Molecular Ecology (2004)

13

, 1231–1240 doi: 10.1111/j.1365-294X.2004.02144.x

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

Temporal and spatial genetic structure in

Vitellaria paradoxa

(shea tree) in an agroforestry system in southern Mali

BOKARY ALLAYE KELLY,

*

OLIVIER J . HARDY

†

and JEAN-MARC BOUVET

‡

*

Institut d’Economie Rurale, Programme Ressources Forestières Centre Régional de la Recherche Agronomique de Sikasso, Sikasso, Mali,

†

Laboratoire de Génétique Evolutive et d’Ecologie vegétale, Université Libre de Bruxelles, Bruxelles, Belgium,

‡

Cirad–Forêt, Campus International de Baillarguet, TA 10C, BP 5035, 34398 Montpellier, cédex, France

Abstract

Ten microsatellite loci were used to investigate the impact of human activity on the spatialand temporal genetic structure of

Vitellaria paradoxa

(Sapotaceae), a parkland tree speciesin agroforestry systems in southern Mali. Two stands (forest and fallow) and three cohorts(adults, juveniles and natural regeneration) in each stand were studied to: (i) compare theirlevels of genetic diversity (gene diversity,

H

E

; allelic richness,

R

s

; and inbreeding,

F

IS

); (ii)assess their genetic differentiation (

F

ST

); and (iii) compare their levels of spatial geneticstructuring. Gene diversity parameters did not vary substantially among stands or cohorts,and tests for bottleneck events were nonsignificant. The inbreeding coefficients were notsignificantly different from zero in most cases (

F

IS

= −−−−

0.025 in forest and 0.045 in fallow),suggesting that the species is probably outbreeding. There was a weak decrease in

F

IS

withage, suggesting inbreeding depression. Differentiation of stands within each cohort was weak(

F

ST

= 0.026, 0.0005, 0.010 for adults, juveniles and regeneration, respectively), suggestingextensive gene flow. Cohorts within each stand were little differentiated (

F

ST

= −−−−

0.001 and0.001 in forest and fallow, respectively). The spatial genetic structure was more pronouncedin fallow than in forest where adults showed no spatial structuring. In conclusion, despitethe huge influence of human activity on the life cycle of

Vitellaria paradoxa

growing inparkland systems, the impact on the pattern of genetic variation at microsatellite lociappears rather limited, possibly due to the buffering effect of extensive gene flow betweenunmanaged and managed populations.

Keywords

: forest tree, gene flow, human impact, inbreeding coefficient, kinship coefficient,microsatellite

Received 15 September 2003; revision received 12 January 2004; accepted 12 January 2004

Introduction

Temporal and spatial genetic structure are influencedby biological aspects of a tree species. Many studies haverevealed that factors such as limited seed and pollendispersal, density, fragmentation, colonization history,isolation into small patches, differential mortality andmicro-environmental selection have an impact on thespatial genetic structure of many forest tree species (Levin& Kerster 1974; Epperson 1993; Austerlitz

et al

. 2000; Petit2001). Of these factors, the pattern of gene dispersal is most

significant (Schaal 1980; Hamrick

et al

. 1993; Ennos 1994).For instance, genetic similarity among progeny spatiallyclustered near their maternal parent because of limitedseed dispersal generates a substantial spatially localizedgenetic structure (Perry & Knowles 1991; Schnabel

et al

.1991; Berg & Hamrick 1994; Geburek & Tripp-Knowles1994; Chung & Epperson 2000a,b). In contrast, if seeds arewidely and independently dispersed, only a weak spatialgenetic structure will result (Dewey & Heywood 1988;Loiselle

et al

. 1995; Chung

et al

. 1999, 2000).Another factor influencing temporal and spatial genetic

structure in tree species may be human activity becausethis leads to many changes in forest community and eco-system processes. Several studies have assessed the impact

Correspondence: J.M. Bouvet. Fax: 33 4 67 59 37 33; E-mail:jean-marc.bouvet@cirad.fr

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Molecular Ecology

, 13, 1231–1240

of human practices on the genetic diversity and geneticstructure of forest tree species (Knowles

et al

. 1992; Young& Merriam 1994; Aldrich

et al

. 1998), but very few havefocused on tree species growing in parkland systems insub-Saharan Africa (Hossaert-Mckey

et al

. 1996; Leonardi

et al

. 1996; Epperson & Alvarez-Buylla 1997; Kitamura

et al

.1997a,b). Measuring and understanding the effects of humanpractices on the genetic structure of tree populations inparkland systems is essential for management strategies inagroforestry ecosystems.

Parkland, defined as a regular, systematic and orderedpresence of trees within fields (Sautter 1968, cited byBagnoud

et al

. 1995), is the result of a long evolutionary pro-cess during which an association between natural elements(trees and shrubs conserved, maintained and enhancedbecause of their utility) and crops happens within a regu-larly exploited space. Parkland includes fields in whichdifferent types of crops (cereals, cotton, peanuts, sorghum)are produced over several years, and fallow, i.e. when cul-tivation activity is stopped on parcels of land in order torestore soil fertility. The length of the fallow period (3–4 to25–30 years) is unique to each farmer, depending upon theland he possesses, the needs of his household and the wayhe manages his land. On these two elements (field and fal-low), there are one or two dominant tree species, as is thecase for

Vitellaria paradoxa

parkland. The latter are the mostrepresented in the soudanian zone, occupying 4.7

×

10

6

hain Mali (Boffa 1998), and are classified as ‘oil parkland’.They are characterized by the dominance of

V. paradoxa

(Sapotaceae) and reflect the presence of stable humanpopulations.

