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Population Biology |
2Department of Systematic and Evolutionary Botany, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; 3Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080-Sevilla, Spain
Received for publication November 22, 2005. Accepted for publication May 31, 2006.
ABSTRACT
Atlas cedar (Cedrus atlantica) is an ecologically and economically important forest tree species of northern Africa and is considered one of the endangered conifer species in the region. Chloroplast microsatellites (cpSSR) were used to study genetic variation within and among populations and geographical structure in natural populations of C. atlantica throughout its entire distribution range in Morocco. A total of 25 chloroplast haplotypes and 66 cpSSR alleles were found among 162 individuals. The cpSSRs indicate that C. atlantica appears to maintain a high level of genetic diversity (mean He = 0.95), as observed in most coniferous species. Values of mean pairwise distance within a population (D2SH) were related to the size and location of the populations. AMOVA analysis showed that most of the variation in C. atlantica occurs within populations and confirmed the general tendency of gymnosperms to display lower values of population differentiation than angiosperms. The distance-based clustering method (PCoA and neighbor-joining analysis) and the geographical structure revealed a poor structure among the six populations of Cedrus atlantica. Also, a Mantel test indicated a weak correlation between geographic and genetic distances (P = 0.106, r = 0.363). These results are also interpreted in the context of postglacial history of the region plus human impacts.
Key Words: Cedrus atlantica • chloroplast microsatellites • conservation • Morocco • Pinaceae • population genetics
The genus Cedrus Mill. includes three species native to Mediterranean mountains, C. atlantica (Endl.) Carr., from NW Africa, C. libani A. Rich. from Asia Minor, C. brevifolia (Hook. fil.) Dole from Cyprus, and one species restricted to the Himalayas, C. deodara (D. Don) G. Don fil. from Afghanistan and the southern slopes of the western Himalayas (Mabberley, 1990
). The Atlas cedar (C. atlantica) is native to the Atlas Mountains of Morocco and Algeria, where it can be found in scattered stands at elevations of 1000 to 2500 m a.s.l. Atlas cedar, the principal timber species in Moroccan forests at c. 100 000 tons per year (AEFCS, 1995
), has been important in the socioeconomy of Morocco, being suitable for furniture, construction materials, and after dry-distillation for tar-making. Benabid (2000)
also mentioned that the leaves (needles) are used in tanning. Like all cedarwoods, the wood is fragrant, insect repellent, and rot-resistant due to the essential oil content. The machining waste, often undervalued, is estimated to be 30% (El Ammari, 1996
); thus sawdust production is estimated to be 18 000 tons. This raw material could be important as a source of essential oils for the medicinal and perfume industries. In Morocco, the Atlas cedar forests constitute some 2.8% of the total area of Moroccan forests and are dispersed in two widely separated geographical areas: the Rif mountains (150 km2) and both the Middle Atlas (1000 km2) and High Atlas mountains (100 km2). In comparison to the estimated original forest cover, Morocco has lost 75% of its cedar forests, namely 3300 km2 between 1940 and 1982 (Benabid and Fennane, 1994
); the Rif (N Morocco) lost 1000 km2 of its high forests between 1956 and 1971. The Atlas cedar forests are characterized by a significant number of unique ecotypes of sub-Mediterranean and Euro-Siberian tree and shrub species, which reach their southernmost range here. These include Taxus baccata L., Sorbus aria Crantz, S. torminalis Garsault, Acer opalum subsp. granatensis (Boiss.) Font Quer & Rothm., A. monspessulanum L., Prunus lusitanica L., P. mahaleb L., P. insititia L., Ilex aquifolium L., Betula pendula subsp. fontqueri (Rothm.) G. Moreno & Peinado, Lonicera etrusca G. Santi, and L. arborea Boiss. (Quézel, 1981
).
Within Pinaceae, opposite uniparental inheritance of mitochondrial and chloroplast genomes provides opportunities to assess discontinuity within an effectively haploid genome. The alleles of loci in an organelle genome can be collectively considered as a haplotype because they are not recombining. In these organelles, effective population size is approximately one half that of nuclear genes (McCauley, 1995
) and, consequently, both the time to allele fixation within populations and the response time to stochastic change are reduced. In plants, mitochondrial genomes have not typically been useful for phylogenetic analyses due to a high rate of sequence reorganization (Sederoff et al., 1981
; Wu et al., 1998
). Mitochondrial haplotype diversity related to sequence rearrangement has, however, been useful to differentiate populations of Pinus and Abies (Strauss et al., 1993
; Tsumura and Suyama, 1998
). In chloroplasts, the conservation and homology of sequences make it possible to compare genes across the plant kingdom (Clegg and Zurawski, 1992
) and examine phylogenetic relationships in taxa that diverged hundreds of thousands to millions of years ago.
