Stomatal density in Pinus sylvestris as an indicator of temperature rather than CO2 : Evidence from a pan-European transect.

The commonly observed negative relationship between stomatal density (SD) and atmospheric CO2 has led to SD being proposed as an indicator of atmospheric CO2 concentration. The use of SD as a proxy for CO2 , however, has been hampered by an insufficient understanding of the intraspecific variation of this trait. We hypothesized that SD in Pinus sylvestris, a widely distributed conifer, varies geographically and that this variation is determined by major climatic variables. By sampling needles from naturally growing trees along a latitudinal range of 32.25°, equivalent to 13.7°C gradient of mean annual temperature (MAT) across Europe, we found that SD decreased from the warmest southern sites to the coldest sites in the north at a rate of 4 stomata per mm2 for each 1°C, with MAT explaining 44% of the variation. Additionally, samples from a provenance trial exhibited a positive relationship between SD and the MAT of the original localities, suggesting that high SD is an adaptation to warm temperature. Our study revealed one of the strongest intraspecific relationships between SD and climate in any woody species, supporting the utility of SD as a temperature, rather than direct CO2 , proxy. In addition, our results predict the response of SD to climate warming.

Adaptive responses to temperature and precipitation variation at the early-life stages of Pinus sylvestris.

Early-stage fitness variation has been seldom evaluated at broad scales in forest tree species, despite the long tradition of studying climate-driven intraspecific genetic variation. In this study, we evaluated the role of climate in driving patterns of population differentiation at early-life stages in Pinus sylvestris and explored the fitness and growth consequences of seed transfer within the species range. We monitored seedling emergence, survival and growth over a 2-yr period in a multi-site common garden experiment which included 18 European populations and spanned 25° in latitude and 1700 m in elevation. Climate-fitness functions showed that populations exhibited higher seedling survival and growth at temperatures similar to their home environment, which is consistent with local adaptation. Northern populations experienced lower survival and growth at warmer sites, contrary to previous studies on later life stages. Seed mass was higher in populations from warmer areas and was positively associated with survival and growth at more southern sites. Finally, we did not detect a survival-growth trade-off; on the contrary, bigger seedlings exhibited higher survival probabilities under most climatic conditions. In conclusion, our results reveal that contrasting temperature regimes have played an important role in driving the divergent evolution of P. sylvestris populations at early-life stages.

Selection patterns on early-life phenotypic traits in Pinus sylvestris are associated with precipitation and temperature along a climatic gradient in Europe.

Understanding the dynamics of selection is key to predicting the response of tree species to new environmental conditions in the current context of climate change. However, selection patterns acting on early recruitment stages and their climatic drivers remain largely unknown in most tree species, despite being a critical period of their life cycle. We measured phenotypic selection on Pinus sylvestris seed mass, emergence time and early growth rate over 2 yr in four common garden experiments established along the latitudinal gradient of the species in Europe. Significant phenotypic plasticity and among-population genetic variation were found for all measured phenotypic traits. Heat and drought negatively affected fitness in the southern sites, but heavy rainfalls also decreased early survival in middle latitudes. Climate-driven directional selection was found for higher seed mass and earlier emergence time, while the form of selection on seedling growth rates differed among sites and populations. Evidence of adaptive and maladaptive phenotypic plasticity was found for emergence time and early growth rate, respectively. Seed mass, emergence time and early growth rate have an adaptive role in the early stages of P. sylvestris and climate strongly influences the patterns of selection on these fitness-related traits.

Higher biomass partitioning to absorptive roots improves needle nutrition but does not alleviate stomatal limitation of northern Scots pine.

Harsh environmental conditions affect both leaf structure and root traits. However, shoot growth in high-latitude systems is predominately under photoperiod control while root growth may occur for as long as thermal conditions are favorable. The different sensitivities of these organs may alter functional relationships above- and belowground along environmental gradients. We examined the relationship between absorptive root and foliar traits of Scots pine trees growing in situ along a temperate-boreal transect and in trees grown in a long-term common garden at a temperate latitude. We related changes in foliar nitrogen, phosphorus, specific leaf area, needle mass and 13 C signatures to geographic trends in absorptive root biomass to better understand patterns of altered tree nutrition and water balance. Increased allocation to absorptive fine roots was associated with greater uptake of soil nutrients and subsequently higher needle nutrient contents in the northern provenances compared with more southern provenances when grown together in a common garden setting. In contrast, the leaf δ13 C in northern and southern provenances were similar within the common garden suggesting that higher absorptive root biomass fractions could not adequately increase water supply in warmer climates. These results highlight the importance of allocation within the fine-root system and its impacts on needle nutrition while also suggesting increasing stomatal limitation of photosynthesis in the context of anticipated climatic changes.

Stronger genetic differentiation among within-population genetic groups than among populations in Scots pine provides new insights into within-population genetic structuring.

