IAWA Journal, Vol. 28 (2), 2007: 125-137

WOOD ULTRASTRUCTURE OF ANCIENT BURIED LOGS OF FITZROYA CUPRESSOlDES

Maria A. Castro1 and Fidel A. Roig2

SUMMARY

The anatomy and ultrastructure of subfossil wood of Fitzroya cup res­ soides from the late Pleistocene (>50,000 14C years before present) were compared with those of extant F. cupressoides trees from southern Chile, using light microscopy (polarized light and ftuorescence), scanning elec­ tron microscopy coupled with an energy dispersive X-ray spectroscopy system, and transmission electron microscopy. The ancient wood showed an unchanged gross wood structure, loss of cell wall birefringence, loss of lignin autoftuorescence, and a loss of the original microfibrillar pat­ tern. The energy dispersive X-ray spectroscopy analysis indicated higher than normal contents of S, Cl, and Na in subfossil wood. Ultrastructural modifications in the cell wall of the subfossil wood could have important implications for further studies involving isotopic and wood anatomical measurements of ancient wood. Key words: Fitzroya cupressoides, Pleistocene subfossil wood, cell wall ultrastructure, TEM, SEM-EDXA analysis, wood anatomy.

INTRODUCTION

The temperate rain forest of South America has a very rich tree species assemblage with a high level of endemism (Arroyo et al. 1993). One of the natural endemies is Fitzroya cupressoides (Molina) I.M.lohnston (alerce, Cupressaceae), a tree species that grows under a relatively low annual mean temperature and high precipitation in areas ofthe southernAndes ofChile and southwesternArgentina. Tree-ring analysis revealed that Fitzroya is a slow-growing tree and is one of the longest-lived tree species in the world, known to reach up to around 3,500 years of age (Lara & Villalba 1993). One record, which was developed from a limited number of pollen assemblages, indicates that Fitzroya forests existed in our study area of southern Chile about 50,000 14C years aga or possibly before (Heusser et al. 1999). Since the end of the 16th century, Fitzroya was intensively exploited for its highly­ prized timber. Today, the species is protected, and the only wood that is harvested is from remnant stumps or logs lying under the upper soillayers. The extraordinary resist-

1) Laboratorio deAnatomia Vegetal, DBBE-FCEN, UBA, Intendente Guiraldes 2620, Ciudad Uni­ versitaria, Pab 11, 4° Piso, (1428) Buenos Aires, Argentina [E-mail: [email protected]]. 2) Laboratorio de Dendrocronologfa e Historia Ambiental, IANIGLA-CRICYT, CC 330 (5500) Mendoza, Argentina [E-mail: [email protected]]. Associate Editor: Lloyd Donaldson

Downloaded from Brill.com10/02/2021 07:21:54PM via free access 126 IAWA Journal, Vol. 28 (2), 2007 ance of the Fitzroya wood to decay, even after thousands of years, as evidenced by the presence of these buried logs, has been repeatedly noted in the literature (Smithüsen 1960; Hück 1978). The low pH values ofthe soil, in addition to the cool, wet, and temperate c1imatic con­ ditions where these logs are found (Roig et al. 1996) may partially facilitate preserva­ tion. As a consequence of the 1960 earthquake in the southem Chilean Lake District, an intertidal area alongside the north shore of Seno Reloncavf and the areas of the eastem co ast of Chiloe Island were eroded. As a result of this erosion, various well-preserved subfossil F. cupressoides stumps were exposed (Klohn 1975, 1976; Heusser 1981; Villa­ gnin et al. 2004). AMS 14C dating revealed that these stumps are around 50,000 14C years old (Roig et al. 2001). However, these dates are at the uppermost limit of 14C dating, and therefore they should be regarded as minimum ages for this subfossil wood. A selection of the wood material from these stumps is the subject of this study. Various authors (Creber & Chaloner 1984; Florian 1990; Hoffman & Iones 1990; Larson & Melville 1996; Schiffer 1987) have evaluated the effects of environmental conditions on the preservation over time of different wood characteristics. These au­ thors generally agree that ancient buried wood is subjected to physical and biological processes that are responsible for both the retention and loss of woody materials, par­ ticularly the removal of the structural carbohydrates followed by the collapse of the lignin skeleton in the cell walls. This study examined changes in structural and ultrastructural characteristics of late Pleistocene (-50,000 14C years BP) Fitzroya cupressoides wood sampies. Eventual changes in chemical or physical characteristics in subfossil wood could have implica­ tions, for example for the conservation of such materials or the derivation of reliable tree-ring data particularly for the development of tree-ring chronologies based on wood density ftuctuations.

