Santiago C. González-Martínez

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.

Capturing neutral and adaptive genetic diversity for conservation in a highly structured tree species

Preserving intraspecific genetic diversity is essential for long-term forest sustainability in a climate change scenario. Despite that, genetic information is largely neglected in conservation planning, and how conservation units should be defined is still heatedly debated. Here, we use maritime pine (Pinus pinaster Ait.), an outcrossing long-lived tree with a highly fragmented distribution in the Mediterranean biodiversity hotspot, to prove the importance of accounting for genetic variation, of both neutral molecular markers and quantitative traits, to define useful conservation units. Six gene pools associated to distinct evolutionary histories were identified within the species using 12 microsatellites and 266 single nucleotide polymorphisms (SNPs). In addition, height and survival standing variation, their genetic control, and plasticity were assessed in a multisite clonal common garden experiment (16 544 trees). We found high levels of quantitative genetic differentiation within previously defined neutral gene pools. Subsequent cluster analysis and post hoc trait distribution comparisons allowed us to define 10 genetically homogeneous population groups with high evolutionary potential. They constitute the minimum number of units to be represented in a maritime pine dynamic conservation program. Our results uphold that the identification of conservation units below the species level should account for key neutral and adaptive components of genetic diversity, especially in species with strong population structure and complex evolutionary histories. The environmental zonation approach currently used by the pan-European genetic conservation strategy for forest trees would be largely improved by gradually integrating molecular and quantitative trait information, as data become available.

Advances in ecological genomics in forest trees and applications to genetic resources conservation and breeding

Forest trees are an unparalleled group of organisms in their combined ecological, economic and societal importance. With widespread distributions, predominantly random mating systems and large population sizes, most tree species harbour extensive genetic variation both within and among populations. At the same time, demographic processes associated with Pleistocene climate oscillations and land‐use change have affected contemporary range‐wide diversity and may impinge on the potential for future adaptation. Understanding how these adaptive and neutral processes have shaped the genomes of trees species is therefore central to their management and conservation. As for many other taxa, the advent of high‐throughput sequencing methods is expected to yield an understanding of the interplay between the genome and environment at a level of detail and depth not possible only a few years ago. An international conference entitled ‘Genomics and Forest Tree Genetics’ was held in May 2016, in Arcachon (France), and brought together forest geneticists with a wide range of research interests to disseminate recent efforts that leverage contemporary genomic tools to probe the population, quantitative and evolutionary genomics of trees. An important goal of the conference was to discuss how such data can be applied to both genome‐enabled breeding and the conservation of forest genetic resources under land use and climate change. Here, we report discoveries presented at the meeting and discuss how the ecological genomic toolkit can be used to address both basic and applied questions in tree biology.

Adaptation and plasticity in aboveground allometry variation of four pine species along environmental gradients

Abstract Plant species aboveground allometry can be viewed as a functional trait that reflects the evolutionary trade‐off between above‐ and belowground resources. In forest trees, allometry is related to productivity and resilience in different environments, and it is tightly connected with a compromise between efficiency‐safety and competitive ability. A better understanding on how this trait varies within and across species is critical to determine the potential of a species/population to perform along environmental gradients. We followed a hierarchical framework to assess tree height‐diameter allometry variation within and across four common European Pinus species. Tree height‐diameter allometry variation was a function of solely genetic components –approximated by either population effects or clinal geographic responses of the populations site of origin– and differential genetic plastic responses –approximated by the interaction between populations and two climatic variables of the growing sites (temperature and precipitation)–. Our results suggest that, at the species level, climate of the growing sites set the tree height‐diameter allometry of xeric and mesic species (Pinus halepensis, P. pinaster and P. nigra) apart from the boreal species (P. sylvestris), suggesting a weak signal of their phylogenies in the tree height‐diameter allometry variation. Moreover, accounting for interpopulation variability within species for the four pine species aided to: (1) detect genetic differences among populations in allometry variation, which in P. nigra and P. pinaster were linked to gene pools –genetic diversity measurements–; (2) reveal the presence of differential genetic variation in plastic responses along two climatic gradients in tree allometry variation. In P. sylvestris and P. nigra, genetic variation was the result of adaptive patterns to climate, while in P. pinaster and P. halepensis, this signal was either weaker or absent, respectively; and (3) detect local adaptation in the exponent of the tree height‐diameter allometry relationship in two of the four species (P. sylvestris and P. nigra), as it was a function of populations latitude and altitude variables. Our findings suggest that the four species have been subjected to different historical and climatic constraints that might have driven their aboveground allometry and promoted different life strategies.