We investigated the presence of spatial genetic groups within forest tree populations and determined if the genetic divergence among these groups is greater than that between populations using Scots pine (Pinus sylvestris) as a model species. We genotyped 890 adult trees of Scots pine in six natural populations in Lithuania at 11 nuclear microsatellite loci. We used a Bayesian clustering approach to identify the within-population genetic groups within each of the six populations. We calculated the differentiation indexes among the genetic groups within each population and among the six populations by ignoring the genetic groups. The Bayesian clustering revealed 2 to 6 distinct genetic groups of varying size as the most likely genetic structures within populations. The genetic differentiation indexes among the genetic groups within populations were nearly tenfold greater (FST = 0.012-0.070) than those between the populations (FST = 0.003). We conclude on the existence of markedly stronger structuring of genetic variation within populations than between populations of Scots pine in large forest tracts of northern Europe. Such genetic structures serve as a contributing factor to large within population genetic diversity in northern conifers. We assume that within population mating in Scots pine is not completely random but rather is stratified into genetic clusters. Our study provides pioneering novel key insights into structuring of genetic variation within populations. Our findings have implications for examining within-population genetic diversity and genetic structure, conservation, and management of genetic resources.

Dendrological Secrets of the Pazaislis Monastery in Central Lithuania: DNA Markers and Morphology Reveal Tilia × europaea L. Hybrids of an Impressive Age.

We benefited from the availability of a species-specific DNA marker to describe the morphometry of T. cordata × platyphyllos hybrids of an impressive age (ca. 150 years) grown in the Pazaislis baroque monastery yard in Central Lithuania. In an earlier study on a country-wide set of 543 T. cordata individuals from natural forest populations in Lithuania, we detected a nuclear microsatellite locus Tc8 well-differentiating between T. cordata and T. platyphyllos. The Tc8 locus contained a 140 bp allele in T. cordata (541 sampled individuals) and alleles above 160 bp in the two trees with a T. platyphyllos-like morphology (sampled in a national park). To verify the Tc8 locus as species specific, we sampled a further four T. platyphyllos-like individuals, which all contained the Tc8 locus alleles above 160 bp. We subsequently genotyped the six old-growth individuals from the Pazaislis monastery with mixed T. cordata × platyphyllos morphology. Results revealed that all six old-growth Tilia individuals from the Pazaislis monastery were heterozygous for the Tc8 locus with alleles of 140 bp (indicative of T. cordata) and 162 bp (indicative of T. platyphyllos). This finding confirms the morphological observations that these individuals are hybrids between T. cordata and T. platyphyllos. Additionally, the genotyping of a set of 14 nuclear microsatellite loci revealed that all six trees from the Pazaislis monastery are clones, possessing identical microsatellite genotypes. After the molecular identification, we morphotyped leaves, bracts, twigs, and nuts of the 6 old-growth T. cordata × platyphyllos hybrids from the Pazaislis monastery, 16 T. cordata old-growth trees, 4 T. × europaea var. europaea 'Pallida' trees growing near the Pazaislis monastery, and 4 mature T. platyphyllos trees from a nearby Girionys park. The morphotyping showed that T. cordata × platyphyllos hybrids may be the easiest to distinguish from T. cordata by raised and horizontally tertiary veins of leaves.

Spontaneous hybridization between Pinus mugo and Pinus sylvestris at the Lithuanian seaside: a morphological survey.

We address the problem of spontaneous hybridization between an exotic species Pinus mugo and the native/local P. sylvestris at the seaside spit of Kursiu Nerija in Lithuania. The objective was to identify spontaneous hybrids between P. mugo and P. sylvestris based on morphology traits among the individuals naturally regenerating at the seaside spit. The field inventory was carried out over the entire Lithuanian part of the spit, and 200 individuals morphologically intermediate between P. sylvestris and P. mugo were identified. Based on a weighted trait index, the intermediate individuals were grouped into two groups, one morphologically close to P. sylvestris and another close to P. mugo. The needle micromorphological traits of the putative hybrids were of intermediate values between P. mugo and P. sylvestris. The results provide a strong evidence of spontaneous hybridization between P. mugo and P. sylvestris in Lithuanian seaside spit of Kursiu Nerija.

Genetic signature of the natural gene pool of Tilia cordata Mill. in Lithuania: Compound evolutionary and anthropogenic effects.

Tilia cordata Mill. is a valuable tree species enriching the ecological values of the coniferous-dominated boreal forests in Europe. Following the historical decline, spreading of Tilia sp. is challenged by the elevated inbreeding and habitat fragmentation. We studied the geographical distribution of genetic diversity of Tilia cordata populations in Lithuania. We used 14 genomic microsatellite markers to genotype 543 individuals from 23 wild-growing populations. We found that Tilia cordata retained high levels of genetic diversity (population F IS = 0-0.15, H o = 0.53-0.69, H e = 0.56-0.75). AMOVA, Bayesian clustering, and Monmonier's barrier detection indicate weak but significant differentiation among the populations (F ST = 0.037***) into geographically interpretable clusters of (a) western Lithuania with high genetic heterogeneity but low genetic diversity, bottleneck effects, (b) relatively higher genetic diversity of Tilia cordata on rich and most soils of midland lowland, and (c) the most differentiated populations on poor soils of the coolest northeastern highland possessing the highest rare allele frequency but elevated inbreeding and bottleneck effects. Weak genetic differentiation among the Tilia cordata populations in Lithuania implies common ancestry, absence of strong adaptive gradients, and effective genetic exchange possible mediated via the riparian networks. A hypothesis on riparian networks as gene flow mediators in Tilia cordata was raised based on results of this study.

