Genetic resources in maritime pine (Pinus pinaster Aiton): molecular and quantitative measures of genetic variation and differentiation among maternal lineages

Forest Ecology and Management 197 (2004) 103–115

Genetic resources in maritime pine (Pinus pinaster Aiton): molecular and quantitative measures of genetic variation and differentiation among maternal lineages Santiago C. Gonza´lez-Martı´neza,*, Ste´phanie Marietteb,c, Maria Margarida Ribeirod, Christian Burbanb, Annie Raffinb, Maria Regina Chambela, Carla Alexandra M. Ribeirod, Alexandre Aguiare, Christophe Plomionb, Ricardo Alı´aa, Luis Gilf, Giovanni Giuseppe Vendraming, Antoine Kremerb a

Unidad de Gene´tica Forestal, CIFOR-INIA, Carretera de La Corun˜a Km 7,5, 28040 Madrid, Spain Equipe de Ge´ne´tique et Ame´lioration des Arbres Forestiers, INRA, 69 route d’Arcachon, 33612 Cestas Cedex, France c Cemagref, Domaine des Barres, 45290 Nogent-sur-Vernisson, France d Unidade Departamental de Silvicultura e Recursos Naturais, Escola Superior Agra´ria, 6001-909 Castelo Branco, Portugal e Departamento de Ecofisiogia e Melhoramento Florestal, Estac¸a˜o Florestal Nacional, 2784-505 Oeiras, Portugal f Unidad de Anatomı´a, Fisiologı´a y Gene´tica, ETSIM, Ciudad Universitaria s/n, 28040 Madrid, Spain g Istituto di Genetica Vegetale, Sezione di Firenze, Via Atto Vannucci 13, 50134 Firenze, Italy b

Abstract Pinus pinaster is a conifer native to western Europe and northern Africa. Following on-going breeding programmes, provenance and progeny trials were established in some of the countries of the species’ range (France, Portugal and Spain) and quantitative traits were measured: growth, stem form, survival and pest and disease resistance, amongst others. Populations from the wide range of P. pinaster were recently screened with molecular markers in order to assess their genetic diversity. Data were obtained using allozymes, chloroplast (cpSSRs) and nuclear (nuSSRs) microsatellites and amplified fragment length polymorphisms (AFLPs). Based on mtDNA-RFLP analysis, three maternal lineages (named ‘‘western’’, ‘‘eastern’’ and ‘‘Moroccan’’) were identified and no population showed a mixed composition. In this study, the imprint that differentiation in putatively isolated glacial refugia (identified by the different maternal lineages) might have left on the nuclear genome was analysed using a wide range of molecular markers and adaptive traits. Multivariate ordination of populations based on nuclear molecular markers (allozymes and nuSSRs) showed a clear clustering of provenances sharing a given mtDNA lineage. However, that clustering was found to be less tight when only quantitative traits were investigated. In P. pinaster, the within-population estimates of gene diversity using different traits were not correlated. Therefore, caution is advisable when designing conservation strategies based on molecular marker studies or a limited number of populations. After these results, we recommend a conservation strategy characterised by gene flow consistent with the current population structure, careful seed transfer among maternal lineages (if any), selection of populations for conservation based on the originality of their allelic composition and definition of Management Units (MUs) based on adaptive traits. # 2004 Elsevier B.V. All rights reserved. Keywords: Pinus pinaster; Genetic resources; Quantitative variation; Molecular markers; Management Units

* Corresponding author. Tel.: þ34-913474161; fax: þ34-913572293. E-mail address: [email protected] (S.C. Gonza´lez-Martı´nez).

0378-1127/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2004.05.008

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S.C. Gonza´ lez-Martı´nez et al. / Forest Ecology and Management 197 (2004) 103–115

