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Brief Communication |
Department of Biological and Environmental Sciences, Georgia College & State University, Milledgeville, Georgia 31062-0001 USA; 3Department of Palaeontology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK; 4School of Life Sciences Faculty & Administration, Arizona State University, Box 85287-4501, Tempe, Arizona 85287-4501 USA; and 5University of Texas X-ray High-Resolution CT Facility (UTCT), Department of Geological Sciences, University of Texas at Austin, Austin, Texas 78712-1100 USA
Received for publication May 4, 2006. Accepted for publication October 6, 2006.
ABSTRACT
High resolution x-ray computed tomography (HRXCT) was used to image pyritized fossil fruits from the Lower Eocene London Clay flora to test the utility of this technique for paleobotanical application. The combination of carbon-pyrite preservation and void spaces between fruit and seed layers within fossils provides differences in density and composition that enable excellent imaging. Fossil fruits of Palaeorhodomyrtus subangulata (Bowerbank) Reid & Chandler (Myrtaceae) were investigated in situ within their silicone fluid conservation medium, which protects these unstable fossils from oxygen and humidity. HRXCT recovers taxonomically informative anatomical and morphological detail and provides a means of nondestructive examination of delicate type materials and other important specimens. These results suggest that HRXCT will be applicable to a broad spectrum of pyritized fossils to record structural details in inherently unstable materials.
Key Words: fossil fruits and seeds • high resolution x-ray computed tomography (HRXCT) • London Clay • paleobotanical techniques • pyrite permineralization
Pyrite (FeS2) is a common permineralizing agent of fossil plants and preserves the detailed structure to the cellular and sometimes subcellular level (Grimes et al., 2002
). Pyritized remains present challenges in both preparing material for study and conservation. Anatomical details are normally observed in this opaque material by reflected light or scanning electron microscopy (Collinson, 1999
; Kenrick, 1999
). However, both of these methods, which involve fracturing or wafering and polishing specimens are inherently destructive and cause loss of material and damage to specimens (Hass and Rowe, 1999
; Jones, 1999
).
Pyrite is unstable in museum collections; even under ambient temperature and humidity, it presents a challenge for long-term conservation. The mineral is prone to oxidation, which leads to deterioration and ultimately to complete loss of specimens (Newman, 1998
). This is particularly problematic for type and figured material. Many approaches to conservation have been tried, with the most successful of these involving the reduction or exclusion of oxygen and the maintenance of low relative humidity (Newman, 1998
; Shute and Foster, 1999
). Inert barriers such as silicone fluid successfully reduce deterioration, but they also pose problems, because removal of specimens from these media for study can result in damage or deterioration. High resolution x-ray computed tomography (HRXCT) has the potential to solve both problems simultaneously by providing a method to image the internal anatomy of pyritized fossils nondestructively without removing them from their conservation medium.
X-ray computed tomography (CT) was developed originally by Cormack and Hounsfield as a medical diagnostic tool (Hounsfield, 1980
). Since then, the method has been extended to additional industrial and scientific applications, as reviewed by Ketcham and Carlson (2001)
. CT creates two-dimensional digital slices based on the attenuation of x-rays as they pass through an object, which is related to density and chemical composition. A three-dimensional digital map is then assembled from the multiple slices. Because x-ray dose is not an issue for fossils, a specialized technique, high resolution x-ray computed tomography (HRXCT), can be used. HRXCT can take advantage of a variety of optimizations such as higher energy x-rays, smaller detectors, and longer exposure times than is practical in conventional medical CT devices. Generally, HRXCT has higher resolving power than CT systems, with a typical scale of resolution of approximately 100 µm and is capable of up to 10 µm or better (Ketcham and Carlson, 2001
). This greater resolution power is clearly within a range that would permit critical studies of plant tissues and, to a lesser degree, cell types.
