The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

Different photorespiratory mechanisms in conifer leaves, where peroxisomes have intrinsically low catalase activity.

Photorespiration is an essential metabolic mechanism associated with photosynthesis; however, little is known about the photorespiratory pathway of conifer gymnosperms. Metabolite analyses of the leaves of 27 tree species showed that the mean glycerate content in conifer leaves was lower than that in angiosperm leaves. We performed experiments where [13 C]-serine was fed to detached shoots of a conifer (Cryptomeria japonica), via the transpiration stream, and compared the labeling patterns of photorespiratory metabolites with those of an angiosperm tree (Populus nigra), because glycerate is produced from serine via hydroxypyruvate in peroxisomes. In P. nigra, hydroxypyruvate, glycerate and glycine were labeled with 13 C, whereas in C. japonica, glycolate and a non-canonical photorespiratory metabolite, formate, were also labeled, suggesting that an H2 O2 -mediated non-enzymatic decarboxylation (NED) reaction occurs in C. japonica. We analyzed changes in the metabolite contents of leaves kept in the dark and leaves exposed to illuminated photorespiration-promoting conditions: a positive relationship between formate and serine levels in C. japonica implied that the active C1 -metabolism pathway synthesizes serine from formate. Leaf gas exchange analyses revealed that CO2 produced through NED was recaptured by chloroplasts. Database analysis of the peroxisomal targeting signal motifs of an H2 O2 -scavenging enzyme, catalase, derived from various species, including nine coniferous species, as well as analyses of peroxisomal fractions isolated from C. japonica and P. nigra leaves indicated that conifer peroxisomes had less catalase activity. These results suggest that NED and the subsequent C1 metabolism are involved in the photorespiratory pathway of conifer leaves, where peroxisomes have intrinsically low catalase activity.

Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene ßglu-1 with large variations in white spruce but not Norway spruce.

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and ßglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of ßglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of ßglu-1 and Ugt5 genes. We observed very large copy numbers of ßglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of ßglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

Genomic selection in western redcedar: from proof of concept to operational application.

Forests face many threats. While traditional breeding may be too slow to deliver well-adapted trees, genomic selection (GS) can accelerate the process. We describe a comprehensive study of GS from proof of concept to operational application in western redcedar (WRC, Thuja plicata). Using genomic data, we developed models on a training population (TrP) of trees to predict breeding values (BVs) in a target seedling population (TaP) for growth, heartwood chemistry, and foliar chemistry traits. We used cross-validation to assess prediction accuracy (PACC) in the TrP; we also validated models for early-expressed foliar traits in the TaP. Prediction accuracy was high across generations, environments, and ages. PACC was not reduced to zero among unrelated individuals in TrP and was only slightly reduced in the TaP, confirming strong linkage disequilibrium and the ability of the model to generate accurate predictions across breeding generations. Genomic BV predictions were correlated with those from pedigree but displayed a wider range of within-family variation due to the ability of GS to capture the Mendelian sampling term. Using predicted TaP BVs in multi-trait selection, we functionally implemented and integrated GS into an operational tree-breeding program.

Global field collection data confirm an affinity of brown rot fungi for coniferous habitats and substrates.

Unlike 'white rot' (WR) wood-decomposing fungi that remove lignin to access cellulosic sugars, 'brown rot' (BR) fungi selectively extract sugars and leave lignin behind. The relative frequency and distribution of these fungal types (decay modes) have not been thoroughly assessed at a global scale; thus, the fate of one-third of Earth's aboveground carbon, wood lignin, remains unclear. Using c. 1.5 million fungal sporocarp and c. 30 million tree records from publicly accessible databases, we mapped and compared decay mode and tree type (conifer vs angiosperm) distributions. Additionally, we mined fungal record metadata to assess substrate specificity per decay mode. The global average for BR fungi proportion (BR/(BR + WR records)) was 13% and geographic variation was positively correlated (R2 = 0.45) with conifer trees proportion (conifer/(conifer + angiosperm records)). Most BR species (61%) were conifer, rather than angiosperm (22%), specialists. The reverse was true for WR (conifer: 19%; angiosperm: 62%). Global BR proportion patterns were predicted with greater accuracy using the relative distributions of individual tree species (R2 = 0.82), rather than tree type. Fungal decay mode distributions can be explained by tree type and, more importantly, tree species distributions, which our data suggest is due to strong substrate specificities.