The recent use of microsatellites, or SSR (simple sequence repeat), markers in plastid DNA (cpDNA), with high levels of variation (Powell et al., 1995
b; Vendramin et al., 1996
; Vendramin and Ziegenhagen, 1997
) has enabled high resolution in differentiating among closely related taxa. Paternal inheritance of chloroplast genomes in most conifers (Neale et al., 1986
; Neale and Sederoff, 1989
) makes chloroplast microsatellites particularly effective markers for studying mating systems, uniparental lineages, and gene flow via both pollen and seeds. Predominant paternal inheritance of chloroplast DNA has been demonstrated in European Abies with previously characterized (Vendramin et al., 1996
) Pinus thunbergii primers at two highly variable microsatellite loci (Vendramin and Ziegenhagen, 1997
; Ziegenhagen et al., 1998
; Vendramin et al., 1999
). Intraspecific diversity and population structure in heterologous amplification with these primers have been demonstrated in many pine species as well (Cato and Richardson, 1996
; Morgante et al., 1998
).
In spite of the biological and economic importance of Cedrus atlantica, only limited information is known about its distribution and genetic variability. This is unfortunate because such information is vital for developing approaches to protect and conserve the genetic resource of the cedar forests. Allozyme variation was studied in the genus Cedrus but without any report on population genetic structure (Panetsos et al., 1992
, 1994
; Scaltsoyiannes, 1999
; Fallour et al., 2001
). Marker-assisted fingerprinting of the offspring resulting from controlled pollination experiments among the Mediterranean cedar taxa was reported by Fady et al. (2003)
, who recommended the use of cpSSRs for monitoring gene flow. Finally, genetic diversity and population differentiation using randomly amplified polymorphic DNA (RAPD) was reported by Renau-Morata et al. (2005)
from natural populations of Cedrus atlantica in Morocco and from a forest plantation in Spain; they revealed the existence of gene flow between very distant populations.
In this paper, we have used chloroplast microsatellites to study natural populations of Cedrus atlantica along the whole distributional area in Morocco to ascertain (1) the levels of genetic diversity within populations and (2) the population genetic structure among populations. The impacts of historical processes are discussed to explain the observed levels of variation and differentiation. The data obtained are basic to establishing a strategy of management and conservation of this species.
MATERIALS AND METHODS
Plant material
Needles were collected from 162 trees that were more than 100 yr old from six natural populations of Cedrus atlantica from the Rif, Middle Atlas, and High Atlas mountains (Fig. 1, Table 1). This sampling strategy avoided the problem of recent reforestation and management during the last 100 yr, mainly by French and Spanish entrepreneurs. For each population, 10 individuals were collected at one edge of the population, 10 from the middle, and the remaining 10 from the other edge; the only exception was population Cedar 6 from Cirque de Jaffar, where we collected only 13 individuals due to the small population size. All vouchers are deposited in the herbarium of the University of Seville (SEV) (Appendix 1).
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. Six primer pairs, originally designed for the Pinus thunbergii chloroplast genome (Wakasugi et al., 1994
), were employed to amplify plastid regions containing short stretches of simple mononucleotide repeats. The primers used were the forward and reverse of the Pt15169, Pt30204, Pt63718, Pt71936, Pt109567 and Pt110048 loci (Vendramin et al., 1996
). The template for PCR amplification consisted of 20 ng genomic DNA. The PCR amplifications were performed in a reaction volume of 10.5 µL containing 9 µL of Reddymix PCR master mix (ABgene Germany, Hamburg, Germany); containing 2.5 mM of MgCl2; dNTPs: 0.2 mM of dATP, dCTP, dGTP, and dTTP; 75 mM Tris HCl, 20 mM (NH4)2SO4; 0.01% Tween 20; 1.25 units Thermoprime Plus DNA polymerase; and precipitant and red dye for electrophoresis), 0.5 µL of fluorescence-labelled forward strand primer (4 pmol) and 0.5 µL of reverse strand primer (4 pmol). The amplification was carried out using 1 cycle of 3 min at 95°C, 25 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and 1 cycle at 72°C.
