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A new taphonomic bioerosion in a Miocene lakeshore environment
Palaeogeography, Palaeoclimatology, Palaeoecology 295 (2010) 192–198
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Palaeogeography, Palaeoclimatology, Palaeoecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a l a e o
A new taphonomic bioerosion in a Miocene lakeshore environment M. Dolores Pesquero a,b,⁎, Carmen Ascaso c, Luis Alcalá a, Yolanda Fernández-Jalvo b a b c
Fundación Conjunto Paleontológico de Teruel-Dinópolis, Avda. Sagunto s/n, 44002 Teruel, Spain Museo Nacional de Ciencias Naturales (CSIC), C/José Gutiérrez Abascal 2, 28006 Madrid, Spain Instituto de Recursos Naturales, Centro de Ciencias Medioambientales (CSIC), C/Serrano 115-bis, 28006 Madrid Spain
a r t i c l e
i n f o
Article history: Received 14 October 2009 Received in revised form 7 May 2010 Accepted 28 May 2010 Available online 9 June 2010 Keywords: Miocene Microboring Bacteria Aquatic ecosystems Concud Teruel Spain
a b s t r a c t This study describes a new type of taphonomic alteration of fossil bone that occurred in a continental carbonate palaeolake environment at the reference Spanish Miocene site of Cerro de la Garita (Concud, Teruel). Scanning electron microscopy showed this type of alteration to be characterized by microtunnels that penetrate inward from the bone surface and by a branching-meandering arrangement of microchannels on the bone surface. These microtunnels had a highly electron dense inner wall, seen as a characteristic rim in transverse section. Microspheres were seen inside the microtunnels. Both this electron dense layer and these microspheres were found to be composed of calcium phosphate. These taphonomic modifications bear some similarities to, but also differs from, those caused by bacterial attack on bone and enamel in marine and terrestrial environments, suggesting the present process to be a new type of bioerosion. The microspheres inside the microtunnels were similar in size, shape and composition to the fossilized bacteria covering fossils from Fossil-Lagersttäten palaeolake sites, such as Libros (Teruel, Spain) and Messel (Germany). Under the transmission electron microscope these structures showed an apparent cell wall, suggesting them to be fossilized coccoid bacteria. © 2010 Elsevier B.V. All rights reserved.
1. Introduction 1.1. Microboring and activity of microorganisms Wedl (1864) was the first to describe microboring affecting the bone structure, a phenomenon he attributed to fungal attack. Hackett (1981) distinguished four types of microscopic focal destruction (MFD) of such bone which he divided into three non-Wedl-types (linear longitudinal, lamellate and budded foci) and one Wedl-type. Hackett (1981) attributed the first three types to bacterial activity and described the areas so attacked to be surrounded by zones of remineralization. Hedges et al. (1995) proposed an index of histological destruction (the Oxford Histology Index, OHI) for describing the overall extent to which the histological integrity of post-mortem bone attacked in this way is lost, from stage 0 (with no original histological features identifiable other than Haversian canals) to stage 5 (with less than 5% of bone affected by bacterial attack). The distribution of modifications affecting the bone matrix made by indigenous bacteria during decay is conditioned by the histological microstructure of the bone in question (Bell, 1990; Jans et al., 2002, 2004; Turner-Walker and Jans, 2008). However, the marks left by bacteria in non-Wedl alteration may appear dispersed, independent of the vascular or cell network; such bacterial activity is post-depositional or post-skeleto-
⁎ Corresponding author. Fundación Conjunto Paleontológico de Teruel-Dinópolis, Avda. Sagunto s/n, 44002 Teruel, Spain. Fax: + 34 978 617 638. E-mail address: [email protected] (M.D. Pesquero). 0031-0182/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.palaeo.2010.05.037