Altitudinal gradients, biogeographic history and microhabitat adaptation affect fine-scale spatial genetic structure in African and Neotropical populations of an ancient tropical tree species

The analysis of fine-scale spatial genetic structure (FSGS) within populations can provide insights into eco-evolutionary processes. Restricted dispersal and locally occurring genetic drift are the primary causes for FSGS at equilibrium, as described in the isolation by distance (IBD) model. Beyond IBD expectations, spatial, environmental or historical factors can affect FSGS. We examined FSGS in seven African and Neotropical populations of the late-successional rain forest tree Symphonia globulifera L. f. (Clusiaceae) to discriminate the influence of drift-dispersal vs. landscape/ecological features and historical processes on FSGS. We used spatial principal component analysis and Bayesian clustering to assess spatial genetic heterogeneity at SSRs and examined its association with plastid DNA and habitat features. African populations (from Cameroon and São Tomé) displayed a stronger FSGS than Neotropical populations at both marker types (mean Sp = 0.025 vs. Sp = 0.008 at SSRs) and had a stronger spatial genetic heterogeneity. All three African populations occurred in pronounced altitudinal gradients, possibly restricting animal-mediated seed dispersal. Cyto-nuclear disequilibria in Cameroonian populations also suggested a legacy of biogeographic history to explain these genetic patterns. Conversely, Neotropical populations exhibited a weaker FSGS, which may reflect more efficient wide-ranging seed dispersal by Neotropical bats and other dispersers. The population from French Guiana displayed an association of plastid haplotypes with two morphotypes characterized by differential habitat preferences. Our results highlight the importance of the microenvironment for eco-evolutionary processes within persistent tropical tree populations.

Spatial genetic structure of an explicit glacial refugium of maritime pine (Pinus pinaster Aiton) insoutheastern Spain

The Iberian Peninsula has been described as a glacial refugium for numerous organisms. In particular, there is evidence both from pollen records and genetic studies that shows the existence of Mediterranean conifers (Pinus halepensis Miller, Pinus pinaster Aiton, and Pinus pinea L) in southeastern Spain during the last glacial stage. Data from eight polymorphic allozyme markers were used to study the spatial genetic structure of 11 native populations of maritime pine, P. pinaster, in this region. Models of isolation by distance were adjusted to different groups of populations to test specific hypotheses about the role of mountain ranges in shaping the spatial genetic structure of maritime pine in southeastern Spain. In addition, pairwise gene interchange was analyzed using migration matrix models and maximum likelihood methods to make joint estimates of dispersal rates and population sizes. A complex pattern in the distribution of gene diversity was found, involving historical isolation due to geographical variables for particular populations. The role of mountain ranges in glacial refugia i) reducing the risk of a population bottleneck by altitudinal migration in response to climatic change, and ii) acting as geographical barriers to gene flow, is discussed.

Biogeography and evolution of seeder and resprouter forms of Erica coccinea (Ericaceae) in the fire-prone Cape fynbos

The genus Erica represents the epitome of plant biodiversity in the South African Cape fynbos with over 700 species. This genus is composed of seeder and resprouter species, but both species diversity and endemism are strongly linked to the seeder habit and concentrated in the southwestern Cape Floristic Region (CFR). Erica coccinea is a relatively abundant and widespread fynbos species whose most remarkable morphological feature is the existence of distinct seeder and resprouter forms, frequently—but not always—in disjunct populations. Both higher within-population genetic diversity and among-population differentiation have been found in seeders, most likely as a consequence of the shorter generation times and faster population turnovers. Resprouters, despite being less diverse, are suspected to be ancestral. However, no solid evidence has yet been provided for the ancestrality of the resprouter form, or for the demographic processes that have determined the current distribution of genetic diversity in both regeneration forms. Here, we used microsatellites and sequences of the nuclear ribosomal internal transcribed spacers to describe the phylogeographic structure of seeder and resprouter E. coccinea populations and provide good evidence for the ancestral status of the resprouter form and the comparatively high rates of molecular evolution in derived seeder populations. We also reveal that mixed populations, where both seeder and resprouter individuals co-occur, were originated by secondary contacts. This study highlights the role of fire in driving accelerated diversification in seeder lineages of highly speciose CFR fynbos taxa.

