Phenological match drives pollen-mediated gene flow in a temporally dimorphic tree.

Variation in flowering phenology is common in natural populations, and is expected to be, together with inter-mate distance, an important driver of effective pollen dispersal. In populations composed of plants with temporally separated sexual phases (i.e. dichogamous or heterodichogamous populations), pollen-mediated gene flow is assumed to reflect phenological overlap between complementary sexual phases. In this study, we conducted paternity analyses to test this hypothesis in the temporally dimorphic tree Acer opalus. We performed spatially explicit analyses based on categorical and fractional paternity assignment, and included tree size, pair-wise genetic relatedness and morph type as additional predictors. Because differences between morphs in flowering phenology may also influence pollination distances, we modelled separate pollen dispersal kernels for the two morphs. Extended phenological overlap between male and female phases (mainly associated with inter-morph crosses) resulted in higher siring success after accounting for the effects of genetic relatedness, morph type and tree size, while reduced phenological overlap (mainly associated with intra-morph crosses) resulted in longer pollination distances achieved. Siring success also increased in larger trees. Mating patterns could not be predicted by phenology alone. However, as heterogeneity in flowering phenology was the single morph-specific predictor of siring success, it is expected to be key in maintaining the temporal dimorphism in A. opalus, by promoting not only a prevalent pattern of inter-morph mating, but also long-distance pollination resulting from intra-morph mating events.

High-density SNP assay development for genetic analysis in maritime pine (Pinus pinaster).

Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.

Field heritability of a plant adaptation to fire in heterogeneous landscapes.

The strong association observed between fire regimes and variation in plant adaptations to fire suggests a rapid response to fire as an agent of selection. It also suggests that fire-related traits are heritable, a precondition for evolutionary change. One example is serotiny, the accumulation of seeds in unopened fruits or cones until the next fire, an important strategy for plant population persistence in fire-prone ecosystems. Here, we evaluate the potential of this trait to respond to natural selection in its natural setting. For this, we use a SNP marker approach to estimate genetic variance and heritability of serotiny directly in the field for two Mediterranean pine species. Study populations were large and heterogeneous in climatic conditions and fire regime. We first estimated the realized relatedness among trees from genotypes, and then partitioned the phenotypic variance in serotiny using Bayesian animal models that incorporated environmental predictors. As expected, field heritability was smaller (around 0.10 for both species) than previous estimates under common garden conditions (0.20). An estimate on a subset of stands with more homogeneous environmental conditions was not different from that in the complete set of stands, suggesting that our models correctly captured the environmental variation at the spatial scale of the study. Our results highlight the importance of measuring quantitative genetic parameters in natural populations, where environmental heterogeneity is a critical aspect. The heritability of serotiny, although not high, combined with high phenotypic variance within populations, confirms the potential of this fire-related trait for evolutionary change in the wild.

Fine-scale spatial genetic dynamics over the life cycle of the tropical tree Prunus africana.

Studying fine-scale spatial genetic patterns across life stages is a powerful approach to identify ecological processes acting within tree populations. We investigated spatial genetic dynamics across five life stages in the insect-pollinated and vertebrate-dispersed tropical tree Prunus africana in Kakamega Forest, Kenya. Using six highly polymorphic microsatellite loci, we assessed genetic diversity and spatial genetic structure (SGS) from seed rain and seedlings, and different sapling stages to adult trees. We found significant SGS in all stages, potentially caused by limited seed dispersal and high recruitment rates in areas with high light availability. SGS decreased from seed and early seedling stages to older juvenile stages. Interestingly, SGS was stronger in adults than in late juveniles. The initial decrease in SGS was probably driven by both random and non-random thinning of offspring clusters during recruitment. Intergenerational variation in SGS could have been driven by variation in gene flow processes, overlapping generations in the adult stage or local selection. Our study shows that complex sequential processes during recruitment contribute to SGS of tree populations.

First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

Admixture mapping of quantitative traits in Populus hybrid zones: power and limitations.