The fluorescence-labelled PCR products were run on a 5% denaturing polyacrylamide gel on an automated sequencer (ABI 377, Perkin Elmer, Massachussets, USA). Before running, 0.8 µL NED- and HEX-labelled, and 0.4 µL FAM-labelled PCR products were mixed with 1 µL loading dye [containing 0.6 µL deionized formamide, 0.27 µL loading buffer, and 0.13 µL GeneScan-500 ROX, PE Applied Biosystems], and denatured at 95°C for 2 min. Raw data were scored and exported using ABI Prism GENESCAN Analysis Software v.2.1 (PE Applied Biosystems, California, USA) and GENOGRAPHER (v.1.1.0; Montana State University, 1998, http://hordeum.msu.montana.edu/genographer/).
Data analyses
Allele and haplotype frequencies within each population were estimated using Arlequin v.2.0 (Schneider et al., 2000
). For each locus, we calculated the number of alleles and the genetic diversity corrected for sample size (He) using the computer program SPAGeDi v.1.1 (Hardy and Vekemans, 2002
). Further, we calculated chloroplast haplotype variation within populations by estimating the total number of haplotypes (No), the effective number of haplotypes (Ne), as well as the unbiased haplotype diversity (He), corrected for sample size (Nei, 1978
), using the following formulas: Ne = 1/(
pi2) and He = [n/(n – 1)](1 – pi2), where n is the number of individuals analyzed in each population and p is the frequency of the ith haplotype in the respective population.
Following Goldstein et al. (1995)
, an estimate of genetic distance among individuals within populations was calculated with the mean pair-wise haplotype distance measure:
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| (1) |
) adjusted for haplotypic data (Goldstein et al., 1995
; Morgante et al., 1998
).
Genetic differentiation among populations was assessed by FST and RST (Slatkin, 1995
), which is the ratio of the between-population variance and the total variance of allele size (bp), adapted to fully linked haplotype markers. Significance testing for variance components in AMOVA was based on 1000 permutations.
Pairwise genetic distances among populations were determined using Ds (Nei et al., 1983
; Takezaki and Nei, 1996
) and (
µ)2 (Goldstein et al., 1995
). Based on the pairwise (µ)2, a neighbor-joining dendrogram (Saitou and Nei, 1987
) of the six cedar populations was constructed with the program SplitsTree 4.0 beta (Huson, 1998
; program available from http://www.splitstree.org). Grouping of the populations was carried out by a principal coordinate analysis (PCoA) performed on Nei's genetic distance matrix (Ds), using the program NTSYSpc 2.02h (Rohlf, 1997
) and the modules "Dcenter" and "Eigen". We measured the goodness of fit of the PCoA by generating a model distance matrix from the eigenvector matrix (using "Simint") and comparing it to the original Ds matrix (with "Mxcomp" and 1000 permutations).
The possible presence of geographic structure of genetic variation in cpSSR C. atlantica was evaluated with three tests. First, we tested for the presence of phylogeographic structure by comparing RST estimates to RST values computed after 10 000 random permutations of allele types among alleles (Hardy et al., 2003
) were calculated using the SPAGeDi program. If RST is >RST (permuted), then there is phylogeographic structure, i.e., on average, phylogenetically similar alleles are found in the same population more often than are randomly chosen alleles. Second, the hypothesis that populations are differentiated due to isolation by distance (Wright, 1965
) was tested by correlating pairwise Ds, using Mantel tests with 1000 permutations as implemented in Arlequin v.2.0. Finally, spatial analysis of molecular variance (SAMOVA) was performed using data from the six populations to identify groups of populations that are phylogeographically homogeneous using the software Samova 1.0 (Dupanloup et al., 2002
). Samova finds the partitioning of populations that maximizes the FCT value when a particular number of groups are specified. SAMOVA can be used to identify the most likely number of groups within the data set from repeated analyses specifying the number of groups and choosing the partitioning of populations that maximizes the FCT value (Dupanloup et al., 2002
). We performed these repeated analyses for 2–4 groups.
RESULTS
Gene diversity
The plastid genome of Moroccan Atlas cedar contains six sites that can be PCR-amplified using the Pinus thunbergii microsatellite primers. Of the six primer pairs, all amplified consistently under standard conditions. All PCR reactions produced a single robust band per primer pair; thus, there was no evidence for heteroplasmy.
Three of the six cpSSR loci analyzed (Pt63718, Pt15169, Pt71936) were polymorphic in all the six Cedrus atlantica populations investigated. A total of 25 different cpSSR alleles among 162 individuals was found. The most variable cpSSR locus was Pt63718 (10 alleles), followed by Pt15169 and Pt71936, with eight and seven alleles, respectively. Of the 25 alleles detected, six were unique to a particular population, in Cedar 1 (Pt15169: 131 bp), Cedar 3 (Pt63718: 96 bp and Pt15169: 123 bp) and Cedar 6 (Pt63718: 95 bp, Pt15169: 104 bp and Pt71936: 146 bp). The gene diversity levels for the three polymorphic loci ranged from 0.708 (Pt71936) to 0.757 (Pt63718).
