Molecular Ecology (2008) 17, 4027–4038
doi: 10.1111/j.1365-294X.2008.03890.x
When dispersal fails: unexpected genetic separation in Gibraltar macaques (Macaca sylvanus) Blackwell Publishing Ltd
L A R A M O D O L O ,* R O B E RT D . M A RT I N ,† C A R E L P. VA N S C H A I K ,* M A R I A A . VA N N O O R D W I J K * and M I C H A E L K R Ü T Z E N * *Anthropological Institute and Museum, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland, †The Field Museum, 1400 South Lake Shore Drive, Chicago, IL 60605, USA
Abstract Barbary macaques (Macaca sylvanus), now restricted in the wild to a few isolated forested areas of Morocco and Algeria, are present in a free-ranging colony on Gibraltar. For many decades, the Gibraltar colony was exposed to multiple bottlenecks due to highly nonrandom removal of animals, followed by repeated introductions of animals from North Africa. Moreover, because of complete isolation, Gibraltar’s several social groups of macaques provide an ideal system to study the genetic consequences of dispersal in cercopithecines in situ. Predictions of genetic consequences due to male-biased dispersal in cercopithecines will be different for autosomal and maternally inherited genetic markers, such as the control region of the mitochondrial DNA. We used a panel of 14 highly polymorphic microsatellite loci and part of the hypervariable region I of the mitochondrial control region to estimate genetic structure between five social groups in Gibraltar. Surprisingly, for autosomal markers, both classical summary statistics and an individual-based method using a Bayesian framework detected significant genetic structure between social groups in Gibraltar, despite much closer proximity than wild Algerian and Moroccan populations. Mitochondrial data support this finding, as a very substantial portion of the total genetic variation (70.2%) was found between social groups. Using two Bayesian approaches, we likewise identified not only a small number of male first-generation immigrants (albeit less than expected for cercopithecines) but also unexpectedly a few females. We hypothesize that the culling of males that are more likely to disperse might slow down genetic homogenization among neighbouring groups, but may also and more perversely produce selection on certain behavioural traits. This may have important repercussions for conservation, as it could lead to evolutionary changes that are not due to inbreeding or genetic drift. Keywords: assignment test, Macaca sylvanus, microsatellites, migration, sex-biased dispersal Received 22 March 2008; revision received 28 June 2008; accepted 10 July 2008
Introduction Sex-biased dispersal is an almost universal characteristic of mammalian life history, yet its evolutionary causes and consequences are still a subject of considerable debate. Several evolutionary models have been developed to explain sex-biased dispersal. The main models invoke inbreeding avoidance, kin-selection effects including cooperative behaviour, and local mate competition as selective pressures promoting or impeding dispersal (Handley & Perrin 2007). Correspondence: Lara Modolo, Fax: +41 44 634 49 62; E-mail:
[email protected] © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
In most systems, stable equilibria are found between immigration and emigration, with equilibrium values being different between females and males in cases with sex-biased dispersal. Cercopithecine primates provide an ideal model to study the proximate genetic effects that dispersal might have on the genetic composition of a population, because in most species, females form strong matrilineal social bonds and spend their entire lives in their natal group, whereas males usually disperse (Pusey & Packer 1987). In most cercopithecine monkey species, dispersal of males seems to be triggered because of their general inability to reproduce within their natal groups (e.g. Packer & Pusey 1979; Cheney & Seyfarth 1983; van Noordwijk & van Schaik
4028 L . M O D O L O E T A L . 1985; Alberts & Altmann 1995). It has generally been acknowledged that the lack of effective mating opportunities and not male–male aggression induces males to disperse (Henzi & Lucas 1980; Cheney & Seyfarth 1983; Berard 1999; van Noordwijk & van Schaik 2001). In contrast to most mammals, however, cercopithecine males may transfer to several different groups during their lifetime (Henzi & Lucas 1980; Melnick et al. 1984; van Noordwijk & van Schaik 1985; Berard 1999). Routine female transfer, on the other hand, appears to be rare in cercopithecines (Pusey & Packer 1987), and has only been described in a few species, e.g. Hamadryas baboons (Papio hamadryas) (Sigg et al. 1982). Predictions of genetic consequences due to male-biased dispersal in cercopithecines will be different for autosomal and maternally inherited genetic markers, such as the control region of the mitochondrial DNA (mtDNA) (Melnick & Hoelzer 1992). Male dispersal will tend to homogenize the genetic differences among neighbouring social groups. The expected amount of genetic differentiation (FST) over generations at nuclear loci among groups due to genetic drift is a function of effective population size Ne of each group and gene flow m between groups (Wright 1943). Differentiation will be counteracted by migration, and it has been suggested that even very small numbers of migrants are sufficient to prevent the effects of drift (Wang 2004). In cercopithecines, almost all males typically immigrate to neighbouring social groups (Di Fiore 2003), providing substantial levels of gene flow per generation that counteract genetic differentiation at autosomal loci. As expected there-