Uncovering the genetic architecture of species differences is of central importance for understanding the origin and maintenance of biological diversity. Admixture mapping can be used to identify the number and effect sizes of genes that contribute to the divergence of ecologically important traits, even in taxa that are not amenable to laboratory crosses because of their long generation time or other limitations. Here, we apply admixture mapping to naturally occurring hybrids between two ecologically divergent Populus species. We map quantitative trait loci for eight leaf morphological traits using 77 mapped microsatellite markers from all 19 chromosomes of Populus. We apply multivariate linear regression analysis allowing the modeling of additive and non-additive gene action and identify several candidate genomic regions associated with leaf morphology using an information-theoretic approach. We perform simulation studies to assess the power and limitations of admixture mapping of quantitative traits in natural hybrid populations for a variety of genetic architectures and modes of gene action. Our results indicate that (1) admixture mapping has considerable power to identify the genetic architecture of species differences if sample sizes and marker densities are sufficiently high, (2) modeling of non-additive gene action can help to elucidate the discrepancy between genotype and phenotype sometimes seen in interspecific hybrids, and (3) the genetic architecture of leaf morphological traits in the studied Populus species involves complementary and overdominant gene action, providing the basis for rapid adaptation of these ecologically important forest trees.

The ancient tropical rainforest tree Symphonia globulifera L. f. (Clusiaceae) was not restricted to postulated Pleistocene refugia in Atlantic Equatorial Africa.

Understanding the history of forests and their species' demographic responses to past disturbances is important for predicting impacts of future environmental changes. Tropical rainforests of the Guineo-Congolian region in Central Africa are believed to have survived the Pleistocene glacial periods in a few major refugia, essentially centred on mountainous regions close to the Atlantic Ocean. We tested this hypothesis by investigating the phylogeographic structure of a widespread, ancient rainforest tree species, Symphonia globulifera L. f. (Clusiaceae), using plastid DNA sequences (chloroplast DNA [cpDNA], psbA-trnH intergenic spacer) and nuclear microsatellites (simple sequence repeats, SSRs). SSRs identified four gene pools located in Benin, West Cameroon, South Cameroon and Gabon, and São Tomé. This structure was also apparent at cpDNA. Approximate Bayesian Computation detected recent bottlenecks approximately dated to the last glacial maximum in Benin, West Cameroon and São Tomé, and an older bottleneck in South Cameroon and Gabon, suggesting a genetic effect of Pleistocene cycles of forest contraction. CpDNA haplotype distribution indicated wide-ranging long-term persistence of S. globulifera both inside and outside of postulated forest refugia. Pollen flow was four times greater than that of seed in South Cameroon and Gabon, which probably enabled rapid population recovery after bottlenecks. Furthermore, our study suggested ecotypic differentiation-coastal or swamp vs terra firme-in S. globulifera. Comparison with other tree phylogeographic studies in Central Africa highlighted the relevance of species-specific responses to environmental change in forest trees.

Nuclear microsatellites for Pinus pinea (Pinaceae), a genetically depauperate tree, and their transferability to P. halepensis.

PREMISE OF THE STUDY: Pinus pinea is one of the few widespread plant species that are also genetically depauperate. It is also an important commercial species with high market value seeds. A deeper knowledge of the existing population genetic variation was needed. METHODS AND RESULTS: Twelve nuclear microsatellites were isolated from genomic and cDNA sequences and screened for variability in 729 individuals from 33 natural populations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11. Hardy-Weinberg equilibrium expectations were not met in only ∼10% of the possible locus/population combinations. All loci were in linkage equilibrium, and the frequency of null alleles was very low (≤1% in 332 out of 396 locus/population combinations). Nine out of the 12 microsatellites were successfully transferred to P. halepensis. CONCLUSIONS: Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability.

Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster.

BACKGROUND AND AIMS: The study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered. METHODS: Male and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers. KEY RESULTS: Trees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size. CONCLUSIONS: The study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.

Recent population decline and selection shape diversity of taxol-related genes.

Taxanes are defensive metabolites produced by Taxus species (yews) and used in anticancer therapies. Despite their medical interest, patterns of natural diversity in taxane-related genes are unknown. We examined variation at five main genes of Taxus baccata in the Iberian Peninsula, a region where unique yew genetic resources are endangered. We looked at several gene features and applied complementary neutrality tests, including diversity/divergence tests, tests solely based on site frequency spectrum (SFS) and Zeng's compound tests. To account for specific demography, microsatellite data were used to infer historical changes in population size based on an Approximate Bayesian Computation (ABC) approach. Polymorphism-divergence tests pointed to positive selection for genes TBT and TAT and balancing selection for DBAT. In addition, neutrality tests based on SFS found that while a recent reduction in population size may explain most statistics' values, selection may still be in action in genes TBT and DBAT, at least in some populations. Molecular signatures on taxol genes suggest the action of frequent selective waves with different direction or intensity, possibly related to varying adaptive pressures produced by the host-enemy co-evolution on defence-related genes. Such natural selection processes may have produced taxane variants still undiscovered.

