New perspectives for wood anatomical analysis in dendrosciences: The GSL1-microtome

The variability of wood anatomical features in the rings of trees and shrubs is known to be dependent on multifaceted environmental parameters. The ability to determine anatomical variations over longer time periods as decades or centuries is a step forward in dendroecological and dendroclimatological research. In this regard micro sectioning is still one of the basic requirements but sectioning devices (microtomes) designed for wood anatomical purposes are rarely available. We present an affordable heavy duty, but light-weight microtome operated with removable blades. This portable device enables the production of large sections for dendroclimatological reconstructions as well as various types of common sections for wood anatomical studies.     

Are climatic factors responsible for the process of oak decline in Poland?

Oak decline, a complex process leading to increased mortality of this species, has been observed in Europe for many years. Previous studies suggest that climate conditions, especially drought, may be one of the most important factors that trigger this phenomenon. The paper investigates the radial growth and wood anatomy features of pedunculate oak (Quercus robur) trees of various health status as well as their response to climate conditions. Wood samples including all annual increments were taken at two sites (western and central Poland, 15 trees each). Based on the crown defoliation level, three health groups (healthy, weakened and dead oaks) were distinguished. Cross-sections were prepared with sliding microtome and Cell P image analysis software was used for the measurements. Tree-ring width (TRW), earlywood vessels density (VDen) and non-weighted vessels diameter (VD) were determined and correlated with mean monthly values of temperature, precipitation, vapour pressure, and Palmer Drought Severity Index (PDSI). Radial increment and anatomical parameters were significantly higher for the healthy oaks than for the weakened and the dead trees. TRW showed smaller dependence on climate than analysed anatomical attributes. No obvious pattern of relationship was found between oak radial growth and climate regarding tree health status. Our results revealed that the drought has a weak impact on the process of oak decline on investigated sites in Poland.     

The advantage of using a starch based non-Newtonian fluid to prepare micro sections

The breakage or distortion of cellular structures is one of the biggest problems in creating micro-sections for wood anatomical analyses in tree-ring as well as other branches of anatomical research. These broken or distorted structures cause artifacts in photomicrographs that require time consuming image manipulation or corrections prior to further analyses. The simple application of a cornstarch, water, and glycerol (CWG) solution (10:8:7 ratio), a so called non-Newtonian fluid to the surface of wooden specimen before sectioning improves the overall quality of the resulting micro-sections. In particular the problem of secondary cell walls splitting off the primary wall while sectioning is drastically reduced. The quality of the sections using this solution is comparable to that obtained from the more laborious and expensive paraffin embedding.     

Improved tree-ring visualization using autofluorescence

The great diversity of wood anatomical features found in trees worldwide results in a broad variety of growth-ring boundary types that are not always easy to recognize, especially in tropical woods. However, the presence of clearly visible limits between tree rings is essential for any tree-ring studies. Here, we propose the use of autofluorescence of wood in order to enhance tree-ring visualization. The multispectral light emitted from the fluorescence stereomicroscope can be filtered in specific wavelengths to improve the visualization of wood anatomical features. To evaluate the effectiveness of this technique, we compared visualization under natural light, GFP (green fluorescent protein) filter, RFP (red fluorescent protein) filter and UV filter. We tested this technique with a set of 38 tree species with different types of growth-ring boundaries. Although results are species-specific, fluorescence has been shown to improve the visualization of growth-ring boundaries by enhancing the contrast among cell types. It may highlight fibrous zones (e.g. Cavanillesia arborea, Aspidosperma polyneuron), different porosity patterns (e.g. Myracrodruon urundeuva), secretory canals (e.g. Copaifera langsdorffii), and parenchyma bands (e.g. Tipuana tipu). Fluorescence allows the visualization of growth-ring boundaries in species that were previously described as having indistinct growth rings under natural light. For species with clear tree-ring boundaries such as Cedrela fissilis and Hymenaea courbaril, this approach aids the identification of false rings. In addition to the visualization of growth-ring boundaries, autofluorescence may be useful for other qualitative and quantitative analyses of wood anatomy, such as wood identification and automated measurements of anatomical features. Scientists struggling with tree-ring counting and cross-dating due to difficult tree-ring visualization may find fluorescence useful. It may also aid to identify new species suitable for tree-ring studies.     

