The oldest known clones of Salix herbacea growing in the Northern Apennines, Italy are at least 2000 years old.

PREMISE: Dominant in many ecosystems around the world, clonal plants can reach considerable ages and sizes. Due to their modular growth patterns, individual clonal plants (genets) can consist of many subunits (ramets). Since single ramets do not reflect the actual age of genets, the ratio between genet size (radius) and longitudinal annual growth rate (LAGR) of living ramets is often used to approximate the age of clonal plants. However, information on how the LAGR changes along ramets and how LAGR variability may affect age estimates of genets is still limited. METHODS: We assessed the variability of LAGR based on wood-section position along the ramets and on the duration of the growing season on three genetically distinct genets of Salix herbacea growing in the Northern Apennines (Italy). We compared genet ages estimated by dividing genet radius by the LAGRs of its ramets. RESULTS: LAGR increased significantly from the stem apex to the root collar; indicating that ramet growth rate decreased with time. Furthermore, a difference of ca. 2 weeks in the onset of the growing period did not impact LAGR. Considering the high LAGR variability, we estimated that the three genets started to grow between ~2100 and ~7000 years ago, which makes them the oldest known clones of S. herbacea even considering the most conservative age estimate. CONCLUSIONS: Our findings indicate that analyzing ramets at the root collar provides an integrative measurement of their overall LAGR, which is crucial for estimating the age of genets.

Bell-shaped tree-ring responses to air temperature drive productivity trends in long-lived mountain Mediterranean pines.

We investigated the dendroclimatic response of a Pinus heldreichii metapopulation distributed over a wide elevation interval (from 882 to 2143 m a.s.l.), spanning from low mountain to upper subalpine vegetation belts in the southern Italian Apennines. The tested hypothesis is that wood growth along an elevational gradient is non-linearly related to air temperature. During three years of fieldwork (2012-2015) at 24 sites, we collected wood cores from a total of 214 pine trees with diameter at breast height from 19 to 180 cm (average 82.7 ± 32.9 cm). We used a combination of tree-ring and genetic methods to reveal factors involved in growth acclimation using a space-for-time approach. Scores from canonical correspondence analysis were used to combine individual tree-ring series into four composite chronologies related to air temperature along the elevation gradient. Overall, the June dendroclimatic response followed a bell-shaped thermal niche curve, increasing until a peak around 13-14 °C. A similarly bell-shaped response was found with previous autumn air temperature, and both dendroclimatic signals interacted with stem size and growth rates, generating a divergent growth response between the top and the bottom of the elevation gradient. Increased tree growth in the upper subalpine belt was consistent with the consequences of increasing air temperature under no drought stress. A positive link was uncovered between pine growth at all elevations and April mean temperature, with trees growing at the lowest elevations showing the strongest growth response. No elevational genetic differences were found, hence long-lived tree species with small geographical ranges may reverse their climatic response between the lower and upper bioclimatic zones of their environmental niche. Our study revealed a high resistance and acclimation capability of Mediterranean forest stands, and such low vulnerability to changing climatic conditions highlights the potential to store carbon in these ecosystems for the coming decades.

Genetic signatures of divergent selection in European beech (Fagus sylvatica L.) are associated with the variation in temperature and precipitation across its distribution range.

High genetic variation and extensive gene flow may help forest trees with adapting to ongoing climate change, yet the genetic bases underlying their adaptive potential remain largely unknown. We investigated range-wide patterns of potentially adaptive genetic variation in 64 populations of European beech (Fagus sylvatica L.) using 270 SNPs from 139 candidate genes involved either in phenology or in stress responses. We inferred neutral genetic structure and processes (drift and gene flow) and performed differentiation outlier analyses and gene-environment association (GEA) analyses to detect signatures of divergent selection. Beech range-wide genetic structure was consistent with the species' previously identified postglacial expansion scenario and recolonization routes. Populations showed high diversity and low differentiation along the major expansion routes. A total of 52 loci were found to be putatively under selection and 15 of them turned up in multiple GEA analyses. Temperature and precipitation related variables were equally represented in significant genotype-climate associations. Signatures of divergent selection were detected in the same proportion for stress response and phenology-related genes. The range-wide adaptive genetic structure of beech appears highly integrated, suggesting a balanced contribution of phenology and stress-related genes to local adaptation, and of temperature and precipitation regimes to genetic clines. Our results imply a best-case scenario for the maintenance of high genetic diversity during range shifts in beech (and putatively other forest trees) with a combination of gene flow maintaining within-population neutral diversity and selection maintaining between-population adaptive differentiation.