fore, very limited genetic differentiation was found between social groups in natural populations of long-tailed (Macaca fascicularis) and Barbary macaques (M. sylvanus), although these studies were based on a small number of loci and/or samples (Kawamoto et al. 1982; de Ruiter 1994; de Ruiter & Geffen 1998; von Segesser et al. 1999). Although a special case, the absence of interspecific differentiation across most contact zones of Sulawesi macaques also supports the hypothesis of genetic homogenization at autosomal loci as a result of male dispersal in cercopithecines (Evans et al. 2001, 2003). For mtDNA, the predictions are different because successful female migration is rare and group fission in cercopithecines generally occurs among matrilines (Di Fiore 2003). Based on these fission patterns, mtDNA haplotypes are expected to be spatially segregated. Haplotype separation between social groups will depend on the genetic history before the fission event, but is expected to be strict, even between neighbouring groups, in cases where the matrilines had different haplotypes. This pattern has been observed in Toque macaques (M. sinica) in Sri Lanka (Hoelzer et al. 1994), where the distribution of two identified haplotypes followed matrilineal group splits and was highly nonrandom within the study area. The goal of this study is to examine, using microsatellites and mtDNA, population structure and levels of dispersal within and between social groups in the Gibraltar colony of Barbary macaques (M. sylvanus), and to compare these features with other natural Barbary macaque populations in Algeria and Morocco (Fig. 1). Since its founding, the
Fig. 1 Map showing the natural distribution of Macaca sylvanus (according to Fa 1984) in North Africa. Samples were obtained from the Djurdjura National Park in Algeria, from the Central Middle Atlas in Morocco and from the free-ranging population in Gibraltar. The Gibraltar colony consists of five social groups distributed on the rocky slopes of the peninsula. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
G E N E T I C S E PA R AT I O N I N G I B R A LTA R M A C A Q U E S 4029 Gibraltar population has been subject to intense human interference. For many years, the British army was in charge of provisioning the macaques and controlling population size by trapping and removing animals. The regulation of population size through culling led to multiple bottlenecks, followed by repeated introductions from Morocco and possibly from Algeria (MacRoberts & MacRoberts 1971; Modolo et al. 2005). The population underwent the last significant decline at the time of World War II, when apparently just a few animals were left (Fa 1984). At this stage, the last documented introductions were made, with animals seemingly originating from Morocco. An additional potentially severe impact on this colony was active limitation of overall population size to about 35 animals up to 1980 (Fa & Lind 1996). This management strategy was implemented by removing mainly males and leaving only one adult male in each group. Between 1946 and 1991, the Gibraltar population was composed of two separate groups, at Apes’ Den (O’Leary & Fa 1993; referred to as Queen’s Gate in their study) and at Middle Hill (Fig. 2). Group fissions subsequently occurred between 1991 and 1993, eventually resulting in six separate groups, living in close proximity to each other. One of these, the Rock Gun group, was removed from Gibraltar in 1998 and translocated to an outdoor enclosure in Germany. Sporadic management decisions did not take genetic aspects into account. Because of regular provisioning and the absence of natural predators, the number of animals has grown continuously (O’Leary & Fa 1993), and haphazard removal of animals by culling was — and still is (Schiermeier 2003) — the predominant approach to restriction of population size. This culling was highly nonrandom, as mainly males were targeted (Fa & Lind 1996). For comparative purposes, two other populations that live in a fairly undisturbed natural setting were included in this study. The first population was Djurdjura, a wild Algerian population living in a nature reserve that, although isolated from other Algerian populations, is not directly affected by human interference (Ménard & Vallet 1993b). The second population inhabits the Middle Atlas region of Morocco. Although these wild populations were potentially exposed to past human pressure and possible bottlenecks, they most probably have not experienced the extensive culling and multiple bottlenecks of the Gibraltar colony.
Materials and methods Study areas The distribution of Macaca sylvanus in Southern Europe and North Africa, together with the sampling sites for the present study, is shown in Fig. 1. There were two main field collection areas: Djurdjura in Algeria (n = 52) and Gibraltar © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
Fig. 2 Group fission in the Gibraltar Barbary macaque population according to Fa & Lind (1996). The Rock Gun group was entirely removed in 1998 and translocated to an outdoor enclosure in Germany and is not included in this study. The pie charts indicate the observed mtDNA haplotype frequencies for each social group. For census and genetic sample sizes see text.