Recent growth increase in endemic Juglans boliviana from the tropical Andes

The spatial coverage of tree-ring chronologies in tropical South America is low compared to the extratropics, particularly in remote regions. Tree-ring dating from such tropical sites is limited by the generally weak temperature seasonality, complex coloration, and indistinct anatomical morphology in some tree species. As a result, there is a need to complement traditional methods of dendrochronology with innovative and independent approaches. Here, we supplement traditional tree-ring methods via the use of radiocarbon analyses to detect partial missing rings and/or false rings, and wood anatomical techniques to precisely delineate tree-ring boundaries. In so doing we present and confirm the annual periodicity of the first tree-ring width (TRW) chronology spanning from 1814 to 2017 for Juglans boliviana (‘nogal’), a tree species growing in a mid-elevation tropical moist forest in northern Bolivia. We collected 25 core samples and 4 cross-sections from living and recently harvested canopy-dominant trees, respectively. The sampled trees were growing in the Madidi National Park and had a mean age of 115 years old, with certain trees growing for over 200 years. Comparison of (residual and standard) TRW chronologies to monthly climate variables shows significant negative relationships to prior year May-August maximum temperatures (r = −0.54, p < 0.05) and positive relationships to dry season May-October precipitation (r = 0.60, p < 0.05) before the current year growing season. Additionally, the radial growth of Juglans boliviana shows a significant positive trend since 1979. Our findings describe a new and promising tree species for dendrochronology due to its longevity and highlight interdisciplinary techniques that can be used to expand the current tree-ring network in Bolivia and the greater South American tropics.     

Dendrochronological evidence of cockchafer (Melolontha sp.) outbreaks in subfossil tree-trunks from Tovačov (CZ Moravia)

In dendrochronological dating, we encounter the trouble that some samples cannot be dated due to the occurrence of anomalies in the tree-ring series. When dating subfossil wood for the extension of existing master chronologies, this may be an especially unwanted circumstance as subfossil wood is scarce and each sample is valuable. In Moravia (Czech Republic) outbreaks of the cockchafer (Melolontha sp.) used to appear with a disastrous effect on agricultural crops, and, for a dendrochronologist, on samples of wood to be dated. Oak ring width reduction caused by cockchafer induced defoliation can superimpose climate induced growth variations and may complicate dating of historical or subfossil samples. For this study, 33 samples of sub-fossil trunks were assessed; 19 of which were attacked by cockchafer. For comparison, we analysed a total of nine living oak trees from the forest district Strá?nice-Vracov, recently attacked by the cockchafer. Occurrence of the cockchafer was reported there in 1999, 2003 and 2007.We found clear growth patterns with cyclic narrow rings every 4 years. This allowed us to create an artificial curve expressing the recurring cycles of cockchafer outbreaks which assisted us in the detection of the same pattern in subfossil tree growth. The tree-ring series of the attacked living oak stand as well as the 19 individual tree-ring series from subfossil oak trunks could not be dated using standard chronologies and showed a high resemblance to the cockchafer life cycle. Additionally, we found out that the living oak stand was attacked in 1983, 1987, 1991 and 1995 as well. The results support the hypothesis that the periodical reduction observed in tree-rings at regular increments is caused by cockchafer infestation. This evidence is further supported by characteristic anatomical features in early wood. A mean tree-ring series was created from the individual tree-ring series of subfossil oaks even though radiocarbon dating showed completely different ages of samples. This means that the affected tree-ring series cannot be used for dendrochronological dating and dendroclimatological analyses. These series cannot be used without preceding mathematical and statistical modification.     

Are radial changes in vascular anatomy mechanically induced or an ageing process? Evidence from observations on buttressed tree root systems

To investigate the effect of changes in mechanical loading conditions on radial anatomical patterns, we here compare the trunk with the roots in two locations of three species of buttressed trees. The proximal part of the buttress roots is highly mechanically loaded throughout juvenile growth whereas the distal part of the buttresses is though to be mechanically unimportant at formation but become progressively more mechanically loaded during growth. We measured the frequency and diameter of the vessels and the vessel area fraction, and from this calculated the specific conductivity of tissue samples of the core-, intermediate- and outer wood. As in previous studies there was an increase in vessel size, vessel area fraction and specific conductivity from the pith to the bark in the trunk. In the proximal part of the buttress roots, however, there was no increase in vessel size and conductivity from core wood and out in agreement with the high mechanical loading found here throughout growth. There was instead a decrease in vessel size, vessel area fraction and specific conductivity from core- to outer wood in the distal part of the buttress roots in accordance with the increase in mechanical loading. Hence, it appears that the radial anatomical patterns are not a passive function of cambial ageing but may be modified in response to local mechanical loading.     