nization (i.e., appearing after the soft tissues have disappeared) (Bell and Elkerton, 2008; Fernández-Jalvo et al., 2010). The most commonly observed type of non-Wedl microbial bone deterioration in terrestrial environments involves discrete zones that in backscattered scanning electron microscopy (BSE-SEM) are seen to contain small pores and thin channels 0.1–1.0 μm in diameter (Hackett, 1981; Bell, 1990; Bell et al., 1991, 1996; Turner-Walker et al., 2002; Jans, 2005). These zones are often surrounded by an electron dense region marking the limit of the affected area. Jackes (1990) hypothesized that this was due to coccoid bacteria removing collagen from the solubilized compactum and then reprecipitating bone mineral in a more dense form. Using the scanning electron microscope (SEM), Jackes et al. (2001) showed a rim surrounding the destructive foci to be much more electron dense than unaltered bone. These authors indicated these rims to contain more mineral (a higher percentage of Ca + P) and less organic matter (collagen) than the surrounding unaltered bone. The remaining type of microscopic focal destruction in post-mortem bones (i.e., Wedl microscopic focal destruction) involves microboring similar to that seen by Wedl (1864). Roux (1887) agreed with Wedl that this bioerosion was the product of fungal activity and assigned such microborings to the ichnogenus Mycelites ossifragus. Arnaud et al. (1978) sectioned medieval human bones from a shipwreck and observed microboring similar to this Wedl type alteration. However, these authors were unable to find any microorganism inside the microtunnels that might have given rise to them. Ascenzi and Silvestrini (1984) extended the study to experimental work and analysed both medieval and modern human bones collected from sea water using the
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transmission electron microscope (TEM) and found electron dense spherical aggregates made of calcium phosphate inside the microtunnels. These authors described a thin rim at the edge of the microtunnels stained by methylene blue, indicating that the bone matrix had been attacked (Ascenzi and Silvestrini, 1984, pg. 532). Bell and Elkerton (2008) described a characteristic post-mortem type of microtunnel on skeletal remains from the Mary Rose shipwreck. The distribution of this alteration was always peripheral, reaching some 300 μm into the bone. These tunnels showed quite constant morphology and dimensions (84% of tunnels were 5–8 μm diameter, with a maximum diameter of 19 μm) (Bell and Elkerton, 2008). No remineralization boundary showing increased density was observed on the internal walls of these microtunnels. The lack of such remineralization suggested to these authors that these microtunnels were different to those that had undergone diagenetic alteration in terrestrial contexts where bacteria are considered the main invading organism (Bell et al., 1991, 1996; Bell and Lee-Thorp, 1998; Turner-Walker and Jans, 2008). It was finally concluded that this characteristic microboring was diagnostic of marine exposure and to be the product of endolithic microorganisms of unknown identity. Turner-Walker and Jans (2008) found similar microtunnels to those described by Bell and Elkerton (2008) in modern bone remains from a gravel beach in Cyprus, as well as in fossil specimens from the littoral zone of an estuary (the West Runton Freshwater Bed, UK), and referred to endolithic filamentous cyanobacteria as strong candidates for having caused this bioerosion. Further experimental work and detailed descriptions referring to the bioerosion of different organic and inorganic substrates caused by cyanobacteria, fungi, algae and other organisms can be found in Golubic et al. (1975) (boring microorganisms and microborings in carbonate substrates), Knoll et al. (1986) (detailed description of bioerosion caused by cyanobacteria in ooliths), García-Pichel (2006) (microboring of cyanobacteria in different substrates) and Ehrlich et al. (2008) (bibliographic summary of bioerosion caused by cyanobacteria, fungi and algae etc).
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Fig. 1. Location of the Cerro de la Garita site.