The Atlantic-Mediterranean watershed, river basins and glacial history shape the genetic structure of Iberian poplars.

Recent phylogeographic studies have elucidated the effects of Pleistocene glaciations and of Pre-Pleistocene events on populations from glacial refuge areas. This study investigates those effects in riparian trees (Populus spp.), whose particular features may convey enhanced resistance to climate fluctuations. We analysed the phylogeographic structure of 44 white (Populus alba), 13 black (Populus nigra) and two grey (Populus x canescens) poplar populations in the Iberian Peninsula using plastid DNA microsatellites and sequences. We also assessed fine-scale spatial genetic structure and the extent of clonality in four white and one grey poplar populations using nuclear microsatellites and we determined quantitative genetic differentiation (Q(ST) ) for growth traits in white poplar. Black poplar displayed higher regional diversity and lower differentiation than white poplar, reflecting its higher cold-tolerance. The dependence of white poplar on phreatic water was evidenced by strong differentiation between the Atlantic and Mediterranean drainage basins and among river basins, and by weaker isolation by distance within than among river basins. Our results suggest confinement to the lower river courses during glacial periods and moderate interglacial gene exchange along coastlines. In northern Iberian river basins, white poplar had lower diversity, fewer private haplotypes and larger clonal assemblies than in southern basins, indicating a stronger effect of glaciations in the north. Despite strong genetic structure and frequent asexual propagation in white poplar, some growth traits displayed adaptive divergence between drainage and river basins (Q(ST) >F(ST)), highlighting the remarkable capacity of riparian tree populations to adapt to regional environmental conditions.

Genetic analysis of post-mating reproductive barriers in hybridizing European Populus species.

Molecular genetic analyses of experimental crosses provide important information on the strength and nature of post-mating barriers to gene exchange between divergent populations, which are topics of great interest to evolutionary geneticists and breeders. Although not a trivial task in long-lived organisms such as trees, experimental interspecific recombinants can sometimes be created through controlled crosses involving natural F(1)'s. Here, we used this approach to understand the genetics of post-mating isolation and barriers to introgression in Populus alba and Populus tremula, two ecologically divergent, hybridizing forest trees. We studied 86 interspecific backcross (BC(1)) progeny and >350 individuals from natural populations of these species for up to 98 nuclear genetic markers, including microsatellites, indels and single nucleotide polymorphisms, and inferred the origin of the cytoplasm of the cross with plastid DNA. Genetic analysis of the BC(1) revealed extensive segregation distortions on six chromosomes, and >90% of these (12 out of 13) favored P. tremula donor alleles in the heterospecific genomic background. Since selection was documented during early diploid stages of the progeny, this surprising result was attributed to epistasis, cyto-nuclear coadaptation, heterozygote advantage at nuclear loci experiencing introgression or a combination of these. Our results indicate that gene flow across 'porous' species barriers affects these poplars and aspens beyond neutral, Mendelian expectations and suggests the mechanisms responsible. Contrary to expectations, the Populus sex determination region is not protected from introgression. Understanding the population dynamics of the Populus sex determination region will require tests based on natural interspecific hybrid zones.

The Strait of Gibraltar as a major biogeographic barrier in Mediterranean conifers: a comparative phylogeographic survey.

The Strait of Gibraltar (SG) is reputed for being both a bridge and a geographic barrier to biological exchanges between Europe and Africa. Major genetic breaks associated with this strait have been identified in various taxa, but it is unknown whether these disjunctions have been produced simultaneously or by independent biogeographic processes. Here, the genetic structure of five conifers distributed on both sides of the SG was investigated using mitochondrial (nad1 b/c, nad5-1, nad5-4 and nad7-1) and chloroplast (Pt1254, Pt15169, Pt30204, Pt36480, Pt71936 and Pt87268) DNA markers. The distribution of genetic variation was partially congruent between types of markers within the same species. Across taxa, there was a significant overlapping between the SG and the genetic breaks detected, especially for the four Tertiary species surveyed (Abies pinsapo complex, Pinus nigra, Pinus pinaster and Taxus baccata). For most of these taxa, the divergence of populations across the SG could date back to long before the Pleistocene glaciations. However, their strongly different cpDNA G(ST) and R(ST) values point out that they have had dissimilar population histories, which might include contrasting amounts of pollen-driven gene flow since their initial establishment in the region. The fifth species, Pinus halepensis, was genetically depauperated and homogenous on both sides of the SG. A further analysis of nuclear DNA sequences with coalescent-based isolation with migration models suggests a Pleistocene divergence of P. halepensis populations across the SG, which is in sharp contrast with the pre-Pleistocene divergence dates obtained for P. pinaster. Altogether, these results indicate that the genetic breaks observed across this putative biogeographical barrier have been produced by independent evolutionary processes related to the biological history of each individual species instead of a common vicariant phenomenon.

