Bridging the genotype-phenotype gap for a Mediterranean pine by semi‐automatic crown identification and multispectral imagery

Summary: Progress in high‐throughput phenotyping and genomics provides the potential to understand the genetic basis of plant functional differentiation. We developed a semi‐automatic methodology based on unmanned aerial vehicle (UAV) imagery for deriving tree‐level phenotypes followed by genome‐wide association study (GWAS). An RGB‐based point cloud was used for tree crown identification in a common garden of Pinus halepensis in Spain. Crowns were combined with multispectral and thermal orthomosaics to retrieve growth traits, vegetation indices and canopy temperature. Thereafter, GWAS was performed to analyse the association between phenotypes and genomic variation at 235 single nucleotide polymorphisms (SNPs). Growth traits were associated with 12 SNPs involved in cellulose and carbohydrate metabolism. Indices related to transpiration and leaf water content were associated with six SNPs involved in stomata dynamics. Indices related to leaf pigments and leaf area were associated with 11 SNPs involved in signalling and peroxisome metabolism. About 16-20% of trait variance was explained by combinations of several SNPs, indicating polygenic control of morpho‐physiological traits. Despite a limited availability of markers and individuals, this study is provides a successful proof‐of‐concept for the combination of high‐throughput UAV‐based phenotyping with cost‐effective genotyping to disentangle the genetic architecture of phenotypic variation in a widespread conifer.This work was supported by the Spanish Government, grant number RTI2018‐094691‐B‐C31 (MCIU/AEI/FEDER, EU)

Inducibility of Plant Secondary Metabolites in the Stem Predicts Genetic Variation in Resistance Against a Key Insect Herbivore in Maritime Pine

Resistance to herbivores and pathogens is considered a key plant trait with strong adaptive value in trees, usually involving high concentrations of a diverse array of plant secondary metabolites (PSM). Intraspecific genetic variation and plasticity of PSM are widely known. However, their ecology and evolution are unclear, and even the implication of PSM as traits that provide direct effective resistance against herbivores is currently questioned. We used control and methyl jasmonate (MJ) induced clonal copies of genotypes within families from ten populations of the main distribution range of maritime pine to exhaustively characterize the constitutive and induced profile and concentration of PSM in the stem phloem, and to measure insect herbivory damage as a proxy of resistance. Then, we explored whether genetic variation in resistance to herbivory may be predicted by the constitutive concentration of PSM, and the role of its inducibility to predict the increase in resistance once the plant is induced. We found large and structured genetic variation among populations but not between families within populations in resistance to herbivory. The MJ-induction treatment strongly increased resistance to the weevil in the species, and the genetic variation in the inducibility of resistance was significantly structured among populations, with greater inducibility in the Atlantic populations. Genetic variation in resistance was largely explained by the multivariate concentration and profile of PSM at the genotypic level, rather than by bivariate correlations with individual PSM, after accounting for genetic relatedness among genotypes. While the constitutive concentration of the PSM blend did not show a clear pattern of resistance to herbivory, specific changes in the chemical profile and the increase in concentration of the PSM blend after MJ induction were related to increased resistance. To date, this is the first example of a comprehensive and rigorous approach in which inducibility of PSM in trees and its implication in resistance was analyzed excluding spurious associations due to genetic relatedness, often overlooked in intraspecific studies. Here we provide evidences that multivariate analyses of PSM, rather than bivariate correlations, provide more realistic information about the potentially causal relationships between PSM and resistance to herbivory in pine trees

Looking for local adaptation:Convergent microevolution in aleppo pine (pinus halepensis)

Finding outlier loci underlying local adaptation is challenging and is best approached by suitable sampling design and rigorous method selection. In this study, we aimed to detect outlier loci (single nucleotide polymorphisms, SNPs) at the local scale by using Aleppo pine (Pinus halepensis), a drought resistant conifer that has colonized many habitats in the Mediterranean Basin, as the model species. We used a nested sampling approach that considered replicated altitudinal gradients for three contrasting sites. We genotyped samples at 294 SNPs located in genomic regions selected to maximize outlier detection. We then applied three different statistical methodologies-Two Bayesian outlier methods and one latent factor principal component method-To identify outlier loci. No SNP was an outlier for all three methods, while eight SNPs were detected by at least two methods and 17 were detected only by one method. From the intersection of outlier SNPs, only one presented an allelic frequency pattern associated with the elevational gradient across the three sites. In a context of multiple populations under similar selective pressures, our results underline the need for careful examination of outliers detected in genomic scans before considering them as candidates for convergent adaptation

The genetic consequences of population marginality: a case study in maritime pine