(n = 127). We also sampled descendants of animals from the Central Middle Atlas in Morocco (n = 23). In Algeria, sampling was conducted in the Djurdjura Nature Reserve, which is inhabited by one of Algeria's seven remaining wild Barbary macaque populations (von Segesser et al. 1999). Samples were collected from individual groups at five different sites [Cheminot (CH), Hotel (HO), Source (SO), Tala Guilef (TG), and Tala Rana (TR)] with intergroup distances ranging between 3 and 12 km. The second population, Gibraltar, is provisioned and completely isolated with no opportunities for natural migration. In 2002, the population consisted of five social groups, of which between 38.8% and 82.4% of all animals in each group had been sampled [Apes’ Den (AD, census size nC = 36; genetic samples n = 14), Farringdon Barracks (FB, nC = 44; n = 19), Middle Hill (MH, nC = 68; n = 56), Prince Philip’s Arch (PPA, nC = 42; n = 19), and Royal Anglian Way (RAW, nC = 35; n = 19)]. Each group had its own provisioning site, separated by distances much smaller than those found in a natural setting, on average between 0.5 and 1.5 km (Fig. 1). In the Middle Atlas of Morocco, Barbary macaques are widely distributed and are found in large numbers. The largest population sizes are found in the Central Middle Atlas, where the most extensive cedar forests occur. Captive animals were originally taken from different social groups in the region of Ain Kahla, where the population density is the highest anywhere in the Barbary macaques’ range (Taub 1982), and samples were obtained from their descendants held in outdoor enclosures in Germany and France.
4030 L . M O D O L O E T A L .
EDTA blood samples were obtained by opportunistic trapping of animals in baited cages, sedating them with a dart from a blowpipe and conducting venipuncture of the femoral vein. Total DNA was extracted from blood using PCI (25: 24:1 mix of phenol, chloroform and isoamylalcohol) and chloroform (Sambrook et al. 1989). Extraction success was judged visually by staining the DNA with ethidium bromide after electrophoresis in a 1% agarose gel.
(van Oosterhout et al. 2004). The program provides estimates of null allele frequency (Brookfield 1996) and adjusts allele and genotype frequencies accordingly. The occurrence of null alleles was also determined empirically by direct comparison of mother–offspring genotypes: mother and offspring will appear to be homozygous for different alleles if the null allele has been passed on from mother to offspring. This method, however, will underestimate the null allele frequency because the null allele is expected to be passed on in 50% of the cases.
Microsatellite analysis
Population differentiation
Because of the limited range of published primers for microsatellite loci in Barbary macaques, the literature was searched for primers in related species. In an initial screening, 28 microsatellite loci were tested in eight randomly selected samples of Barbary macaques from two distant populations (Gibraltar and Algeria). All of these loci are known to be polymorphic in different Macaca species, vervet monkeys or humans. Of the 28 primer pairs tested, 14 loci successfully amplified and were selected for this study. Eight systems (D3S1279, D4S243, D6S311, D7S503, D8S1106, D11S925, D16S420 and D17S791) had already been shown to be variable in Barbary macaques (von Segesser et al. 1999; Lathuillière et al. 2001; Kümmerli & Martin 2005). D2S144, D6S493, D8S72 and D19S582 (Nürnberg et al. 1998; Nair et al. 2000), as well as D10S611 and D13S894 (Smith et al. 2000), had previously been applied to M. nemestrina and M. mulatta, respectively. The 14 polymorphic microsatellites were amplified in an Applied Biosystems 9700 thermal cycler in final volume of 10 μL using 0.06 M Tris, 0.015 m (NH4)2SO4, 1.5 mm MgCl2, 2% dimethyl sulfoxide (DMSO), 0.02 mm of each dNTP, 0.25 μM of each primer and 0.5 U of Promega Taq polymerase. The fluorescent tag FAM was added to the 5′-end of the forward primer. The general polymerase chain reaction (PCR) amplification conditions involved an initial denaturing at 94 °C for 5 min, followed by the appropriate number of cycles of denaturation at 94 °C for 30 s, annealing at the appropriate temperature for 50 s, and extension at 72 °C for 1 min. Final extension was at 72 °C for 30 min. Negative controls were included in all amplification series. PCR products were separated using capillary electrophoresis on an ABI 3730 Genetic Analyzer. Alleles were sized relative to an internal size standard (LIZ) using GeneMapper version 3.5 (Applied Biosystems). Potential contamination with human DNA was tested by genotyping all laboratory personnel. Furthermore, PCR amplifications were repeated in cases where negative control showed some products. For about 10% of the samples, analyses were repeated to confirm genotypes (Hoffman & Amos 2005). The occurrence of null alleles was tested for each social group as a single unit using micro-checker, version 2.2.3
Genetic variability was also estimated for each population by calculating microsatellite allele frequencies, the number of alleles per locus (A), observed (HO) and expected (HE) heterozygosities, as well as rarefacted allelic richness (AR, El Mousadik & Petit 1996). Deviations from Hardy–Weinberg equilibrium for each locus and population were assessed using the exact probability tests implemented in GenePop version 3.4 (Raymond & Rousset 2001). Allelic linkage disequilibrium for all pairwise locus combinations within each population was tested using the exact probability test in GenePop. Critical levels of significance for multiple testing were adjusted according to the Bonferroni correction (Rice 1989). Genetic differentiation between sampling localities was measured using both summary statistics and individualbased approaches. FST estimates (Weir & Cockerham 1984) were calculated in fstat version 2.9.3.2 (Goudet 1995). The software package Structure version 2.1 (Pritchard et al. 2000) was used to determine population structure between all samples and potential source populations for the Gibraltar colony, and to detect potential genetic clustering and affiliation of individuals within Gibraltar social groups following fission events. Structure divides sampled individuals into a number of clusters (K) independent of locality information, in order to minimize deviations from Hardy–Weinberg and linkage equilibrium. The program uses a Markov chain Monte Carlo (MCMC) procedure to estimate P(X|K), the posterior probability that the data fit the hypothesis of K clusters. The program also calculates the fractional membership of each individual in each cluster (Q). Three different analyses were conducted, for which the admixture model with correlated allele frequencies was chosen. The length of the burnin period was set to 105, followed by 106 MCMC steps. For each K, the analysis was run at least three times. The first analysis was performed with all individuals, while only animals from Gibraltar were considered in the second. In the third step, only Algerian samples were analysed.