1. Introduction The main goal in conservation genetics is the preservation of genetic traits that enable evolutionary lineages to cope with changing environments while, at the same time, preventing the fixation of deleterious alleles (Lynch, 1996). Identification of Evolutionary Significant Units (ESUs) has attracted considerable attention up to date and is subjected to controversial discussions (Moritz, 1994; Crandall et al., 2000). ESU concepts combine information on neutral genetic variation with ecological data and genetic variation of adaptive significance (see review in Fraser and Bernatchez, 2001). An increasing amount of population-based data with different traits is becoming available. Common analysed traits differ greatly in several features including type of inheritance, mutation rates and evolutionary significance. For instance, typical mutation rates for molecular markers range from 108 to 105 per year and locus, whereas variation for quantitative traits (heritability) is typically introduced at a rate of 103 to 102 per generation (Lynch, 1996). Traits also differ in evolutionary significance. Most molecular markers used in conservation genetic studies are assumed to be neutral or quasi-neutral. Moreover, when multiple loci affect a quantitative trait, selection on the trait is diluted over many loci and each locus behaves as if it was nearly neutral (McKay and Latta, 2002). On the other side, common garden experiments (provenance and progeny tests) provide direct information about adaptive variation in controlled environments and some studies assessing variation of genes or gene families of known function have been recently published in different species, including some forest trees such as Picea abies (L.) Karst. (Schubert et al., 2001; see a recent review in van Tienderen et al., 2002). Molecular markers based on organelle genomes (chloroplast and mitochondria) have received much attention in conservation genetics of forest trees and, in some cases, have been used to identify ESUs (see review in Newton et al., 1999). Maternal lineages were reconstructed for many forest trees, including several pine species (e.g. Pinus flexilis James, Latta and Mitton, 1997; Pinus sylvestris L., Sinclair et al., 1999; Pinus pinaster Aiton, Burban and Petit, 2003). Studies comparing population genetic structure using both organelle- and nuclear-based markers have

shown a certain degree of correlation, which varies depending on the species and the scale of the study (Latta and Mitton, 1997; Kremer et al., 2002). Extensive gene flow by pollen (or human-mediated seed transfer) can erode divergence among lineages identified using maternally inherited organelle markers, even when boundaries among maternal lineages were sharply defined (Liepelt et al., 2002). Hu and Ennos (1999) showed that, at migration-drift equilibrium, genetic differentiation for maternally inherited genes is greater than for paternally inherited genes, which in turn is higher than for biparentally inherited nuclear genes. A basic assumption, often not tested, underlying the use of molecular markers in conservation genetics is that the groups identified using molecular markers are associated with specific adaptive responses and gene complexes, behaving as different evolutionary units. However, most adaptive characters appear to be products of ten to perhaps hundreds of genetic loci, which are differentially expressed in response to environmental factors. A direct association between singlelocus markers, specially those based on organelle genomes, and complex polygenic trait variation is unlike to occur. Therefore, the confidence attached to genetic conservation inferences is limited in absence of complementary quantitative genetic studies. For instance, Kremer et al. (2002), in a wide range study on European oaks, hypothesised a gradual erosion of the original nuclear differentiation due to pollen flow and local selection to account both for the lack of association between chloroplastic divergence and phenotypic traits and the correlation that still remains within cpDNA lineages for those nuclear gene markers that are less affected by selection. In the case of maritime pine (P. pinaster Aiton), a widespread forest tree in the western Mediterranean Basin, three maternal lineages (named ‘‘western’’, ‘‘eastern’’ and ‘‘Moroccan’’), without a single population having a mixed composition (GST ¼ 1), were recently identified based on mtDNA-RFLP analysis (Burban and Petit, 2003). The eastern mtDNA lineage is distributed in northeastern Spain (only one population, located at Catalonia), southeastern France, Corsica, Italy (including Pantelleria island), Tunisia and Algeria. The western mitotype is found in the remaining Iberian Peninsula, continental France and Punta Cires (northern Morocco). The Moroccan lineage is

S.C. Gonza´ lez-Martı´nez et al. / Forest Ecology and Management 197 (2004) 103–115

specific of Morocco, where this species covers approximately 14,000 ha. Populations belonging to the western mtDNA lineage, which covers approximately 89% of the native range of the species, display different local adaptations in quantitative traits. Danjon (1994) showed that provenances from the Atlantic range of the species (southwestern France and Leiria, Portugal) had higher growth (although frost tolerance is significantly different among them; Alı´a et al., 1997 and references therein) than Mediterranean provenances, including those belonging to the western mtDNA lineage located in the Iberian Peninsula. The Atlantic provenances’ higher biomass yield increases their salinity tolerance, which is an important adaptive trait (Nguyen et al., 1995). Data from pest and fungi resistance also suggest a different performance of provenances included in the same mtDNA lineage. Provenances from Tamjout (Moroccan mtDNA lineage) and Serranı´a de Cuenca (western mtDNA lineage) are less susceptible to Matsucoccus feytaudi than provenances from Italy, France or Portugal (including populations from both western and eastern mitotypes). Nevertheless, all populations showing the eastern mitotype were highly susceptible to the pest (Schvester, 1982; Burban et al., 1999). Desprez and Baradat (1991) showed that provenances from Italy (eastern mtDNA lineage) and Landes (western mtDNA lineage) did not differ in susceptibility to Melampsora pinitorqua, while Tamjout (Moroccan mtDNA lineage) was highly susceptible. In maritime pine, several studies using nuclear molecular markers have been conducted at local and wide range scales (Derory et al., 2002 and references therein). In spite of the increasing amount of data available, no study has questioned whether the sharp differentiation of maternal lineages in this species, commonly associated with the existence of differentiated refugia during the last glaciation, have led to divergences in the nuclear genome. The extensive data recently gathered using different nuclear molecular markers, in combination with already available measurements of quantitative traits in common garden experiments, provide an excellent opportunity to test this hypothesis, both at the molecular and quantitative trait levels. Once different evolutionary units are identified, the amount of within-population genetic variation found in each unit is relevant to the design of conservation