•Aim: Marginal tree populations, either those located at the edges of the species' range or in suboptimal environments, are often a valuable genetic resource for biological conservation. However, there is a lack of knowledge about the genetic consequences of population marginality, estimated across entire species' ranges. Our study addresses this gap by providing information about several genetic indicators and their variability in marginal and core populations identified using quantitative marginality indices. •Location: Southwestern Europe and North Africa. •Methods: Using 10,185 SNPs across 82 populations of maritime pine (Pinus pinaster Ait.), a widespread conifer characterised by a fragmented range, we modelled the relationship of seven genetic indicators potentially related to population evolutionary resilience, namely genetic diversity (based on both all SNPs and outlier SNPs), inbreeding, genetic differentiation, recessive genetic load and genomic offset, with population geographical, demo-historical and ecological marginality (as estimated by nine quantitative indices). Models were constructed for both regional (introducing gene pool as a random factor) and range-wide spatial scales. •Results: We showed a trend towards decreasing overall genetic diversity and increasing differentiation with geographic marginality, supporting the centre-periphery hypothesis (CPH). However, we found no correlation between population inbreeding and marginality, while geographically marginal populations had a lower recessive genetic load (only models without the gene pool effect). Ecologically marginal populations had a higher genomic offset, suggesting higher maladaptation to future climate, albeit some of these populations also had high genetic diversity for climate outliers. •Main Conclusions: Overall genetic diversity (but not outlier-based estimates) and differentiation patterns support the CPH. Ecologically marginal populations and those at the southern edge could be more vulnerable to climate change due to higher climate maladaptation, as predicted by genomic offsets, and/or lower potentially adaptive genetic diversity. This risk is exacerbated by typically small effective population sizes and increasing human impact in marginal populations

Foraging patterns of acorn woodpeckers (Melanerpes formicivorus) on valley oak (Quercus lobata Née) in two California oak savanna-woodlands

Landscape characteristics and social behavior can affect the foraging patterns of seed-dependent animals. We examine the movement of acorns from valley oak (Quercus lobata) trees to granaries maintained by acorn woodpeckers (Melanerpes formicivorus) in two California oak savanna-woodlands differing in the distribution of Q. lobata within each site. In 2004, we sampled Q. lobata acorns from 16 granaries at Sedgwick Reserve in Santa Barbara County and 18 granaries at Hastings Reserve in Monterey County. Sedgwick has lower site-wide density of Q. lobata than Hastings as well as different frequencies of other Quercus species common to both sites. We found acorn woodpeckers foraged from fewer Q. lobata seed source trees (Kg = 4.1 ± 0.5) at Sedgwick than at Hastings (Kg = 7.6 ± 0.6) and from fewer effective seed sources (Nem* = 2.00 and 5.78, respectively). The differences between sites are due to a greater number of incidental seed sources used per granary at Hastings than at Sedgwick. We also found very low levels of seed source sharing between adjacent granaries, indicating that territoriality is strong at both sites and that each social group forages on its own subset of trees. We discovered an interesting spatial pattern in the location of granaries. At Sedgwick, acorn woodpeckers situated their granaries within areas of higher-than-average tree density, while at Hastings, they placed them within areas of lower-than-average tree density, with the outcome that granaries at the two sites were located in areas of similar valley oak density. Our results illustrate that landscape characteristics might influence the number of trees visited by acorn woodpeckers and the locations of territories, while woodpecker social behavior, such as territoriality, shapes which trees are visited and whether they are shared with other social groups

Polygenic adaptation and negative selection across traits, years and environments in a long-lived plant species (Pinus pinaster Ait., Pinaceae)

17 Pág.A decade of genetic association studies in multiple organisms suggests that most complex traits are polygenic; that is, they have a genetic architecture determined by numerous loci, each with small effect-size. Thus, determining the degree of polygenicity and its variation across traits, environments and time is crucial to understand the genetic basis of phenotypic variation. We applied multilocus approaches to estimate the degree of polygenicity of fitness-related traits in a long-lived plant (Pinus pinaster Ait., maritime pine) and to analyse this variation across environments and years. We evaluated five categories of fitness-related traits (survival, height, phenology, functional, and biotic-stress response) in a clonal common-garden network planted in contrasted environments (over 20,500 trees). Most of the analysed traits showed evidence of local adaptation based on Qst -Fst comparisons. We further observed a remarkably stable degree of polygenicity, averaging 6% (range of 0%-27%), across traits, environments and years. We detected evidence of negative selection, which could explain, at least partially, the high degree of polygenicity. Because polygenic adaptation can occur rapidly, our results suggest that current predictions on the capacity of natural forest tree populations to adapt to new environments should be revised, especially in the current context of climate change.This study was funded by the Spanish Ministry of Economy and Competitiveness through projects RTA2010-00120-C02- 02 (CLONAPIN), CGL2011-30182- C02- 01 (AdapCon) and AGL2012-40151- C03- 02 (FENOPIN). The study was also supported by the “Initiative d’Excellence (IdEx) de l’Université de Bordeaux - Chaires d'installation 2015” (EcoGenPin) and the European Union's Horizon 2020 research and innovation programme under grant agreement No 773383 (B4EST)Peer reviewe