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view.

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long-term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species-specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold-enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold-tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.

sofsog: a suite of programs to avoid inbreeding in plantation designs.

Cost-effective ways of controlling inbreeding in conservation or productive plantations imply the allocation of individuals reducing the possibility of close relatives' mating and, consequently, limiting inbreeding. sofsog is a suite of programs, which helps to design plantation sites. First, if the plantation scheme involves several plots, it allows distribution of individuals available among different sites minimizing within-site global coancestry. Then, it yields a plantation design for each site, either following the classical permutated neighbourhood strategy or the recently developed method by Fernández and González-Martínez. This new method allows the implementation of different pollen dispersion kernels, and to include in the designing strategy any available information on individual relationships, reproductive success, differences in phenology, etc., via weighting or penalization matrices. Additionally, the package includes a tool for calculating the molecular coancestry (Identity By State) from codominant marker data.

Spatial genetic structure in continuous and fragmented populations of Pinus pinaster Aiton.

Habitat fragmentation, i.e., the reduction of populations into small isolated remnants, is expected to increase spatial genetic structure (SGS) in plant populations through nonrandom mating, lower population densities and potential aggregation of reproductive individuals. We investigated the effects of population size reduction and genetic isolation on SGS in maritime pine (Pinus pinaster Aiton) using a combined experimental and simulation approach. Maritime pine is a wind-pollinated conifer which has a scattered distribution in the Iberian Peninsula as a result of forest fires and habitat fragmentation. Five highly polymorphic nuclear microsatellites were genotyped in a total of 394 individuals from two population pairs from the Iberian Peninsula, formed by one continuous and one fragmented population each. In agreement with predictions, SGS was significant and stronger in fragments (Sp = 0.020 and Sp = 0.026) than in continuous populations, where significant SGS was detected for one population only (Sp = 0.010). Simulations suggested that under fat-tailed dispersal, small population size is a stronger determinant of SGS than genetic isolation, while under normal dispersal, genetic isolation has a stronger effect. SGS was always stronger in real populations than in simulations, except if unrealistically narrow dispersal and/or high variance of reproductive success were modelled (even when accounting for potential overestimation of SGS in real populations as a result of short-distance sampling). This suggests that factors such as nonrandom mating or selection not considered in the simulations were additionally operating on SGS in Iberian maritime pine populations.

Estimating gametic introgression rates in a risk assessment context: a case study with Scots pine relicts.

The estimation of recent gene immigration is fundamental to a wide range of evolutionary and conservation studies. In a risk assessment context, gene flow estimation procedures are needed that are both accurate and readily amenable to formal evaluation of statistical uncertainty. However, genetic methods for estimating recent migration rates that are specific and have been thoroughly evaluated are scarce. Here we use an original and straightforward maximum-likelihood method to estimate recent uniparental gametic immigration from non-local plantations into an endangered population of the Iberian relict pine variety Pinus sylvestris var. nevadensis D. H. Christ. Our approach is not intended to ascertain population membership of individuals, but rather to obtain accurate immigration rate estimates with reliable confidence limits. We found very high (approximately 40%) pollen introgression at the seed-crop level into the Scots pine relict, and substantial (10-15%) male gametic introgression among naturally regenerated recruits. Using numerical simulation, we show that our method yields uniparental gametic immigration estimates that are expected to be virtually unbiased and usually accurate under our sampling conditions. Among four tested methods to estimate the confidence intervals for immigration estimates, the profile-likelihood method was the best, as it outperformed bootstrapping procedures and yielded coverage close to nominal limits under different sample sizes and migration rates. This study presents a method by which researchers can facilitate decision making within a gene flow risk assessment context.

Is Cupressus sempervirens native in Italy? An answer from genetic and palaeobotanical data.