Both medical CT and industrial HRXCT systems are used increasingly by vertebrate and invertebrate paleontologists in place of destructively sectioning rare and valuable specimens (e.g., Conroy and Vannier, 1984
; Haubitz et al.,
1988; Rowe et al., 1993
; Zinsmeister and DeNooyer, 1996
; Alonso et al., 2004
; Clarke et al., 2005
). Botanists have used the method to measure density of extant woods (Taylor et al., 1984
; Funt and Bryant, 1987
; Lindgren, 1991
; Fromm et al., 2001
) and to quantify spatial distribution of rooting systems in trees (Heeraman et al., 1997
; Pierret et al., 1999
). Floral structures and tissue distribution (e.g., endosperm, endocarp, embryo) in extant fruits and seeds can also be quantified in cases with significant variation in tissue density (Stuppy et al., 2003
). Experiments with silicified fossil plant remains of Cycadeoidea stems and Araucaria mirabilis cones have had some successful results (Pika-Biolzi et al., 2000
). Here we extend the application of HRXCT to fossil plants preserved in pyrite and to the resolution of objects an order of magnitude smaller.
Our investigation is based on small (ca. 5 mm diameter) pyritized fruits from the London Clay Formation of southern England, the primary reference material for the Lower Eocene of northern Europe (Reid and Chandler, 1933
; Collinson, 1983
; Collinson and Cleal, 2001
). These fossils are highly unstable and must be preserved in small vials of silicone fluid conservation medium. Long-term conservation of important collections poses significant problems (Shute and Foster, 1999
). We use the London Clay material to show how HRXCT can be used to obtain taxonomically significant, high-resolution images of pyritized fruits without removing them from their preservation fluid.
MATERIALS AND METHODS
Scans of a fragmented fruit of Palaeorhodomyrtus subangulata (Bowerbank) Reid & Chandler (Myrtaceae), Natural History Museum, London no. V 62263, were acquired at the University of Texas X-ray High-Resolution CT Facility (UTCT). The facility equipment and methods are described in detail by Ketcham and Carlson (2001)
. The specimen was scanned in a small cylinder partially filled with silicone fluid to prevent degradation. X-rays were set to 180 kV and 0.133 mA, resulting in a focal spot size of approximately 0.03 mm. A series of 27 transverse slices was acquired in a single turntable rotation, in which 2000 angular projections (views) were obtained over 400 s. This permitted the examination of approximately three rows of seeds. Slice spacing was 0.01297 mm, and each 1024 x 1024 pixel slice image had a field of view of 12.45 mm, resulting in a pixel spacing of 0.01216 mm. Image gray levels were normalized to silicone fluid using a wedge calibration, and beam-hardening artifacts were reduced by applying a polynomial correction during reconstruction. Images were produced as 16-bit TIFF files, which were then reduced to 8-bit format (Figs. 1–3, 5). The image in Fig. 4 was acquired at slightly lower resolution (0.027 mm) in a reconnaissance scan of the fruit in longitudinal view. Resulting images were imported into Photoshop version 9.0 CS2 (Adobe, San Jose, California, USA), sized, and sharpened. The images are stored at UTCT and maintained by the facility. Palaeorhodomyrtus fruits are housed in the Department of Palaeontology, Natural History Museum, London.
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The HRXCT scans are presented as two-dimensional transverse and longitudinal sections (Figs. 1–5) and in a three-dimensional reconstruction of a transverse segment through the central area of the fragmented fruit (Fig. 6). Palaeorhodomyrtus is a loculicidal capsule 12.5 mm high x 15 mm wide enclosed within a persistent calyx. The calyx is missing along one margin (Fig. 4, bottom), and the apical portion of the locules are incompletely preserved (Fig. 1, at top). The variation in gray-scale in the scanned image corresponds to several factors, but generally the fruit wall, locules, and calyx lobes appear light gray and the vascular strands darker gray (Fig. 1, dark arrows). Boundaries between locules (Figs. 1–4), as well as cracks in the fruit (Figs. 1, 4, at left), appear gray and represent small void spaces between adjacent fruit and seed layers.
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Four calyx lobes are preserved surrounding the fruit. They tend to be abutted in some areas and overlapping in others, and there is an open area where a fifth one, now missing, was presumably attached (Figs. 4, 6). Sepals are comparatively thick (ca 1.4 mm), the fruit wall correspondingly thin (ca 250 µm), yet discernable as a distinct entity in the scans (Figs. 1, 4, 6). Vascular tissue is evident in both longitudinal and transverse section (Figs. 1, 4, dark arrows).