105

programs. In general, mean heterozygosity measured by molecular markers (i.e. gene diversity) is a poor indicator of the variance of a polygenic trait (see, for instance, Pfrender et al., 2000), although a linear relationship is to be expected if all gene action was additive. A typically high non-additive variance and the role of natural selection were reported to explain this divergence in forest trees (see Gonza´ lez-Martı´nez et al., 2002a for maritime pine). Several other reasons explain why genetic variation estimates using molecular markers and quantitative traits may differ (see reviews in Lynch (1996) and Reed and Frankham, 2001). For instance, the recover of the genetic variance in quantitative traits after a reduction in population size is higher than the increase in mean heterozygosity (Willis and Orr, 1993). In maritime pine, spreads and retreats of the species range during glacial times have (putatively) produced frequent changes in effective population sizes promoting differentiation among quantitative and molecular variation. However, in this species, some correlation between different types of traits (organelle/ nuclear or molecular/quantitative) were found at a local scale (Ribeiro et al., 2002; M.M. Ribeiro, unpublished results) or along postglacial migration pathways (Gonza´ lez-Martı´nez et al., 2002a). Two were the main objectives of this work. Firstly, we analysed the imprint that differentiation in putatively isolated glacial refugia (identified by different maternal lineages) has left on the chloroplast (paternally inherited in this species) and nuclear genomes, including a wide range of molecular markers and adaptive traits. Secondly, we studied the amount of within-population genetic variation for each maternal (mtDNA) lineage and correlations between molecular and quantitative genetic diversity at the wide range of the species were reported. The discussion about the identification and characterisation of Evolutionary Significant Units for maritime pine was done using all the information available up to date for this ecologically and economically important species.

2. Material and methods 2.1. Molecular markers Molecular marker data from 97 populations analysed with different types of markers (cpSSRs, allo-

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Table 1 Number of populations belonging to each maternal lineage (‘‘western’’, ‘‘eastern’’, and ‘‘Moroccan’’) as detected by mtDNA-RFLP patterns, for which there are also available data with other types of molecular markers (cpSSRs, allozymes, nuSSRs, and AFLPs) and quantitative traits (provenance trials) Type of trait

Western lineage

Eastern lineage

Moroccan lineage

Traits used in this study

Molecular markers cpSSRs

37

11

2

Allozymes

39

9

1

nuSSRs

37

15

1

AFLPs

25

10

0

HE, number of haplotypes, frequency of the nine most common haplotypes HE, A, allele frequencies of 13 alleles with frequencies between 5 and 95% in at least one of the mtDNA lineages HE, A, FIS, allele frequencies of 29 alleles with frequencies between 5 and 95% in at least one of the mtDNA lineages HE

7

3

Quantitative traits Trials at southwestern France Cestas 6 Isaac

5

5

1

Lacanau

4

3

1

HT9, CIR9, VOL9, ECV9, cvHT9, cvCIR9, cvVOL9, cvECV9 HT9, CIR9, VOL9, ECV9, cvHT9, cvCIR9, cvVOL9, cvECV9 HT9, cvHT9

Trials at central Spain Acebo 12 Caban˜ eros 12 Pen˜ a Negrillas 12 Riofrı´o 12

3 5 3 5

2 2 2 2

HT12, HT12, HT12, HT12,

CIR12, CIR12, CIR12, CIR12,

cvHT12, cvHT12, cvHT12, cvHT12,

cvCIR12 cvCIR12 cvCIR12 cvCIR12

HE ¼ Nei’s gene diversity; A ¼ allele richness; FIS ¼ fixation index; HT9 ¼ total height at age 9; CIR9 ¼ circumference at age 9; VOL9 ¼ volume at age 9; ECV9 ¼ deviation from straightness at age 9; HT12 ¼ total height at age 12; CIR12 ¼ circumference at age 12; a parameter preceded by cv means the phenotypic coefficient of variation of the given parameter, this coefficient was calculated using population means and within-plot standard deviations