This study represents the first large-scale analysis using nuclear molecular markers to assess genetic diversity and structure of Cupressus sempervirens L.. Genetic and fossil data were combined to infer the possible role of human activity and evolutionary history in shaping the diversity of cypress populations. We analysed 30 populations with six polymorphic nuclear microsatellite markers. Dramatic reductions in heterozygosity and allelic richness were observed from east to west across the species range. Structure analysis assigned individuals to two main groups separating central Mediterranean and eastern populations. The two main groups could be further divided into five subgroups which showed the following geographical distributions: Turkey with the Greek islands Rhodes and Samos, Greece (Crete), Southern Italy, Northern Italy, Tunisia with Central Italy. This pattern of genetic structure is also supported by SAMOVA and Barrier analyses. Palaeobotanical data indicated that Cupressus was present in Italy in the Pliocene, Pleistocene and Holocene. Furthermore, our molecular survey showed that Italian cypress populations experienced bottlenecks that resulted in reduced genetic diversity and allelic richness and greater genetic differentiation. Recent colonization or introduction may also have influenced levels of diversity detected in the Italian populations, as most individuals found in this range today have multilocus genotypes that are also present in the eastern range of the species. The data reveal a new interpretation of the history of cypress distribution characterized by ancient eastern populations (Turkey and Greek islands) and a mosaic of recently introduced trees and remnants of ancient, depauperate populations in the central Mediterranean range.

Association genetics in Pinus taeda L. II. Carbon isotope discrimination.

Dissection of complex traits that influence fitness is not only a central topic in evolutionary research but can also assist breeding practices for economically important plant species, such as loblolly pine (Pinus taeda L). In this study, 46 single nucleotide polymorphisms (SNPs) from 41 disease and abiotic stress-inducible genes were tested for their genetic association with carbon isotope discrimination (CID), a time-integrated trait measure of stomatal conductance. A family-based approach to detect genotype/phenotype genetic association was developed for the first time in plants by applying the quantitative transmission disequilibrium test on an association population of 961 clones from 61 families (adopted from previous breeding programs) evaluated for phenotypic expression of CID at two sites. Two particularly promising candidates for their genetic effects on CID are: dhn-1, involved in stabilization of cell structures, and lp5-like, a glycine rich protein putatively related to cell wall reinforcement proteins, both of which were shown in previous studies to be water-deficit inducible. Moreover, association in lp5-like involves a nonsynonymous mutation in linkage disequilibrium with two other nonsynonymous polymorphisms that could, by acting together, enhance overall phenotypic effects. This study highlights the complexity of dissecting CID traits and provides insights for designing second-generation association studies based on candidate gene approaches in forest trees.

Mating system and pollen gene flow in Mediterranean maritime pine.

Mating systems define the mode of gene transmission across generations, helping to determine the amount and distribution of genetic variation within and among populations of plant species. A hierarchical analysis of Mediterranean maritime pine mating system (61 mother trees from 24 plots, clustered in three populations) was used to identify factors affecting mating patterns and to fit pollen dispersal kernels. Levels of ovule and seed abortion, multi- and single-locus outcrossing rates and correlated paternity were estimated from progeny arrays and correlated with ecological stand variables and biometric tree measures. Pollen dispersal kernels were fitted using TwoGener and KinDist indirect methods and simulations were carried out to identify relevant factors affecting correlated paternity. Maritime pine showed high outcrossing rates (t(m) and t(s) approximately 0.96) and relatively low levels of correlated paternity [an r(p) of 0.018 (Ritland's estimate) or 0.048 (Hardy's estimate)], although higher than in other anemophilous tree species. Mating system parameters had high variation at the single-tree level (99-100%) but no stand or population effect was detected. At the single-tree level, outcrossing rates were correlated with tree (diameter and height) and crown size. In addition, correlated paternity showed a significant negative correlation with tree height, height to crown base and height to the largest crown width, probably reflecting the importance of the trees' 'ecological neighborhoods'. Indirectly estimated pollen dispersal kernels were very leptokurtic (exponential-power distributions with beta<<0.5), with mean dispersal distances from 78.4 to 174.4 m. Fitted dispersal kernels will be useful in building explicit simulation models that include dispersal functions, and which will contribute to current conservation and management programs for maritime pine. Nevertheless, the numerical simulations showed that restricted dispersal, male fertility and phenological overlap could only partially explain the observed levels of correlated paternity; so other factors may also be relevant for the management of this valuable forest tree species.