When alleles at each of the six loci were jointly analyzed, 66 different haplotypes were identified among the 162 individuals. The majority of these haplotypes (71%) was detected only once, 17% were detected twice and the rest were found in 3–5 individuals. We found 19 haplotypes common to more than one of the six populations studied, and seven haplotypes (h29 to h51) were not found in any individuals of the two Rif's populations (Bou-Hachem and Ketama).
Table 1 shows the genetic characteristics of chloroplast haplotypes based on six cpSSR loci in the six Atlas cedar populations analyzed. The lowest value of haplotype diversity was observed in Cedar 1 (He = 0.88) and the highest (He = 1) in population (Cedar 6). Estimates of the effective number of haplotypes (Ne) and haplotype diversity (He) averaged across all the populations were 12.4 and 0.91, respectively. Values of mean pairwise distances within populations (D2SH) varied greatly among populations, from a minimum of 7.15 in the population Cedar 1 to 18.46 in the population Cedar 6. The proportion of distinguishable haplotypes was high and ranged between 41.4% in population Cedar 3, to 100% in population Cedar 6, where all individuals had unique haplotypes. When considering cpSSR allele frequencies, the proportion of genetic differentiation residing among populations, RST, was 22.32%, and FST, was 10.74% (Table 2).
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µ)2, in which populations Cedar 1 and 2 from the Rif mountains are grouped, as well as populations Cedar 3 and 4 from the Middle Atlas mountains. Population Cedar 6 from High Atlas Mountains was the most separated. Population Cedar 5 is genetically closer to Rif than to the other populations from the Middle Atlas.
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DISCUSSION
Genetic diversity
The cpSSRs indicate that Cedrus atlantica appears to maintain a high level of genetic diversity (PD = 65.2%; He = 0.95), as observed in most coniferous species based on cpSSR (Vendramin et al., 1998
; Clark et al., 2000
; Parducci et al., 2001
; Ribeiro et al., 2001
; Gómez et al., 2003
; Hansen et al., 2005
). Whereas the genetic differentiation of Atlas cedar was found to be much higher than those previously reported by other authors using allozymes (He = 0.147, Panetsos et al., 1992
; He = 0.183, Scaltsoyiannes, 1999
; He = 0.158, Lefèvre et al., 2004
; He = 0.231, Fady, 2005
), and RAPD markers (H = 0.191, Renau-Morata et al., 2005
), these differences can be due to the methodology applied and to the different regions of sampling. Similar conclusions were found in Pinus pinaster using cpSSR and isozymes (Salvador et al., 2000
; Ribeiro et al., 2001
).
We found a high level of variation among the D2SH values observed in the six C. atlantica populations. The level of within-population stepwise haplotypic diversity of C. atlantica (mean D2SH =11.46) was similar to that observed for the genus Abies (Clark et al., 2000
; Parducci et al., 2001
) (mean D2SH =18.58 and 11, respectively), but much higher than those observed in P. halepensis or in P. resinosa (mean D2SH =0.154 and 3.58, respectively; Echt et al., 1998
; Morgante et al., 1998
; Vendramin et al., 1998
). With the exception of population Cedar 6 and consistent with results of Parducci et al. (2001)
and Clark et al. (2000)
, the D2SH values found were related to population size (see Table 1) and location of populations. Low D2SH values were found in Cedar 1 and 3 populations, both being small and isolated (Cedar 1 in Jbel Bou-Hachem with ~10 ha of cedar, and Cedar 3 in Jbel Tazzeka with ~ 25 ha of cedar). That the D2SH value in Cedar 6 shows no correlation with population size could be due to fewer trees sampled (13), less than half than in the other five populations.
As for the proportion of distinguishable genotypes, all individuals of population Cedar 6 were clearly differentiated from each other, i.e., 100% of them could be genotypically identified. This result could be due to the high number of fathers (pollinators) in this population. Such an increase in the number of pollinators would decrease the number of individuals with identical haplotypes in a few generations and affect the overall level of genetic variation of the population. In contrast, low values were found in populations 1 and 3. In recent decades, low vitality and pronounced signs of decline have been observed in Bou-Hachem cedar individuals (cedar 1, S. Talavera, personal observation). It is possible that this decline could be linked to reduction in number of fathers in the population (Parducci et al., 2001
).