zymes, nuSSRS and AFLPs) were used in this study (Table 1). The sample covered the complete native range of the species but allozyme studies were mainly focused on the Iberian Peninsula and eastern Mediterranean range while AFLP studies were focused on western France, Corsica and Portugal. Detailed description of molecular methods and location of populations can be found elsewhere (cpSSRs, Vendramin et al., 1998; Ribeiro et al., 2001; G.G. Vendramin, unpublished results; allozymes, Salvador et al., 2000; Gonza´ lez-Martı´nez, 2001; Barba et al., 2001; nuSSRs, Derory et al., 2002; AFLPs, Mariette et al., 2001; Ribeiro et al., 2002). Six new populations from the Iberian Peninsula (Figueira da Foz, Manteigas, Arenas de San Pedro, San Leonardo de Yagu¨ e, Tabuyo del Monte and Fuencaliente) were analysed in order to complete the wide range nuSSR study of Derory et al. (2002).

Unbiased genetic diversity estimates based on chloroplast haplotype (cpSSRs) or allele (allozymes, nuSSRs and AFLPs) frequency, HE, was computed for each population and molecular marker (Nei, 1987). For AFLP markers, the frequencies of the genotypes (AA, Aa and aa) were deduced assuming that the true value of FIS, the deficiency in heterozygotes, was known (Chong et al., 1994). Available data from codominant markers (in this case nuSSRs with FIS ¼ 0:05; Mariette et al., 2001) was used to correct the unknown deviation from the Hardy– Weinberg equilibrium when dealing with dominant markers (see details in Ribeiro et al., 2002). Nei’s estimator of gene diversity was corrected to account for unequal sample sizes. For allozymes and nuSSRs, allelic richness (A), observed heterozygosity (HO) and fixation index ½FIS ¼ 1  ðHO =HE Þ were also computed.

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2.2. Quantitative traits Quantitative traits were measured in two multi-site provenance tests and three progeny tests located in Portugal, central Spain and southwestern France. The Spanish provenance field tests have been previously described by Alı´a et al. (1995, 1997) and correspond to five trials following a Randomised Complete Block (RCB) design with four replicates and 16 tree-plots. The French provenance tests included three trials planted in 1970; the first site (Cestas) had a Randomised Incomplete Block design (RIB) with 80 blocks of 4 plots and a random composition of provenances per block; the other two sites (Issac and Lacanau) had a RCB design with 10–20 repetitions and a variable number of trees per plot (see Kremer, 1981; Guyon and Kremer, 1982). In central Spain, total height (HT) and diameter at breast height (transformed to circumference to be comparable with French provenance tests, CIR) were measured at age 12 (average total height 5.16 m). Measurements in southwestern France were done at age 9 (average total height 4.17 m) on two trees per plot including the following traits: total height (HT), circumference at breast height (CIR), volume (VOL) and deviation from straightness (ECV). The quantitative variability within a given population was estimated using the coefficient of variation for the phenotypic value (cv). Coefficients of variation were obtained from population means and within-plot standard deviations. This parameter is an overestimation of the additive variance and was suggested as useful when estimating within-population quantitative genetic variation when more suitable data were not available (Kremer, 1994). Narrow-sense heritability estimates (h2), a more adequate estimate of within-population quantitative genetic variation, were based on measures of total height and stem straightness at ages 9–12 in two progeny test and a combined provenance-progeny test located in Portugal (Leiria), central Spain (Valverde del Fresno) and southwestern France (Gironde), respectively. Populations included in the progeny trials were: Leiria (evaluated at Leiria, Portugal), Arenas de San Pedro (evaluated at Valverde del Fresno, central Spain) and Landes and Corsica (evaluated at Gironde, southwestern France). The progeny test in Portugal included 46 open-pollinated families planted in 8 RCB and 8 treeplots (see Aguiar et al., 2003). In central Spain, 87

107

open-pollinated families were planted in seven RCB and four tree-line plots (only five blocks used in this analysis). The combined provenance-progeny test at southwestern France included 365 open-pollinated families (179 from Landes and 186 from Corsica) in 28 incomplete blocks with four replicates and 3–100 tree-plots. Plant materials from Arenas de San Pedro and Leiria progeny tests were phenotypically selected for stem straightness and for both growth and stem straightness, respectively. Analyses of progeny tests were carried out using REML multivariate mixed models and standard computation of variance components. 2.3. Correlation among traits Each population/provenance was assigned to a mtDNA type, i.e. maternal lineage (western, eastern or Moroccan) following Burban and Petit (2003). Unbalanced univariate analysis of variance was used to detect significant differences among maternal lineages for different molecular and quantitative traits (Table 1). The Bonferroni correction for multiple tests was applied to determine significance levels (Rice, 1989). A general multivariate ordination method, the canonical discriminant analysis (CDA), was applied to groups based on maternal pools and to both nuclear molecular and quantitative data. This multivariate method maximises the variance that discriminates among groups. For allozymes and nuSSRs, CDA was conducted on allele frequencies of alleles polymorphic at 95% in at least one of the lineages (13 allozyme and 29 nuSSR alleles, respectively). AFLPs were excluded from this and further analyses (unless stated otherwise) due to an incomplete coverage of the range of the species. In the case of quantitative traits, two different analyses were performed for trials established in Spain and France. Four (H9, CIR9, VOL9, ECV9) and two (H12, CIR12) phenotypic traits were, respectively, included in discriminant analyses of French and Spanish provenance tests. Provenance tests located at Isaac and Lacanau (France) were excluded from this analysis due to incomplete datasets. Correlations among (a) quantitative estimates of within-population variation (coefficient of variation, cv, and narrow-sense heritability, h2) and molecular gene diversity (HE) and (b) gene diversity estimates