Pointer years in tree-ring width and earlywood-vessel area time series of Quercus robur—Relation with climate factors near its northern distribution limit

For a long time, radial growth of pedunculate oak (Quercus robur) in relation to environmental factors has been studied in Central Europe. However, there is insufficient information on oak growth in the Baltic region. Climate–growth interactions have been mostly investigated by correlation/response analysis between ring width and climatic factors. Other wood anatomical proxies, and also pointer year analysis, which focuses on weather extremes, can be sources of additional information. Wood samples were taken from 40 sites across Latvia. Tree-ring width (TRW) and mean area of earlywood vessels (EVA) were measured. To assess differences in wood formation among sites, a PCA was performed based on pointer year series of TRW and EVA per site. The relation with climate was assessed by Pearson's correlation. Patterns of wood formation differed along west/east gradient, so that two regions (western and eastern) of Latvia were distinguished. Pointer year values were higher for TRW than for EVA and higher in the eastern than in the western region. However, the pointer year values were moderate, suggesting that there was no strict limiting factor for their formation. Pointer years of EVA were strongly related with temperature during the dormant period in both regions; June precipitation had a significant effect in the eastern region. Pointer years of TRW were correlated with spring and summer temperature in the western region and with February temperature in the eastern region; precipitation in autumn showed a negative effect. These differences can be explained by a gradient from maritime to continental climate, leading to shifts in the growth limiting factors. Extremely cold winters resulted in most of the negative pointer years of TRW and EVA.     

Applying methods of hard tissues preparation for wood anatomy: Imaging polished samples embedded in polymethylmethacrylate

Cambial activity records short and long-term environmental signals in xylem anatomy, creating a permanent archive. Quantitative wood anatomy deciphers the relationship between cell structure and function in a spatiotemporal context. Obtaining high-resolution images of wood anatomical preparations is a critical stage in the process of decoding this information. Damage to cellular structures when sectioning by microtome is one of the main problems in the preparation of high-quality micro-sections. Cell damage leads to the occurrence of artifacts – most often related to broken cell walls – hindering the performance of image recognition programs, and increasing the time spent on the manual editing of images. In this work, we propose an alternative method to microtomy, based on embedding-polishing protocols established for hard tissue preparation. Wood samples are embedded in a transparent and non-reactive resin as polymethylmethacrylate (PMM) that is subsequently ground and polished. Being able to acquire images from the stained or unstained polished surfaces of the PMM-blocks and sections (thinner than 100 μm) by using a wide range of optical methods such as reflected polarizing microscopy, epifluorescence microscopy, bright-field microscopy with diffuse illumination and circularly polarizing microscopy. This embedding method improves the mechanical integrity and quality of wood anatomical preparations, eliminating the problem of broken cell walls. Furthermore, this technique allows the preparation and analysis of large tissue surfaces.     

Wood anatomy and carbon‐isotope discrimination support long‐term hydraulic deterioration as a major cause of drought‐induced dieback

Hydraulic impairment due to xylem embolism and carbon starvation are the two proposed mechanisms explaining drought‐induced forest dieback and tree death. Here, we evaluate the relative role played by these two mechanisms in the long‐term by quantifying wood‐anatomical traits (tracheid size and area of parenchyma rays) and estimating the intrinsic water‐use efficiency (iWUE) from carbon isotopic discrimination. We selected silver fir and Scots pine stands in NE Spain with ongoing dieback processes and compared trees showing contrasting vigour (declining vs nondeclining trees). In both species earlywood tracheids in declining trees showed smaller lumen area with thicker cell wall, inducing a lower theoretical hydraulic conductivity. Parenchyma ray area was similar between the two vigour classes. Wet spring and summer conditions promoted the formation of larger lumen areas, particularly in the case of nondeclining trees. Declining silver firs presented a lower iWUE than conspecific nondeclining trees, but the reverse pattern was observed in Scots pine. The described patterns in wood anatomical traits and iWUE are coherent with a long‐lasting deterioration of the hydraulic system in declining trees prior to their dieback. Retrospective quantifications of lumen area permit to forecast dieback in declining trees 2–5 decades before growth decline started. Wood anatomical traits provide a robust tool to reconstruct the long‐term capacity of trees to withstand drought‐induced dieback.     

Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana

Temperature is a primary environmental cue for seed germination of many weeds and vegetables. To investigate the mechanism of germination regulation by temperature, we selected five high temperature (thermoinhibition)-resistant germination mutants (TRW lines) from 20,000 T-DNA insertion lines of Arabidopsis. Segregation analyses indicated that each of the five lines had single locus recessive mutations. The seeds of TRW134-15 and TRW187 showed reduced sensitivity to ABA and also to the gibberrellin biosynthesis inhibitor, paclobutrazol. Genetic and nucleotide sequencing analyses indicated that TRW187 is a new allele of abi3 (abi3-14). TRW71-1 exhibited a maternal effect for both thermoinhibition-resistant and transparent testa phenotypes, and genetic analysis revealed that the mutation was allelic to tt7 (tt7-4 sib). Interestingly, the seeds of reduced dormancy mutants rdo1, rdo2, rdo3 and rdo4 were also thermoinhibition tolerant, and all the TRW seeds showed reduced dormancy. Like rdo3, TRW13-1 had shorter siliques and slightly shorter stems than the wild type. The mutation of TRW13-1 was mapped to the bottom arm of chromosome 1 where rdo3 has also been mapped, but the two mutants are not allelic. We designated TRW13-1 as thermoinhibition-resistant germination 1 (trg1). We also mapped the ABA-insensitive mutation of TRW134-15 to the bottom arm of chromosome 5 and named it trg2. These results show that both embryo/endosperm and maternal factors contribute to germination inhibition at supraoptimal temperatures in Arabidopsis. In addition, we confirm the role of ABA in thermoinhibition of seed germination and a link between seed physiological dormancy and response to high temperature.     

Beetles provide directed dispersal of viable spores of a keystone wood decay fungus

Wood decay fungi are considered to be dispersed by wind, but dispersal by animals may also be important, and more so in managed forests where dead wood is scarce. We investigated whether beetles could disperse spores of the keystone species Fomitopsis pinicola. Beetles were collected on sporocarps and newly felled spruce logs, a favourable habitat for spore deposition. Viable spores (and successful germination) of F. pinicola were detected by dikaryotization of monokaryotic bait mycelium from beetle samples. Viable spores were on the exoskeleton and in the faeces of all beetles collected from sporulating sporocarps. On fresh spruce logs, nine beetle species transported viable spores, of which several bore into the bark. Our results demonstrate that beetles can provide directed dispersal of wood decay fungi. Potentially, it could contribute to a higher persistence of some species in fragmented forests where spore deposition by wind on dead wood is less likely.     

Moisture‐driven shift in the climate sensitivity of white spruce xylem anatomical traits is coupled to large‐scale oscillation patterns across northern treeline in northwest North America

Tree growth at northern treelines is generally temperature‐limited due to cold and short growing seasons. However, temperature‐induced drought stress was repeatedly reported for certain regions of the boreal forest in northwestern North America, provoked by a significant increase in temperature and possibly reinforced by a regime shift of the pacific decadal oscillation (PDO). The aim of this study is to better understand physiological growth reactions of white spruce, a dominant species of the North American boreal forest, to PDO regime shifts using quantitative wood anatomy and traditional tree‐ring width (TRW) analysis. We investigated white spruce growth at latitudinal treeline across a >1,000 km gradient in northwestern North America. Functionally important xylem anatomical traits (lumen area, cell‐wall thickness, cell number) and TRW were correlated with the drought‐sensitive standardized precipitation–evapotranspiration index of the growing season. Correlations were computed separately for complete phases of the PDO in the 20th century, representing alternating warm/dry (1925–1946), cool/wet (1947–1976) and again warm/dry (1977–1998) climate regimes. Xylem anatomical traits revealed water‐limiting conditions in both warm/dry PDO regimes, while no or spatially contrasting associations were found for the cool/wet regime, indicating a moisture‐driven shift in growth‐limiting factors between PDO periods. TRW reflected only the last shift of 1976/1977, suggesting different climate thresholds and a higher sensitivity to moisture availability of xylem anatomical traits compared to TRW. This high sensitivity of xylem anatomical traits permits to identify first signs of moisture‐driven growth in treeline white spruce at an early stage, suggesting quantitative wood anatomy being a powerful tool to study climate change effects in the northwestern North American treeline ecotone. Projected temperature increase might challenge growth performance of white spruce as a key component of the North American boreal forest biome in the future, when drier conditions are likely to occur with higher frequency and intensity.     