1.2. The Cerro de la Garita site and taphonomic context The site examined in the present work, that of Cerro de la Garita (Concud, Teruel, Spain), is a reference site for the continental Euroasiatic Neogene. Located in northeastern Spain a few kilometres outside Teruel (Fig. 1), the site is around 7 million years old and has yielded many of the mammals that characterise the middle Turolian (Upper Miocene) (MN zone 12 sensu Mein, 1990, or local zone L sensu Dam et al., 2001). A National Heritage site, Cerro de la Garita is of special historic value and has yielded a large number of type and paratype specimens (Hernández Pacheco, 1924; see also Alcalá, 1992 and Alcalá, 1994). The historic collection recovered since the 1920 s and held at the Museo Nacional de Ciencias Naturales (MNCN) consists of 5230 fossils. Detailed taphonomic studies have been possible in more recent systematic excavations undertaken by the MNCN and the Fundación Conjunto Paleontológico de Teruel-Dinópolis between 1997 and 2003. Taphonomic analyses have now also been performed on the historic collection for comparison with the surface alterations found in the modern collection. Unlike in recent excavations, no 3D field coordinates of fossils nor detailed descriptions of finds in the excavation area were recorded during the gathering of the historic collection. The Cerro de la Garita site formed during a transitional stage occurring between sediment levels deposited in alluvial complexes and a sporadic lacustrine system. The site represents a shallow, highly alkaline lake shore. The area was almost certainly a water hole, and therefore a good hunting area for predators and scavengers. Hyena coprolites have been found at the site, and tooth marks have been observed on bone surfaces, the size and shape of some suggesting them to have been made by hyenids. Several bone fragments some 2–5 cm in length show clear evidence of heavy digestion and probable regurgitation; all have a characteristic highly rounded and polished surface. All
these traits coincide with hyenid activity, although other tooth marks may have been made by other carnivores. Abundant trampling marks can be seen on the surface of these bone fossils, congruent with their having been deposited in a damp, lakeshore environment. Most of the remains are broken, and only a few anatomical elements from the same individual have been found close to one another, but not in anatomical connection. No hydrological selection (neither by shape nor size) nor signs of abrasion have been observed in these fossils, although in the ground they showed a certain orientation that suggests the influence of a low energy stream. Despite being an open air site, none of these fossils appears weathered, which is also suggestive that the surrounding environment was a damp lakeshore. Detailed taphonomic analyses and descriptions of the site can be found in Pesquero (2006, in preparation) and Pesquero et al. (2005a,b). 2. Materials and methods A total of 4790 bone fossils were collected during the systematic excavations of the site between 1997 and 2003. All were examined under the binocular light microscope (10× to 60×). However, given the special historic and palaeontological value of the Cerro de la Garita site, only a limited number of fossils were sectioned and processed for further analysis. Twenty-four specimens, 13 of which were sectioned, were examined by environmental SEM (QUANTA 200) at the MNCN. Histological analyses were made of both natural fractures and of the cut sections after embedding in resin. Analyses were performed in secondary electron emission (SE-SEM) and BSE-SEM modes using Oxford energy dispersive spectrometry/wavelength dispersive spectrometry (EDS/WDS) detectors. EDS analyses provided the element composition of specific areas of interest. SEM analysis of the fossil
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Table 1 Samples analysed and techniques used. Label
Surface SEM cut EDS TEM Petrographic X-ray sections microscope Diffraction Fluorescence
CG97-65 CG97-66 CG97-98 CG97-128 CG97-638 CG97 639 CG97-687 CG98 1365 CG98 1809H CG98 1809P CG98-1832 CG98-2014 CG99 2461 CG99-2537 CG99 2601 CG99-2607 CG99-2619 CG99-2728 CG99-2998 CG99-3045 CG99-3081 CG99-3285 CG03-3320 CG03-3436 CG03-3619 CG03-3640 CG03-3676 CG03-3923 CG03-4137a CG03-4137b CG03-4409 CG03-4481 CG03-4538 CG03-4607 CG03-4724 SED
X X X
X
X
X
X
X
X X
X X
X
X
X
X
X X X X X X X X X X X X X X X X X X X X
X X X
X X X
X
X X X
X X
X
X
X
X
X
X
X
X X X X
X X X X X X X
X X
X X
X X
X X
X
X Fig. 2. Scanning electron photomicrographs (backscattered mode) showing a typical terrestrial bacterial attack. (a) Incipient bacterial attack dispersed according to histological traits. (b) Incipient bacterial attack, some signs near Haversian canals. (c) Close-up of previous photomicrograph. (d) Bacterial attack distributed along fissures. Note that cracks are filled with characteristic pores produced by bacteria, indicating bacteria invaded these postdepositional cracks.