Fine-scale spatial genetic structure across the species range reflects recent colonization of high elevation habitats in silver fir (Abies alba Mill.).

Variation in genetic diversity across species ranges has long been recognized as highly informative for assessing populations' resilience and adaptive potential. The spatial distribution of genetic diversity within populations, referred to as fine-scale spatial genetic structure (FSGS), also carries information about recent demographic changes, yet it has rarely been connected to range scale processes. We studied eight silver fir (Abies alba Mill.) population pairs (sites), growing at high and low elevations, representative of the main genetic lineages of the species. A total of 1,368 adult trees and 540 seedlings were genotyped using 137 and 116 single nucleotide polymorphisms (SNPs), respectively. Sites revealed a clear east-west isolation-by-distance pattern consistent with the post-glacial colonization history of the species. Genetic differentiation among sites (FCT = 0.148) was an order of magnitude greater than between elevations within sites (FSC = 0.031), nevertheless high elevation populations consistently exhibited a stronger FSGS. Structural equation modelling revealed that elevation and, to a lesser extent, post-glacial colonization history, but not climatic and habitat variables, were the best predictors of FSGS across populations. These results suggest that high elevation habitats have been colonized more recently across the species range. Additionally, paternity analysis revealed a high reproductive skew among adults and a stronger FSGS in seedlings than in adults, suggesting that FSGS may conserve the signature of demographic changes for several generations. Our results emphasize that spatial patterns of genetic diversity within populations provide information about demographic history complementary to non-spatial statistics, and could be used for genetic diversity monitoring, especially in forest trees.

Evolutionary history of the mediterranean Pinus halepensis-brutia species complex using gene-resequencing and transcriptomic approaches.

KEY MESSAGE: Complementary gene-resequencing and transcriptomic approaches reveal contrasted evolutionary histories in a species complex. Pinus halepensis and Pinus brutia are closely related species that can intercross, but occupy different geographical ranges and bioclimates. To study the evolution of this species complex and to provide genomic resources for further research, we produce and analyze two new complementary sets of genetic resources: (i) a set of 172 re-sequenced genomic target loci analyzed in 45 individuals, and (ii) a set of 11 transcriptome assemblies. These two datasets provide insights congruent with previous studies: P. brutia displays high level of genetic diversity and no genetic sub-structure, while P. halepensis shows three main genetic clusters, the western Mediterranean and North African clusters displaying much lower genetic diversity than the eastern Mediterranean cluster, the latter cluster having similar genetic diversity to P. brutia. In addition, these datasets provide new insights on the timing of the species-complex history: the two species would have split at the end of the tertiary, and the changing climatic conditions of the Mediterranean region at the end of the Tertiary-beginning of the Quaternary, together with the distinct species tolerance to harsh climatic conditions would have resulted in different geographic distributions, demographic histories and genetic patterns of the two pines. The multiple glacial-interglacial cycles during the Quaternary would have led to the expansion of P. brutia in the Middle East, while P. halepensis would have been through bottlenecks. The last glaciations, from 0.6 Mya on, would have affected further the Western genetic pool of P. halepensis.

Functional Relationships of Wood Anatomical Traits in Norway Spruce.

The quantitative assessment of wood anatomical traits offers important insights into those factors that shape tree growth. While it is known that conduit diameter, cell wall thickness, and wood density vary substantially between and within species, the interconnection between wood anatomical traits, tree-ring width, tree height and age, as well as environment effects on wood anatomy remain unclear. Here, we measure and derived 65 wood anatomical traits in cross-sections of the five outermost tree rings (2008-2012) of 30 Norway spruce [Picea abies (L.) H. Karst.] trees growing along an altitudinal gradient (1,400-1,750 m a.s.l.) in the northern Apennines (Italy). We assess the relationship among each anatomical trait and between anatomical trait groups according to their function for (i) tree-ring growth, (ii) cell growth, (iii) hydraulic traits, and (iv) mechanical traits. The results show that tree height significantly affects wood hydraulic traits, as well as number and tangential diameter of tracheids, and ultimately the total ring width. Moreover, the amount of earlywood and latewood percentage influence wood hydraulic safety and efficiency, as well as mechanical traits. Mechanically relevant wood anatomical traits are mainly influenced by tree age, not necessarily correlated with tree height. An additional level of complexity is also indicated by some anatomical traits, such as latewood lumen diameter and the cell wall reinforcement index, showing large inter-annual variation as a proxy of phenotypic plasticity. This study unravels the complex interconnection of tree-ring tracheid structure and identifies anatomical traits showing a large inter-individual variation and a strong interannual coherency. Knowing and quantifying anatomical variation in cells of plant stem is crucial in ecological and biological studies for an appropriate interpretation of abiotic drivers of wood formation often related to tree height and/or tree age.