V. paradoxa

is conserved and maintained in thefield by farmers because of its economical importance. Itsfruit pulp is consumed by humans and animals, and butteris extracted from the seed kernel for local consumption (oilfor cooking and ointment in traditional medicine) and fortrade on local and international markets.

The species faces a high degree of thinning, selectionand natural mortality, leading to a noticeable reduction indensity. Because the field/fallow cycle may contrast withnatural forest, especially for the long-term evolutionarydynamics of the tree species, the impact of human practiceson the genetic diversity and genetic structure of

V. paradoxa

raises many questions

.

Genetic markers and spatial statis-tics are efficient tools to assess the impact of evolutionaryforces on plant genetic structure (Loiselle

et al

. 1995; Doligez& Joly 1997; Chung

et al

. 2000; Isagi

et al

. 2000). It is,therefore, appealing to apply these methods to study theimpact of human practices on the genetic structure of

V. paradoxa

.Our main objectives were to characterize the level and

organization of genetic diversity within

V. paradoxa

popu-lations, and to assess the impact of human practices on thespatial and temporal genetic structure. We attempted toanswer the following questions:

1

What is the impact of farming practices on the level ofgenetic diversity?

2

Does human activity lead to genetic differentiationamong cohorts?

3

How has human activity influenced the spatial patternof genetic diversity?

To this end, we analysed the pattern of variation in micro-satellite markers in three cohorts, comparing a populationlocated in forest, which should be representative of naturalconditions, with a population subject to the field/fallowcycle in the parkland system.

Materials and methods

Model species

The main parkland tree species in soudanian zones of sub-Saharan Africa,

Vitellaria paradoxa

reaches 20 m in heightand 1 m in diameter at chest height. Recognized as a long-living savannah tree species (> 200 years), its natural rangeextends from the eastern part of Senegal to the high plateauof Uganda forming an almost unbroken belt 5000 km longand 500 km wide. Its population density varies greatlyaccording to land use, locality and ecological conditions. Itmostly reproduces sexually and is mainly insect pollinated,flowering and fruiting from December to May with somegeographical variations. It fruits more in fields and youngfallow than in forest because of reduced competition,ploughing and crop fertilization, and shows intense regenera-tion in fallow. Harvesting of fruit occurs mainly betweenJuly and September The species is barochore but its seedscan also be dispersed by animals such as birds, monkeysand rodents, and also by humans.

Site and experimental design

The study was carried out in Sikasso, southern Mali, onterritory belonging to Mperesso village. This village islocated in the north of the region (12

°

16

′

N, 5

°

19

′

W) wherethe climate is south soudanian. During the decade 1982–92 mean annual rainfall was 680 mm with a minimum of435 mm and a maximum of 950 mm.

Two parcels of land, 2 and 7 ha, separated by 1.2 km,were selected on the basis of two criteria: land use (fallowand forest stands) and a relatively high density of adulttrees. Each stand is part of two different cycles. In the first,there is a succession of cultivation (field), regeneration andgrowth (fallow), and a reduction in tree density by thin-ning in preparation for re-use as a field. This cycle is shownin Fig. 1(a) by the distribution of tree circumference atchest height (CBH) which exhibits two modes: the first(15 cm) reflecting the distribution of regeneration since thefield was left as fallow, and the second (95–105 cm) that of

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, 13, 1231–1240

adult trees already present during the field stage, the lackof intermediate sizes reflects the low regeneration poten-tial during field stages. In forest, the cycle corresponds tothat for the classical dry forest with weak but constantregeneration and slow growth, as well as selective tree cut-ting for various purposes (mainly for local craft). This cycleresults in fewer individuals in the first size classes com-pared with fallow and the absence of individuals in sizeclasses > 125 cm (Fig. 1b). In each stand, individuals with aCBH > 10 cm were marked, mapped and their CBH meas-ured. Populations from the two stands were divided intothree cohorts according to flowering and fruiting ability,which correlated well with tree size (Kelly, personal commun-ication). Therefore, we recognized: (i) adults (CBH > 30 cm),(ii) juveniles (CBH 10–30 cm); and (iii) natural regeneration(from height > 1 cm to CBH < 10 cm; which include seed-lings and saplings). The samples were exhaustive for adultsand juveniles and the sampled regeneration (not mapped)was collected under marked adult trees. Samples size, meanCBH, density and the area of each parcel are given in Table 1.