108

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provided by different kind of molecular markers (including, in this case, also AFLPs) were done using Kendall’s t statistics, a non-parametric correlation coefficient suitable for datasets with a low number of observations. Data were analysed using procedures CORR, GLM and CANDISC of SAS v. 8.0 statistical package.

3. Results Differences between chloroplast haplotype and nuclear-marker allele frequencies among maternal lineages were high. Thirty-three ANOVA tests were significant (P < 0:05) and 17 remained significant when the Bonferroni correction for multiple tests was applied. One chloroplast haplotype, four allozyme allele and 11 nuSSR allele frequencies were different among maternal lineages. Gene diversity, estimated by mean heterozygosity (HE), only showed significant differences among lineages when nuSSRs were used. The alleles and haplotypes that proved to be more useful in distinguishing lineages are shown in Table 2. With respect to quantitative traits, only 6 out of 34 ANOVA tests were significant (P < 0:05) but only one remained significant when the Bonferroni correction for multiple tests was applied. Trees from eastern and Moroccan mtDNA lineages had smaller diameter than trees from the western mtDNA lineage in Cestas provenance test. Differences in diameter growth were also marginally significant (P < 0:10) in two other provenance tests (one in southwestern France and one in central Spain) following the same trend as described above. In addition, differences in total height (without Bonferroni correction) were significant in two provenance tests, Cestas (southwestern France) and Rı´ofrio (central Spain), and marginally significant in other two, Acebo and Caban˜ eros (both located in central

Spain). With respect to total height, provenances from the western mtDNA lineage had, on average, higher growth than provenances from the other lineages. Multivariate ordination of populations based on nuclear molecular markers showed a clear distinction among mtDNA lineages, both with allozymes and nuSSRs, although the latter produced the sharpest differences among groups (Fig. 1). Remarkably, canonical discriminant analysis based on quantitative traits also showed some clustering of provenances sharing a given mtDNA lineage. Differentiation among groups using quantitative traits was not as strong as differentiation among groups using nuclear markers. In particular, the analysis conducted in the provenance tests from central Spain, under Mediterranean conditions, showed an ordination of the lineages along the canonical axis 1 (CANq1; Fig. 2). This result is not surprising, due to the fact that CANq1, which explained 93.7% of the variance, was well correlated with height and circumference growth, and provenances were aligned from Moroccan (class mean ¼ 4:22) to western (class mean ¼ 1:04) lineages; the eastern lineage being positioned between them (class mean ¼ 1:71). No differences in within-population genetic variation (except for nuSSRs) were found for the different maternal lineages, when a wide range of traits (molecular and quantitative) was used. Our study showed no correlation in genetic diversity between molecular markers and quantitative traits, or even amongst different types of molecular markers (cpSSRs, allozymes, nuSSRs and AFLPs). In fact, only allozymes and nuSSRs showed a moderate (0.231), though not significant (P ¼ 0:272), correlation. With respect to adaptive traits, no correlation was detected between the coefficient of phenotypic variation (cv) for different growth and stem form traits (see Table 1) analysed in two environmental conditions (Atlantic

Table 2 Diagnostic loci to distinguish among maternal lineages detected by mtDNA-RFLP patterns and population-average frequency of the marker within the lineage Type of marker

Allele/haplotype code

Average frequency in the western lineage (%)

Average frequency in the eastern lineage (%)

Average frequency in the Moroccan lineage (%)

cpSSRs nuSSRs nuSSRs Allozymes

Haplotype 64 Allele 13 (173 bp) of locus FRPP91 Allele 19 (159 bp) of locus ITPH4516 Allele a of GDH (EC 1.4.1.3)

2 2 5 98

14 58 31 88

21 13 10 100 (fixed)

109

CAN2 (9.84%)

S.C. Gonza´ lez-Martı´nez et al. / Forest Ecology and Management 197 (2004) 103–115

CAN2 (5.76%)

CAN1 (90.16%)

CAN1 (94.24%)

Fig. 1. Canonical discriminant analysis (CDA) of mtDNA lineages based on allozyme (top) and nuSSR (bottom) allele frequencies; circles: western lineage; triangles: eastern lineage; squares: Moroccan lineage.

in southwestern France and Mediterranean in central Spain) and molecular gene diversity (HE), estimated using cpSSRs, allozymes and nuSSRs. A more accurate estimate of quantitative genetic variability, the narrow-sense heritability (h2), also failed to show any correlation with molecular gene diversity, for both total height (Fig. 3) and stem straightness (Fig. 4).