Sampling and DNA extraction
Detection of migrants Two Bayesian approaches, implemented in GeneClass 2.0 (Piry et al. 2004) were used to identify migrants and those © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
G E N E T I C S E PA R AT I O N I N G I B R A LTA R M A C A Q U E S 4031 individuals with migrant or mixed ancestry (Rannala & Mountain 1997; Baudouin & Lebrun 2001). GeneClass uses a suite of likelihood-based statistics, in combination with resampling methods, to calculate probabilities that individuals are first-generation migrants. The ratio of Lh (the likelihood of detecting a given individual in the population from which it was sampled) and Lmax (the greatest likelihood among all sampled populations) was used, as it is most informative when all source populations have been sampled (Paetkau et al. 2004). The ‘detect migrants’ function was selected as it is explicitly designed to identify first-generation migrants (Paetkau et al. 2004; Piry et al. 2004), i.e. individuals born in a population other than the one in which they were sampled. Given the fairly recent splits of most of the social groups (Fig. 2), which predated genetic sampling by only 7–9 years, data on recent migration will be more representative of current population processes. In order to reach an appropriate balance between stringency and power, the alpha level was set to 0.01 in order to determine critical values (Paetkau et al. 2004). For a conservative estimate, and in order to avoid type I errors, animals were only considered to be true migrants if they were unambiguously identified by both Structure and GeneClass analyses.
Matrilineal group structure Matrilineal group structure was assessed by sequencing 468 base pairs of the hypervariable region I (HVR-I) of the control region according to Modolo et al. (2005). In most cases, a larger number of individuals was available from each group compared to the microsatellite analysis (AD, nHVR-I = 21; FB, nHVR-I = 22; MH, nHVR-I = 30; PPA, nHVR-I = 17; RAW, nHVR-I = 27). Sequences were aligned by eye using the program Sequencher version 4 (GenCodes), and different haplotypes were identified using collapse version 1.1 (available from http://darwin.uvigo.es/). The degree of population structure within Gibraltar was tested by an analysis of molecular variance (amova; Excoffier et al. 1992), using the program Arlequin, version 3.1 (Excoffier et al. 2005). Genetic variance components were calculated among and within social groups. We also used Arlequin to calculate pairwise FST values for haplotype frequencies. The null distribution of pairwise FST values under the hypothesis of panmixia was obtained by 10 000 permutations.
Results Suitability of genetic markers Considering 10 subpopulations in Algeria and Gibraltar, all of the loci across populations and all of the populations across loci displayed genotypes in Hardy-Weinberg equilibrium after Bonferroni correction for multiple testing (Rice 1989). However, there were departures from Hardy© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
Weinberg expectation in the Moroccan population for two loci (D6S493 and D19S582). Both cases involved homozygote excess compared to expected numbers, suggesting a Wahlund effect, probably a result of pooling genetically differentiated samples. None of the 31 analysed mother–offspring pairs showed any indication of mismatches, indicating no or very limited evidence for the presence of null alleles. The estimates derived from the Brookfield (1996) method suggest null allele frequencies of 0.129 at a single locus in Gibraltar and of 0.176 at another locus in Algeria. However, the effect of null alleles in the present data set can be assumed to be negligible, as both Algerian and the Gibraltar populations were in Hardy-Weinberg equilibrium. Hence, all loci were included in the genetic analyses, as there were also no significant departures from linkage equilibrium after Bonferroni correction for multiple testing between pairs of loci (P < 0.001). For all measures of genetic diversity, the Gibraltar colony showed uniformly low values at all sites compared to Algeria and Morocco (Table 1). A Wilcoxon signed-rank test indicated that HE in Gibraltar was significantly lower than in Algeria (P = 0.0029), with mean values per population of 0.55 and 0.70, respectively, while the difference between Gibraltar and Morocco was not significant. The population with the largest sample size, Gibraltar, showed a significantly lower allelic richness (P = 0.004) compared to Algeria. Most private alleles were detected in Algeria (29), followed by Morocco (5). For Gibraltar, only four unique alleles were found, despite this being by far the largest sample size.