DNA extraction and microsatellite-enriched library development

Two or three leaves were harvested from each tree anddried in silica gel. DNA was extracted from dried leavesfollowing the Matab method developed for

Theobromacacao

(Bousquet

et al

. 1990). Dried leaf samples (150 mg)were flash-frozen in a tube filled with liquid nitrogen andmixed to a homogenous slurry with 5 mL DNA extractionbuffer (100 m

m

Tris–HCl, pH 8.0, 20 m

m

EDTA, 1.4

m

NaCl,1% PEG 6000, 2% mixed alkyl trimethyl amonium bromide,0.5% sodium sulphite). The tube was then incubated at74

°

C for 30 min. Samples were washed with wet chloroform(CIAA, 24:1) to remove cellular debris and protein. Followingcentrifugation at 9000

g

for 15 min, the liquid phase was trans-ferred into 15 mL tubes, and 5 mL of ice-cold isopropanolwas added to precipitate the DNA. The resulting DNApellets were resuspended in 400

µ

L sterile water and thenstored at 4

°

C until required.Microsatellite-enriched libraries were achieved follow-

ing the method developed for SSR markers in tropicalcrops; the two principles of the technique are hybridizationwith biotin-labelled oligorobe followed by the capture ofselected sequences using streptavidin-coated magnetic beads.The procedure is detailed in (Billotte

et al

. 1999). After shear-ing the DNA with an endonuclease

Rsa

I, fragments wereligated to adaptators Rsa21 5

′

-CTCTTGCTTACGCGTGGA-CTA-3

′

and Rsa25 5

′

-TAGTCCACGCGTAAGCAAGAG-CACA-3

′

. Fragments of DNA containing (GA) microsatellitewere selected and PCR-amplification was performedusing primers Rsa21 and purified PCR products werecloned into pGEM-T Easy vector. Cloned fragmentswere then used to transform competent DH5

µ

Escherichiacoli

strain. Plasmids from positive clones containing amicrosatellite sequence were extracted and sequencing wasperformed using the universal T7 and M13 reverse sequen-cing primers and the dideoxydye terminator method.

Primers complementary to flanking regions of therepeats were designed for 96 clones containing micro-satellite sequence using

oligo

v4 software (NationalBioscience, Inc). Primers pairs were generated to produce

Fig. 1 Distribution of the tree circumference at chest height in the(a) fallow and (b) forest stands.

Stand CohortSample size (N)

Mean CBH (cm)

CV of CBH (%)

Area (m2)*

Density (N/ha)

Fallow AD (CBH > 30 cm) 68 78.57 43.46 21 000 32JUV (10 cm ≥ CBH ≥ 30 cm) 63 17.79 28.04 30NR (CBH < 10 cm) 90 — — 3305†

Forest AD (CBH > 30 cm) 78 51.66 42.21 73 500 11JUV (10 cm ≥ CBH ≥ 30 cm) 52 19.12 28.50 7NR (CBH > 30 cm) 90 — — 218†

*Parameter is given for the whole stand and not by cohort.†Density for natural regeneration is estimated including seedlings.AD: adults; JUV: juveniles, NR: natural regeneration.

Table 1 Mean circumference (CBH), approx-imate area and estimates of density perstand and cohort

1234

B . A . K E L L Y , O . H A R D Y and J . - M . B O U V E T

© 2004 Blackwell Publishing Ltd,

Molecular Ecology

, 13, 1231–1240

amplified DNA fragments between 95 and 350 bp inlength. Thirteen polymorphic nuclear loci with sizesranging from 109 to 226 bp were obtained and sampledpopulations were screened using these loci (

B5

,

E4

,

E11

,

E6a

,

E6b

,

B3

,

H4

,

D10

,

G7

,

D6

,

F1

,

F5

and

E5

). This numberof microsatellite loci allows a good assessment of popu-lation genetic structure.

Data analysis

Six loci (

E6b

,

E4

,

D6

,

D10

,

G7

and

F1

) showed suspiciousresults regarding

F

IS

values in a primary analysis.

E6b

,

E4

,

D6

,

D10

and

G7

gave systematically positive and highvalues of

F

IS

which could be due to null alleles effect,whereas

F1

gave systematically negative and very lowvalues which may result from gel misreading. Congruently,when checking individuals that were undetermined at oneor two loci, we found that

E6b

,

G7

and

F1

had the highestrate of missing data (42, 24 and 19%, respectively, com-pared with a mean of 5% for the other loci), suggesting thepresence of null alleles at fairly high frequency (resultingin frequent homozygotes for null alleles). Therefore, thesethree loci were eliminated.

E4

,

D6

and D10 presented anormal rate of missing data, suggesting a frequency ofnull alleles < 10–20%, and numerous alleles. Hence, it wasdecided to remove them when calculating multilocus FIS,but to use them to calculate the diversity parameters, theFST and spatial genetic structure parameter in order to keepa good precision in the estimates and sufficient testingpower. Whereas a low frequency of null alleles has asignificant impact on FIS, it has a weak effect on FST or Fijestimates (see below) (Hardy 2000), so that removing E4,D6 and D10 was not necessary.

Genetic diversity analysis

Standard genetic diversity parameters were determinedfor each cohort (adults, juveniles and regeneration) inforest and fallow: number of alleles per locus (A0), allelicrichness (Rs), which measures the number of alleles inde-pendent of sample size (Petit et al. 1998), and the expectedheterozygosity or gene diversity (HE) using an unbiasedestimator (see Nei 1987).