4. Discussion Global oscillations in climate during the quaternary period, including glacial events and warmer interglacials, have strongly influenced the present-day population genetic structure of European temperate forest trees (Hewitt, 1996, 2001; Newton et al., 1999).

Location of former glacial refugia and the description of migration pathways have been conducted for several forest species. In maritime pine, wide range chloroplast variation studies showed three main refugial areas: the Atlantic coast of Portugal, southwestern Iberia and Pantellaria and Sardinia in Italy (Ribeiro, 2001 and references therein; G.G. Vendramin, unpublished results). Salvador et al. (2000) have described the P. pinaster’s inland migration pathways from southern and eastern Iberia using allozyme markers. Burban and Petit (2003) found a private mtDNA haplotype in Morocco, which may be associated with a new putative refugium for the species, and gave further evidence of the existence of glacial refugia in the Iberian Peninsula (related to the western mtDNA lineage) and southern Italy (related to the eastern mtDNA lineage).

S.C. Gonza´ lez-Martı´nez et al. / Forest Ecology and Management 197 (2004) 103–115

CANq2 (12.45%)

110

CANq2 (6.30%)

CANq1 (87.55%)

CANq1 (93.70%)

Fig. 2. Canonical discriminant analysis (CDA) of mtDNA lineages based on quantitative traits measured at southwestern France (top) and central Spain (bottom); circles: western lineage; triangles: eastern lineage; squares: Moroccan lineage.

In this work, we have shown that groups of populations based on mtDNA (maternal) lineages can be distinguished using a wide range of nuclear traits. A high geographical genetic structure was associated with nuclear molecular markers, such as nuSSRs, and a low, but still present, with quantitative traits. This evidence may be understood as the imprint on the nuclear genome left by among-refugia divergence during glacial times, lately eroded by gene flow and natural (or human-mediated) selection. Correlated patterns of differentiation among organelle- and nuclear-based molecular markers are common in forest trees, although differentiation is much higher in cytoplasmic markers (see review in Newton et al., 1999). Latta and Mitton (1997) found concordant groups of populations when using both mtDNA and

RAPD markers in P. flexilis James. In European white oaks, Kremer et al. (2002) found significant differences in allozyme frequencies among four maternal lineages characterised by cpDNA-RFLP patterns. In the present study, a slight concordance was found in P. pinaster between quantitative and molecular variation, which has only been reported in few other forest trees (Pseudotsuga menziesii (Mirb.) Franco, El-Kassaby, 1982; Picea abies (L.) Karst., Lagercrantz and Ryman, 1990; Alnus rubra Bong., Hamman et al., 1998). In wide range species, concordant patterns of molecular and quantitative differentiation have often been associated with clinal structures formed after a rapid glacial spread (Lagercrantz and Ryman, 1990). Long periods of geographical isolation are likely to enhance genetic differentiation not only on uniparen-

1.00 0.80 0.60 0.40

cpSSRs

0.20

nuSSRs

0.00 0.20

0.25

0.30

0.35

0.40

0.45

0.50

Nei's gene diversity

Nei's gene diversity

S.C. Gonza´ lez-Martı´nez et al. / Forest Ecology and Management 197 (2004) 103–115

0.80 0.60 0.40

cpSSRs

0.20

nuSSRs

0.00 0.10

0.15

0.20

0.25

0.30

narrow-sense heritability

0.25 0.20 0.15 0.10 0.05

Allozymes AFLPs

0.00

0.20

0.25

0.30

0.35

0.40

0.45

0.50

narrow-sense heritability

Nei's gene diversity

Nei's gene diversity

1.00

0.05

narrow-sense heritability

111

0.25 0.20 0.15 0.10 0.05

Fig. 3. Correlations between narrow-sense heritability (h ) for total height and Nei’s gene diversity (HE) estimated using chloroplast and nuclear microsatellites (top), and allozymes and AFLPs (bottom).