Individual reproductive success in Norway spruce natural populations depends on growth rate, age and sensitivity to temperature.

Quantifying the individual reproductive success and understanding its determinants is a central issue in evolutionary research for the major consequences that the transmission of genetic variation from parents to offspring has on the adaptive potential of populations. Here, we propose to distil the myriad of information embedded in tree-ring time series into a set of tree-ring-based phenotypic traits to be investigated as potential drivers of reproductive success in forest trees. By using a cross-disciplinary approach that combines parentage analysis and a thorough dendrophenotypic characterisation of putative parents, we assessed sex-specific relationships between such dendrophenotypic traits (i.e., age, growth rate and parameters describing sensitivity to climate and to extreme climatic events) and reproductive success in Norway spruce. We applied a full probability method for reconstructing parent-offspring relationships between 604 seedlings and 518 adult trees sampled within five populations from southern and central Europe. We found that individual female and male reproductive success was positively associated with tree growth rate and age. Female reproductive success was also positively influenced by the correlation between growth and the mean temperature of the previous vegetative season. Overall, our results showed that Norway spruce individuals with the highest fitness are those who are able to keep high-growth rates despite potential growth limitations caused by reproductive costs and climatic limiting conditions. Identifying such functional links between the individual ecophysiological behaviour and its evolutionary gain would increase our understanding on how natural selection shapes the genetic composition of forest tree populations over time.

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.

Isolation, characterization, and cross-amplification of 20 microsatellite markers for Conospermum undulatum (Proteaceae).

PREMISE: Recent habitat fragmentation is posing a risk to the wavy-leaved smokebush, Conospermum undulatum (Proteaceae), a rare plant species endemic to southwestern Western Australia. Microsatellite markers are required to characterize the genetic diversity and structure of the species for conservation purposes and to facilitate ecological studies. METHODS AND RESULTS: Illumina MiSeq high-throughput sequencing was used to develop 20 novel microsatellite markers for C. undulatum. Polymorphism at each locus was assessed using 72 individuals from three natural populations. Nineteen markers were polymorphic, with the number of alleles per locus ranging from two to 21, and observed and expected heterozygosity ranging from 0.000 to 1.000 and 0.117 to 0.919, respectively. All markers successfully amplified in three congeneric species (C. stoechadis, C. canaliculatum and C. triplinervium). CONCLUSIONS: The microsatellite markers will be useful for revealing patterns of genetic diversity, dispersal dynamics, and hybridization events for C. undulatum to inform future conservation efforts.

Disentangling the effects of spatial proximity and genetic similarity on individual growth performances in Norway spruce natural populations.

Cambial growth is a phenotypic trait influenced by various physiological processes, numerous biotic and abiotic drivers, as well as by the genetic background. By archiving the outcome of such complex interplay, tree-rings are an exceptional resource for addressing individual long-term growth responses to changing environments and climate. Disentangling the effects of the different drivers of tree growth, however, remains challenging because of the lack of multidisciplinary data. Here, we combine individual dendrochronological, genetic and spatial data to assess the relative importance of genetic similarity and spatial proximity on Norway spruce (Picea abies (L.) Karst.) growth performances. We intensively sampled five plots from two populations in southern and central Europe, characterizing a total of 482 trees. A two-step analytical framework was developed. First, the effects of climate and tree age on tree-ring width (TRW) were estimated for each individual using a random slope linear mixed-effects model. Individual parameters were then tested against genetic and spatial variables by Mantel tests, partial redundancy analyses and variance partitioning. Our modelling approach successfully captured a large fraction of variance in TRW (conditional R2 values up to 0.94) which was largely embedded in inter-individual differences. All statistical approaches consistently showed that genetic similarity was not related to variation in the individual parameters describing growth responses. In contrast, up to 29% of the variance of individual parameters was accounted by spatial variables, revealing that microenvironmental features are more relevant than genetic similarity in determining similar growth patterns. Our study highlights both the advantages of modelling dendrochronological data at the individual level and the relevance of microenvironmental variation on individual growth patterns. These two aspects should be carefully considered in future multidisciplinary studies on growth dynamics in natural populations.

Genetic diversity and divergence at the Arbutus unedo L. (Ericaceae) westernmost distribution limit.

Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species' distribution range in the region. We applied Bayesian analysis and identified four clusters (north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species' genetic structure.

Local Adaptation in European Firs Assessed through Extensive Sampling across Altitudinal Gradients in Southern Europe.

BACKGROUND: Local adaptation is a key driver of phenotypic and genetic divergence at loci responsible for adaptive traits variations in forest tree populations. Its experimental assessment requires rigorous sampling strategies such as those involving population pairs replicated across broad spatial scales. METHODS: A hierarchical Bayesian model of selection (HBM) that explicitly considers both the replication of the environmental contrast and the hierarchical genetic structure among replicated study sites is introduced. Its power was assessed through simulations and compared to classical 'within-site' approaches (FDIST, BAYESCAN) and a simplified, within-site, version of the model introduced here (SBM). RESULTS: HBM demonstrates that hierarchical approaches are very powerful to detect replicated patterns of adaptive divergence with low false-discovery (FDR) and false-non-discovery (FNR) rates compared to the analysis of different sites separately through within-site approaches. The hypothesis of local adaptation to altitude was further addressed by analyzing replicated Abies alba population pairs (low and high elevations) across the species' southern distribution range, where the effects of climatic selection are expected to be the strongest. For comparison, a single population pair from the closely related species A. cephalonica was also analyzed. The hierarchical model did not detect any pattern of adaptive divergence to altitude replicated in the different study sites. Instead, idiosyncratic patterns of local adaptation among sites were detected by within-site approaches. CONCLUSION: Hierarchical approaches may miss idiosyncratic patterns of adaptation among sites, and we strongly recommend the use of both hierarchical (multi-site) and classical (within-site) approaches when addressing the question of adaptation across broad spatial scales.

Within-population genetic structure in beech (Fagus sylvatica L.) stands characterized by different disturbance histories: does forest management simplify population substructure?

The fine-scale assessment of both spatially and non-spatially distributed genetic variation is crucial to preserve forest genetic resources through appropriate forest management. Cryptic within-population genetic structure may be more common than previously thought in forest tree populations, which has strong implications for the potential of forests to adapt to environmental change. The present study was aimed at comparing within-population genetic structure in European beech (Fagus sylvatica L.) plots experiencing different disturbance levels. Five plot pairs made up by disturbed and undisturbed plots having the same biogeographic history were sampled throughout Europe. Overall, 1298 individuals were analyzed using four highly polymorphic nuclear microsatellite markers (SSRs). Bayesian clustering within plots identified 3 to 11 genetic clusters (within-plot θ ST ranged from 0.025 to 0.124). The proportion of within-population genetic variation due to genetic substructuring (F CluPlot = 0.067) was higher than the differentiation among the 10 plots (F PlotTot = 0.045). Focusing on the comparison between managed and unmanaged plots, disturbance mostly explains differences in the complexity of within-population genetic structure, determining a reduction of the number of genetic clusters present in a standardized area. Our results show that: i) genetic substructuring needs to be investigated when studying the within-population genetic structure in forest tree populations, and ii) indices describing subtle characteristics of the within-population genetic structure are good candidates for providing early signals of the consequences of forest management, and of disturbance events in general.

Effect of habitat fragmentation on the genetic diversity and structure of peripheral populations of beech in Central Italy.

Fragmentation can affect the demographic and genetic structure of populations near the boundary of their biogeographic range. Higher genetic differentiation among populations coupled with lower level of within-population variability is expected as a consequence of reduced population size and isolation. The effects of these 2 factors have been rarely disentangled. Given their high gene flow, anemophilous forest trees should be more affected, in terms of loss of genetic diversity, by small population size rather than geographic isolation alone. We studied the impact of distance from the main range (a measure of isolation) and reduced population size on the within-population and among population components of genetic variability. We assayed 11 isozyme loci in a total of 856 individuals in 27 marginal populations of European beech (Fagus sylvatica L.) in Central Italy. Populations were divided into 3 groups with an increasing level of fragmentation. In the most fragmented group, the within-population genetic variability was slightly smaller and the among population differentiation significantly larger than in the other 2 groups. Isolation-by-distance was lost when only pairs of populations involving at least one from the most fragmented group were considered and maintained in the other groups. These results support the role of random genetic drift having a larger impact on the most fragmented group, whereas gene flow seems to balance genetic drift in the 2 less fragmented ones. Given that average distance from the main range is not different between the intermediate and the most fragmented group, but average population size is smaller, we can conclude that gene flow is effective, even at relatively long distances, in balancing the effect of fragmentation if population size is not too small.