MATERIAL AND METHODS

The subfossil wood sampies of Fitzroya cupressoides used in the study were obtained from stumps located at different sites in the Reloncavi Bay and on the eastem shore of Chiloe Island (Roig et al. 2001; Villagran et al. 2004). For comparison,wood sampies from F. cupressoides were taken from extant trees growing in the vicinity of the same areas (Roig et al. 1996). For light microscopic study (LM), small wood blocks were sectioned (10-15 I-lm thick), stained with 1% safranin in 50 % alcohol, dehydrated, and then mounted in artifi­ cial balsam. An additional set of unstained trans verse sections was prepared for polarized light microscopy (LMPL) and ftuorescence (LMUV) microscopy. Phloroglucinol/HCl was used to detect the lignification level in xylem cell walls (D' Ambrogio de Argüeso 1986). A scanning electron microscope (ESEM-Philips XL30, Eindhoven-Holland) cou­ pled with an energy dispersive X-ray spectroscopy analysis system (EDXA) was used to evaluate the inorganic constituents of the cell walls of the wood specimens. For

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ESEM-EDXA, longitudinal sections of wood sampIes were analyzed without previous treatment. For observations using transmission electron microscopy (TEM), blocks of 2 x 3 mm were fixed in 3.5 % glutaraldehyde in 0.1 M phosphate buffer (pR 7.2-7.5), re-fixed in 1.5 % osmium tetroxide in buffer solution, dehydrated in acetone series and then embedded in Spurr's low viscosity epoxy resin (Spurr 1969). Blocks were cut into ultra-thin sections using a Sorvall MT 2-13 ultramicrotome with a diamond knife, stained with uranyl acetate and lead citrate prior to exarnination by a Siemens Elmiskope microscope (Siemens GA, Karlsruhe, Germany). Furthermore, ultrarnicrotome sections of about 1 !Am thick were double stained with fuchsin-toluidine blue for LM.

RESULTS In trans verse section, LM observations of both extant and subfossil sampIes reveal the well- known non structure recorded for Fitzroya cupressoides wood (Roig 1992). The growth rings are normally very narrow (30 cells wide) with a distinct ring bound­ ary marked by radial ftattening of the latewood tracheids (Fig. 2); sometimes the ring boundaries are slightly undulated. The axial parenchyma is scarce and diffuse, and occasionally loosely grouped in tangential bands near or at the beginning of the late­ wood zone. The dimension and shape of cells of the subfossil wood do not appear to be different from those of the wood from extant trees. Rowever, the subfossil tracheids show conspicuous holes or hemispherical cavities in their cell walls, probably resulting from localized activity of micro-organisms (Fig. 1 & 2D).

Figure 1. Subfossil wood of Fitzroya cupressoides: general aspect in trans verse seetion. - Scale bar = I nun.

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Figure 2. Light micrographs of transverse seetions of subfossil wood. - A-D: general organiza­ tion of the tissue is unaffected. - D: Partial decay in earlywood tracheids as a result of attack by micro-organisms; see the presence of micro-organisms in the lumina (arrow) and cell wall cavi­ ties of tracheids. - Scale bars = A: 100 !lm, B-D: 50 !lm.

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The analysis of the lignin contents of the subfossil wood cell walls, using the staining method and light microscopy, showed varying amounts of lignin for different regions of the ring. The brownish color of the earlywood tracheid walls after applying the phloroglucinol / HCl reaction test indicates a severe loss of lignin. The loss of lignin in the earlywood tracheids is greater than that in the latewood tracheid cell walls (stained purple after the phloroglucinol/HCl test), suggesting that the latewood tracheids main­ tained a similar lignification level as cells in recent material. In addition, observations using LMUV showed a loss of lignin autofiuorescence of the earlywood tracheid cell walls (Fig. 3), and an autofiuorescence ofthe latewood tracheid cell walls that is some­ what similar to that observed in equivalent cells of extant trees. Furtherrnore, under

Figure 3. Transverse sections showing lignin autoftuorescence level in both early- and latewood zones. - A: Intense autoftuorescence (normal lignin distribution in the middle lamella and sec­ ondary wall) in extant wood. - B: Weak autoftuorescence (abnormallignification ofboth middle lamella and secondary wall) in subfossil wood. - Scale bars = 100 !-lm.

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Figure 4. Cell wall anisotropie properties seen in polarized light. - A & B: Birefringence of extant wood. - C & D: Loss of cell wall birefringence in subfossil wood. - Scale bars = 100 !Am.

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Figure 5 - For the legends, see the next page.