Rs is based on the rarefaction index and consists ofestimating the expected number of alleles in a subsampleof 2n genes, given that 2N genes have been sampled (N ≥ n).n was fixed as the smallest number of individuals typed fora locus in a sample and Rs was calculated as:

where Ni is the number of alleles of type i among the 2Ngenes.

Comparisons of cohorts within and among standsaccording to these parameters (A0, Rs, HE), were madeusing the software fstat v2.9.3 (Goudet 2001). The sameprogram was used to test Hardy–Weinberg equilibrium,heterozygote deficit and population differentiation. Allthese tests are randomization based, i.e. datasets fitting thenull hypotheses to be tested are generated by randomisingthe appropriate units (alleles or genotypes) and statisticsare calculated on these randomised datasets to assess theirfrequency distributions. To test for heterozygote deficitrelative to Hardy–Weinberg expectations, alleles werepermuted among individuals within samples and FIS wasused as a statistic for comparison. To test for populationdifferentiation, genotypes were permuted among samples.

To check if there is a signature for recent bottleneckevents, we used the software bottleneck (Cornuet &Luikart 1996) which compares gene diversity observed(HE) with that expected from the number of alleles perlocus (A0) when population size remains constant and fora given mutation model. After a bottleneck, one expects A0to decrease more than HE, so that the observed HE shouldbe higher than expected HE on basis of A0. As the micro-satellite mutation model is thought to be intermediatebetween a stepwise mutation model and an infinite allelemutation model, we tested bottleneck events assumingeach of these models. We used the Wilcoxon sign-rank testas suggested by Cornuet & Luikart (1996).

Genetic structure analysis

The genetic structure of populations was assessed by F-statistics computed using a nested anova (Michalakis &Excoffier 1996). The level of significance of F-statistics wasobtained after 10 000 permutations of individuals (FST), orgenes (FIS) using spagedi v1.0 (Hardy & Vekemans 2002).

Spatial genetic structure analysis

The spatial distribution of alleles in adults and juveniles ofeach stand (fallow and forest) was assessed using pairwisekinship coefficient Fij (Ritland 1996) between individuals.

where xlcia is an indicator variable (xlcia = 1 if the allele onthe chromosome c at locus l for individual i is a, otherwisexlcia = 0), pla is the frequency of allele a at locus l in the referencesample (fallow or forest), ml is the number of different

R

N Nn

Nn

i

i

nu

s

,= −

−

=∑ 1

22

22

1

F

x x p

mij

lcia lcja lacjcia

cjcil

ll

( / )

( ),=

−

−

∑∑∑∑∑∑

∑1

1

1

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© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1231–1240

alleles found in the sample at locus l, and Σci stands for thesum over homologous chromosomes of individual i.

The spatial structure was assessed: (i) by averaging Fijvalues over pairs of individuals separated by a given dis-tance class in a way equivalent to a spatial autocorrelationanalysis; (ii) by regressing Fij values on ln(dij), where dij isthe Euclidian distance between i and j. To compare cohortswithin each stand, we distinguished the following types ofpairwise comparisons: adult–adult, juvenile–juvenile andadult–juvenile. The averaged Fij estimators per distanceclass and the regression slopes were tested against the nullhypothesis of random distribution of genotypes by random-ising 1000 times the spatial positions using spagedi v1.0(Hardy & Vekemans 2002).

Results

Genetic diversity of the markers

Loci E4, D10 and D6 were the most polymorphic, displaying5–10 alleles (Table 2). For each locus, one or two alleles hada higher frequency. In most cases, these dominant alleleswere the same for cohorts and stands. The distribution ofrare alleles (frequency < 0.01) was different for the twostands. In the forest the highest number of rare alleles (5)was found in adults and no rare allele was found injuveniles, whereas in fallow the reverse was observed (i.e.three rare alleles in juveniles and no rare allele in adults).

Genetic diversity and inbreeding: comparison between stands

Table 3 shows the averaged value over loci of four parametersof genetic diversity within each stand. Considering theadults, the expected heterozygosity (HE) was higher inforest, whereas the inbreeding coefficient (FIS) was higherin fallow. The difference (Table 4) was significant for HEbut not for FIS. Over the pooled sample of cohorts, the testwas not significant for Rs, HE or FIS (Table 4).

Genetic diversity and inbreeding: comparison among cohorts

The total number of alleles (all loci) was similar for thethree cohorts within each stand. In forest, the highest meannumber of alleles (A0) and mean allelic richness (Rs) wereobserved in the regeneration and the highest mean genediversity (HE) was observed in juveniles. In fallow, thethree parameters were higher for juveniles (Table 3).Randomization-based tests (Table 4) showed that, in forest,Rs and HE were not significantly different between cohorts.In fallow, HE was significantly different between adultsand juveniles and marginally significant differences werefound in the comparisons adult cohort vs. juvenile cohort

according to Rs and juvenile cohort vs. regeneration cohortaccording to HE (P = 0.05).

