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 Downloaded from Brill.com10/02/2021 07:21:54PM via free access Castro & Roig - Subfossil Fitzroya cupressoides 127 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. Downloaded from Brill.com10/02/2021 07:21:54PM via free access 128 IAWA Journal, Vol. 28 (2), 2007 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. Downloaded from Brill.com10/02/2021 07:21:54PM via free access Castro & Roig - Subfossil Fitzroya cupressoides 129 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. Downloaded from Brill.com10/02/2021 07:21:54PM via free access 130 IAWA Journal, Vol. 28 (2), 2007 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. Downloaded from Brill.com10/02/2021 07:21:54PM via free access Castro & Roig - Subfossil Fitzroya cupressoides 131 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.