Scots pine - panmixia and the elusive signal of genetic adaptation.

Scots pine is the foundation species of diverse forested ecosystems across Eurasia and displays remarkable ecological breadth, occurring in environments ranging from temperate rainforests to arid tundra margins. Such expansive distributions can be favored by various demographic and adaptive processes and the interactions between them. To understand the impact of neutral and selective forces on genetic structure in Scots pine, we conducted range-wide population genetic analyses on 2321 trees from 202 populations using genotyping-by-sequencing, reconstructed the recent demography of the species and examined signals of genetic adaptation. We found a high and uniform genetic diversity across the entire range (global FST 0.048), no increased genetic load in expanding populations and minor impact of the last glacial maximum on historical population sizes. Genetic-environmental associations identified only a handful of single-nucleotide polymorphisms significantly linked to environmental gradients. The results suggest that extensive gene flow is predominantly responsible for the observed genetic patterns in Scots pine. The apparent missing signal of genetic adaptation is likely attributed to the intricate genetic architecture controlling adaptation to multi-dimensional environments. The panmixia metapopulation of Scots pine offers a good study system for further exploration into how genetic adaptation and plasticity evolve under gene flow and changing environment.

Cone allometry and seed protection from fire are similar in serotinous and nonserotinous conifers.

Serotiny is an adaptive trait that allows certain woody plants to persist in stand-replacing fire regimes. However, the mechanisms by which serotinous cones avoid seed necrosis and nonserotinous species persist in landscapes with short fire cycles and serotinous competitors remain poorly understood. To investigate whether ovulate cone traits that enhance seed survival differ between serotinous and nonserotinous species, we examined cone traits in 24 species within Pinaceae and Cupressaceae based on physical measurements and cone heating simulations using a computational fluid dynamics model. Fire-relevant cone traits were largely similar between cone types; those that differed (e.g. density and moisture) conferred little seed survival advantage under simulated fire. The most important traits influencing seed survival were cone size and seed depth within the cone, which was found to be an allometric function of cone mass for both cone types. Thus, nonserotinous cones should not suffer significantly greater seed necrosis than serotinous cones of equal size. Closed nonserotinous cones containing mature seeds may achieve substantial regeneration after fire if they are sufficiently large relative to fire duration and temperature. To our knowledge, this is the most comprehensive study of the effects of fire-relevant cone traits on conifer regeneration supported by physics-based fire simulation.

Bark structure is coordinated with xylem hydraulic properties in branches of five Cupressaceae species.

The properties of bark and xylem contribute to tree growth and survival under drought and other types of stress conditions. However, little is known about the functional coordination of the xylem and bark despite the influence of selection on both structures in response to drought. To this end, we examined relationships between proportions of bark components (i.e. thicknesses of tissues outside the vascular cambium) and xylem transport properties in juvenile branches of five Cupressaceae species, focusing on transport efficiency and safety from hydraulic failure via drought-induced embolism. Both xylem efficiency and safety were correlated with multiple bark traits, suggesting that xylem transport and bark properties are coordinated. Specifically, xylem transport efficiency was greater in species with thicker secondary phloem, greater phloem-to-xylem thickness ratio and phloem-to-xylem cell number ratio. In contrast, species with thicker bark, living cortex and dead bark tissues were more resistant to embolism. Thicker phellem layers were associated with lower embolism resistance. Results of this study point to an important connection between xylem transport efficiency and phloem characteristics, which are shaped by the activity of vascular cambium. The link between bark and embolism resistance affirms the importance of both tissues to drought tolerance.

Terpenoids are involved in the expression of systemic-induced resistance in Austrian pine.