Population differentiation FST values were used to provide quantitative information on population differentiation among social groups from Algeria and Gibraltar. In Algeria, the level of genetic differentiation was significant in all possible comparisons except for two sites (CH and TR, Table 2). Ln P(X|K) suggested a subdivision of K = 2 (data not shown), with SO and TR groups being different from all others. Among all populations, examination of Ln P(X|K) values in the structure analysis suggested a subdivision of K = 4 (Fig. 3), clearly placing Algeria and Morocco into two different groups (Fig. 4). Surprisingly, strong grouping patterns were also found among the five Gibraltar social groups (Fig. 4), despite the close proximity in which the groups occur. For K = 5, the overall pattern was the same, but some of the Algerian samples started to show mixed origin (Fig. 4). In cases where the Structure program finds clustering solutions with similar probabilities at different values of K, the lowest value is typically the most accurate (Pritchard et al. 2000; Lindholm et al. 2005). Within Gibraltar, significant population structure between social groups was evident irrespective of marker system or analytical method used. These findings suggest that group
4032 L . M O D O L O E T A L . Table 1 Gene diversities in Barbary macaque populations Algeria (n = 52)
Morocco (n = 23)
Gibraltar (n = 127)
Locus
A
AR
HO
HE
A
AR
HO
HE
A
AR
HO
HE
D6S311 D6S493 D10S611 D8S1106 D11S925 D7S503 D13S894 D16S420 D4S243 D3S1279 D19S582 D18S72 D2S144 D17S791 Mean SD
8 5 4 4 5 5 7 6 8 3 4 4 23 9 6.6 4.8
6.5 4.4 3.9 4.0 4.7 4.7 5.6 5.7 7.1 3.0 3.3 3.9 18.1 8.6 5.9 3.8
0.788 0.577 0.558 0.731 0.654 0.615 0.731 0.692 0.827 0.481 0.404 0.538 0.904 0.820 0.666 0.139
0.821 0.601 0.598 0.636 0.637 0.715 0.700 0.766 0.833 0.592 0.506 0.634 0.937 0.832 0.701 0.116
6 3 5 4 5 4 5 3 4 3 3 2 11 7 4.6 2.2
6.0 3.0 5.0 4.0 5.0 4.0 5.0 3.0 4.0 3.0 3.0 2.0 10.9 7.0 4.6 2.2
0.609 0.348 0.696 0.435 0.727 0.696 0.783 0.591 0.318 0.565 0.391 0.409 0.739 0.773 0.577 0.161
0.700 0.603 0.738 0.540 0.637 0.687 0.765 0.588 0.322 0.643 0.631 0.426 0.799 0.729 0.629 0.126
6 3 6 3 6 5 3 5 8 3 3 3 6 3 4.4 1.4
4.9 2.2 4.8 2.2 4.4 4.6 3.0 4.1 5.9 3.0 2.6 2.2 5.3 2.2 3.7 1.3
0.638 0.465 0.622 0.175 0.520 0.543 0.654 0.740 0.742 0.661 0.433 0.464 0.627 0.442 0.552 0.145
0.693 0.423 0.668 0.174 0.550 0.514 0.625 0.679 0.791 0.630 0.454 0.495 0.649 0.406 0.554 0.151
Number of alleles detected per locus (A), rarefacted allelic richness (AR), observed (HO) and expected (HE) heterozygosities as well as number of individuals scored (n). SD, standard deviation.
Table 2 Pairwise FST estimates based on microsatellite and mtDNA data (above and below the diagonal, respectively). Significant values (P = 0.017 after Bonferroni correction for microsatellites, and after 10 000 permutations for the mtDNA data set) are marked with an asterisk Algeria
CH
CH HO SO TG TR Gibraltar
MH
MH RAW FB AD PPA
0.154* 0.109 0.827* 1.000*
HO
SO
TG
TR
0.110*
0.078* 0.041*
0.072* 0.081* 0.089*
0.008 0.085* 0.056* 0.079*
RAW
FB
AD
PPA
0.121*
0.051* 0.066*
0.165* 0.025* 0.121*
0.071* 0.026* 0.048* 0.068*
0.000 0.537* 0.771*
0.547* 0.833*
0.835*
Cheminot (CH); Hotel (HO); Source (SO); Tala Guilef (TG); Tala Rana (TR); Middle Hill (MH); Royal Anglian Way (RAW); Farringdon Barracks (FB); Apes’ Den (AD); Prince Philip’s Arch (PPA).
