Automatic identification of cell files in light microscopic images of conifer wood

International audienceIn this paper, we present an automatic method to recognize cell files in light microscopic images of conifer wood. This original method is decomposed into three steps: the segmentation step which extracts some anatomical structures in the image, the classification step which identifies in these structures the interesting cells, and the cell files recognition step. Some preliminary results obtained on several species of conifers are presented and analyzed

Intra-annual fluctuation in morphology and microfibril angle of tracheids revealed by novel microscopy-based imaging

Woody cells, such as tracheids, fibers, vessels, rays etc., have unique structural characteristics such as nano-scale ultrastructure represented by multilayers, microfibril angle (MFA), micro-scale anatomical properties and spatial arrangement. Simultaneous evaluation of the above indices is very important for their adequate quantification and extracting the effects of external stimuli from them. However, it is difficult in general to achieve the above only by traditional methodologies. To overcome the above point, a new methodological framework combining polarization optical microscopy, fluorescence microscopy, and image segmentation is proposed. The framework was tested to a model softwood species, Chamaecyparis obtusa for characterizing intra-annual transition of MFA and tracheid morphology in a radial file unit. According our result, this framework successfully traced the both characteristics tracheid by tracheid and revealed the high correlation (|r| > 0.5) between S2 microfibril angles and tracheidal morphology (lumen radial diameter, tangential wall thickness and cell wall occupancy). In addition, radial file based evaluation firstly revealed their complex transitional behavior in transition and latewood. The proposed framework has great potential as one of the unique tools to provide detailed insights into heterogeneity of intra and inter-cells in the wide field of view through the simultaneous evaluation of cells’ ultrastructure and morphological properties

Open tools for dendrochronology. Advances in sample digitization and deep learning methods for image segmentation

Dendrochronological techniques are paramount in forest research. The current climate change scenario and the central role of forests in biogeophysical cycles enforce the importance of novel techniques to get accurate data from trees and their relationship with the environment in faster ways. Recent technological advances and the place of open source software and hardware are making free, user-developed tools for forest research available to the research community. The aim of this Ph.D. thesis is the development of tools for image acquisition and data collection in dendrochronology based on open source software and hardware. Thus, four different tools for dendrochronological research are presented in five different chapters. The first chapter focuses on the development of a do-it-yourself tool based on open source hardware for image acquisition and wood sample digitization at high resolution. We used open hardware equipment from Arduino and Python programming to develop CaptuRING and published the entire free open source tool as: "CaptuRING: A Do-It-Yourself tool for wood sample digitization" in Methods in Ecology and Evolution, 2022; 13:1185-1191. Furthermore, the original software was registered in the Registro General de Propiedad Intelectual (00/2022/737) of Ministerio de Cultura y Deporte (Spain). The second chapter presents "How to build and install your own CaptuRING". This contribution presents a series of videos with a step-by-step guide to promote the use of CaptuRING in the research community. The manuscript and related videos have been submitted for publication. The third chapter describes ρ-MtreeRing. This free and open-source software, which is written in R, analyzes X-ray films from dendrochronological samples to get microdensity values automatically segmented through a graphical user interface. The open source tool and manuscript are published as: "ρ-MtreeRing. A graphical user interface for X-ray microdensity analysis" in Forests. 2021; 12(10):1405. The fourth chapter describes the potential of deep learning methods to automatically segment xylem vessels. We trained three different convolutional neural networks to segment vessels in stained wood microsections using the Keras framework in Python. Our results demonstrate the potential of these techniques to automatically segment xylem vessels and overcome derived problems from image illumination, which hamper segmentation using classical image segmentation methods. The manuscript is published as "Convolutional neural networks for segmenting xylem vessels in stained cross-sectional images" in: Neural Computing & Applications, 2020; 32:17927-17939. The fifth chapter develops an algorithm to delineate annual ring limits in stained wood microsections of a species with diffuse porous wood using convolutional neural networks. We used Python for image processing and the Keras framework for the algorithm training. The results show the ability of this techniques to obtain accurate tree ring segmentation for quantitative wood anatomy, reaching similar or even outperforming conventional manual delimitation in most of the evaluated cases. The results of this chapter will be presented in the manuscript "Deep Learning for ring bordering in stained cross-sectional images". This PhD Thesis presents four open source tools to get accurate information from wood features to unveil how trees respond to the environment. From digitization at macroscopic perspective, automatic data collection and the development of feature segmentation on microscopic samples. The presented four novel dendrochronological tools based on open source software facilitates forest research in the current climate change scenario.Las técnicas dendrocronológicas son fundamentales en la investigación forestal. El escenario actual de cambio climático y el papel central de los bosques en los ciclos biogeofísicos subrayan la necesidad de nuevas técnicas para obtener de un modo ágil datos precisos de los árboles y de su relación con el medio ambiente. Los recientes avances tecnológicos, además de la disponibilidad actual del software y el hardware de código abierto están poniendo a disposición de la comunidad investigadora herramientas gratuitas desarrolladas por los usuarios para la investigación forestal. El objetivo de esta tesis doctoral es el desarrollo de herramientas para la adquisición de imágenes y la recogida de datos basadas en software y hardware de código abierto para el estudio dendrocronológico. Esta tesis presenta cuatro herramientas diferentes para esta rama científica en cinco capítulos diferentes. El primer capítulo se centra en el desarrollo de una herramienta "hágalo usted mismo" basada en hardware de código abierto para la adquisición de imágenes y la digitalización de muestras de madera a alta resolución. Usamos equipos de hardware abierto de Arduino y programación de Python para desarrollar CaptuRING y publicamos la herramienta completa de código abierto como: "CaptuRING: A Do-It-Yourself tool for wood sample digitization" en Methods in Ecology and Evolution, 2022; 13:1185-1191. Además, el software original fue registrado en el Registro General de Propiedad Intelectual (00/2022/737) del Ministerio de Cultura y Deporte (España). El segundo capítulo presenta "Cómo construir e instalar su propio CaptuRING" ("How to build and install your own CaptuRING"). Esta contribución presenta una serie de vídeos con una guía paso a paso para promover el uso de CaptuRING en la comunidad investigadora. El manuscrito y los vídeos relacionados se han enviado para su publicación. El tercer capítulo describe ρ-MtreeRing. Este software gratuito y de código abierto, que está escrito en R, analiza imágenes de rayos X de muestras dendrocronológicas para obtener valores de microdensidad automáticamente segmentados a través de una sencilla interfaz gráfica de usuario. La herramienta de código abierto y el manuscrito se publicaron como: "ρ-MtreeRing. A graphical user interface for X-ray microdensity analysis" en Forests. 2021; 12(10):1405. El cuarto capítulo describe el potencial de los métodos de aprendizaje profundo para segmentar automáticamente los vasos del xilema. Entrenamos tres redes neuronales convolucionales diferentes para segmentar vasos en cortes histológicos de madera utilizando el marco Keras en Python. Nuestros resultados demuestran el potencial de estas técnicas para segmentar automáticamente los vasos del xilema y superar los problemas derivados de la iluminación de la imagen, que dificultan la labor de métodos clásicos de segmentación de imágenes. El manuscrito se publicó como "Convolutional neural networks for segmenting xylem vessels in stained cross-sectional images" en: Neural Computing & Applications. 2020; 32:17927-17939. El quinto capítulo desarrolla un algoritmo para delinear los límites anuales de los anillos en cortes histológicos de una especie con madera difuso-porosa utilizando redes neuronales convolucionales. Se utilizó Python para el procesamiento de imágenes y el marco Keras para el entrenamiento del algoritmo. Los resultados muestran la capacidad de estas técnicas para obtener una segmentación precisa de los anillos de los árboles para la anatomía cuantitativa de la madera alcanzando, en la mayoría de los casos evaluados, un rendimiento similar o incluso superior a la delimitación manual convencional. Los resultados de este capítulo se presentarán en el manuscrito "Deep Learning for ring bordering in stained cross-sectional images". Esta Tesis Doctoral presenta cuatro herramientas de código abierto para obtener información precisa de las características de la madera investigar cómo los árboles responden al entorno facilitando la investigación en el actual escenario de cambio climático.Escuela de DoctoradoDoctorado en Conservación y Uso Sostenible de Sistemas Forestale

CHARACTERIZING INTRA-ANNUAL XYLEM CELL FORMATION AND CIRCADIAN CYCLE DYNAMICS OF JACK PINE (PINUS BANKSIANA) IN THE NORTHERN BOREAL FOREST, YELLOWKNIFE, NORTHWEST TERRITORIES

Xylem cell formation and stem radial fluctuations for jack pine in Yellowknife, NT were assessed during the 2017 season. The characterization of the timing of the onset of xylogenesis reactivation, period(s) of rapid cell development, and termination of seasonal stem growth were recorded over the estimated growing season. Two methods, microcores and automatic point dendrometers were used to better understand what the characteristics of a growth season looks like for jack pine at its northern limit within the boreal forest. It was demonstrated that onset of cell development at the study site “Treeville” occurred once temperatures had exceeded the 4-5ºC temperature threshold, the period of rapid growth was observed around June 21 for all phases of xylogenesis, aligning with the timing of annual longest photoperiod during summer solstice. It is demonstrated that jack pines in this region are much slower growing, with cell development at ~0.28 cells/day during the period of rapid growth, and site wide average of ~13 cells. Precipitation was highly correlated with the observation of the SRI phase throughout the season, most notably in the period prior to cell wall-thickening. A multi-year cell development assessment indicated a relationship with June precipitation, demonstrating lower cell counts in years with low June precipitation. Cell growth was recorded to end in late August, although some trees demonstrated cells in developing phases into early September. Overall, results suggest (1) strong relationship between the onset of jack pine wood formation when temperatures exceed established thresholds for growth, (2) demonstrated that the period of rapid growth aligns with longest daylength June, and (3) annual cell count is controlled by June precipitation

Evaluation of Skylab (EREP) data for forest and rangeland surveys

The author has identified the following significant results. Four widely separated sites (near Augusta, Georgia; Lead, South Dakota; Manitou, Colorado; and Redding, California) were selected as typical sites for forest inventory, forest stress, rangeland inventory, and atmospheric and solar measurements, respectively. Results indicated that Skylab S190B color photography is good for classification of Level 1 forest and nonforest land (90 to 95 percent correct) and could be used as a data base for sampling by small and medium scale photography using regression techniques. The accuracy of Level 2 forest and nonforest classes, however, varied from fair to poor. Results of plant community classification tests indicate that both visual and microdensitometric techniques can separate deciduous, conifirous, and grassland classes to the region level in the Ecoclass hierarchical classification system. There was no consistency in classifying tree categories at the series level by visual photointerpretation. The relationship between ground measurements and large scale photo measurements of foliar cover had a correlation coefficient of greater than 0.75. Some of the relationships, however, were site dependent

Environmental effects and biophysical constraints on xylem physiology and tree growth in conifers in the Alps

Trees are impressive long-living organisms that continuously increase in size by many orders of magnitude during ontogeny by accumulating xylem biomass in stem, branches and roots. While growing taller, trees continuously adjust the xylem structure to achieve an optimal balance of carbon costs for the competing biomechanical and hydraulic requirements. One of the main function of the xylem structure is the delivery of the water from the roots up to the leaves. This must be maintained during the ontogeny, when the hydrodynamic resistance increase due to the increase in the xylem path length. However, by widening the diameter of xylem conduit (from the stem apex downwards), trees are able to minimize the negative effect of height growth. Additionally, this widening is stable during ontogeny, thus determining the radial change in conduit dimension with cambial age (from the pith outwards), implying a dependency between the variation of conduit-lumen diameter with cambial age and the rates of stem elongation. These adjustments in the xylem structure remain permanently fixed and chronologically archived in the secondary xylem, and, given the tight link between structures and functions, these provide a ‘time component’ to functional responses induced by xylem plasticity, thus allowing to reconstruct growth dynamics under different environmental conditions. However, there is a lack of detailed information and standardized procedures to explore, at the intra-specific level, the long-term modifications of xylem traits over the full life-span of trees, together with their variability along axial and radial profiles. Additionally, little is known about the relationships between the structures and functions in a view of exploring the future challenges in how a plant’s hydraulic architecture may respond to the ongoing climate change. This thesis, represent a set of studies based on dendro-anatomical and physiological approaches aimed to: - identify priorities and trade-offs among xylem functions; - determine the anatomical traits responsible for them; - retrospectively analyze how these relationships vary during ontogeny under different environmental condition; - analyze the functional response to xylem modifications occurring during ontogeny; - investigate the possibility of retrospectively analyzed height growth based on hydraulic radial profiles. Furthermore, a guidance from sample collection to xylem anatomical data and a new approach to customize cell wall thickness measurements according to the specific aims of the study, were developed. This thesis has highlighted that the xylem anatomical structure of conifer trees (Larix decidua, Picea abies, Pinus cembra) showed a high priority and biophysical determination of traits linked to hydraulic efficiency, such as conduit size, to efficiently support assimilation necessary for tree growth. Besides, other functional traits linked to mechanical support and metabolic xylem functions showed more plastic responses to intrinsic and extrinsic factors. Due to the ontogenetic stability of axial patterns of conduit size, it was possible, based on radial profiles of xylem conduit diameter of tree rings, to estimate tree growth rate, even if species-site specific, and make comparison between trees living in different epochs. In addition, despite the risk of becoming more vulnerable to air seeding cavitation, trees showed to prioritize of hydraulic efficiency vs. safety during the ontogenetic development, as the increase in xylem conductance with tree height determined a contextual decrease in the hydraulic safety margin. This study showed the importance of taking into account the three dimensional anatomical trends to better understand of the trade-offs of hydraulic safety vs. efficiency shape up the tree architecture and affect its adjustments occurring during ontogeny to cope with the arising intrinsic (i.e., size-related) and extrinsic (i.e., environmental) constraints to growth

Scientific merits and analytical challenges of tree-ring densitometry

R.W. was supported by NERC grant NE/K003097/1.X-ray microdensitometry on annually-resolved tree-ring samples has gained an exceptional position in last-millennium paleoclimatology through the maximum latewood density parameter (MXD), but also increasingly through other density parameters. For fifty years, X-ray based measurement techniques have been the de facto standard. However, studies report offsets in the mean levels for MXD measurements derived from different laboratories, indicating challenges of accuracy and precision. Moreover, reflected visible light-based techniques are becoming increasingly popular and wood anatomical techniques are emerging as a potentially powerful pathway to extract density information at the highest resolution. Here we review the current understanding and merits of wood density for tree-ring research, associated microdensitometric techniques, and analytical measurement challenges. The review is further complemented with a careful comparison of new measurements derived at 17 laboratories, using several different techniques. The new experiment allowed us to corroborate and refresh ?long-standing wisdom?, but also provide new insights. Key outcomes include; i) a demonstration of the need for mass/volume based re-calibration to accurately estimate average ring density; ii) a substantiation of systematic differences in MXD measurements that cautions for great care when combining density datasets for climate reconstructions; and iii) insights into the relevance of analytical measurement resolution in signals derived from tree-ring density data. Finally, we provide recommendations expected to facilitate future inter-comparability and interpretations for global change research.Publisher PDFPeer reviewe

Simulating Wood Properties in the Context of a Growth and Yield Model for Planted Douglas-fir

Forest growth and yield models are critical to supporting decision making in forestry, but often lack considerations for wood properties. The feasibility of simulating wood properties in the context of a Douglas-fir individual tree growth and yield model was evaluated. This assessment explored the effect of predicted sapwood width, stem taper, branch size and juvenile wood proportion on recovery of utility poles and sawn lumber. Results from the assessment indicated wood properties can be simulated in the context of growth and yield modeling systems with biologically reasonable results. The same assessment indicated recovery of wood products may be overestimated in the absence of penalties for stem defects. Two critical Douglas-fir wood attributes, knot geometry and wood density, were targeted for modeling after the feasibility analysis. A model for Douglas-fir knot pith curvature was developed based on predicted branch angle and growth ring position over time. Based on the predicted knot pith curvature, knot geometry could be implied from branch radius predicted at the stem surface. Medical computed tomography was identified as one way to rapidly and efficiently estimate Douglas-fir wood density from the proxy of X-ray attenuation. In support of deploying this technology, the effect of wood moisture content and other scanner settings on the relationship of X-ray attenuation and wood density was modeled. Results indicated that X-ray attenuation explains most of the variation in predicting wood density, but moisture content and other scanner settings have a small but significant effect. A number of knowledge gaps should be resolved to sufficiently and robustly simulate wood properties in the context of Douglas-fir growth and yield simulation. Models for sawing simulation and for addressing deficits in existing wood properties models should be an important focus for future research in this field

Functional wood traits: xylogenesis, tree rings and intra-annual density fluctuations (IADFs) in Mediterranean species

The occurrence of long and severe summer drought periods, forecasted for the Mediterranean basin (IPCC, 2013), is expected to affect cambial activity and wood formation, thus altering tree growth and vegetation dynamics thus affecting the productivity of forests (Sarris et al., 2007; Martinez del Castillo et al., 2016). In such a context, is important to understand the strategies in water utilisation exploited by different Mediterranean tree and shrub species in response to variation in the climate conditions. Such knowledge would allow to predict the response of Mediterranean ecosystems to climate change and help in decision-making for a focused forest management. The general aim of the thesis is to acquire knowledge on the adaptation strategies operated at the xylem level by Mediterranean species to cope with the intra-annual variation of environmental conditions, and in particular with summer drought. This main aim has been pursued through the study of wood functional traits within tree-ring series, focusing in tree rings presenting Intra-annual density fluctuations (IADFs). The starting hypothesis of this Doctoral Thesis is that the Mediterranean woody species could respond to the seasonal distribution of precipitations and temperature patterns, showing a bimodal model of xylem growth identifiable by main trends in the wood functional traits, attested by the occurrence of IADFs within tree rings

Sareomycetes: more diverse than meets the eye

Since its resurrection, the resinicolous discomycete genus Sarea has been accepted as containing two species, one with black apothecia and pycnidia, and one with orange. We investigate this hypothesis using three ribosomal (nuITS, nuLSU, mtSSU) regions from and morphological examination of 70 specimens collected primarily in Europe and North America. The results of our analyses support separation of the traditional Sarea difformis s.lat. and Sarea resinae s.lat. into two distinct genera, Sarea and Zythia. Sarea as circumscribed is shown to conservatively comprise three phylospecies, with one corresponding to Sarea difformis s.str. and two, morphologically indistinguishable, corresponding to the newly combined Sarea coeloplata. Zythia is provisionally maintained as monotypic, containing only a genetically and morphologically variable Z. resinae. The new genus Atrozythia is erected for the new species A. klamathica. Arthrographis lignicola is placed in this genus on molecular grounds, expanding the concept of Sareomycetes by inclusion of a previously unknown type of asexual morph. Dating analyses using additional marker regions indicate the emergence of the Sareomycetes was roughly concurrent with the diversification of the genus Pinus, suggesting that this group of fungi emerged to exploit the newly-available resinous ecological niche supplied by Pinus or another, extinct group of conifers. Our phylogeographic studies also permitted us to study the introductions of these fungi to areas where they are not native, including Antarctica, Cape Verde, and New Zealand and are consistent with historical hypotheses of introduction