Values of FIS varied according to cohorts in the twostands and positive or negative values per locus (not shown)were met without any real trend. In forest, the lowest multi-locus FIS was observed for the regeneration (FIS = −0.002)

Table 2 Allele frequencies per locus and cohort in forest andfallow. The different alleles are expressed in base pairs

Forest Fallow

Locus Alleles AD JUV NR AD JUV NR

B5 157 0.270 0.217 0.283 0.286 0.270 0.176161 0.730 0.783 0.717 0.714 0.730 0.824

E4 112 0.016114 0.008 0.012 0.007 0.019116 0.065 0.071 0.033 0.020 0.083 0.077118 0.016 0.020119 0.032 0.202 0.100 0.039 0.065 0.070120 0.169 0.179 0.180 0.167 0.102 0.049121 0.645 0.488 0.600 0.745 0.694 0.775123 0.008 0.036 0.040 0.020 0.028 0.028125 0.008 0.010127 0.032 0.012 0.020 0.009

E11 224 0.676 0.625 0.655 0.680 0.639 0.701228 0.324 0.375 0.331 0.300 0.333 0.278230 0.014 0.020 0.028 0.021

E6a 120 0.008 0.013 0.039 0.027 0.007122 0.142 0.156 0.133 0.275 0.282 0.201124 0.708 0.700 0.713 0.618 0.509 0.634126 0.142 0.144 0.140 0.069 0.182 0.157

B3 151 0.011 0.013 0.037 0.025 0.021155 1.000 0.989 0.987 0.963 0.975 0.979

H4 214 0.346 0.219 0.306 0.147 0.209 0.188216 0.615 0.703 0.625 0.809 0.745 0.715218 0.038 0.078 0.069 0.044 0.036 0.097222 0.009

D10 218 0.032 0.097 0.049 0.053 0.028 0.021220 0.016 0.153 0.069 0.224 0.148 0.336222 0.177 0.167 0.389 0.211 0.361 0.192224 0.371 0.444 0.382 0.395 0.315 0.288226 0.226 0.083 0.069 0.039 0.093 0.082228 0.177 0.056 0.042 0.079 0.056 0.082

D6 114 0.007116 0.026 0.015 0.027 0.015 0.111 0.068117 0.092 0.197 0.062 0.015 0.102 0.096118 0.158 0.167 0.226 0.147 0.157 0.158119 0.592 0.545 0.610 0.721 0.565 0.596120 0.092 0.061 0.048 0.074 0.056 0.082124 0.039 0.015 0.021 0.029 0.009

F5 201 0.539 0.679 0.582 0.676 0.649 0.658205 0.008 0.021 0.029 0.018 0.041209 0.453 0.321 0.397 0.294 0.333 0.301

E5 240 1.000 0.974 0.987 1.000 1.000 1.000242 0.026 0.013

N (78) (52) (90) (68) (63) (90)

N: sample size for adults (AD), Juveniles (JUV) and natural regeneration (NR).

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but the FIS of the three cohorts were not significant. Infallow, the lowest FIS was observed for the adults (FIS = 0.024)but the three cohorts did not exhibit FIS values significantlydifferent from zero. When the whole population was con-sidered, fallow displayed the highest multilocus FIS (FIS =0.045), which was significant at the 10% level (Table 3). Nosignificant difference in FIS was found between cohorts withinthe two stands and between stands within cohorts (Table 4).

Bottleneck effects

Whatever the mutation model assumed for the micro-satellites (infinite allele mutation model or stepwise mutationmodel), gene diversity found in each cohort and each standwas consistent with the number of alleles per locus for a

demographically stable population (Wilcoxon sign-ranktests were all nonsignificant at the 5% level). Hence, thereis no sign of the occurrence of recent bottleneck events.

Temporal genetic structure — F-statistics among cohorts within each stand

Average within-cohort inbreeding level (FIS) was higherin the fallow (FIS = 0.045) than in the forest (FIS = –0.025)(Table 5). Although FST values were estimated with highprecision (SE = 0.003 and 0.004 for forest and fallow stands,respectively), differentiation among cohorts was weakand not significant (Table 5). This result was confirmed inforest when computing pairwise FST between cohorts; onlythe comparison of adults and juveniles was marginallysignificant: FST (P-value) = 0.0114 (0.0521), 0.0042 (0.2177)and 0.0079 (0.0717) for adult–juvenile, adult–regenerationand juvenile–regeneration, respectively. In fallow, FST amongcohorts was not significant, but pairwise FST values, estimatedwith 10 loci, suggest that juveniles and regeneration weredifferentiated: FST (P-value) = 0.0056 (0.2099), 0.0027 (0.4075)and 0.0081 (0.0413) for adult–juvenile, adult–regenerationand juvenile–regeneration, respectively.

Genetic structure — F-statistics between stands within each cohort

All FIS and FST values were positive and gave low values.Only FST for adults (FST = 0.026) was significant (Table 6).Thus, cohorts of the two stands did not display any hetero-zygote deficit and were weakly genetically differentiated.