Terpenoids are defense metabolites that are induced upon infection or wounding. However, their role in systemic-induced resistance (SIR) is not known. Here, we explored the role of terpenoids in this phenomenon at a very early stage in the interaction between Austrian pine and the tip blight and canker pathogen Diplodia pinea. We induced Austrian pine saplings by either wounding or inoculating the lower stems with D. pinea. The seedlings were then challenged after 12 h, 72 h, or 10 days with D. pinea on the stem 15 cm above the induction. Lesion lengths and terpenoids were quantified at both induction and challenge locations. Key terpenoids were assayed for antifungal activity in in vitro bioassays. SIR increased with time and was correlated with the inducibility of several compounds. α-Pinene and a cluster of ß-pinene, limonene, benzaldehyde, dodecanol, and n-dodecyl acrylate were positively correlated with SIR and were fungistatic in vitro, while other compounds were negatively correlated with SIR and appeared to serve as a carbon source for D. pinea. This study shows that, overall, terpenoids are involved in SIR in this system, but their role is nuanced, depending on the type of induction and time of incubation. We hypothesize that some, such as α-pinene, could serve in SIR signaling.

DAL10 is a direct target of putative DAL1-mediated age pathway in conifers.

The optimal timing of the transition from vegetative growth to reproductive growth is critical for plant reproductive success, and the underlying regulatory mechanisms have been well studied in angiosperm model species, but relatively little in gymnosperms. DAL1, a MADS domain transcription factor (TF) gene that shows a conserved age-related expression profile in conifers, may be an age timer. However, how the DAL1 mediates the onset of reproductive growth remains poorly understood. Here, we have shown that the PtDAL1 directly regulates the PtDAL10 transcription by binding to its promoter region in vitro. PtDAL1, forms ternary complexes in vitro and in N. benthamiana with PtDAL10 and PtMADS11, two potential candidate regulators of the vegetative to reproductive transition in Chinese pine (Pinus tabuliformis). The PtDAL10 was progressively induced in new shoots with age and highly accumulated in male and female cones. Overexpression of PtDAL10 rescued the flowering of ft-10 and soc1-1-2 mutants in Arabidopsis. We provide insight into the molecular components associated with the PtDAL1, which integrates the vegetative to reproductive phase transition into age-mediated progressive development of the whole plant in conifers.

A dwarf conifer tree from the Triassic of Antarctica: the first fossil evidence of suppressed growth in a favorable climate?

BACKGROUND AND AIMS: The complexity of fossil forest ecosystems is difficult to reconstruct due to the fragmentary nature of the fossil record. However, detailed morpho-anatomical studies of well-preserved individual fossils can provide key information on tree growth and ecology, including in biomes with no modern analog such as the lush forests that developed in the polar regions during past greenhouse climatic episodes. METHODS: We describe an unusual-looking stem from Middle Triassic (ca 240 Ma) deposits of Antarctica with over 100 very narrow growth-rings and conspicuous persistent vascular traces through the wood. Sections of the specimen were prepared using the cellulose acetate peel technique to determine its systematic affinities and analyse its growth. KEY RESULTS: The new fossil shows similarities with the form genus Woodworthia and with conifer stems from the Triassic of Antarctica, and is assigned to the conifers. Vascular traces are interpreted as those of small branches retained on the trunk. Growth-ring analyses reveal one of the slowest growth rates reported in the fossil record, with an average of 0.2 mm/season. While the tree was growing within the Triassic polar circle, sedimentological data and growth-ring information from other fossil trees, including from the same locality, support the presence of favorable conditions in the region. CONCLUSIONS: The specimen is interpreted as a dwarf conifer tree that grew under a generally favorable regional climate but whose growth was suppressed due to stressful local site conditions. This is the first time that a tree with suppressed growth is identified as such in the fossil record, providing new insights on the structure of polar forests under greenhouse climates and, more generally, on the complexity of tree communities in deep time.

Temperature dependence of pollen germination and tube growth in conifers relates to their distribution along an elevational gradient in Washington State, USA.

