often reveal the contrary. Most recently, Balloux et al. (2000)
reported severe underestimation of divergence between two chromosomal races of a common shrew based on microsatellites. However, this example is somewhat unique in that there were sex-biased viability differences between the races, for which the evolutionary implications are not yet entirely clear. Thus, while there is ample evidence of homoplasy affecting divergence estimates, and most recently several mechanistic explanations of the dynamics of homoplasy (Harr and Schlötterer 2000
; Xu et al. 2000)
, there is no empirical study yet that has revealed the stationary distributions predicted by Nauta and Weissing (1996)
that will result from even moderate population sizes and some level of divergence. Our data provide a clear example of these predictions, where the pattern of homoplasy is most heuristically explained by considering the mutational spectrum as being filled between two boundaries, one representing the minimal repeat size below which no more slippage occurs, and the other representing a constraint on the maximum size of an allele. The evolutionary history of rabbits in Iberia has been sufficiently long, and population sizes sufficiently large to produce such a phenomenon.
Despite extensive homoplasy, we were nonetheless able to distinguish between major geographic regions, because of fluctuating population sizes in one geographic unit (NEIP) which promoted sufficient drift of some intermediately sized alleles. While our pattern of private allele distributions between areas of refuge (SWIP and NEIP) and expansion (FR) can be seen as being analogous to those obtained for human populations (Perez-Lezaun et al. 1997
) it may be dangerous to draw conclusions concerning private alleles when the nature of their evolution in terms of drift, mutation, and shifts in allele size distributions over time is not known.
The clearest example of microsatellites effectively differentiating populations in a phylogeographic context involves a shallow history and constant and relatively small population sizes with Ne's on the order of hundreds of individuals (Goldstein and Schlötterer 1999
). In our study, microsatellites were also most effective in the periphery of the rabbit's native range, where a hypothesized expansion and colonization scenario through the Pyrenees into southern France was well supported. However, even at the intraspecific level, an increasing number of studies are revealing complex evolutionary histories and population dynamics across broad temporal scales (Avise 2000)
. In such contexts, the sole use of microsatellites may mislead as often as inform on patterns of genetic differentiation and gene flow among populations. In our study, as in that of Calafell et al. (1998)
, there were large differences in the ability of various genetic distance measures to distinguish known phylogeographic pattern. We suggest that without some a priori understanding of historical complexity, in terms of fluctuating population sizes and divergence among populations, the application of various microsatellite-based distance measures may become arbitrary, especially in supporting explicit interpretations as to why populations do or do not appear differentiated.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Present address: Population Génétique et Evolution (PGE), CNRS, Gif sur Yvette cedex, France.
Keywords: European rabbit
phylogeography
microsatellite
homoplasy
allele size constraints
Address for correspondence and reprints: Guillaume Queney, Centre de Génétique Moléculaire, CNRS, 91198 Gif sur Yvette cedex, France. queney{at}cgm.cnrs-gif.fr
.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allendorf F., L. Seeb, 2000 Concordance of genetic divergence among Sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers Evolution 54:640-651[ISI][Medline]
Angers B., L. Bernatchez, 1998 Combined use of SMM and non-SMM methods to infer fine structure and evolutionary history of closely related brook charr populations from microsatellites Mol. Biol. Evol 15:143-159
Archibald A. L., 1994 Mapping the pig genome Curr. Opin. Genet. Dev 4:395-400[Medline]
Avise J., 2000 Phylogeography The history and formation of species. Harvard University Press, Cambridge, Mass
Balloux F., H. Brunner, N. Lugon-Moulin, J. Hausser, J. Goudet, 2000 Microsatellites can be misleading: an empirical and simulation study Evol. Int. J. Org. Evol 54:1414-1422
Belkhir K., P. Borsa, J. Goudet, L. Chikhi, F. Bonhomme, 1996 Genetix, logiciel sous Windows pour la génétique des populations Laboratoire Génome et Populations, Montpellier, France
Biju-Duval C., H. Ennafaa, N. Dennebouy, M. Monnerot, F. Mignotte, R. Soriguer, A. E. Gaaïed, A. E. Hili, J. Mounolou, 1991 Mitochondrial DNA evolution in lagomorphs: origin of systematic heteroplasmy and organization of diversity in european rabbits J. Mol. Evol 33:92-102[ISI]
Bowcock A. M., A. Ruiz-Linares, J. Tomfohrde, E. Minch, J. R. Kidd, L. L. Cavalli-Sforza, 1994 High resolution of human evolutionary trees with polymorphic microsatellites Nature 368:455-457[ISI][Medline]
Branco M., N. Ferrand, M. Monnerot, 2000 Phylogeography of the European rabbit (Oryctolagus cuniculus) on the Iberian Peninsula inferred from RFLP analysis of the cytochrome b gene Heredity 85:307-317[ISI][Medline]
Calafell F., A. Shuster, W. C. Speed, J. R. Kidd, K. K. Kidd, 1998 Short tandem repeat polymorphism evolution in humans Eur. J. Hum. Genet 6:38-49[Medline]