Spatial genetic structure

The spatial genetic structure within and between cohortswithin the forest and fallow stands are depicted in Fig. 2.Globally, the genetic structure was more pronounced in thefallow stand, where it appeared within as well as between

Table 3 Levels of gene diversity and individual inbreeding within cohorts and stands. The multilocus averages of the following statisticsare reported: number of alleles detected (A0), allelic richness (Rs), expected heterozygosity (HE), and inbreeding coefficient (FIS). Arandomization procedure was used to test if FIS deviated significantly from zero

Forest Fallow

AD JUV NR Total AD JUV NR Total

N 78 52 90 220 68 63 90 221A0 3.8 3.5 4 4.2 3.6 4.2 3.4 4.0Rs 3.48 3.39 3.63 3.51 3.52 3.93 3.29 3.45HE 0.42 0.44 0.42 0.43 0.38 0.44 0.39 0.41FIS −0.018 ns −0.075 ns −0.002 ns −0.026 ns 0.024 ns 0.073 ns 0.036 ns 0.045†

N: sample size for adults (AD), Juveniles (JUV), natural regeneration (NR) and the pooled sample (TOTAL). Levels of significance: †P < 0.10; ns, not significant.

Table 4 Differences in levels of gene diversity and inbreedingamong cohorts and among stands as measured by Rs, HE and FIS.Levels of significance were obtained after 5000 permutationsusing fstat (Goudet 2001)

ComparisonsMean Diff. (Rs)

Mean Diff. (HE)

Mean Diff. (FIS)

Differences among cohorts within standForest AD vs. NR −0.15 ns 0.00 ns 0.007 ns

AD vs. JUV 0.09 ns −0.02 ns 0.073 nsJUV vs. NR −0.24 ns 0.02 ns −0.080 ns

Fallow AD vs. NR 0.23 ns −0.01 ns −0.014 nsAD vs. JUV −0.41† −0.06** 0.050 nsJUV vs. NR 0.64 ns 0.05† 0.038 ns

Differences among stands within cohort or for the pooled cohorts (Total)AD FO vs. FA −0.04 ns 0.04* −0.035 nsJUV FO vs. FA −0.54 ns 0.00 ns −0.151 nsNR FO vs. FA 0.34 ns 0.03 ns −0.042 nsTOTAL FO vs. FA 0.06 ns 0.02 ns −0.070 ns

AD, adults; JUV, juveniles; NR, natural regeneration; FO, forest stand; FA, fallow stand. Levels of significance: †P = 0.05; *P < 0.05; **P < 0.01; ns, not significant.

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cohorts, than in the forest stand, where adults showed nospatial structuring. This is confirmed by the regressionslopes of Fij on ln(distance) which were equal to: (i) in forest,−0.00005 (P = 0.97) within adults, −0.00697 (P = 0.012) withinjuveniles and −0.00653 (P = 0.003) for adult–juvenile pairs;(ii) in fallow, −0.01141 (P = 0.013) within adults, −0.02147(P < 0.001) within juveniles and −0.02034 (P < 0.001) for adult–juvenile pairs.

Curiously, in the fallow stand, Fij values decreased sharplyat distances > 120 m, whatever the pairwise comparisons. Acloser look at the data showed that these values resulted froma subgroup of six adults and eight juveniles located at oneextremity of the stand. When these 14 individuals wereremoved from the sample, the genetic structure appearedvery similar to that in the forest stand (Fig. 2, lower), althoughit remained somewhat more marked: regression slopes of−0.00645 (P = 0.055) within adults, −0.01177 (P = 0.003) withinjuveniles and −0.00721 (P = 0.018) for adult–juvenile pairs.

It is noteworthy that, in both stands, mean Fij at short dis-tance (< 15 m) was higher for adult–juvenile pairs thanwithin adults or juveniles. This makes sense under limitedseed dispersal because a proportion of such adult–juvenilepairs may correspond to mother–offspring pairs.

Discussion

Genetic diversity in Vitellaria paradoxa

To our knowledge, this is the first study using micro-satellites to investigate the genetic diversity and spatial

genetic structure of a savannah woody tree species. In thetwo stands, genetic diversity parameters of Vitellaria paradoxawere low compared with microstaellite marker results forother tropical tree species with the same pollination andseed-dispersal systems. For instance, the mean allelenumber, A0, varied from 3.4 to 4.2 for cohorts in the twostands, while Aldrich et al. (1998) observed for Symphoniaglobulifera, a tropical forest tree species with a 4–5 cmdiameter, single-seeded fruit mainly dispersed by batsin a forest stand, a mean allele number of 11.66, 13 and16 for adults, saplings and seedlings, respectively. Forthe same cohorts, S. globulifera had a mean expectedheterozygosity (HE) of 0.819, 0.823 and 0.775, which weremuch higher than those observed for V. paradoxa in thetwo stands. In tropical forest trees (Hall et al. 1994), as wellas in temperate evergreen woody plants (Soo Oh et al.1996), genetic diversity parameters did not vary greatlyaccording to cohorts, and this was also observed forV. paradoxa.