Marginality indices for biodiversity conservation in forest trees

Marginal and peripheral populations are important for biodiversity conservation. Their original situation in a species’ geographic and ecological space often confers them genetic diversity and traits of high adaptive value. Yet theoretical hypotheses related to marginality are difficult to test because of confounding factors that influence marginality, namely environment, geography, and history. There is an urgent need to develop metrics to disentangle these confounding factors. We designed nine quantitative indices of marginality and peripherality that define where margins lie within species distributions, from a geographical, an environmental and a historical perspective. Using the distribution maps of eight European forest tree species, we assessed whether these indices were idiosyncratic or whether they conveyed redundant information. Using a database on marginal and peripheral populations based on expert knowledge, we assessed the capacity of the indices to predict the marginality status of a population. There was no consistent pattern of correlation between indices across species, confirming that the indices conveyed different information related to the specific geometry of the species distributions. Contrasting with this heterogeneity of correlation patterns across species, the relative importance of the indices to predict the marginality status of populations was consistent across species. However, there was still a significant country effect in the marginality status, showing a variation in expert opinion of marginality vis-á-vis the species distribution. The marginality indices that we developed are entirely based on distribution maps and can be used for any species. They pave the way for testing hypotheses related to marginality and peripherality, with important implications in quantitative ecology, genetics, and biodiversity conservation

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (<it>Pinus pinaster </it>Ait.), the main conifer used for commercial plantation in southwestern Europe.</p> <p>Results</p> <p>We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 <it>in vitro </it>SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 <it>in silico </it>SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for <it>in silico </it>and <it>in vitro </it>SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a <it>Pinus taeda </it>linkage map, made it possible to align the 12 linkage groups of both species.</p> <p>Conclusions</p> <p>Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.</p

The GenTree Platform: growth traits and tree-level environmental data in 12 European forest tree species

Background: Progress in the field of evolutionary forest ecology has been hampered by the huge challenge of phenotyping trees across their ranges in their natural environments, and the limitation in high-resolution environmental information. Findings: The GenTree Platform contains phenotypic and environmental data from 4,959 trees from 12 ecologically and economically important European forest tree species: Abies alba Mill. (silver fir), Betula pendula Roth. (silver birch), Fagus sylvatica L. (European beech), Picea abies (L.) H. Karst (Norway spruce), Pinus cembra L. (Swiss stone pine), Pinus halepensis Mill. (Aleppo pine), Pinus nigra Arnold (European black pine), Pinus pinaster Aiton (maritime pine), Pinus sylvestris L. (Scots pine), Populus nigra L. (European black poplar), Taxus baccata L. (English yew), and Quercus petraea (Matt.) Liebl. (sessile oak). Phenotypic (height, diameter at breast height, crown size, bark thickness, biomass, straightness, forking, branch angle, fructification), regeneration, environmental in situ measurements (soil depth, vegetation cover, competition indices), and environmental modeling data extracted by using bilinear interpolation accounting for surrounding conditions of each tree (precipitation, temperature, insolation, drought indices) were obtained from trees in 194 sites covering the species’ geographic ranges and reflecting local environmental gradients. Conclusion: The GenTree Platform is a new resource for investigating ecological and evolutionary processes in forest trees. The coherent phenotyping and environmental characterization across 12 species in their European ranges allow for a wide range of analyses from forest ecologists, conservationists, and macro-ecologists. Also, the data here presented can be linked to the GenTree Dendroecological collection, the GenTree Leaf Trait collection, and the GenTree Genomic collection presented elsewhere, which together build the largest evolutionary forest ecology data collection available

Between but not within species variation in the distribution of fitness effects

New mutations provide the raw material for evolution and adaptation. The distribution of fitness effects (DFE) describes the spectrum of effects of new mutations that can occur along a genome, and is therefore of vital interest in evolutionary biology. Recent work has uncovered striking similarities in the DFE between closely related species, prompting us to ask whether there is variation in the DFE among populations of the same species, or among species with different degrees of divergence, i.e., whether there is variation in the DFE at different levels of evolution. Using exome capture data from six tree species sampled across Europe we characterised the DFE for multiple species, and for each species, multiple populations, and investigated the factors potentially influencing the DFE, such as demography, population divergence and genetic background. We find statistical support for there being variation in the DFE at the species level, even among relatively closely related species. However, we find very little difference at the population level, suggesting that differences in the DFE are primarily driven by deep features of species biology, and that evolutionarily recent events, such as demographic changes and local adaptation, have little impact