Cellular level detail was not resolved in the calyx either in transverse section or on the outer surface of the fruit. The cellular detail typically preserved in permineralizations is likely to be absent from the scans due to the resolving power of the HRXCT at the settings used in this study. At this time, we elected to focus the use of the methodology for determining three-dimensional organization of the seeds within the fruit and other gross morphological features of the fruit.
DISCUSSION
The ability to resolve internal features using HRXCT is clearly related to the property of tissue-density heterogeneity. The technique works well with hard objects of low water content and variable tissue density. In living plants, previous studies obtained good results for dicot wood and palm fruits, but less success with organs of higher water content and more uniform tissue densities, such as flowers and inflorescences (Stuppy et al., 2003
). In a similar manner, HRXCT successfully images pyritized fossil fruits and seeds, despite the additional variables introduced by taphonomy and permineralization. In an earlier study of silicified fossil plant remains, good spatial resolution of tissue systems was achieved in Araucaria mirabilis cones and Cycadeoidea stems (Pika-Biolzi et al., 2000
), even though cellular level detail was not resolved. Our own analysis of Palaeorhodomyrtus demonstrates that a remarkable level of anatomical detail is retrievable from small pyritized fruits.
We chose the fruit of Palaeorhodomyrtus for this study because of our familiarity with the anatomical structure and organization of similar berry-like, myrtaceous fruits preserved as permineralizations (Pigg et al., 1993
). Additionally, seeds of this fruit have a distinctive morphology that provides an excellent test of how well the HRXCT 3-D imaging would resolve details over a range of scales. Pseudolocule morphology, fruit wall morphology, and vascular tissues were recovered, as were seed shape, arrangement, and attachment. Hence, features of critical taxonomic value can be resolved by HRXCT. At a finer scale, cellular level detail, such as the comparatively large (300 µm wide), distinct bulbous cells of the outer seed coat are discernable (Figs. 1, 5). We were able to resolve cellular level detail to about 100 µm, but theoretically greater resolutions are possible with additional optimizations (e.g., higher energy x-rays, smaller detectors, and longer exposure times).
The resolution of internal structures in pyritized fruits of Palaeorhodomyrtus appears to be related to several factors including density of tissues, distribution of organic inclusions, and presence of void space between adjacent fruit and seed layers. Pyritized plant fossils typically contain a high proportion of organic material, which represents the coalified remnants of the original tissues (Kenrick, 1999
). Frequently, these soft tissues are preserved at the cellular level, so tissue density heterogeneity may be conserved and reflected in the proportion of organics present within the pyrite fabric. This holds true even if individual cells cannot be resolved. The amount and distribution of internal void space can also be informative. Voids can occur at the boundaries of tissue systems, delimiting the various fruit and seed layers.
From our brief study, we were able to successfully recover taxonomically useful information with a 100-µm level of resolution. Information obtained in the course of a morning's work, and for a price somewhat comparable to that of SEM beam time, provided us with more three-dimensional detail than would have been possible from thin sectioning or fracturing the same fruits. This nondestructive method can be used in a variety of situations where destructive sampling is prohibitive (e.g., unique specimens, type specimens). HRXCT furnishes a means of investigating pyritic material in situ in its conservation medium. Alternatively, it provides a means of capturing basic data from specimens with a short shelf life. Because pyrite is a very common permineralizing agent, HRXCT is quite likely to find applications in the study of a wide range of fossil materials throughout the Phanerozoic.
FOOTNOTES
1 This research was funded by National Science Foundation Grant EAR-0345569 and a Faculty Research Grant, Georgia College & State University (M.L.D.), and National Science Foundation Grant EAR-0345838 (K.B.P). Operation of the University of Texas High-Resolution X-ray CT Facility is supported by National Science Foundation Grant EAR-0345710. The authors thank N. D. Wilkens and B. H. Tiffney for their comments on a draft of the manuscript. 
2 Author for correspondence (e-mail: melanie.devore{at}gcsu.edu
)
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