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.

Genetic variation in early fitness traits across European populations of silver birch (Betula pendula).

Given that the ecological niche of tree species is typically narrower for earlier life stages, intraspecific genetic variation at early fitness traits may greatly influence the adaptive response of tree populations to changing environmental conditions. In this study, we evaluated genetic variation in early fitness traits among 12 populations of Betula pendula from a wide latitudinal range in Europe (41-55°N). We first conducted a chamber experiment to test for population differences in germination and the effect of pre-chilling treatment on seed dormancy release. We then established three common gardens spread across the species latitudinal range in order to evaluate levels of quantitative genetic variation and genotype-by-environment interaction at different early life traits. Our results showed significant variation in chamber germination rates among populations (0-60 %), with southern populations exhibiting lower germination. Pre-chilling treatments did not generally improve germination success. Population seedling emergence rates in the field were correlated with chamber germination rates, though being an order of magnitude lower, with an average ranging from 0 to 1.3 % across gardens. Highly significant variation was found in field emergence rates among populations, and between seed-crop years within populations, but not among families within populations. Populations differed in seedling height, diameter, slenderness and budburst date, with significant among-family variation. Population latitude was positively associated with chamber germination rate and with seedling emergence rate in one of the central field sites. Overall, genetic, environmental and demographic factors seem to influence the observed high levels of variation in early fitness traits among B. pendula populations. Our results suggest limited regeneration capacity for the study species under drier conditions, but further field trials with sufficient replication over environments and seed crops will improve our understanding of its vulnerability to climate change.

Author Correction: The GenTree Dendroecological Collection, tree-ring and wood density data from seven tree species across Europe.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The GenTree Dendroecological Collection, tree-ring and wood density data from seven tree species across Europe.

The dataset presented here was collected by the GenTree project (EU-Horizon 2020), which aims to improve the use of forest genetic resources across Europe by better understanding how trees adapt to their local environment. This dataset of individual tree-core characteristics including ring-width series and whole-core wood density was collected for seven ecologically and economically important European tree species: silver birch (Betula pendula), European beech (Fagus sylvatica), Norway spruce (Picea abies), European black poplar (Populus nigra), maritime pine (Pinus pinaster), Scots pine (Pinus sylvestris), and sessile oak (Quercus petraea). Tree-ring width measurements were obtained from 3600 trees in 142 populations and whole-core wood density was measured for 3098 trees in 125 populations. This dataset covers most of the geographical and climatic range occupied by the selected species. The potential use of it will be highly valuable for assessing ecological and evolutionary responses to environmental conditions as well as for model development and parameterization, to predict adaptability under climate change scenarios.

Does climate-related in situ variability of Scots pine (Pinus sylvestris L.) needles have a genetic basis? Evidence from common garden experiments.

The correlations of phenotypic traits with environmental drivers suggest that variability of these traits is a result of natural selection, especially if such trait correlations are based on genetic variability. We hypothesized that in situ correlations of structural needle traits of Scots pine (Pinus sylvestris L) with minimal winter temperature (Tmin) reported previously from a temperate/boreal transect would be conserved when plants are cultivated under common conditions. We tested this hypothesis by analyzing needles from two common gardens located in the temperate zone, one including adult trees and the other juvenile seedlings. The majority of adult needle traits for which correlations with Tmin were found in the field turned out to be under environmental influence. In contrast, the majority of traits studied in juvenile needles were correlated with the original Tmin suggesting the role of past natural selection in shaping their variability. Juvenile needles thus appeared to be inherently less plastic than adult needles, perhaps reflecting the stronger selective pressure acting during juvenile, as compared with adult, ontogenetic stage. Genetically based cold-climate adaptation in either juvenile or adult needles, or both, involved an increase in leaf mass per area and leaf density, decrease in needle length, reduction in the amount of xylem and phloem, increase in thickness of epidermis, decrease in tracheid diameter and increase in tracheid density, and increase in diameter and volume fraction of resin ducts. We also show that at least some traits, such as transverse xylem and phloem areas and number of fibers, scale with needle length, suggesting that climate-related trait variation may also be mediated by changes in needle length. Moreover, slopes of these allometric relationships may themselves be plastically modified. The phenotypic syndrome typical of needles from cold environments may thus be under environmental, genetic and allometric control.