X
X
X
X
X
X
X
X
X
surface was complemented by thin section observations made under a petrographic microscope (Nikon YM-EPI) using parallel and crossed nichols. Imaging analyses were completed by transmission electron microscopic (TEM) (Zeiss EM 910) examination of ultra-thin preparations at the Electron Microscopy Unit of the Institute of Environmental Sciences of the CSIC. Some samples were analysed by X-ray diffraction (Philips PW-1830) and fluorescence measurements (Philips PW-1404) to obtain their mineral and element compositions (Table 1).
for analysis under the petrographic microscope (Fig. 4a). Both techniques revealed microtunnels that penetrated 200–300 μm into the bone; all contained highly electronic dense spherical aggregates or ‘microspheres’. The distribution of this microboring in the bone sections was peripheral (OHI 0, according to Hedges et al., 1995), with no histologically-orientated organization (Figs. 3 and 4a). The diameters of these tightly clustered microtunnels ranged from 7 to 18 μm (N= 73; mean = 9.84 ± 2.6), their dimensions and morphology remaining constant (Fig. 4b). The microtunnel walls showed a rim of highly electron dense material with a width of 0.1–1.5 μm (N = 45;
3. Results Some of the Cerro de la Garita fossils showed typical signs of terrestrial bacterial attack (Fig. 2). The signs of this bacterial attack in the histologically-studied samples showed three types of distribution, although all were very incipient (OHI 4, according to Hedges et al., 1995). Some specimens showed bacterial alteration around the osteons (Fig. 2a), others showed dispersed attacks near the Haversian canals (Figs. 2b and c) and others still showed signs of attack that occurred post-depositionally following the cracking of the specimens due to sediment compaction (Fig. 2d). Well according to other authors this alteration show small pores with diameters of some 0.9 μm. The precise number of fossils affected by microorganisms remains unknown since histological sections were not prepared of all 4790 specimens. About a dozen of the examined fossils showed a characteristic form of macroscopic damage undescribed until now (Fig. 3a). In order to identify the origin of this alteration and to determine its intensity, the surface and natural fractures of one of these fossils (CG03-4538) were analysed by SEM (Fig. 3b) after transverse sectioning of the specimen and embedding in resin (Figs. 3c and d). Thin sections were also made
Fig. 3. New type of taphonomic alteration. (a) Fossil fragment from the Cerro de la Garita site (CG03-4538), the surface of which has been intensely affected by bacteria. (b) SEM photomicrograph of a natural fracture in fossil CG03-4538. (c) SEM micrograph of the same specimen, cut and embedded in resin; note the signs of intense attack in the cortical layer contrasting with the deeper bone. (b) Close-up of previous picture showing distinctive microboring.
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grooves (Fig. 5c white arrows) or branching-meandering arrangements (Fig. 5c black arrows) were distinguished in less altered areas. Microspheres with a diameter of 1–2.8 μm (N = 49; mean = 2.01 ± 0.44) were observed inside the tunnels (Fig. 6). EDS analysis of these microspheres showed them to have a calcium phosphate composition (Fig. 7) in similar proportion to non-affected bone. Fig. 8 shows ultrathin sections of bone observed under the TEM. Fig. 8a shows dark circles (black arrows) representing the microspheres shown in Fig. 6a under SEM at a similar magnification. Fig. 8b and d show further microtunnels containing these microspheres that appear mineralized at the interior (asterisks). Fig. 8b, d, e and f show the cell walls of what appear to be mineralized microorganism (white arrows). 4. Interpretation and discussion Some of the components of the Cerro de la Garita fossil association showed typical signs of incipient terrestrial bacterial attack characterized by microscopic focal destruction involving nanometric-sized pores within the bone. Some of these alterations followed a Fig. 4. New type of taphonomic alteration. Alteration in specimen CG03-4538. (a) Thin section under the petrographic microscope showing invasive microtunnels penetrating into the bone from the outer cortical lamellae (bottom of the picture). (b) SEM photo micrograph of invasive microtunnels containing microspheres. The walls of these tunnels show a higher electron density than the unaltered bone. (c) EDS analyses of the highly electron dense microtunnel wall (transverse section; spectrum 3, right side top diagram) and non-affected bone (spectrum 4, right side bottom diagram).