Fig. 3 Ln P(D) as a function of K, the number of putative populations. The top panel shows Ln P(D) for all populations in this study, the bottom panel only for the social groups of Gibraltar.
fission occurred along maternal lines, assuming that no sampling bias of certain matrilines was introduced due to the trapping regime. However, dispersal does not seem to have a sufficiently strong enough effect to genetically
homogenize the groups. For mtDNA, three different haplotypes were found in Gibraltar (Fig. 2). At 70.2%, a very substantial portion of the total genetic variation was found between social groups (Table 3). Pairwise mtDNA © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
G E N E T I C S E PA R AT I O N I N G I B R A LTA R M A C A Q U E S 4033
Fig. 4 Estimated population structure in macaques. Each individual is represented by a vertical bar. The individual’s estimated membership fraction Q to the Kth cluster is indicated through the segmentation of each vertical line. The gender symbols above the panel ‘Gibraltar, K = 3’ denote the individuals identified as potential first-generation migrants using GeneClass. Animals were only considered to be true migrants if they were unambiguously identified by both Structure and GeneClass analyses (see text).
FST estimates were high and significant between all groups except FB and MH, as well as FB and RAW (Fig. 2, Table 2). A previously published unrooted network of mtDNA haplotypes (Modolo et al. 2005) indicates that Gibraltar haplotypes originated in Morocco and Algeria. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
Autosomal markers support mtDNA results of genetic separation among Gibraltar groups, as pairwise FST values were significant between all five social groups (Table 2). Examination of Ln P(X|K) values suggested a subdivision of K = 3 (Figs 3 and 4). Two social groups, RAW and AD,
AD FB RAW MH AD PPA FB
PPA
9.354
AD FB RAW MH AD PPA PPA PPA PPA
Adult male Adult male 1996 1998 2000 1992 1999 1998 1994 1995 1986 1999 W16 A1 A8 C10 C15 C9 F16 F6 W4 W6 Groove H57
DOB, date of birth; SP, potential source group (site codes are as in Table 2); –log(L), L_home/L_max (Paetkau et al. 2004). Criteria for computation: (1) (Baudouin & Lebrun 2001); (2) (Paetkau et al. 1995); (3) (Rannala & Mountain 1997). Probability simulation algorithm: (1) (Cornuet et al. 1999); (2) (Rannala & Mountain 1997). *See text for reasons for exclusion and mixed ancestry assignment. NS, not significant.
MH Excluded* RAW AD AD mixed ancestry?* FB Unclear Excluded* Excluded* PPA PPA FB? 8.511 7.791 8.123 NS 8.874 5.344 7.480 7.213 11.162 9.030 NS NS MH RAW RAW
8.511 7.791 8.123 NS 8.874 5.344 7.480 7.213 11.162 9.030 NS NS MH RAW RAW
8.561 8.165 8.522 10.617 9.220 5.595 7.740 7.374 NS 8.690 MH RAW RAW AD AD FB RAW MH
SP Sex
Group
SP
–log(L)
SP
–log(L)
SP
–log(L)
SP
–log(L)
SP
–log(L)
(3),(2) (3),(1) (2),(2) (2),(1) (1),(2)
Criteria for computation, probability simulation algorithm
(1),(1)
Year sampled
Significant population differentiation was found between Algerian social groups (range 0.008–0.110, mean 0.067) as well as between groups in Gibraltar (range 0.025–0.165, mean 0.064). However, the area covered by Gibraltar macaques is very small compared to that of natural populations (Fig. 1). Previous studies have pointed out that interpretation of FST estimates per se might be of little biological relevance (Balloux & Lugon-Moulin 2002). What is widely accepted, however, is the suggestion that a value
DOB
Persistent differentiation among social groups within populations
Animal
Discussion
Table 4 Potential first-generation migrants in Gibraltar macaques, identified at the α < 0.01 level
which originated from a split in 1991, seem to share common ancestry, as their overall Structure profile is similar (Fig. 2), and their differentiation at 14 microsatellite loci is the smallest found in the data set (Table 2). However, the mtDNA profiles of the two groups differ significantly (Fig. 2, Table 2), suggesting that the splits occurred largely along mtDNA lineages. The MH and PPA groups, which split off from the AD group in 1946 and 1993, respectively, appear to be of different genetic origin from each other and the AD group (Fig. 2). In Gibraltar, the proportion as well as the sex ratio of potential first-generation immigrants was different between groups (Fig. 4, panel ‘Gibraltar, K = 3’). Interestingly however, both male and female migrants were found. With a few exceptions, there is generally good concordance between the Structure and GeneClass analyses (Fig. 4, Table 4). While most of the animals could be genuine immigrants, at least three animals appear to be spurious (female in RAW, first female in FB, first male in AD, Fig. 4, panel ‘Gibraltar, K = 3’), as the Structure analysis does not indicate immigration status, but GeneClass does. For one additional female (F16), the GeneClass analysis was ambiguous, as depending on the method, two potential source groups were found (Table 4). Furthermore, animal C15 was only 1-year-old when sampled, suggesting that it might be an animal of mixed ancestry. Hence, for a conservative estimate and in order to reduce type I errors, the total number of potential migrants in Gibraltar was regarded to be five males and two females.