BACKGROUND AND AIMS: Pollen germination and tube growth are essential processes for successful fertilization. They are among the most temperature-vulnerable stages and subsequently affect seed production and determine population persistence and species distribution under climate change. Our study aims to investigate intra- and inter-specific variations in the temperature dependence of pollen germination and tube length growth and to explore how these variations differ for pollen from elevational gradients. METHODS: We focused on three conifer species, Pinus contorta, Picea engelmannii, and Pinus ponderosa, with pollen collected from 350 to 2200m elevation in Washington State, USA. We conducted pollen viability tests at temperatures from 5 to 40°C in 5°C intervals. After testing for four days, we took images of these samples under a microscope to monitor pollen germination percentage (GP) and tube length (TL). We applied the Gamma function to describe the temperature dependence of GP and TL and estimated key parameters, including the optimal temperature for GP (Topt_GP) and TL (Topt_TL). KEY RESULTS: Results showed that pollen from three species and different elevations within a species have different GP, TL, Topt_GP, and Topt_TL. The population with a higher Topt_GP would also have a higher Topt_TL, while Topt_TL was generally higher than Topt_GP, i.e., a positive but not one-to-one relationship. However, only Pinus contorta showed that populations from higher elevations have lower Topt_GP and Topt_TL and vice versa. The variability in GP increased at extreme temperatures, whereas the variability in TL was greatest near Topt_TL. CONCLUSIONS: Our study demonstrates the temperature dependences of three conifers across a wide range of temperatures. Pollen germination and tube growth are highly sensitive to temperature conditions and vary among species and elevations, affecting their reproduction success during warming. Our findings can provide valuable insights to advance our understanding of how conifer pollen responds to rising temperatures.

Forest restoration and fuels reduction work: Different pathways for achieving success in the Sierra Nevada.

Fire suppression and past selective logging of large trees have fundamentally changed frequent-fire-adapted forests in California. The culmination of these changes produced forests that are vulnerable to catastrophic change by wildfire, drought, and bark beetles, with climate change exacerbating this vulnerability. Management options available to address this problem include mechanical treatments (Mech), prescribed fire (Fire), or combinations of these treatments (Mech + Fire). We quantify changes in forest structure and composition, fuel accumulation, modeled fire behavior, intertree competition, and economics from a 20-year forest restoration study in the northern Sierra Nevada. All three active treatments (Fire, Mech, Mech + Fire) produced forest conditions that were much more resistant to wildfire than the untreated control. The treatments that included prescribed fire (Fire, Mech + Fire) produced the lowest surface and duff fuel loads and the lowest modeled wildfire hazards. Mech produced low fire hazards beginning 7 years after the initial treatment and Mech + Fire had lower tree growth than controls. The only treatment that produced intertree competition somewhat similar to historical California mixed-conifer forests was Mech + Fire, indicating that stands under this treatment would likely be more resilient to enhanced forest stressors. While Fire reduced modeled wildfire hazard and reintroduced a fundamental ecosystem process, it was done at a net cost to the landowner. Using Mech that included mastication and restoration thinning resulted in positive revenues and was also relatively strong as an investment in reducing modeled wildfire hazard. The Mech + Fire treatment represents a compromise between the desire to sustain financial feasibility and the desire to reintroduce fire. One key component to long-term forest conservation will be continued treatments to maintain or improve the conditions from forest restoration. Many Indigenous people speak of "active stewardship" as one of the key principles in land management and this aligns well with the need for increased restoration in western US forests. If we do not use the knowledge from 20+ years of forest research and the much longer tradition of Indigenous cultural practices and knowledge, frequent-fire forests will continue to be degraded and lost.

The non-flowering plants of a near-polar forest in East Gondwana, Tasmania, Australia, during the Early Eocene Climatic Optimum.