Callou C., 1995 Modifications de l'aire de répartition du lapin (Oryctolagus cuniculus) en France et en Espagne, du Pléistocène à l'époque actuelle Etat de la question. Anthropozoologica 21:95-114
Dobson M., 1998 Mammal distributions in the western mediterranean: the role of human intervention Mammal Rev 28:77-88[ISI]
Donard E., 1982 Recherches sur les Léporinés quaternaires (Pléistocène moyen et supérieur, Holocène) PhD thesis, Université Bordeaux I
Estoup A., L. Garnery, M. Solignac, J.-M. Cornuet, 1995 Microsatellite variation in honey bee (Apis mellifera L.) populations: hierarchical genetic structure and test of the infinite allele and stepwise mutation models Genetics 140:679-695
Felsenstein J., 1993 PHYLIP (phylogeny inference package) Version 3.5. Distributed by the author, Department of Genetics, University of Washington, Seattle
Ferrand N., 1995 Variação genética de proteinas em populações de coelho (Oryctolagus cuniculus) PhD thesis, Universidade do Porto
Flux J., P. Fullagar, 1983 World distribution of the rabbit (Oryctolagus cuniculus) Acta Zool. Fennica 174:75-77
Garza J., M. Slatkin, N. Freimer, 1995 Microsatellite allele frequencies in humans and chimpanzees with implications for constraints on allele size Mol. Biol. Evol 12::594-603[Abstract]
Goldstein D., A. Linares, L. Cavalli-Sforza, M. Feldman, 1995 An evaluation of genetic distances for use with microsatellite loci Genetics 139:463-471
Goldstein D., C. Schlötterer, 1999 Microsatellites Evolution and applications. Oxford University Press, Oxford
Goudet J., 1995 F-STAT version 1.2: a computer program to calculate F-statistic J. Hered 86:485-486[ISI]
Hardy C., C. Callou, J. Vigne, D. Casane, N. Dennebouy, J. Mounolou, M. Monnerot, 1995 Rabbit mitochondrial DNA diversity from prehistoric to modern times J. Mol. Evol 40:227-237[ISI][Medline]
Harr B., C. Schlötterer, 2000 Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation Genetics 155:1213-1220
Harr B., S. Weiss, J. David, G. Brem, C. Schlötterer, 1998 A microsatellite-based multilocus phylogeny of the Drosophila melanogaster genome Curr. Biol 8:1183-1186[ISI][Medline]
Kimura M., J. Crow, 1964 The number of alleles that can be maintained in a finite population Genetics 49:725-738
Lopez-Martinez N., 1989 Revision sistematica y biostratigrafica de los lagomorphos (Mammalia) del terciaro u cuaternario de Espana. Memorias del Muséo Paleontológico de la Universidad de Zaragoza. Diputación general de Aragón
Minch E., 1996 Microsatellite distance program, http://lotka.stanford.edu/microsat/
Monnerot M., J.-D. Vigne, C. Biju-Duval, D. Casane, C. Callou, C. Hardy, F. Mougel, R. C. Soriguer, N. Dennebouy, J.-C. Mounolou, 1994 Rabbit and man: genetic and historic approach Genet. Select. Evol 26: (Suppl. 1) 167s-182s
Mougel F., J. Mounolou, M. Monnerot, 1997 Nine polymorphic microsatellite loci in the rabbit, Oryctolagus cuniculus Anim. Genet 28:58-71[ISI][Medline]
Nauta M., F. Weissing, 1996 Constraints on allele size at microsatellite loci: implications for genetic differentiation Genetics 143:1,021-1,032
Nei M., 1987 Molecular evolutionary genetics Columbia University Press, New York
Ohta T., M. Kimura, 1973 A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population Genet. Res., Cambridge 22:201-204
Pages M.-V., 1980 Essai de reconstitution de l'histoire du lapin de garenne en Europe Bull. Mens. Off. Natl. Chasse, Sp. Scien. Techn., Décembre 1980:1321
Perez-Lezaun A., F. Calafell, E. Mateu, D. Comas, R. Ruiz-Pacheco, J. Bertranpetit, 1997 Microsatellite variation and the differentiation of modern humans Hum. Genet 99:1-7[ISI][Medline]
Queney G., 2000 Histoire des populations et organisation sociale du lapin européen (Oryctolagus cuniculus) à travers l'étude de marqueurs microsatellites Denis Diderot University, Paris
Raymond M., F. Rousset, 1995 GENEPOP (version 1.2): a population genetics software for exact tests and ecumenicism J. Hered 86:248-249[ISI]
Rice W., 1989 Analyzing tables of statistical tests Evolution 43:223-225[ISI]
Shriver M., L. Jin, E. Boerwinkle, R. Deka, R. Ferrel, R. Chakraborty, 1995 A novel measure of genetic distance for highly polymorphic tandem repeat loci Mol. Biol. Evol 12:914-920[Abstract]
Slatkin M., 1985 Rare alleles as indicators of gene flow Evolution 39:53-65[ISI]
. 1995 A measure of population subdivision based on microsatellite allele frequencies Genetics 139:457-462
Takezaki N., M. Nei, 1996 Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA Genetics 144:389-399
Threadgill D., J. Womack, 1990 Genomic analysis of the major bovine milk protein NAR 18:6,935-6,942[Abstract]
van der Loo W., N. Ferrand, R. Soriguer, 1991 Estimation of gene diversity at the b locus of the constant region of the immunoglobulin light chain in natural populations of european rabbit (Oryctolagus cuniculus) in Portugal, Andalusia and on the Azorean Islands Genetics 127:789-799
van der Loo W., F. Mougel, C. Bouton, M. Sanchez, M. Monnerot, 1999 The allotypic patchwork pattern of the rabbit IGKC1 allele b5wf: genic exchange or common ancestry? Immunogenetics 49:7-14[ISI][Medline]
Vigne J.-D., 1988 Données préliminaires sur l'histoire du peuplement mammalien de l'îlot de Zembra (Tunisie) Mammalia 52:567-574[ISI]
Xu X., M. Peng, Z. Fang, X. Xu, 2000 The direction of microsatellite mutations is dependant upon allele length Nat. Genet 24:396-399[ISI][Medline]