With regard to the impact of human practices, it is inter-esting to note that genetic diversity parameters in the twostands were not significantly different (Table 4). Hence,human practices do not cause a significant bottleneck,and/or gene flow between forest stands and field/fallow

Table 5 Temporal genetic structure as measured by F-statisticsamong cohorts within each stand. Significance levels are obtainedafter 10 000 permutations. Standard errors (SE) of the multilocusestimates were obtained by jack-knifing over loci

Locus

Forest (3 cohorts: AD, JUV and NR)

Fallow (3 cohorts: AD, JUV and NR)

FIS FST FIS FST

B5 −0.0121 ns −0.0027 ns 0.0107 ns 0.0107 nsE4‡ 0.1221** 0.0114 ns 0.2640*** 0.0049 nsE11 −0.0631 ns −0.0056 ns −0.0259 ns −0.0043 nsE6a −0.0593 ns −0.0076** 0.0146 ns 0.0092 nsB3 −0.0042 ns −0.0019 ns −0.0214 ns −0.0055 nsH4 −0.0069 ns 0.0000 ns 0.0843 ns −0.0005 nsD10‡ 0.1934*** 0.0356** 0.1869*** 0.0197*D6‡ 0.1561** 0.0007 ns 0.1720** 0.0047 nsF5 −0.0153 ns 0.0077 ns 0.1445* −0.0081 nsE5 0.4974* −0.0045 nsAll −0.025 ns −0.001 ns 0.044† 0.001 nsSE 0.014 0.003 0.032 0.004

Levels of significance: †P < 0.10, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.‡Loci suspected to display null alleles that were not used for the calculation of the multilocus FIS. Fig. 2 Mean pairwise kinship coefficients between individuals

according to distance in the forest stand (upper), the fallow stand(middle), and in the fallow stand when a subsample of individualswere removed (lower, see text). Adult–adult (square), juvenile–juvenile (circle) and adult–juvenile (diamond) pairs are distinguished.Filled symbols show values departing significantly (α = 5%) froma random spatial distribution of genotypes.

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stands is sufficient to compensate any human-mediatedbottleneck effect.

Inbreeding level of Vitellaria paradoxa

The multilocus FIS values for each cohort were close to0 and not significant at the 5% level in forest and fallow,suggesting that selfing is weak or absent in this treespecies. In fallow, however, for the total population, FISwas positive and significant at the 10% level. Many authorshave attributed significantly positive FIS values to populationsubstructure and inbreeding (Ueno et al. 2002). Positive FIScould be explained by more selfing in fallow than in forest,but we do not have reliable information on the matingsystem of this tree species in the two stands to confirm this.FIS values close to 0 were similar to those observed for othertree species with the same pollination and seed-dispersalsystems such as S. globulifera (FIS = 0.02; Aldrich et al. 1998)and Cecropia obtusifolia (FIS = −0.081; Alvarez-Buylla et al. 1996),a pioneer tropical tree species with bird- and mammal-distributed seeds and some gravity-dispersed seed.

Although the multilocus FIS values over the seven lociwere not significantly different from zero, they decreasedweakly with age in the two stands (Table 3). This trendis similar to that found in S. globulifera (FIS = 0.20, 0.10 and0.02 for seedlings, saplings and adults, respectively) andC. obtusifolia (FIS = 0.065, −0.005 and −0.081 for the samecohorts). For these species, however, FIS values decreasedmore strongly with age than was the case for V. paradoxa.Hamrick et al. (1993) attributed a decrease in FIS with age asbeing primarily the result of a loss of fine-scale geneticstructure with age. Such a pronounced effect does notoccur in V. paradoxa, unless it happens at an earlier stage,from seed to seedling. In the case of V. paradoxa, FIS valuesclose to zero for juveniles and the regeneration might bea consequence of a recent increase in gene flow fromfields surrounding the two stands, which show a higherflowering and fruiting yield than the forest. Indeed, 100–150 years ago, conditions were very different as there werefew cultivated areas (less pollen and fewer seeds produced),more abundant vegetation (high density per hectare), andfewer dissemination agents, such as humans, resulting inless gene flow and weaker seed dispersal than today.

Genetic differentiation between stands

The multilocus FST values between stands were signific-antly positive, whatever the cohort (Table 6), suggesting thatthe two stands were differentiated, but this differentiationwas very weak (low FST and two of three not significantlydifferent from zero). This suggests that these populationshave the same genetic basis and have shared the sameevolutionary processes, being in the same geographicalspace. Moreover, one cannot exclude that the forest wasalso used as a field a long time ago. This is the case for mostof the stands considered as ‘forest’ in soudanian andsoudano–sahelian zones, and so this term should be usedwith caution. In addition, gene flow via pollen and/orseeds may be sufficient to compensate for differentiationby drift. Note that low FST values are usually noted for treespecies, most of them exhibiting > 90% of the total geneticvariation within a population (Ledig 1986; Cheliak et al.1988).

Temporal genetic structure

Differentiation among cohorts within each stand was weak.Significantly positive FST values among cohorts wouldhave been expected if: (i) young cohorts had many genescoming from a differentiated population; and/or (ii) youngcohorts showed a marked reduction in gene diversityrelative to the parental population. The absence of a cleardifferentiation among cohorts is thus consistent with thelow differentiation observed among stands and the absenceof a reduction in gene diversity in young cohorts.

Spatial genetic structure

In studies of spatial genetic structure, many authors havereported either a weak or even a lack of spatial structure forvarious tropical or temperate tree species, e.g. Quercus spp.(Berg & Hamrick 1994; Streiff et al. 1998), Psychotria officinalis(Loiselle et al. 1995), Rhus spp. (Chung et al. 1999). Theirresults are explained by limited seed dispersal and extensivegene flow. Other authors (Dewey & Heywood 1988;Loiselle et al. 1995; Chung et al. 1999, 2000) reported that forwoody insect-pollinated species with seeds widely andindependently dispersed by birds, only weak spatialgenetic structure will result. For C. obtusifolia, Epperson &Alvarez-Buylla (1997) found a very weak spatial structurein juveniles and adults but strong genetic spatial auto-correlations among seedlings, which they explain as aconsequence of thinning processes.

A lack of spatial structure was found for other tree speciesby Sokal & Oden (1978), Waser (1987), Dewey & Heywood(1988), Doligez & Joly (1997) and Chung et al. (2000). Theseauthors explained their results by extensive gene flow, wideseed dispersal, self-incompatibility and dispersal agents.

Table 6 F-statistics for comparison between stands within eachcohort. Significance levels are obtained after 5000 permutations

Cohort FIS FST

AD 0.001 ns 0.026**JUV 0.014 ns 0.0005 nsNR 0.016 ns 0.010 ns

Level of significance: **P < 0.01; ns, not significant.AD: adults; JUV: juveniles; NR: regeneration.

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In the forest stand of V. paradoxa, no significant spatialstructure was found for adults, but juveniles showed a spa-tial structure and were also more closely related to nearbyadults, demonstrating the impact of limited seed dispersal(Fig. 2). In the fallow stand, a spatial structure was foundfor both adults and juveniles, and juveniles were also moreclosely related to nearby adults (Fig. 2). The extent of struc-turing was more pronounced in the fallow stand than inthe forest stand, although this difference was attenuatedwhen a differentiated subsample of individuals from thefallow was removed. Different factors could be respons-ible for a stronger spatial genetic structure in the fallowstand: the high degree of fruiting (no crown competitionbetween trees, good soil conditions) favouring the importantnatural regeneration of siblings in the neighbourhood ofthe mother trees as seeds of V. paradoxa are mostly dis-persed by gravity, and the restricted seed dissemination bysecondary dispersal agents such as farmers who aban-doned the fallow and devoted their time to the exploitationof fields. By contrast, in forest, the low degree of fruiting oftrees (strong competition) reduced the number of siblingsin the neighbourhood of the mother trees. The higher spe-cies richness of the forest ecosystem and the habit of thedispersal agents may also lead to a reduction in spatialgenetic structure. For example V. paradoxa fruit eaters (birds,bats and even humans working in forest gathering fire-wood) pick fruits from different mother trees and dispersethem in the forest. Important regeneration of V. paradoxafound under other tree species in the forest supports thishypothesis. As seed dissemination of V. paradoxa occursduring the rainy season, another possible factor enhancingseed dispersal is surface water flow mixing seeds severalmetres apart. We observed this phenomenon during field-work, particularly in the forest stand where a smooth slopefavours surface water flow.

The impact of humans on the pattern of genetic variation in Vitellaria paradoxa

The field/fallow cycle was heavily influenced by humanpractices, leading to low species diversity, high tree selection,low density and high fruit production. Nevertheless, theimpact of these practices on the pattern of genetic variationat microsatellite loci in V. paradoxa appears to be limited.Genetic diversity indicators were not reduced in the fallowstand, inbreeding level was weakly affected, the fallowstand was little differentiated from the forest stand andcohorts were also little differentiated. There was, however,somewhat more spatial genetic structuring in the fallowstand, which might reflect a difference in seed dispersalpatterns due to human practices. Frequent gene flow viaseed and/or pollen is likely to be a determining factorbuffering the possible impact of human practices on geneticdifferentiation. It must, however, be kept in mind that

microsatellites are representative only of neutral geneticvariation. Hence, they can be powerful for investigating theimpact of humans on neutral diversity, mating system, driftand gene flow, but not on phenotypic characters that are subjectto selection. Human practices surely changed substantiallythe nature of the selection processes acting on V. paradoxatrees in the field/fallow cycle. More research is needed toexplore this impact using larger samples and modellingon longer cycles. Other markers associated with adaptivephenotypic difference may also be used in future (SNPs).

Acknowledgements

We are grateful to the two anonymous reviewers for their com-ments to improve the manuscript. We are very grateful to CélineCardi for having carried out a huge laboratory work for the develop-ment of microsatellites for the first time regarding the tree species(Vitellaria paradoxa). Many thanks to EU for financial support, toIER (Mali) and Cirad-forêt (Montpellier, France) for all logistic sup-port which allowed us to undertake field and laboratory studies ina convivial atmosphere. Our thanks are also addressed to all thosewho contributed at any time, in any way to this work and particularlyto the farmers of Mperesso who allowed us to carry out this studyon their land and who participated in field activities.

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This study is part of BA Kelly’s PhD thesis on the impact ofagroforestry practices on genetic structure in Vitellaria paradoxa.This study was part of the INCO project founded by EuropeanUnion for the ‘Improved management of agroforestry parklandsystem in Sub-Saharan Africa’. Institut d’Economie Rurale (IER) inMali where the study was undertaken was one of the contractorsof the INCO project in addition to Cirad-forêt where laboratorywork was done and where JM Bouvet is the responsible of geneticresources programme. OJ Hardy is a postdoctoral researcher fromthe Belgian National Fund for Scientific Research (FNRS).