mean= 0.81± 0.25). EDS analysis showed the chemical composition of the unaltered bone and these rims to be the same (Fig. 4c). i.e., calcium phosphate. BSE-SEM images showed this rim to have a bright white colour, indicating it to be denser than unaltered bone. The distribution of the surface alteration of the fossil bones was heterogeneous (see Fig. 5a in contrast with Fig. 5b). Individual
Fig. 5. New type of taphonomic alteration. SEM photomicrographs of fossil bone surfaces showing distribution of alteration produced by invasive microboring (CG034538). (a) Some areas appear affected by an intense pitting, increasing the bone porosity. (b) In contrast, some areas are less affected. (c) In areas where surface microboring is less intense, individual grooves (white arrows) can be distinguished in contrast to areas where these channels form a ramified arrangement (black arrow). (d) Longitudinal grooves on bone surface (black arrows) that may be related to the incipient stage of this alteration; photographed at a higher magnification than in the previous picture.
Fig. 6. (a), (b), (c) New type of taphonomic alteration. SEM image of fossilized microspheres (black arrows) inside a microtunnel. These photomicrographs represent a naturally, transversally broken surface of the same specimen at different magnifications. (d) SEM photomicrograph of a fossil from the Fossil-Lagersttäten site of Libros (Teruel, Luque et al., 1996). The picture, taken at a magnification similar to that used in (c), represents the preserved soft tissues of an amphibian and shows a diatom and abundant fossil bacteria covering the fossil surface. (e) SEM photomicrograph of a section of a modern bone collected from Neuadd (Wales, UK), showing a typical terrestrial bacterial attack with mineral redeposition rims around individual colonies. EDS analysis returned a calcium phosphate chemical composition for the bone and the outer ring. (f) New type of taphonomic alteration. Section of a fossil showing mineral redeposition on the internal wall of microtunnels, seen as a rim in transverse section (high electron density). The EDS mineral composition is shown in Fig. 3 (spectrum 3).
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experimental work by Davis (1997). The latter author reported strong similarities with the microboring described by Wedl (1864) and Roux (1887), but assigned this type of alteration to endolithic cyanobacteria or algae based on the microorganisms found in the examined bone. Underwood and Mitchell (2004) reported branching-meandriform microtunnels affecting the surface of the tooth roots of Mesozoic fossil sharks identical to that observed by Ascenzi and Silvestrini (1984) and Davis (1997) on human and bird bones respectively. Underwood and Mitchell (2004) concluded that the causative agent was Mycelites ossifragus. Table 2 lists the similarities and differences between these various types of alteration, suggesting that the second type of microboring seen in the present work represents a formerly unknown type of bioerosion. Chemical analysis of the inner wall of the new type of microboring showed it to be similar in chemical composition (calcium phosphate) to unaffected bone, suggesting this high density structure represents bone remineralization. The highly electron dense microspheres inside the tunnels have identical calcium phosphate composition. These microspheres are similar in shape, size and chemical composition to those found covering amphibian fossils with preserved soft tissues (Luque et al., 1996; Meyer and Milsom, 2001; McNamara et al., 2003, 2006) from the Fossil-Lagerstätten site of Libros (Teruel). The latter authors interpret the microspheres they observed to be bacteria preserved in calcium phosphate. Liebig (1998) also documented that a large percentage of the bacteria fossilized in the Messel Oil Shale (Germany) were preserved in amorphous calcium phosphate or that cryptocrystalline apatite had replaced them. When observed under TEM the present microspheres showed strong similarities in size and structure to those reported by Ascenzi and Silvestrini (1984). Some of the present microspheres show a preserved cell wall; this, plus their size and shape suggests them to be coccoidal forms in the range of bacteria. TEM-SEM and EDS analyses ruled out any possibility of modern bacterial contamination; the present microspheres were clearly mineralized and preserved in calcium-phosphate, and found inside tunnels located around 300 μm into the bone, ruling out secondary infilling from the external environment (see Figs. 3d and 4b). Fig. 7. Above: SEM picture of the microspheres inside microtunnels (specimen CG03538), showing the specific area analysed (+) using EDS detectors. Below: Element composition of the microspheres as determined by EDS.