PREMISE: The Cenozoic Macquarie Harbour Formation (MHF) hosts one of the oldest and southernmost post-Cretaceous fossil plant assemblages in Australia. Coinciding with the Early Eocene Climatic Optimum (EECO) and predating the breakup of Australia from Antarctica, it offers critical data to study the diversity and extent of the Austral Polar Forest Biome, and the floristic divergence between Australasia and South America resulting from the Gondwana breakup. METHODS: The micromorphology and macromorphology of new fossil plant compressions from the MHF were described and systematically analyzed. Previously published non-flowering plant records were reviewed and revised. Macrofossil abundance data were provided. The flora was compared with other early Paleogene assemblages from across the Southern Hemisphere. RESULTS: Twelve species of non-flowering plants were identified from the macrofossil record. Conifers include Araucariaceae (Araucaria macrophylla, A. readiae, A. timkarikensis sp. nov., and Araucarioides linearis), Podocarpaceae (Acmopyle glabra, Dacrycarpus mucronatus, Podocarpus paralungatikensis sp. nov., and Retrophyllum sp.), and Cupressaceae (Libocedrus microformis). Dacrycarpus linifolius was designated a junior synonym of D. mucronatus. Further components include a cycad (Bowenia johnsonii, Zamiaceae), a pteridosperm (Komlopteris cenozoicus, Umkomasiaceae), and a fern (Lygodium dinmorphyllum, Schizaeaceae). CONCLUSIONS: The fossil assemblage represents a mixed near-polar forest with a high diversity of conifers. The morphology and preservation of several species indicate adaptations to life at high latitudes. The coexistence of large- and small-leaved conifers implies complex, possibly open forest structures. Comparisons with contemporaneous assemblages from Argentina support a circumpolar biome during the EECO, reaching from southern Australia across Antarctica to southern South America.

Water quality effects of peat rewetting and leftover conifer brash, following peatland restoration and tree harvesting.

Harvesting of plantation conifers on peatlands is carried out as part of restoration and forestry operations. In particular, in the UK and Ireland, conifer plantations on drained ombrotrophic blanket and raised bogs are increasingly being removed (by harvesting), along with blocking of drainage ditches to help raise water tables to reinitiate and restore bog vegetation and function. However, both tree harvesting and peatland restoration operations can have significant impacts on water quality at local and catchment scales. Previous research has suggested that leaching from leftover decomposing brash (tree tops and branches, including wood and needles) is the primary cause, while other work has suggested that release from rewetted peat also contributes to water quality changes. This research investigates the relative importance of peat rewetting, needles and branches on water quality using mesocosm experiments, to help elucidate the mechanisms behind water quality changes following restoration and harvesting operations. Peat and brash were collected from a drained afforested blanket bog in the Flow Country, Scotland. Short-term mesocosm experiments were conducted by incubating peat, peat + needles and peat + needles + branches with rainwater in quadruplicate. Brash from Sitka spruce (Picea sitchensis) and lodgepole pine (Pinus contorta) was investigated separately, while we also conducted experiments with fresh and aged (∼18 months) brash. Peat, needles and branches all significantly impacted water quality in the order of branches > needles > peat, while concentrations of DOC, PO43-, NH4+, K and Mn were most impacted. Water quality impacts of spruce brash appeared generally greater than pine, while fresh brash had larger effects than aged brash. In our mesocosms, relative contributions to water quality changes were estimated by elemental yields. On average, peat contributed 25.4% (range 0.6-72.3%), while needles and brash contributed 19.7% (range 3.0-37.0%) and 54.9% (range 22.1-70.2%) to yields, respectively. We further estimate that 267 kg C ha-1 (255.8 kg as DOC, 10.7 kg as DIC), 27.4 kg K ha-1, 5.8 kg P ha-1 (as PO43-) and 0.5 kg N ha-1 (as NH4+) could be released from brash, over nine days.

Tree richness increased biomass carbon sequestration and ecosystem stability of temperate forests in China: Interacted factors and implications.