tally inherited organelle markers but also on the nuclear genome. Genetic differentiation may be diluted over time by two main evolutionary forces: gene flow and natural (or human-mediated) selection (Kremer et al., 2002). Assuming an island model, Ennos (1994) estimated in 24–64 the ratio of pollen to seed flow in four Pinus species, corresponding to a genetic differentiation (FST) sixfold higher in organelle inherited compared to nuclear-inherited markers. In a maritime pine stand located in central Spain, restricted seed gene flow resulted in the clustering of sib-families, whereas no fine-scale structure due to limited pollen flow was found (Gonza´ lez-Martı´nez et al., 2002b, 2003). Additional evidence of high pollen to seed flow ratio in this species arises from differential dispersion of mtDNA (maternally inherited) and cpDNA (paternally inherited) haplotypes (Burban and Petit, 2003). For instance, the pattern of distribution of chlorotype K (see details in Burban and Petit, 2003), fixed in Morocco and also present in the Iberian Peninsula (where no population with a Moroccan mitotype has been found), could reflect

AFLPs

0.00 0.05

2

Allozymes

0.10

0.15

0.20

0.25

0.30

narrow-sense heritability Fig. 4. Correlations between narrow-sense heritability (h2) for stem straightness and Nei’s gene diversity (HE) estimated using chloroplast and nuclear microsatellites (top), and allozymes and AFLPs (bottom).

migration across the Gibraltar straight exclusively through pollen. Different types of selection also appear to have influenced the present-day population genetic structure of maritime pine. Diversifying selection may have resulted in different local adaptive responses of groups spreading from the same glacial refugia. Atlantic provenances of maritime pine have typically high growth, survival rate and salinity tolerance when tested in Atlantic conditions (western France, Danjon, 1994; Portugal, Aguiar et al., 1999). However, survival of Atlantic provenances decreased considerably when tested in Mediterranean conditions (central Spain), where local-adapted provenances displayed similar levels of growth and higher survival rate (Alı´a et al., 1995, 1997). Conversely, Mediterranean provenances tested under Atlantic climates usually show lower performance than Atlantic provenances. Convergent selection can promote similar quantitative adaptive responses under similar environmental pressures, even when populations have a different historical

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background. For instance, many mountain provenances of maritime pine originating in different regions (central Spain, western mtDNA lineage; Corsica; eastern mtDNA lineage) have straight stems. Competition for light and adaptation to snow and dominant winds in mountain environments have seemingly resulted in a similar phenotype. In maritime pine, previous work on correlations among different kind of traits using within-population gene diversity estimates showed positive results at local scales under high gene flow and low environmental variation (Portugal; M.M. Ribeiro, unpublished results) but not in other areas of the species (Gonza´ lez-Martı´nez, 2001; Mariette et al., 2001). At the broader scale of this study, no correlation using within-population genetic diversity parameters was found for quantitative traits and different nuclear molecular markers. Our study is limited by small sample sizes, in particular, narrow-sense heritability estimates, which are difficult to obtain in long-lived organisms such as trees, and might fail to uncover typically weak associations between quantitative and molecular estimates of gene diversity. However, similar studies in species with short life-span (e.g. Daphnia) and large sample sizes have concluded that measures of molecular heterozygosity provide little information about genetic variation for quantitative traits within natural populations (Morgan et al., 2001; Pfrender et al., 2000) and this might also be the case in maritime pine. Considering a neutral model, Mariette et al. (2002) identified three cases where no correlation in gene diversity estimates, when using different molecular makers, is expected: (i) in large populations with extensive gene flow and, consequently, poorly differentiated levels of diversity, (ii) in highly heterogeneous genomes, in particular when a low number of markers are sampled and (iii) in non-equilibrium populations. Temperate forest trees are likely to display low differences in diversity levels due to large population sizes and to the relatively recent colonisation of temperate regions (a few hundreds of generations). Moreover, genetic equilibrium among drift, mutation and migration may not have been reached in most maritime pine populations, since few generations have passed after the postglacial spread (as shown in other species, such as oaks; Kremer, 1994). This might help in explaining the lack of correlation in diversity estimates among different