distribution around the Haversian canals, while others showed a more dispersed distribution or occurred in postdepositional cracks (Fig. 2). A second type of alteration was also observed (Figs. 3–8), characterized by a peripheral microboring intensively affecting the outer compact bone layer. These microtunnels and their internal microspheres differed from the typical damage caused by terrestrial bacterial attack. In transverse section, the rim at the edge of these microtunnels was highly electron dense, similar to the hypermineralized zones surrounding the microscopic focal destruction described in terrestrial environments. This second type of alteration also shows similarities and differences to the bacterial microboring of bones that occurs in marine environments. Certainly, the present microtunnels are similar in size and distribution to those described in bones collected in marine environments (Bell and Elkerton, 2008; Turner-Walker and Jans, 2008), but the latter authors observed no distinct, highly electron dense rim at the edge of the microtunnels they examined. The presence or absence of an outer rim appears to differentiate between lacustrine and marine bacterial microboring. On the bone surface, this microboring showed a meandriformbranching arrangement of channels similar to that reported in marine
5. Conclusions The microboring-modified fossils of the Cerro de la Garita show similarities and differences to taphonomic modifications described by other authors in skeletal remains collected from marine and terrestrial environments. As seen in terrestrial bacterial attacks, the new type of microboring in the present specimens showed characteristic remineralizations, forming highly electron dense microtunnel walls of calcium phosphate (appearing as rims in transverse section). Unlike in terrestrial bacterial attack, however, this highly electron dense layer covers the entire microtunnel inner wall; in terrestrial microscopic focal destruction it appears in discrete zones around nanometric pores and channels. Further, the present modification is characterized by microspheres some 1–2.8 μm in diameter made of calcium phosphate inside the microtunnels. The new type of microboring also showed a meanderingbranching arrangement at the bone surface, similar to that seen in bones collected form marine environments. TEM analyses of the microspheres inside the tunnels showed them to be the same as those described by Ascenzi and Silvestrini (1984). However similar bioerosion in marine environments is not associated with the remineralized, highly electron dense calcium phosphate microtunnel walls seen in the present fossils. The cocci-like microspheres detected in this work had shape, size and a distribution similar to the spherical structures described in fossils from the lacustrine Fossil-Lagersttäten deposits of Libros
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Fig. 8. TEM images of ultrathin bone sections. (a) The dark grey circles (black arrows) are the microspheres shown in the previous figures. (b), (c), (d) (e), (f) Closer views of these microspheres; a mineralized cell wall (white arrows) can be distinguished. Asterisks indicate mineralized microspheres.
(Spain) and Messel (Germany) (Luque et al., 1996; Liebig, 1998; McNamara et al., 2003, 2006). The latter microspheres are composed of calcium phosphate, as are those described in the present work. The latter authors considered these structures to be fossilized bacteria.
The present microspheres observed in Cerro de la Garita may also be fossilized bacteria, or at least, coccoidal forms in the range of bacteria. The present work provides evidence that a type of bone alteration, different to that seen in marine and terrestrial environments, may
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Table 2 Main features of the taphonomic structures discussed in the present work.