8.472 7.665 8.056 10.969 8.847 5.225 7.320 7.160 NS 8.683 8.464
70.2*** 29.8
MH RAW RAW AD AD FB PPA MH
0.2216 0.0941
8.561 8.165 8.522 10.617 9.220 5.595 7.740 7.374 NS NS NS NS
4 88
MH RAW RAW AD AD FB RAW MH
Among groups Within groups
8.561 8.165 8.522 10.617 9.220 5.595 7.740 9.071 NS 8.690 NS NS
Percentage of variation
MH RAW RAW AD AD FB RAW MH
Variance components
RAW AD AD PPA PPA PPA FB FB RAW RAW MH MH
d.f.
–log(L)
Source of variation
M M M F F F F F F M M M
Table 3 Sources of genetic mtDNA variation among all five Gibraltar social groups
2001 2000 2001 2001 2001 2001 2000 2000 2000 2000 1998 2002
Immigrated from group
4034 L . M O D O L O E T A L .
© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
G E N E T I C S E PA R AT I O N I N G I B R A LTA R M A C A Q U E S 4035 between 0.15 and 0.25 indicates marked differentiation (Hartl & Clark 1997), which was found between the two primary splitting troops on Gibraltar: Middle Hill and Apes’ Den (FST = 0.165). Population structure is expected to increase due to genetic drift according to the amount of time that two populations are separated. Indeed, the most genetically differentiated groups — MH and AD — were the first to separate around 50 years ago. Remarkably, the Structure analysis also revealed that, despite very close proximity, genetic differentiation between Gibraltar groups is either maintained or established after fission events. This is an interesting conundrum, as macaques show male dispersal as well as extra-group paternity (Di Fiore 2003), which should contribute to sufficient genetic exchange to prevent genetic drift at autosomal loci. One would expect an increased level of gene flow between social groups in Gibraltar because of the small geographical distance between them. Surprisingly, tests for genetic differentiation in the Gibraltar population indicate that groups are genetically differentiated at similarly high levels compared to the widely separated Algerian social groups. Mean FST values for Gibraltar macaques were greater than those observed among wild neighbouring groups in M. fuscata (mean 0.008: Nozawa et al. 1982), M. fascicularis (mean 0.022: Kawamoto et al. 1984) and M. nigra (nearly zero: Kawamoto 1996), although different marker systems were used in these studies. However, two studies of M. maura showed similarly high levels of FST between neighbouring groups (0.06: Evans et al. 2001; 0.067: Kawamoto 1996), which could indicate similar processes as operate in Gibraltar. The pronounced population structuring is consistent with reduced migration rates of dispersing males. Group sizes in Gibraltar macaques are large, and it has been shown that, in macaque groups with female philopatry, males migrate less as the number of females in a group increases (van Noordwijk & van Schaik 2004). Moreover, in semicaptive conditions, only one-third of males leave their natal group, and secondary transfers are rare (Kuester & Paul 1999). However, even if group size has affected male dispersal, a considerable proportion of males would always be expected to migrate and should permit a certain level of gene flow, as it clearly did in the wild Algerian population sampled. Moreover, for wild Barbary macaques, Mehlman (1986) reported 12 natal dispersals from six groups in a 2-year study, but did not report rates of transfer. Ménard & Vallet (1993b) showed that approximately 50% of natal males leave their groups, with the remainder staying in their natal group at least beyond 9 years of age. In Gibraltar, male dispersal is limited to at most a few individuals each year and is significantly reduced compared to both wild and semicaptive populations (Mehlman 1986; Ménard & Vallet 1993a; Kuester & Paul 1999). The few identified male migrants in this study were potentially old enough to have dispersed by the time when they were sampled (Table 4). © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
Our genetic data support the behavioural observations that male transfer within Gibraltar is significantly reduced due to targeted culling relative to expectation for cercopithecines, which in turn would increase genetic drift in each group, causing the observed high levels of population structure. On a proximate level, the lack of male immigrants may have consequences for female mating behaviour and migrations. Females of many primate species generally show preferences for novel, i.e. immigrant, males as mates (Manson & Perry 1993; Manson 1994, 1995; Berard 1999), which could provide an indirect genetic benefit by reducing the risk of inbreeding. However, Gibraltar females must reproduce with natal males within their social unit. This may have had two consequences. First, although lower paternity concentration in the top-ranking male is expected for seasonal breeders such as Barbary macaques (Takahata et al. 1994), the effect of dominance rank on paternity should always remain positive (van Noordwijk & van Schaik 2004). Yet, 59% of the offspring in Gibraltar were sired by two low-ranking males, whereas the two top-ranking males sired only one-fifth (Modolo & Martin 2007). This unusual finding might have arisen because females may have attempted to avoid mating with familiar males, as the successful low-ranking males were likely to be immigrants and hence ‘new’ to the females as partners. Moreover, the lack of unfamiliar prime-age males (Modolo & Martin 2007) as mates may have even driven some females to emigrate form their natal group, as some of the migrants identified by both the Structure and GeneClass analyses are female (Fig. 3). This is rather unexpected for cercopithecine monkeys, where females have a strong tendency to remain in their natal groups (Pusey & Packer 1987), and only leave