nuclear molecular markers. Correlations between molecular and quantitative genetic variability estimates are also expected to be weak. Reed and Frankham (2001), in a meta-analysis covering a wide range of organisms, found that the correlation between molecular heterozygosity and phenotypic variance was moderate (0.217) and significantly different from zero. Yet this correlation was not significant when the meta-analysis was reduced to datasets reporting heritability, which is considered the best estimator of adaptive potential. This discrepancy could be explained by the fact that processes affecting the variation in quantitative traits are usually related to natural selection and adaptation to ecological factors, whereas molecular marker diversity is caused by historical factors, usually more homogeneous over large areas. Caution is always required when using molecular studies to design conservation programs (Newton et al., 1999). For instance, Bekessy et al. (2003) found that neutral DNA markers failed to detect an important quantitative genetic divergence related to drought tolerance in the monkey-puzzle tree (Araucaria araucana). Cluster analysis based on RAPD markers grouped populations from eastern and western Andes, suggesting a common gene pool. However, common garden experiments showed high differences between provenances from these two regions in root mass ratio and d13C, both measures being related to adaptation to drought. Therefore, risks of planting failure in A. araucana, undetected by molecular markers, exists when seed is transferred across the Andes. The foregoing study shows a similar pattern in maritime pine where the estimates of within-population gene diversity using different traits were not correlated and different performance in quantitative traits is found within groups scarcely differentiated at the molecular marker level. In maritime pine, the combination of genetic (molecular and quantitative) and ecological data seems mandatory in the definition of conservation programmes (Derory et al., 2002). Fraser and Bernatchez (2001) pointed out that ESUs can be designated using genetic markers alone but in the absence of either evaluation of adaptive divergence or historical isolation, limitations to make recommendations for conserving genetic resources should be acknowledged. In addition, selection of populations for conservation based on allelic richness is preferable to

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selection based on gene diversity estimates (Petit et al., 1998). Following Crandall et al. (2000), maritime pine structuring in maternal lineages, also imprinted in nuclear molecular and quantitative traits, could be considered as an evidence of historical and (partially) recent genetic inexchangeability among lineages. In addition, partial ecological exchangeability and a similar level of within-population gene diversity for each lineage are present in this species. In these cases, a strategy that allows gene flow consistent with current population structure and careful seed transfer among lineages (if any) are recommended (Crandall et al., 2000). Instead of ESUs, Newton et al. (1999) suggested the definition of Management Units (MUs) in forest trees. Potentially, Management Units could be based on any population differentiation detected, irrespectively to the phylogenetic background. As the maternal lineages found in maritime pine can be further subdivided attending to adaptive traits, the definition of within-lineage MUs relating to adaptive traits, such as drought or pest tolerance, are highly recommended. Another conservation issue to consider is the lack of knowledge about maritime pine populations in Morocco. Genetic sampling of this region was poor and based on few populations (typically Tamjout and in some cases Tamrabta, both located at the Middle Atlas). Although maritime pine populations in Morocco nowadays accounts for as few as 14,000 ha (0.61% of maritime pine range in the western Mediterranean Basin), they constitute an independent evolutionary lineage as shown by mtDNA-RFLP patterns, and a considerable amount of genetic variation seems to remain in this region.

5. Conclusion Data from various sources, including organelle and nuclear molecular markers and growth traits, allowed the detection of a phylogenetic pattern in maritime pine. Three broad regions: (i) the ‘‘western’’ region including most Iberian Peninsula and continental France, (ii) the ‘‘eastern’’ region including southeastern France, Corsica, Italy, Tunisia and Algeria, and (iii) the ‘‘Moroccan’’ region, restricted to Morocco and two putative hybrid zones: (i) the Gibraltar

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straight, and (ii) northeastern Iberia/southeastern France, could be differentiated. Diagnostic loci that allow to distinguish among regions using a variety of markers (cpSSRs, allozymes, and nuSSRs) have been identified. Estimation of genetic diversity using molecular markers was not useful to define conservation strategies in this species. Instead, specific strategies within broader regions using differentiation measures to detect singular populations and the definition of Management Units based on adaptive traits, such as drought or pest resistance, were recommended. To achieve this aim, more genetic information, principally about adaptive traits and the genes underlying these traits but also about molecular markers in regions incompletely sampled (e.g. Morocco), would be needed. Detailed studies of gene flow in the hybrid zones would allow knowing the present-day levels of gene flow among regions. Finally, the integration of genetic, ecological and cost–benefit information at a supranational scale seems mandatory to define a global programme to preserve forest genetic resources in maritime pine.

Acknowledgements ´ lvaro Soto and Ana A ´ lvarez We are grateful to A who provided unpublished cpSSR data from San Leonardo de Yagu¨ e and Arenas de San Pedro populations. Thanks are extended to P.C. Grant who reviewed the English language. This work was supported by EU sponsored projects (FOSSILVA, EVK2-1999-00015P and INCO, ERBIC-08CT-970200) and grants from France (Ministe`re de l’Agriculture et de la Peˆ cheDERF no. 61.21.04/98 and DERF no. 61.45.0401), Spain (Convenios DGCN-INIA CC95-0097 and CC00-0035, and Proyecto Sectorial SC97-118) and Portugal (Fundac¸a˜ o Calouste Gulbenkian, and projects PIDDAC 238/88 and 214/99).

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