Elsevier

Dendrochronologia

Volume 69, October 2021, 125878
Dendrochronologia

Technical Note
Growth-ring boundaries of tropical tree species: Aiding delimitation by long histological sections and wood density profiles

https://doi.org/10.1016/j.dendro.2021.125878Get rights and content

Highlights

  • A protocol for obtaining long histological sections of medium-high-density wood is presented.

  • A multi-proxy approach is used to analyse growth-ring boundaries in six tropical species.

  • Visualization, delimitation and characterization were improved at a microscopic scale.

  • Our methodology opens new perspectives for dendrochronological studies in the tropics.

Abstract

Recent methodological advances have opened new perspectives for tropical dendrochonological studies by facilitating the visualization, delimitation, and analyses of tree-rings. One of those improvements was brought by X-ray densitometry, which allows building radial wood density profiles at microscopic scale. Furthermore, recent methods allow for cutting long histological sections to study anatomical variations along the entire radius of trees. These techniques have mainly been applied to low wood-density species from temperate and Mediterranean regions, with only limited applications in the tropics. Here we provide an improved protocol that allows for obtaining long histological sections of tropical woods, apply it to six species with varying wood densities 0.45−0.85 g cm−3 (Eucalyptus grandis, Tectona grandis, Acacia mangium, Cedrela fissilis, Hymenaea courbaril, and Copaifera duckei), and explore potential applications for tree-ring analyses. We provide instructions on core-microtome knife adjustments, procedures for softening and sectioning long histological samples of high wood-density species. We also present a multi-proxy approach that combines X-ray density profiles with the histological sections that improve the characterization and distinction of the various and complex tropical growth rings anatomical markers (fibre zone, marginal parenchyma, and ring porosity). This multi-proxy approach also opens the door for obtaining quantitative anatomy and physical parameters of tropical species with (intra-annual resolution. Our proposed approach is thus not only an additional tool to improve ring-boundary delimitation of tropical species, but it also paves the way to more innovative, borderline approaches in tropical dendrochronology.

Introduction

In plants that present secondary growth, the formation of secondary xylem is regulated by genetic expression, tree age and environmental conditions (De Micco et al., 2019). The vascular cambium drives this secondary growth by cell-division and differentiation processes (De Micco et al., 2014; Lupi et al., 2010; Schweingruber, 2007). These processes can be temporarily interrupted under unfavourable environmental conditions (e.g., cold, drought, flooding) or during specific phenological phases (e.g., leafless periods) in these plants (Fritts 1976). The dormancy of the cambium layer may induce the formation of annual tree rings and the information stored in subsequent wood layers (e.g., ring width, wood density, isotope composition, etc.) is crucial to understand tree functioning and provides unique long-term data useful for global-change research (Babst et al., 2017; Björklund et al., 2019). In tropical forests, however, tree-ring analyses or dendrochronology is often precluded by the absence of clearly identifiable annual growth rings, or by the difficulty in finding common growth signals between trees due to weak effects of environmental seasonality and variability (Fichtler et al., 2003; Groenendijk et al., 2014; Worbes, 2002).

In general, the transverse surface wood samples of tropical species is polished with increasingly finer sandpaper to allow for the visualization and measurement of ring-widths (Orvis and Grissino-Mayer, 2002). This method is simple and affordable, but the delimitation of ring boundaries in some tropical species may still present difficulties and limitations, such as the rupture of thin cell walls, lumen obstruction, and difficulties visualizing narrow growth layers (Nath et al., 2016). Careful identification of what constitutes a ring boundary, preferably based on anatomical thin-sections, is an important first step to aid the macroscopic visualization, correct identification and measurement of tropical tree rings (Aragão et al., 2019; Fichtler and Worbes, 2012; Islam et al., 2018a). The realization of periodic cambial marks on the tree trunk (Mauriaux's windows), the use of dendrometer bands and radiocarbon analysis are also tools that assist in the identification of annual tree rings (Brienen et al., 2016; Groenendijk et al., 2014; Worbes, 2002). Moreover, recent methods using fluorescent light of different colours has been shown to further aid in ring-boundary identification on polished surfaces (Godoy-Veiga et al., 2019) and to reduce measurement errors. Clearly, extra care is needed when working with tropical species and multi-faceted approaches are important to guarantee the quality of tropical tree-ring research.

To aid in ring boundary identification of tropical species, additional approaches have been applied. Among them, X-ray densitometry has been used for improving the visualization of tropical tree rings (Gaitan-Alvarez et al., 2019; Tomazello Filho et al., 2008; Vetter and Botosso, 1989). X-ray densitometry allows for estimating wood density profiles ​​with high sensitivity at intra-annual resolution (e.g. Oliveira et al., 2017; Moreno-Fernández et al., 2018; Ortega Rodriguez and Tomazello-Filho, 2019) and marked density variations often coincide with annual ring boundaries (Pagotto et al., 2017). This technique was initially described as laborious and expensive compared to anatomical and dendrometric techniques, likely due to the lack of specialized equipment for cutting samples, the time needed to reveal and scan the X-ray films, and the measurements using non-specialized software (Jacquin et al., 2017). Although still requiring costly equipment and careful sample preparation to obtain wood sections of constant thickness (Jacquin et al., 2017), the procedure for X-ray density data acquisition nowadays is fast and sample preparation straightforward (Hervé et al., 2014) allowing for a wider application of the method. In spite of the high potential for X-ray densitometry to aid in characterizing tree rings of tropical woody species, the method has only occasionally been applied (Vetter and Botosso, 1989; Roque and Tomazelo-Filho, 2007; Nock et al., 2009; Oliveira et al., 2011; Pagotto et al., 2017; De Mil et al., 2018; Gaitan-Alvarez et al., 2019).