when groups fission. For instance, in female captive rhesus macaques (M. mulatta), the intensity of inbreeding avoidance was positively correlated with the degree of matrilineal male relatedness (Smith et al. 2000). On an ultimate level, a possible explanation for the lack of male migration, and thus the remarkable genetic differentiation among the Gibraltar groups, is provided by continuous culling of parts of the Gibraltar colony since its foundation, as culling is decidedly nonrandom, and mainly targeted at males who have wandered into human-inhabited areas (Fa & Lind 1996). Such culling may therefore represent a human-induced ‘selection for timidity’ in the males of this population. There have been a number of studies linking behavioural traits to functional variation in different genes, which would provide an effective mechanism of natural selection on behavioural variation. For example, in rhesus macaques and other primates, functional allelic variation in the transcriptional control region of the serotonin transporter and monoamine oxidase A gene has been associated with anxiety- and aggression-related behaviour (Wendland et al. 2006). Furthermore, age at natal dispersal is related to VNTR variation in the 5′ upstream regulatory region of the
4036 L . M O D O L O E T A L . serotonin transporter (SLC6A4) gene (Heils et al. 1995; Lesch et al. 1997). When VNTR genotypes were related to age at male natal dispersal in free-ranging and provisioned rhesus macaques, animals homozygous for a certain VNTR variant were found to have left their natal groups significantly earlier than carriers that were homozygous for another allele (Trefilov et al. 2000). Animals that were heterozygous for both variants migrated at an intermediate age. Such a system could be a target of overdominant selection, as migration at an intermediate age appears to have conferred a heterozygote advantage (Trefilov et al. 2000). If the more daring or less timid males are also the most likely to take risks during transfer between groups, they may be removed at a higher rate from the gene pool without having reproduced. As a consequence, artificial directional selection through human culling could have led to a situation in which male migration between social groups is now highly limited, hence counteracting genetic homogenization due to dispersal. This hypothesis predicts significant differences in allele frequencies of the genomic regions involved between the Gibraltar Barbary macaques and their conspecifics living in undisturbed wild populations. To our knowledge, the pattern of persistent genetic structure despite natal dispersal has been described in no cercopithecine species to date. In one other mammal species, the common vole (Microtus arvalis), significant genetic structure was found between six neighbouring populations (Schweizer et al. 2007). Yet, assignment tests revealed a relatively high proportion of first-generation immigrants in each population, suggesting that although immigration was common, their per capita reproductive success was poor.
Conservation implications The finding of a relatively low level of genetic variation in Gibraltar supports the notion that human intervention in regulating population size has had significant impacts on levels of genetic diversity. Artificial selection due to targeted culling should be avoided in Gibraltar in the future, particularly because of the absence of natural migrants from other populations. Gene flow plays a central role in the genetic management of conserved animal populations (Frankham 1995). It can increase the effective population size and thus reduce the rate of inbreeding and loss of genetic variation. Genetic management in Gibraltar should routinely consider the possibility of introducing animals, for example from other outdoor enclosures, to generate at least a minimal level of gene flow. The culling of males more likely to disperse might slow down genetic homogenization among neighbouring groups, but may also and more perversely produce selection on certain behavioural traits. This may have important repercussions for conservation, as it could lead to evolutionary changes that are not due to inbreeding or genetic drift.
Acknowledgements We gratefully acknowledge F. Botte-von Segesser, N. Ménard and W. Scheffrahn for kindly providing samples from Algeria. We thank K. Hodges, R. Kümmerli, J. Kuester, U. Möckli, U. Möhle, C. Roos for assistance in field sample collection or for providing samples from outdoor enclosures, John Cortes and Eric Shaw from the Gibraltar Ornithological and Natural History Society and Mark Pizarro from the Gibraltar Veterinary Clinic for research permission and collaboration. We would also like to thank M. Bruford, J. Pastorini and H. Zischler for valuable assistance in laboratory work and L. Keller and A. Bissonnette for many helpful comments that improved this manuscript. This research was supported by grants from the A. H. Schultz Foundation and the Swiss National Foundation (5001-034878 and 3100-045923).
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This study formed part of the doctoral thesis of Lara Modolo on phylogeography and population genetics of Barbary macaques and issues related to the conservation of the primates. Robert Martin works on various aspects of primate evolution and has special interests in primate conservation. Carel van Schaik has strong interest in the social and cognitive evolution of primates. Maria van Noordwijk is interested in male–female relationships and reproductive strategies in primates. Michael Krützen’s work is concerned with social and gene-culture co-evolution in both cetaceans and primates.
© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd
