Chloroplast DNA phylogeography of the argan tree of Morocco

Introduction

Phylogeography, the study of the ‘principles and process-es governing the geographical distributions of genealogi-cal lineages’ (Avise 1994), developed when data on thenon-recombining mitochondrial DNA (mtDNA) of ani-mals became available since about 1980. In plants, howev-er, chloroplast DNA (cpDNA) and mtDNA sequences aremuch more conserved (Wolfe et al. 1987), a serious limita-tion for intraspecific studies. This is unfortunate, becauseplant organelles, when maternally inherited, are movedonly by seeds and not by pollen, and hence are predictedto be highly structured, as compared with nuclear genes(Petit et al. 1993a). Nevertheless, in some plant species,enough variability could be detected to build intraspecific

phylogenies and to relate these phylogenies to the geo-graphical distribution of the haplotypes (Soltis et al. 1989,1991, 1992; Byrne & Moran 1994; Demesure et al. 1996).Although more difficult to obtain than in animals, suchdata on the phylogeography of plants could help in ‘sepa-rating population structure from population history’(Templeton et al. 1995).

The argan tree Argania spinosa (L.) Skeels is an endan-gered multipurpose species endemic to western Morocco,in the Agadir region (see Nouaïm et al. 1991; for a reviewof the species biology). Its complete range covers onlyabout 800 000 ha (Ayad 1989), allowing a fine-scale analy-sis of its whole range. Furthermore, a description of thegeographical structure of this species is urgently needed,because argan tree forests are presently shrinking due toovergrazing by livestock and human pressures, causingthe desertification of large areas.

In addition to its ecological role (conservation of thesoil and improvement of the microclimate), this species

Chloroplast DNA phylogeography of the argan tree ofMorocco

A. EL MOUSADIK* † and R. J . PETIT** Laboratoire de génétique et d’amélioration des arbres forestiers, Institut National de la Recherche Agronomique, Pierroton, BP 45,F-33611 Gazinet cedex, France, and †Laboratoire d’Agroforesterie, Faculté des Sciences, Département de Biologie, Université IbnouZohr, BP 28S, Agadir, Morocco

Abstract

Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemicspecies of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of 12 chloroplast DNA (cpDNA) and two mitochondrialDNA (mtDNA) fragments were amplified and digested with a single restriction enzyme(HinfI). Polymorphisms were identified in six of the cpDNA fragments, whereas nomtDNA polymorphisms were detected in a survey of 95 individuals from 19 populationsencompassing most of the natural range of the species. The cpDNA polymorphismsallowed the identification of 11 haplotypes. Two lineages, one in the south-east and theother in the north-west, divide the range of the argan tree into two distinct areas. The levelof genetic differentiation measured at the haplotype level (GSTc = 0.60) (i.e. withunordered haplotypes) was smaller than when phylogenetic relationships were takeninto account (NSTc = 0.71–0.74) (ordered haplotypes), indicating that population historymust be considered in the study of the geographical distribution of cpDNA lineages inthis species. If contrasted with the level of nuclear genetic differentiation measured in aprevious study with isozymes (GSTn = 0.25), the results indicate a relatively high level ofgene flow by seeds, or conversely a relatively low level of gene flow by pollen, as com-pared with other tree species. Goats and camels could have played an important role indisseminating the fruits of this tree.

Keywords: Argania spinosa, chloroplast DNA (cpDNA), differenciation, ordered alleles, seed dis-persal, universal primers

Received 20 January 1996, revision accepted 27 March 1996

Molecular Ecology 1996, 5, 547–555

Correspondence: Dr R. J. Petit. Tel.: +33-57-97-90-87, Fax: +33-57-97-90-88. E-mail: [email protected]

© 1996 Blackwell Science Ltd

has economic importance and constitutes a strong poten-tial for the sustainable development of the region. The oilextracted from the seeds has high nutritional and pharma-ceutical values. The argan tree ‘forest’ is, in fact, perhapsbest described as a large natural ‘orchard’ that is also usedfor wood production and grazing by livestock (sheep,goats and camels). However, precisely because of over-grazing, natural regeneration is now virtually non-existentover the majority of the species range. This is not due tolimited seed production or to germination problems but isa consequence of the absence of enclosed areas protectedfrom the animals where the young trees could reach a suf-ficient age (about 10 years) to be able to resist by them-selves to grazing pressures. As a consequence of this gen-eral failure of regeneration, the factors which wereinvolved in shaping the present genetic structure of thespecies may no longer be operating.

In the Sapotaceae, a primarily tropical tree family, theargan tree is the single species of a monotypic genus.However, a species belonging to a related genus(Sideroxylon marmulano Banks) is present in the CanaryIslands, Madeira and the Cape Verde Islands (Montoya

1984). Argania spinosa is a monoecious, allogamous(Msanda et al. 1994), and apparently insect pollinatedspecies. A previous study using isozyme markers hasrevealed a significant level of population differentiationfor the loci analysed (El Mousadik & Petit 1996).

This study investigates the cpDNA diversity and thegeographical structure of the argan tree using a PCR-derived technique which takes advantage of the numerousuniversal cytoplasmic primers which were recentlydescribed (Demesure et al. 1995).

Materials and Methods

Populations samples and DNA isolation

A total of 19 populations of Argania spinosa were sampledacross its range (Fig. 1 and Table 1). Ten of these 19 popu-lations had been previously studied with isozymes (ElMousadik & Petit 1996), including two disjunct popula-tions located about 700 and 400 km north of the mainrange of the species. Sampled area was 1–2 ha, except forthe low-density Saharan population of Goulimine (cf. El

548 A . E L M O U S A D I K and R . J . P E T I T

© 1996 Blackwell Science Ltd, Molecular Ecology, 5, 547–555

Fig. 1 Map of the sampled populationsof the argan tree in Morocco and theirchloroplast DNA haplotypes. A phylo-genetic tree of the 11 haplotypes is illus-trated for comparison (see Fig. 3 for thedetails concerning this tree).

Mousadik & Petit 1996). At each site, seeds were collectedfrom 20 to 50 nonadjacent trees, as separate seed lots. Asingle fruit from each of five trees per population wasselected, one seed was extracted from each fruit and ger-minated on wet paper in a Petri dish. Total DNA wasextracted from single root tips (1–2 cm long) following theprocedure described in Dumolin et al. (1995). Overall, 95individuals (19 × 5) were studied, along with one oak sam-ple as an amplification control.

DNA amplification and digestion

The PCR method was as detailed in Demesure et al. (1995).We used 12 pairs of chloroplast primers, one described byTaberlet et al. (1991), nine described in Demesure et al.(1995), two described in Dumolin-Lapègue et al. (submit-ted) and two pairs of mitochondrial primers described inDemesure et al. (1995) (Table 2). Microplates containingthe 96 diluted DNA samples were prepared in advance forthe 14 amplifications using an 8-channel micropipette andstored at –18 °C before use. The PCR products (5 µL) weredigested overnight with 5 units of HinfI. Restriction frag-ments were separated by electrophoresis in 8% polyacry-lamide gels using Tris Borate EDTA buffer (1×) at 300 V for2 h. The gels were stained with ethidium bromide andphotographed under UV light with Polaroid 665 filmsusing an MP4 Polaroid camera. The negatives werescanned and analysed with Whole Band AnalyserSoftware (version 3.2) of the Bio Image system in order to

estimate precisely the size of the fragments, using the 1-kbladder of Gibco BRL (Life Technologies) as molecularweight marker.

Analysis of data

The total diversity (hT), the average intrapopulation diver-sity (hS), the level of subdivision of chloroplast diversity(GSTc) and their standard deviations were estimated fol-lowing Pons & Petit (1995), with the computer programH A P L O D I V (available from R. J. Petit). To derive theintraspecific phylogenetic trees, the Wagner parsimonymethod was used. The molecular data were scored asunordered multistate characters and the equally most par-simonious trees were identified using the branch andbound algorithm of P A U P version 3.1 (Swofford 1993).Then, the level of subdivision for ordered alleles (NSTc)modified from Lynch & Crease (1990) was computed fol-lowing Pons and Petit (submitted): if the distancesbetween the haplotypes i and j are noted πij (for example,the number of mutational steps separating the two haplo-types), xki is the observed frequency of allele i in the kthpopulation, and n and nk are the number of sampled pop-ulations and the number of sampled individuals in popu-lation k, then the average within-population diversity is:

c p D N A P H Y L O G E O G R A P H Y O F T H E A R G A N T R E E 549


Table 1 List and location of the populations used in this study

Longitude AltitudePopulation Abbr. Geographical origin (west) Latitude (m) Climate

Ademine AD Souss plain 9°22’ 30°20’ 90 AridAghroud AG Atlantic coast 9°47’ 30°37’ 40 SubaridAït Baha AB Northern side of the Anti Atlas 9°12’ 30°06’ 550 AridArgana AR Southern side of the High Atlas 9°11’ 30°47’ 600 AridBeni-Snassen BS North-East of Oujda 2°36’ 34°51’ 260 AridBouzakaren BZ Southern plateau of the Anti Atlas 9°47’ 29°08’ 510 AridEssaouira ES Atlantic coast 9°42’ 31°25’ 80 SubaridGoulimine GO Northern limit of the Sahara 010°04’ 28°58’ 530 AridMijji MI Chichaoua plateau 9°19’ 31°33’ 350 AridOued Grou OG South-East of Rabat 6°35’ 33°43’ 400 SubaridOuled Berhil OB Plateau of the Souss valley 8°43’ 30°31’ 220 AridPiedmont Tizint’est PT Southern Piedmont of the High Atlas 8°25’ 30°43’ 600 AridSidi Ifni SI Atlantic coast 010°07’ 29°26’ 20 AridSmimou SM Atlantic coast 9°43’ 31°13’ 210 SubaridTafraout TA Northern side of the Anti Atlas 8°58’ 29°46’ 1150 AridTamanar TM Atlantic coast 9°36’ 31°06’ 170 SubaridTensift TE Atlantic coast 9°25’ 31°54’ 110 SubaridTizint’est TT Southern side of the High Atlas 8°22’ 30°50’ 1200 AridTlat Lakhssas TL South-western side of the Anti Atlas 9°44’ 29°21’ 890 Arid

v̂

nn

nx xs

k

kkij ki

ijkj=

−∑ ∑11

π

and the total diversity becomes:

Finally

This value can be compared with GST using the test statis-tic described in Pons and Petit (submitted):

The seed/pollen migration ratio was obtained with theformula

valid for outcrossing diploid and hermaphrodite plants ifthe cytoplasmic genome is maternally inherited (Petit1992; Petit et al. 1992). The notations are as follows: mp isthe pollen migration rate, ms the seed migration rate, GSTc

the cytoplasmic GST and GSTn the nuclear GST.

Results

After digestion with HinfI, 116 restriction fragments wereobtained with the 12 cpDNA PCR fragments and 12 with

the two mtDNA PCR fragments. Monomorphic patternswere obtained with six pairs of cpDNA primers and withthe two pairs of mtDNA primers. The six remainingcpDNA PCR fragments allowed the detection of 11 poly-morphisms separating 11 haplotypes (Table 3 and Fig. 2).Complete data were obtained from 90 individuals. Most ofthese mutations were apparently insertions/deletions(indels), although this is difficult to ascertain given that asingle restriction enzyme was used for each fragment.

Phylogenetic analysis was conducted on the 11 haplo-types that were detected. Sixteen most parsimonious treesof length twelve were obtained, i.e. a single homoplasticmutation was inferred. The trees differ in whether thehomoplastic mutation is in fragment AS11 or VL9, and alsoin the position of the haplotypes 7 and 11, due to theabsence of outgroup and to the occurrence of severallength variants for the same restriction fragment. One ofthese trees is illustrated in Fig. 3. Table 4 gives the molec-ular weights of all polymorphic fragments. There is a sin-gle mutation in fragment AS11, an indel of about 16 bases(or possibly a point mutation, because a fragment of 16bases would not have been detected), whereas in fragmentVL9 there are already three different length variants whichdiffer by less than 10 bases from each other. Hence, weconsider it more likely that the homoplastic mutation islocated in fragment VL9. There are nine trees (out of 16) forwhich the homoplastic mutation is in fragment VL9 andnot in fragment AS11. In these nine trees therefore twogroups of haplotypes can be distinguished, separated bythe mutation in the fragment AS11. The first group



Table 2 List of primers used in this study

Molecular Primer 1 Primer 2 Abbr. References weight in A. spinosa

Chloroplast primerstrnH [tRNA-His (GUG)] trnK [tRNA-Lys (UUU) exon 1] HK Demesure et al. 1995 1670 bptrnK [tRNA-Lys (UUU) exon 1] trnK [tRNA-Lys (UUU) exon 2] K Demesure et al. 1995 2610 bptrnQ [tRNA-Gln (UUG)] trnR [tRNA-Arg (UCU)] QR Dumolin et al. unpubl. data 3380 bptrnC [tRNA-Cys (GCA)] trnD [tRNA-Asp (GUC)] CD Demesure et al. 1995 3470 bptrnD [tRNA-Asp (GUC)] trnT [tRNA-Thr (GGU)] DT Demesure et al. 1995 1250 bptrnT [tRNA-Thr (GGU)] psbC [rpsII 44 kd protein] TC Dumolin et al. unpubl. data 3510 bppsbC [rpsII 44 kd protein] trnS [tRNA-Ser (UGA)] CS Demesure et al. 1995 1640 bptrnS [tRNA-Ser (UGA)] trnfM [tRNA-fMet (CAU)] SfM Demesure et al. 1995 1230 bppsaA [PS I (P 700 apoprotein A1)] trnS [tRNA-Ser (GGA)] AS Demesure et al. 1995 3550 bptrnS [tRNA-Ser (GGA)] trnT [tRNA-Thr (UGU)] ST Demesure et al. 1995 1370 bptrnT [tRNA-Thr (UGU)] trnF [tRNA-Phe (GAA)] TF Taberlet et al. 1991 2160 bptrnV [tRNA-Val (UAC) 3′ exon] rbcL [RuBisCO large subunit)] VL Dumolin et al. unpubl. data 3860 bpTotal cpDNA 29 700 bp

Mitochondrial primersnad1 exon B nad1 exon C N1BC Demesure et al. 1995 1430 bpnad4 exon 1 nad4 exon 2 N412 Demesure et al. 1995 2000 bpTotal mtDNA 3430 bp

ˆ .. . . .v x xn n

x x x xT ij i jij

ij ki i kj jijk

= −−( ) −( ) −( )∑ ∑∑π π1

1

ˆ ˆ

ˆ.N

vvST

S

T

= −1

UN G

Var N Var G Cov N G

ST ST

ST ST ST ST

=−

( ) + ( ) − ( ){ }ˆ ˆ

ˆ ˆ ˆ ˆ ˆ ˆ , ˆ/ .

21 2

r

m

m

G G

Gp

s

STc STn

STc= =

−( )− −( )−( )

2 1 1 1 1

1 1

/ /

/

comprises haplotypes 1, 2, 3 and 4 and the second theseven remaining haplotypes. The genetic composition ofall populations studied is provided in Table 5 and is illus-trated in Fig. 1. Two regions can be distinguished, with sixpopulations in the north-west having exclusively haplo-types from the first group and nine populations from thesouth-east having haplotypes from the other group. Thegeographically intermediate population of Argana has amixture of haplotypes from both groups, as the southerncoastal population of Sidi Ifni. Both disjunct populationsfrom the north of the country have haplotypes from thesecond (south-eastern) group.

A total of 63% (12/19) of the populations were poly-morphic. Two haplotypes were restricted to a single pop-ulation, including one case where the population wasmonomorphic for this private haplotype (Tensift, see

Table 5). In contrast to the situation for nuclear genes(isozymes) where GSTn = 25% (El Mousadik & Petit 1996),most of the total chloroplast diversity is distributed amongpopulations: GSTc = 60% (Table 6).

For ordered haplotypes, we measured the level of sub-division of diversity for the nine most parsimonious treesin which AS11 was not homoplastic. In all these cases πij

was deduced from the trees by considering the number ofmutational steps separating all pairs of haplotypes. Wealso considered an additional matrix of distances betweenhaplotypes, derived simply by counting the number of dif-ferences between each pair of haplotype directly in theoriginal data matrix. NSTc varied between 71.0 and 74.0%in the first case, and was equal to 71.3% in the second. Allthese values differ significantly (P < 0.01) from GSTc asjudged from the value of the test statistic U (cf. Table 6).



Polymorphic fragments

Haplotypes AS11* TF3 CD1 CD2 QR6 VL9 K6 K7 HK4

1 1 2 1 1 2 2 0† 1 22 1 1 1 1 2 1 0 1 23 1 1 2 1 2 1 0 1 24 1 1 1 1 2 1 1 1 25 2 1 1 1 2 2 0 1 26 2 1 1 2 2 2 0 1 27 2 1 1 1 2 3 0 1 28 2 1 1 1 2 2 2 1 29 2 1 1 1 1 2 0 1 2

10 2 1 1 1 1 2 0 0 211 2 1 1 1 2 1 0 1 1

* For each primers/enzyme combination the restriction fragments were labelled by decreasing order ofmolecular weights.†Length variants of a given fragment are also labelled by decreasing order of molecular weights (1–3), with0 indicating the absence of the fragment (as far as could be judged).

87pb E81pb E

M 1 2 3 4 5 6 7 8 9 10 11 12 13 M 14 15 16 17 18 19 20 21 22 23 24 25 M

Fig. 2 Polymorphism (insertions/deletions) detectedafter amplification with the primers trnH and trnKand digestion by the endonuclease HinfI. Samples 9,10, 11, 16 and 17 have an insertion of 6 bp relative tothe other individuals. M, molecular weight marker.

Table 3 Description ofthe 11 haplotypesidentified

If we assume that cpDNA is maternally inherited in theargan tree, such an asymmetry in the levels of differentia-tion of the two genomes probably indicates a higher levelof pollen flow (mp) as compared with seed flow (ms):r = mp/ms = 2.5.

Discussion

Approximately half of all cpDNA mutations are due toshort indels (1–10 bases), located primarily in the noncod-ing regions of the chloroplast genome (Zurawski, et al.1984; Kanno & Hirai 1992). Studies of noncoding chloro-plast DNA sequences have shown that insertions/dele-tions of more than two bases that do not belong to tandemrepetitions are good phylogenetic markers (Gielly &Taberlet 1994a, 1994b 1996). Previous work (Demesure etal.), using the same approach, has shown that these indelsmay be detected within species belonging to families, suchas the Fagaceae, where cpDNA is known to evolve at a rel-atively reduced rate (Frascaria et al. 1993). A single 4-cut-ter restriction enzyme such as HinfI will readily detectmost indels larger than 3–6 bp with the gel system used.Because many kilobases of cpDNA amplified with the con-sensus primers can now be ‘scanned’ to identify such poly-morphisms, population studies using cpDNA as a markercan now be conducted in a limited amount of time and atlow cost, as demonstrated in this study.

The level of differentiation for isozymes and forcpDNA in the argan tree (GSTn = 25%, GSTc = 60%) can becompared with the values obtained from the literature forother forest trees (Table 7). The tree which has the mostsimilar partitioning of diversity (for both isozymes andcpDNA) is Eucalyptus nitens, an insect-pollinated tree withno obvious adaptations for seed dispersal (Byrne & Moran

1994). In all the cases reported in Table 7, as expected ontheoretical grounds (Petit, Kremer, & Wagner 1993a), thelevel of subdivision of cpDNA diversity is higher than forisozymes (i.e. nuclear) markers. However, this contrastseems stronger among those species with higher levels ofcpDNA differentiation such as the beech or the oaks.Actually, the chloroplast and nuclear Gst values are notindependent and they may be used to derive thepollen/seed migration ratio (Petit 1992; Petit et al. 1992,1993a; Ennos 1994) if a set of assumptions are met. In par-ticular, the island model of population structure and equi-librium between migration and drift are assumed.Moreover, in the absence of any knowledge concerningthe mode of inheritance of cpDNA in the argan tree, wehad to consider that it is maternal, as in a majority (but notin all) of the angiosperms studied (Birky 1995). Despitethese limitations, the migration ratio could be a usefuldescriptor for species comparisons. Its value is lowest forthe argan tree (2.5) and for Eucalyptus nitens (1.80). It isintermediate for the two conifers (which have wind-dis-persed seeds and pollen) and highest for the beech and theoaks, characterized by bird-dispersed nuts and wind-dis-persed pollen. Hence, the argan tree, like the eucalyptus, ischaracterized by relatively high seed flow and low pollenflow among populations. Livestock (especially goats andcamels) can disperse the nuts of the argan tree, probablyup to at least a few tens of kilometres, within the limits ofthe villages for the goats and probably at longer distancesin the case of camels. Both swallow the fleshy fruits andregurgitate the kernel which contains the seeds, when theyare ruminating, later in the day or during the night. Beforethey became exterminated, antelopes could also haveplayed this role of seed dissemination. However, thismechanism of seed dispersal is not sufficient to preventthe establishment of a well-marked cpDNA geographicalstructure. Pollen flow, on the other hand, may be quitelimited, as in the eucalyptus, due to limited dispersal byinsects and to selfing, which is possible in this species (ElMousadik, unpublished data).

Table 4 Molecular weights in base pairs of the polymorphicfragments

Polymorphic Fragmentfragments size in bp

AS11 108 (1), 92 (2)TF3 266 (1), 261 (2)CD1 458 (1), 451 (2)CD2 422 (1), 403 (2)QR6 220 (1), 206 (2)VL9 255 (1)*, 247 (2), 237 (3)K6 272 (1), 247 (2)K7 240 (1), 224 (2HK4 87 (1), 81 (2)



Fig. 3 One of the phylogenetic trees of the 11 cpDNA haplotypesof the argan tree found in Morocco. The symbols correspond toFig. 1.

7

4

The phylogenetic relationships between cpDNA haplo-types (and hence history) can be accounted for in the mea-sure of subdivision by computing NST, an analogue of GST

for ordered alleles (O. Pons and R. J. Petit, unpubl. data).This approach is similar to the methods outlined byExcoffier et al. (1992) and Lynch & Crease (1990), whichwere recently applied to cpDNA data by Mason-Gamer etal. (1995) and by Byrne & Moran (1994). Similar to theparameter ΦST in the work of Excoffier and co-workers,our NST is a measure of subdivision based on the networkobtained by parsimony analysis. However, like in Lynch &Crease (1990) approach, it is not based on the analysis ofvariance. Interestingly, the values found here for NST

(71–74%) are significantly higher than the GST value (60%),suggesting that the relative distribution of phylogenetical-ly related haplotypes contributes to the overall geograph-ical structure of the species. Actually, this is quite clearfrom Fig. 1 and Table 5. Intrapopulation polymorphismsusually involve related haplotypes, belonging to the samelineage (in nine out of 11 cases). Excoffier & Smouse (1994)suggest to use allele frequencies and other geographicalcriteria to select the most probable intraspecific gene trees.However, they propose that their ΦST should, in fact, beused in a second step, to characterize the trees obtained,rather than as a criteria to select one of them. Here, the dif-ferent trees found did not yielded very different NST val-ues and we followed their advice and refrained fromselecting the tree having the highest NST value.

The two populations disconnected from the main range(Beni-Snassen and Oued Grou), often described as ‘relic’populations, do not have particular haplotypes. Rather,they have haplotypes of the south-eastern lineages, and inparticular one (no. 7) present in the High Atlas region, andanother (no. 11) (in the Beni-Snassen mountains) presentin the south of the country, close to the Sahara. If thesepopulations were indeed old relic populations, separatedfrom the main range of the species during several thou-sands years, we may expect to find unique haplotypesthere, given the overall level of intraspecific polymor-phism detected in this study. Because this is not the case,and given their small sizes, seed transfer(s) from the mainrange followed by successful establishment during histor-ical times seem more likely. Their reduced allelic richness



Haplotypes

Population 1 2 3 4 5 6 7 8 9 10 11 Total

Ademine 5 5Aghroud 5 5Aït Baha 2 3 5Argana 3 1 1 5Beni-Snassen 1 4 5Bouzakaren 1 4 5Essaouira 2 3 5Goulimine 1 4 5Mijji 3 1 4Oued Grou 5 5Ouled Berhil 4 4Piedmont Tizint’est 1 3 1 5Sidi Ifni 1 2 2 5Smimou 2 2 4Tafraout 4 4Tamanar 5 5Tensift 4 4Tizint’est 4 1 5Tlat Lakhssas 3 2 5Total 4 10 3 14 3 4 21 6 9 3 13 90

Table 6 Results of the analysis of diversity and standard errors ofthe estimates. The figures for NSTc presented in this table corre-spond to the tree illustrated in Fig. 3

Number of populations: 19Arithmetic mean: 4.74Harmonic mean: 4.69

hs 0.36 (0.07)ht 0.90 (0.02)GSTc 0.60 (0.08)

vs 0.74 (0.16)vt 2.75 (0.15)NSTc 0.73 (0.06)U 4.68

Table 5 Geneticcomposition of the 19populations

(El Mousadik & Petit 1996) would then be due to thisrecent founder effect. Note, however, that in other plantspecies there are examples of disjunct populations whichare not due to anthropogenic transfers but rather to natur-al transfers during the Pleistocene. In some of these cases,no cpDNA differences could be detected between the dis-junct populations, as a consequence of the low mutationrate of this molecule (see, e.g. Soltis et al. 1991; Liston &Kadereit 1995).

Overall, the results demonstrate that this threatenedspecies is strongly structured geographically. They showthe efficiency of cpDNA to recover a genetic pattern whichreflects the combined effect of the past history of thespecies together with the more recent seed dispersal byanimals or by humans. Such information can now beincorporated into efficient conservation policies.

Acknowledgements

We thank Rémi Chaussod, Sylvie Dumolin-Lapègue, Saïd Fakirand Rachida Nouaïm for their encouragement and help. The com-ments of three anonymous referees were also greatly appreciated.Our study was supported by a grant to A. El Mousadik from theFrench embassy in Morocco (co-operation programme).

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Table 7 Comparison of genetic differentiation for nuclear (isozymes) and maternally inherited cytoplasmic markers, in various foresttree species

Species GSTn Reference GSTc Reference mp/ms

Argania spinosa 25% El Mousadik & Petit, 1996 60% This study 2.5*Eucalyptus nitens 30% Moran, 1992 62% Byrne & Moran, 1994 1.8Fagus sylvatica 5.4% Comps et al. 1990 83% Demesure et al. 1996 84*Pinus contorta 9% Wheeler & Guries, 1982 72%† Dong & Wagner, 1993 24Pseudotsuga menziesii 24% Li & Adams, 1988 73%‡ Aagaard et al. 1995 7Quercus petraea 2.4% Zanetto et al. 1994 90% Petit et al. 1993b 500Quercus robur 3.2% Zanetto et al. 1994 92% Petit et al. 1993b 286

* In these species inheritance of cpDNA is unknown but was supposed to be maternal.† Mitochondrial DNA estimate, since it is the only organelle maternally inherited in this species, recomputed from Dong and Wagner(1993).‡ Estimate obtained from maternally inherited RAPD fragments (probably mtDNA).

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Abdelhamid El Mousadik is a geneticist working in a multidisci-plinary group involved in a long-term programme aimed at pre-serving and extolling a unique Moroccan resource, the argan tree.During a 6-week visit to France, he completed this study ofcpDNA diversity as part of his State Doctorate Thesis. In the for-est genetics laboratory of INRA, near Bordeaux, a comprehensiveprogramme on the analysis of cytoplasmic diversity of forest treeshas been undertaken for several years now, using both empiricaland theoretical approaches.



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Chloroplast DNA phylogeography of the argan tree of Morocco