In addition to the visualization of tropical tree rings, the construction of long temporal series of anatomical features (especially diameter and vessel frequency) is currently a challenge for dendrochronological studies in the tropics (Islam et al., 2018b). Methods such as darkening the polished transversal wood surface with ink and filling the lumen of vessels with white chalk have been proposed to improve the macroscopic visualization of cell structures (especially xylem vessels) for their subsequent quantification (Fonti et al., 2010; Gärtner and Schweingruber, 2013; Islam et al., 2019a, 2018b; Rosell et al., 2017). However, tropical species presents a complex structure, where small vessels, for example, can be confused with other cellular elements, like axial parenchyma (Ewers and Carlquist, 2006) when prepared using this protocol. Working with diffuse-porous species requires a more refined method to be able to correctly distinguish and measure anatomical wood elements. In recent years, the use of the core-microtome (Gärtner and Nievergelt, 2010) allowed to improve the surface preparation process of core samples and to obtain long histological sections up to 40 cm in length (Ivanova et al., 2015) that can be used for a detailed characterization of tree rings at microscopic scale (Gärtner and Nievergelt, 2010). Long histological sections allow for the analysis of microscopic wood features throughout the plant's life and provide crucial insights into tree functioning and responses to past climate (e.g. Fonti and Jansen, 2012; Gärtner et al., 2014; von Arx et al., 2012). Long histological sections and related dendrochronological applications have been mainly conducted in boreal, Mediterranean and temperate species (e.g., Akhmetzyanov et al., 2019; Gärtner and Nievergelt, 2010; Gärtner et al., 2015a,b; Ivanova et al., 2015), but to our knowledge have not yet been applied in tropical species. Developing this field requires the development of adequate protocols to deal with the high wood density and the high diversity of anatomical structures of tropical tree species.

In this study, we aimed to (i) describe a protocol allowing to obtain long histological sections of six tropical species of contrasting wood densities (Eucalyptus grandis W. Hill ex Maiden, Tectona grandis L.f., Acacia mangium Willd., Cedrela fissilis Vell., Hymenaea courbaril L., Copaifera duckei Dwyer.), (ii) explore the potential of a multi-proxy approach, combining X-ray density with quantitative wood anatomy, to improve growth-ring boundary delimitation of species with varying anatomical markers (fibre zone, marginal parenchyma and ring porosity – (semi-)ring-porous). Along with a textual description of each steps, accompanied by detailed notes, we also provide ample visual material (photos and video) illustrating them. We expect that using long histological sections with X-ray densitometry will improve the visualization, delimitation, and characterization of growth-ring boundaries in tropical tree species. Additionally, we expect that the multi-proxy approach proposed here will facilitate expanding dendroscience research in the tropics, to further improve our understanding of the dynamics and functioning of tropical forests (Zuidema et al., 2013).

Section snippets

Species selection and preliminary sample preparation

We selected six species to be analysed based on their anatomical characteristics, and we included native and non-native species, as well as trees from plantation and form natural forests. With these species we cover the main growth-ring boundary (anatomical markers) types observed for tropical species (Fichtler and Worbes, 2012), we include some of the most valuable plantation species/genera in the tropics (Eucalyptus grandis, Tectona grandis and Acacia mangium), and include important

Long histological sections of tropical species of medium-high wood density

Using the core-microtome improves core-surface preparation (Gärtner and Nievergelt, 2010) and allows for the production of 15–20 μm thick sections of entire increment cores (Ivanova et al., 2015; von Arx et al., 2016). The increased stability of the core-microtome has been indicated as one of the main characteristics to obtain sections of up to 40 cm in length (Gärtner and Nievergelt, 2010; Gärtner et al., 2015a,b; Ivanova et al., 2015). We attest both improvements when working with six

Final remarks

Compared to sanding, using the core microtome to prepare the surfaces to be measured improves the visualization of wood structures as it provides a plane measuring surface with cells not obstructed by dust (Gärtner and Nievergelt, 2010). This improvement mainly aided working with diffuse-porous species that present a slight variation in the size and number of vessels along the transverse plane. Working with long histological radial sections also improves the visualization of tree-ring

Conclusions

The evaluation of tree-rings in tropical species reveals important insight into the dynamics of tropical forests and their relationship with the environment. The protocol described here allows for obtaining long histological sections of high-density tropical woods. Combined with X-ray density profiles and quantitative wood anatomy analyses, this approach provides improved visualization, delimitation and characterization of tropical tree rings, as well as a multi-proxy toolbox to advance

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the Laboratory of Wood Anatomy, Tree-Ring and X-ray Densitometry (LAIM) of the Department of Forest Sciences of Luiz de Queiroz College of Agriculture (ESALQ) (FAPESP project: 2009/53951-7), and the Itatinga Experimental Station of Forest Sciences. We acknowledge support from the EUCFLUX project. Also acknowledged are Francisco de Figueiredo, Pedro Vieira, Júlia Gil and Alinne Santos for their help and for providing some of the material used in this work. This research was supported by

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