Nucleotide polymorphisms in a pine ortholog of the Arabidopsis degrading enzyme cellulase KORRIGAN are associated with early growth performance in Pinus pinaster

We have carried out a candidate-gene-based association genetic study in Pinus pinaster Aiton and evaluated the predictive performance for genetic merit gain of the most significantly associated genes and single nucleotide polymorphisms (SNPs). We used a second generation 384-SNP array enriched with candidate genes for growth and wood properties to genotype mother trees collected in 20 natural populations covering most of the European distribution of the species. Phenotypic data for total height, polycyclism, root-collar diameter and biomass were obtained from a replicated provenance-progeny trial located in two sites with contrasting environments (Atlantic vs Mediterranean climate). General linear models identified strong associations between growth traits (total height and polycyclism) and four SNPs from the korrigan candidate gene, after multiple testing corrections using false discovery rate. The combined genomic breeding value predictions assessed for the four associated korrigan SNPs by ridge regression-best linear unbiased prediction (RR-BLUP) and cross-validation accounted for up to 8 and 15% of the phenotypic variance for height and polycyclic growth, respectively, and did not improve adding SNPs from other growth-related candidate genes. For root-collar diameter and total biomass, they accounted for 1.6 and 1.1% of the phenotypic variance, respectively, but increased to 15 and 4.1% when other SNPs from lp3.1, lp3.3 and cad were included in RR-BLUP models. These results point towards a desirable integration of candidate-gene studies as a means to pre-select relevant markers, and aid genomic selection in maritime pine breeding programs.

Local effects drive heterozygosity-fitness correlations in an outcrossing long-lived tree.

Heterozygosity–fitness correlations (HFCs) have been used to understand the complex interactions between inbreeding, genetic diversity and evolution. Although frequently reported for decades, evidence for HFCs was often based on underpowered studies or inappropriate methods, and hence their underlying mechanisms are still under debate. Here, we used 6100 genome-wide single nucleotide polymorphisms (SNPs) to test for general and local effect HFCs in maritime pine (Pinus pinaster Ait.), an iconic Mediterranean forest tree. Survival was used as a fitness proxy, and HFCs were assessed at a four-site common garden under contrasting environmental conditions (total of 16 288 trees). We found no significant correlations between genome-wide heterozygosity and fitness at any location, despite variation in inbreeding explaining a substantial proportion of the total variance for survival. However, four SNPs (including two non-synonymous mutations) were involved in significant associations with survival, in particular in the common gardens with higher environmental stress, as shown by a novel heterozygosity–fitness association test at the species-wide level. Fitness effects of SNPs involved in significant HFCs were stable across maritime pine gene pools naturally growing in distinct environments. These results led us to dismiss the general effect hypothesis and suggested a significant role of heterozygosity in specific candidate genes for increasing fitness in maritime pine. Our study highlights the importance of considering the species evolutionary and demographic history and different spatial scales and testing environments when assessing and interpreting HFCs.

Inferring selection in instances of long-range colonization: The Aleppo pine (Pinus halepensis) in the Mediterranean Basin

Teasing apart the effects of natural selection and demography on current allele frequencies is challenging, due to both processes leaving a similar molecular footprint. In particular, when attempting to identify selection in species that have undergone a recent range expansion, the increase in genetic drift at the edges of range expansions (“allele surfing”) can be a confounding factor. To address this potential issue, we first assess the long‐range colonization history of the Aleppo pine across the Mediterranean Basin, using molecular markers. We then look for single nucleotide polymorphisms (SNPs) involved in local adaptation using: (a) environmental correlation methods (bayenv2), focusing on bioclimatic variables important for the species’ adaptation (i.e., temperature, precipitation and water availability); and (b) FST‐related methods (pcadapt). To assess the rate of false positives caused by the allele surfing effect, these results are compared with results from simulated SNP data that mimics the species’ past range expansions and the effect of genetic drift, but with no selection. We find that the Aleppo pine shows a previously unsuspected complex genetic structure across its range, as well as evidence of selection acting on SNPs involved with the response to bioclimatic variables such as drought. This study uses an original approach to disentangle the confounding effects of drift and selection in range margin populations. It also contributes to the increased evidence that plant populations are able to adapt to new environments despite the expected accumulation of deleterious mutations that takes place during long‐range colonizations.