Biodiversity loss and forest degradation have received increasing attention worldwide, and their effects on forest biomass carbon storage and stability have not yet been well defined. This study examined 1275 tree plots using the field survey method to quantify the effects of tree diversity, tree sizes, and mycorrhizal symbiont abundance on biomass carbon storages (Cs) and NDVI (Normalized Difference Vegetation Index)-based ecosystem stability (standard deviation/mean NDVI = NDVI_S) during the field survey period from 2008 to 2018. Our data showed Cs and NDVI_S averaged at 31-108 t ha-1 and 32.04-49.28, respectively, and positive relations between Cs and NDVI_S were observed (p < 0.05). Large forest-type and regional variations were found in these two parameters. Broadleaf forests had 74% of Cs (p < 0.05) of the conifer forests, but no differences were in NDVI_S. Cold regions at high latitudes had 71% of NDVI_S in the warm regions at low latitudes, while no differences were in Cs. Moist regions at high longitudes had 2.04 and 1.28-fold higher Cs and NDVI_S (p < 0.05). The >700 m a.s.l. regions had 1.24-fold higher Cs (p < 0.01) than the <700 m a.s.l. regions, but similar NDVI_S (p > 0.05). Nature Reserves had 1.94-fold higher Cs but 30% lower NDVI_S than outside Reserves (p < 0.001). > 40-year-old forests had 1.3- and 2-fold higher Cs and NDVI_S than the young forests. Structural equation modeling and hierarchical partitioning revealed the driving paths responsible for these variations. Tree richness was positively associated with Cs and ecosystem stability, contributing 21.6%-30.6% to the total effects on them; tree sizes significantly promoted the Cs, but had negligible impacts on NDVI_S. MAT's total effects on NDVI_S of conifer forests were 40% higher than that of broadleaf forests, MAP's total effects on Cs varied with forest types; arbuscular mycorrhizal tree dominance exhibited a smaller positive impact on Cs and ecosystem stability in comparison to other factors. Our findings underscore that the significance of climatic-adapted forest management, diversity conservation, and big-sized tree protections can support the achievement of carbon neutrality in China from biomass carbon sequestration and ecosystem stability.

From common gardens to candidate genes: exploring local adaptation to climate in red spruce.

Local adaptation to climate is common in plant species and has been studied in a range of contexts, from improving crop yields to predicting population maladaptation to future conditions. The genomic era has brought new tools to study this process, which was historically explored through common garden experiments. In this study, we combine genomic methods and common gardens to investigate local adaptation in red spruce and identify environmental gradients and loci involved in climate adaptation. We first use climate transfer functions to estimate the impact of climate change on seedling performance in three common gardens. We then explore the use of multivariate gene-environment association methods to identify genes underlying climate adaptation, with particular attention to the implications of conducting genome scans with and without correction for neutral population structure. This integrative approach uncovered phenotypic evidence of local adaptation to climate and identified a set of putatively adaptive genes, some of which are involved in three main adaptive pathways found in other temperate and boreal coniferous species: drought tolerance, cold hardiness, and phenology. These putatively adaptive genes segregated into two 'modules' associated with different environmental gradients. This study nicely exemplifies the multivariate dimension of adaptation to climate in trees.

Transcriptome features of stone cell development in weevil-resistant and susceptible Sitka spruce.

Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.

Tracing the opposing assimilate and nutrient flows in live conifer needles.

The vasculature along conifer needles is fundamentally different from that in angiosperm leaves as it contains a unique transfusion tissue inside the bundle sheath. In this study, we used specific tracers to identify the pathway of photoassimilates from mesophyll to phloem, and the opposing pathway of nutrients from xylem to mesophyll. For symplasmic transport we applied esculin to the tip of attached pine needles and followed its movement down the phloem. For apoplasmic transport we let detached needles take up a membrane-impermeable contrast agent and used micro-X-ray computed tomography to map critical water exchange interfaces and domain borders. Microscopy and segmentation of the X-ray data enabled us to render and quantify the functional 3D structure of the water-filled apoplasm and the complementary symplasmic domain. The transfusion tracheid system formed a sponge-like apoplasmic domain that was blocked at the bundle sheath. Transfusion parenchyma cell chains bridged this domain as tortuous symplasmic pathways with strong local anisotropy which, as evidenced by the accumulation of esculin, pointed to the phloem flanks as the preferred phloem-loading path. Simple estimates supported a pivotal role of the bundle sheath, showing that a bidirectional movement of nutrient ions and assimilates is feasible and emphasizing the role of the bundle sheath in nutrient and assimilate exchange.