Downloaded from Brill.com10/02/2021 07:21:54PM via free access 132 IAWA Journal, Vol. 28 (2), 2007 polarized LM, we observed a loss of the wall birefringence of latewood tracheids and an almost complete loss of birefringence of earlywood tracheids (Fig. 4), which indi­ cates a deterioration of the cell wall anisotropic properties. At higher magnifications, we observed uneven staining ofthe cell walllayers. This is probably because oflower lignin content in the SI and innermost parts of the S2' and practically no lignin content in a small part of the outermost wall sector of S2' the S3 and the warty layer. TEM micrographs ofliving Fitzroya wood showed an electron-dense cell wall formed by packed microfibrils with a more or less uniform lignification pattern. In addition, the cellulose microfibrils have variable orientations within the tracheid cell wall (Fig. 5 A, B). ConverseIy, TEM micrographs of the subfossil wood showed a tracheid cell wall ultrastructure composed of a loose cellulose microfibrillar pattern and microcapilaries (Fig. 5C-F). This altered ultrastructural fibrillar pattern is observed throughout the secondary wall. EDXA indicated that the elemental (inorganic) components of cell walls for subfossil wood specimens were different from those of extant wood (Fig. 6). EDXA spectra from subfossil wood showed significant peaks for Cl, Na and S, and minor peaks for Mg, Ca and Si (Fig. 6A, B). EDXA spectra for extant wood yielded a substantially differ­ ent result with the absence of most of the previously mentioned inorganic constituents (Fig.6C).

DISCUSSION

The literature provides evidence that wet anoxie conditions (e.g. Hoffman & Iones 1990; Larson & Melville 1996) or exceptionally dry environments (e.g. Schweingruber 1983; Larson & Melville 1996) can support the long-term preservation ofwood. Such environmental conditions are necessary to prevent or diminish the biologie al activity of micro-organisms that effect wood properties. In particular, the wood of several Cu­ pressaceae including Cupressus, Juniperus, Chamaecyparis, Austrocedrus, Fitzroya, and Thuja show a considerable resistance to decay (Schweingruber 1983; Le Quesne et al. 2000; Roig et al. 2001). A non-mineralized waterlogged Cupressaceae log from central Germany that was preserved in lignite sediments since the Miocene (20-25 million years BP; Hoffmann & Blanchette 1997) is evidence of how many years wood properties can be preserved under certain biogeochemical conditions. Archaeologists and dendrochronologists are especially interested in using these very old preserved logs to research aspects of the past environment.

-Figure 5. TEM micrographs of trans verse sections of tracheid walls, uranyl acetate and lead citrate staining. - A & B: TEM micrographs of extant wood. - A: Tracheids showing a well­ developed middle lamella and secondary cell wall. - B: Cell wall tracheids in transverse section; note the electron-dense wall with a packed microfibrillar arrangement and variable orientation of cellulose fibrils. - C-F: TEM micrographs of subfossil wood. - C: Tracheids showing a well­ developed middle lamella and a less electron-dense secondary cell wall. - D-F: Progressive higher magnifications showing a loose fibrillar organization and microcapilaries. - A & C = x4,OOO; B = x31,500; D = x25,OOO; E & F = x50,OOO.