Life-span growth dynamics and xylem anatomical patterns of diffuse-porous Afzelia africana Sm. (Fabaceae) in different ecological zones in Burkina Faso

Tropical West African savannas are exposed to high climatic variability with potential impacts on tree growth, forest dynamics and ecosystem productivity. In such context, understanding the long-term ecological responses of savanna trees to changing environmental conditions is of great relevance for taking appropriate conservation actions. We conducted the first study on tree-ring analysis and quantitative wood anatomy on Afzelia africana Sm. in Burkina Faso, to investigate the life-span growth trajectories and wood anatomical adjustment to site and to climate variations. A total of 24 stem discs was collected in four protected forests along the Sudano-sahelian and the Sudanian climatic zones. Wood samples were analyzed using standard dendrochronological methods and quantitative wood anatomy. The mean annual growth rates varied from 1.002 (± 0.249) mm. year⁻¹ in the Sudanian zone to 1.128 (± 0.436) mm. year⁻¹ in the Sudano-sahelian zone. Analysis of growth trajectories showed high variations within sites and between climatic zones. Wood anatomical traits significantly varied between sites. Principal Component Analysis revealed strong relationships between ring width, wood density and vessel traits, with 82.81 % of the total variance explained. Vessel size significantly increased from the pith to the bark, highlighting the ontogenetic effects on xylem anatomical variations. Inverse relationships were found between vessel size and vessel density across the driest site and the wettest site, suggesting that the higher the rainfall, the taller the tree, the larger vessel size, but the lower vessel density. By contrast, more arid conditions and high evapotranspiration lead to smaller vessel sizes and higher vessel density. Such anatomical adjustments highlight the trade-offs between water conductance efficiency and hydraulic safety, and emphasize physiological responses to climate variability. These variations on the long-term dynamics and xylem anatomical patterns underline complex interactions between ontogenetic effects and contrasting environmental factors that affect the eco-physiological functioning of A. africana throughout the Sudanian region.     

翅梗石斛组织培养与快繁技术(简报)

以云南野生翅梗石斛Dendrobium trigonopus种子无菌萌发的试管苗为材料,初步探讨翅梗石斛的组织培养和快速繁殖技术。结果表明：B5 + NAA 0.5 mg·L^-1 + 10%马铃薯汁和B5 + NAA 0.5 mg·L^-1 + 6-BA 0.5 mg·L^-1 + 10%马铃薯汁培养基分别对翅梗石斛种子的萌发与原球茎的增殖效果明显;B5 + NAA 0.5 mg·L^-1 + 10%马铃薯汁+云南松树皮粉末2 g·L^-1培养基对翅梗石斛的生根及壮苗效果较好。成苗后进行简单的药剂灭菌,便可直接移栽至锯末与刨花或锯末与树皮比例为1∶1的基质中,25 d后,成活率达95%以上。     

Effects of flooding on wood and bark anatomy of four species in a mangrove forest community

Variation in the wood and bark anatomy of the dominant species of a mangrove forest community in Mexico was evaluated in relation to some environmental factors, and their physiological adaptations to salinity and flooding period are discussed. The forest is characterized by three zones according to the presence of dominant tree species and flooding periodicity. Vessel arrangement and wood and bark ray height are strongly associated with flooding zones where trees are growing. Variance analyses revealed significant differences among zones for these anatomical characteristics. Soil texture and water salinity were the most useful parameters for the prediction of values of anatomical characteristics. More abundant vessels in radial multiples in a shorter flooding period suggest a functional advantage of multiple vessel groups. Taller wood and bark rays in response to prolonged flooding period can be attributed to anoxic conditions. Among zones, significant differences in the vulnerability index of the species were detected, but not with respect to relative conductivity. Significant differences among zones exist for wood and bark characteristics involved in vertical and horizontal water transport, photosynthates and gas exchange.     

Size of conducting phloem: The “key” factor for bark recovery of 12 tropical medicinal tree species

For rural populations in Africa, tree bark is widely used for medicinal purposes. Despite its importance, no anatomical studies exist which detail the recovery of medicinal tree species in Africa to bark harvesting. This present study aims to determine the anatomical variable(s) that could help to predict the differing recovery rates of 12 medicinal tree species. Discs of branches were collected from non-harvested trees of 12 different African species. A total of 12 anatomical variables were measured in the wood, the cambium and the phloem zone, and the correlation between the bark recovery rate and each variable was tested. Among the 12 anatomical variables tested, the thickness of the conducting phloem zone emerged as the most important one to explain the bark recovery rate. The presence of sclereids within the conducting phloem zone was also found to be an explanatory variable and was negatively correlated with the bark recovery rate. For 10 out of the 12 species, the thickness of the cambial zone varied significantly with the season. Nevertheless, this variable did not contribute significantly to the explanation of the bark recovery rate. Given that the 12 studied species showed a large range of bark recovery rates (0.1–10.0 cm y^?1), we assume that they may be representative of the variety of wound healing responses (i.e. wood and bark tissue production) in many of African tree species. Consequently, our results could offer the advantage to foresee the potential of wound closure in any tree from which bark could be harvested.     

Colonization of disturbed Scots pine trees by bark‐ and wood‐boring beetles

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