Genetic differentiation among populations
The AMOVA analysis shows that most of the variation in C. atlantica lies within populations, a result compatible with those from RAPD studies and those involving other woody perennial, outbreeding plant species, especially conifers (Hamrick et al., 1992
). In a review, Nybom and Bartish (2000)
assembled AMOVA-derived FST values for about 100 plant species and confirmed the tendency of gymnosperms to have lower values of population differentiation than angiosperms. The mean proportion of chloroplast diversity that we found among populations (FST = 0.107) was lower than that reported for a Moroccan natural population of C. atlantica using RAPD markers (FST = 0.309) and indicates moderate genetic differentiation (Hartl and Clark., 1997
). The relatively low genetic differentiation among populations for cpSSR investigated in this study might be due to gene flow through pollen among populations, i.e., absence of strong barriers to gene exchange. Only a few migrants per generation are necessary to inhibit differences accumulating between populations. In general, the hypothesis of C. atlantica acting as one single large population does not seem plausible, taking into account the scattered occurrence of the species in mountainous areas. The several populations are separated by valleys and confront considerable barriers to gene flow, such as the main Rif and Atlas mountain ranges. If we accept gene flow as one of the causes for only moderate genetic differences among populations, we can infer that the Rif and Atlas Mountains may be playing a slight role in preventing gene flow through pollen in this species. The presence of Cedrus pollen in sedimentary sequences at various sites in southern Europe has already shown the capacity of this species for long-distance pollen transport from North Africa (De Beaulieu and Reille, 1973
; Reille, 1990
).
Geographical relationships
The distance-based clustering method (PCoA and neighbor-joining analysis, see Fig. 2) and the geography of genetic variation revealed poor geographic structure among the six populations of C. atlantica analyzed. Similarly, Renau-Morata (2005) found three clusters; two of them had individuals from the three Moroccan regions (Rif, Middle, and High Atlas).
These results suggest that possibly the Rif and Middle Atlas populations were in the past genetically isolated, and thus, only recently gene flow between them became possible. Both the postglacial history of C. atlantica and the continuous presence of Cedrus pollen during the Late Pleistocene to early Holocene (Cheddadi et al., 1998
; Magri and Parra, 2002
) suggest that cedars were present during the full-glacial in the N African region rather than in distant refugia. Therefore, the genetic similarity of population 5 with the Rif populations (see Figs. 1 and 2), more than 220 km distant, can be explained by gene flow between the populations. On the other hand, anthropogenic exploitation since c. 1300 BP has also had a marked effect on the forest ecosystems, perhaps greater than any impacts from the Holocene and intervals (Lamb and van der Kaars, 1995
).
Workers have generally considered the level of homoplasy to be low enough to permit population genetic analysis. Even when homoplasy has been identified, it has been considered "moderate" and its potential for confounding results disregarded (Cuenca et al., 2003
). Estoup et al. (2002)
, using simulations, concluded that the large amount of variability at microsatellite loci often largely compensates for homoplasious evolution. The cases in which size homoplasy may be a problem involve high mutation rates. There are no estimates of mutation rates in Cedrus, but Provan et al.
(1999) found a low mutation rate for other conifers.
Conservation implications for C. atlantica
In this study, genetic diversity was analyzed for six populations of C. atlantica over the species' distributional range. The results have several implications for the development of conservation strategies for Moroccan Atlas cedar. The detection of population differentiation using techniques such as cpSSR can help define appropriate units for conservation, providing a good focus for conservation management (Newton et al., 1999
). All the populations studied therefore deserve some attention in genetic conservation programs for Moroccan Atlas cedar, especially population Cedar 2 (Ketama) in the Rif Mountains, Cedar 4 (Ain Kahla) and Cedar 5 (Col de Zad) in the Middle Atlas Mountains, and Cedar 6 (Cirque de Jaffar) in the High Atlas Mountains. The neighbor-joining tree indicates that there is no clear center of genetic diversity in C. atlantica. Isolation between the populations should be encouraged to avoid gene flow between them and to preserve their genetic distinctiveness.
This study is the first to investigate the population genetic structure of C. atlantica using wide sampling and chloroplast microsatellites. It also highlights the importance of molecular analyses for effective conservation of genetic resources in the Moroccan Atlas cedar.
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1 The authors thank the Junta de Andalucía (Spain) for a postdoctoral fellowship for A. T., the Ministerio de Educación y Ciencias (REN2002-04634-C05-03 to S. T. and REN2002-04354-C02-02 to M. A.), the Junta de Andalucía (Group RNM-204 to S. T.), and the Austrian Science Foundation (FWF P13055 BIO to T. S.) for grant support. 
4 Author for correspondence (anass{at}us.es
)
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