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In this research concerning Fitzroya transverse sections under LM showed that the gross anatornical features of subfossil wood are similar to those of extant wood. This suggests that cell dimensions and shape have not been modified. Deformation of the stern body by pressure is a comrnon feature observed in horizontally positioned archaeologicallogs (Hoffmann & Blanchette 1997). However, because the tree from which these subfossil remnants were collected were buried in their upright position, their original size and shape were basically preserved, even though they were under the pressure of thousands of tons of glacial deposits (mostly accumulated during the Last Glacial Maximum in the region of Seno Reloncavf and Isla Grande de Chiloe between 29,400 14C years and 14,500 14C years BP; Denton et al. 1999). However, at the ultrastructurallevel the subfossil tracheid cell wall seems to have undergone severe modifications, as indicated by chemical alteration and modification of the original cellulose microfibrillar pattern. Throughout its history underground, the Fitzroya subfossil wood has probably been subjected to variable conditions ofhurnidity, inc1uding exposure to salt water (Roig et al. 2001). The literature suggests that wood decay is provoked by both microbial and chemical factors when exposed to long-term underwater conditions (Kim & Singh 2000). The hernispherical cavities or openings in the subfossil cell walls suggest that bacteria or marine fungi could have caused wood biodegradation. Under anoxic conditions or low levels of oxygen, such as water­ saturated environments or deep layers of fine-textured sediments, wood is primarily degraded by bacteria that seem to be highly tolerant to such conditions (Singh et al. 1990; Kim et al. 1996; Kim & Singh 2000). Because chernical analysis of the wood was not inc1uded in this study, we cannot be certain that biological processes modified the chemical composition of the cell walls. Wood decay as evidenced by a decrease in lignin autofluorescence could be caused by physical environmental actions over long periods of time. The loss of lignin auto­ fluorescence could result from the collapse of the lignin skeleton of the cell walls. Although many authors support the idea that lignin is not extensively degraded in wet environments, some authors reported some kind of modifications in lignin structure (Zeikus 1980; Brenner et al. 1984). Fengel (1991) suggested that guiacyllignin is more stable that syringyllignin in aging wood subfossils. Less intense staining can indicate modifications in the lignin content of the cell wall (Kim & Singh 2000). The following characteristics suggest modifications in the lignin properties of Fitz­ raya subfossil woods: brownish color acquired by the cell walls of the earlywood tra­ cheids after applying the phloroglucinol/HCI reaction test; the higher autofluorescence of earlywood tracheids versus latewood tracheid cell walls; and decreased electron­ density in the middle lamella, and even the breakdown of cell walls. Similarly, chernical cell wall alterations and changes in the orientation of the cel­ lulose microfibrils could be the cause of loss of cell wall birefringence. In waterlogged environments, hemicellulose is much more readily degraded than cellulose (Hoffmann & Jones 1990; Kim 1990) and in extreme conditions, waterlogged wood may be almost totally depleted of polysaccharides. In order to fully understand the molecular and biochernical characteristics ofburied and waterlogged woods subjected to decay, X-ray diffraction techniques should be

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C CI A

o Na S

Mg Si K Ca

0.70 1.40 2.10 2.80 3.50 4.20 4.90 5.60 6.30 7.00 C B

0 S CI Mg K Ca Na Si

0.70 1.40 2.10 2.80 3.50 4.20 4.90 5.60 6.30 7.00 C c

o

Mg S. Na I SCI K Ca

0.70 1.40 2.10 2.80 3.50 4.20 4.90 5.60 6.30 7.00 Energy (keV)

Fig. 6. SEM-EDXA surface analysis. Energy spectra of subfossil (A, earlywood tracheid cell walls; B, latewood tracheid cell walls) and extant (e, earlywood tracheid cell wall) woods.

Downloaded from Brill.com10/02/2021 07:21:54PM via free access Castro & Roig - Subfossil Fitzroya cupressoides 135 used to compare the cellulose crystallinity of living and subfossil woods. Analytical studies of cell wall chemical composition would provide estimations of the cellulose, hemicellulose, lignin and pectic contents. This would help to explain the process of cell wall degradation as evidenced by changes in anisotropie wall properties, loss or decrease of lignin autofluorescence and alterations of cell wall ultrastructure. This study provides evidence of decay of subfossil wood. Under wet environmental conditions, physical and chemical interactions should be considered among the many potential causes of wood degradation. But bacteria and soft rot fungi should be taken into account as important agents of decay. Under wet conditions, the lack of oxygen may restriet decay by more aggressive basidiomycetes. Experimental data are needed to determine the exact level of oxygen that is required for decay by bacteria and fungi. It is known that, under wet conditions, bacterial erosion is the most common type of decay. The fact that bacteria are sometimes the only cause of decay under wet condi­ tions may be evidence that they have the highest tolerance to low oxygen availability. No doubt, there may be factors others than oxygen availability that influence the type and rate of wood decay under wet conditions (Kim & Singh 2000). Some observations indicate that wood exposed to wet environments deteriorates more from the action of biological factors than from non-biological factors (Kim & Singh 2000). The cavities found in the cell walls of the subfossil Fitzroya wood indicate that bacteria have a major role in the degradation of this species under wet conditions. Regarding the inorganic composition ofthe cell walls, the X-ray analysis provided different spectral patterns between subfossil (Fig. 6A, B) and living (Fig. 6C) wood. The analysis indicated that proportions of carbon and oxygen are the same for subfossil and living woods. However, there are many other elements that are always found in subfossil woods, as indicated by the high spectral energy levels of Cl and Na, and lower levels of S, Mg and Si in these woods. The analysis indicates that there are relatively higher levels of Cl and Na in subfossil earlywood cell walls compared with latewood cell walls. The presence of these elements in the subfossil cell walls is probably related to prolonged exposure to salt ocean water.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to Ing. Andres Pinto and Sandra Romano from INTI-SEG­ EMAR for their assistanee in the SEM-EDXA analysis. Thanks to Judy Boshoven who helped us to improve the manuseript. This work was supported by a National Geographie Soeiety grant 7345-02 to F.A. Roig.

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