Cerro de la Garita Terrestrial change Turner Walker et al. (2002) Mary Rose (Bell and Elkerton, 2008) Hackett tunnels (Davis, 1997)
Distribution of this alteration
Depth into the cortical layer
Diameter
Rim
Peripheral Discrete zones Peripheral Parallel to the periosteal surface
200–300 μm Variable N 300 μm 0.2 mm
7–18 μm 0.1–1.0 μm 5–8 μm 0.25 mm
Yes Yes No No
occur in aquatic continental environments. Experimental work, especially in lacustrine environments, is needed to identify the origin of this type of alteration and differentiate it from others environments. Acknowledgements The excavations at the Cerro de la Garita site, the preparation of the present fossils and their examination was made possible by support from the Departamento de Educación Cultura y Deporte, the Dirección General de Patrimonio Cultural (projects 022/97, 133/98, 142/99 and 197/00) and the Departamento de Ciencia, Tecnología y Universidad (Grupo de Investigación Emergente E-62) of the Regional Government of Aragón (Spain), and the Spanish Ministry of Science and Innovation (Project CGL2007-66231). M.D.P was the recipient of a Juan de la Cierva contract. (Ref. JCI-2007-132-565). We are grateful to the Laboratories of Electron Microscopy at the Museo Nacional de Ciencias Naturales (MNCN) and the Centro de Ciencias Medioambientales (CCMA), as well as the Laboratory of Fluorescence and X-ray diffraction at the MNCN. We also thank microbiologists Blanca Pérez Uz and Lynne Bell for their comments and suggestions. References Alcalá, L., 1992. Macromamíferos neógenos de la fosa de Alfambra-Teruel. Ph.D Thesis, Univ. Complutense Madrid, Spain. Alcalá, L., 1994. Macromamíferos neógenos de la fosa de Alfambra-Teruel. Instituto de Estudios Turolenses, Teruel. Arnaud, G., Arnaud, S., Ascenzi, A., Bonucci, E., Graziani, G., 1978. On the problem of the preservation of human bone in sea-water. Journal of Human Evolution 7, 409–420. Ascenzi, A., Silvestrini, G., 1984. Bone-boring Marine Micro-organisms: an experimental investigation. Journal of Human Evolution 13, 531–536. Bell, L.S., 1990. Palaeopathology and diagenesis: an SEM evaluation of structural changes using backscattered electron imaging. Journal of Archaeological Science 17, 85–102. Bell, L.S., Elkerton, A., 2008. Unique marine taphonomy in human skeletal material recovered from the Medieval warship Mary Rose. International Journal of Osteoarchaeology 18 (5), 523–535. Bell, L.S., Lee-Thorp, J.L., 1998. Advances and constraints in the study of human skeletal remains: a joint perspective. In: Cox, M. (Ed.), Grave Concerns. York, CBA, pp. 238–246. Bell, L.S., Boyde, A., Jones, S.J., 1991. Diagenetic alteration to teeth in situ illustrated by backscattered electron imaging. Scanning 13, 173–183. Bell, L.S., Skinner, M.F., Jones, S.J., 1996. The speed of post-mortem change to the human skeleton and its taphonomic significance. Forensic Science International 82, 129–140. Dam, J.A., Alcalá, L., Alonso Zarza, A.M., Calvo, J.P., Garcés, M., Krijgsman, W., 2001. The upper Miocene mammal record from the Teruel-Alfambra region (Spain): biostratigraphy and chronology. Journal Vertebrate Paleontology 21 (2), 367–385. Davis, P.G., 1997. The bioerosion of bird bones. International Journal of Osteoarchaeology 7, 388–401. Ehrlich, H., Koutsoukos, P.G., Demadis, K.D., Pokrovsky, O.S., 2008. Principles of demineralization: modern strategies for the isolation of organic frameworks. Part. 1. Common definitions and history. Micron 39, 1062–1091. Fernández-Jalvo, Y., Andrews, P., Pesquero, D., Smith, C., Marín-Monfort, D., Sánchez, B., Geigl, E.M., Alonso, A., 2010. Early bone diagenesis in temperate environments Part I: Surface features and histology. Palaeogeography, Palaeoclimatology, Palaeoecology 288, 62–81. García-Pichel, F., 2006. Plausible mechanisms for the boring in carbonates by microbial phototrophs. Sedimentary Geology 185, 205–213. Golubic, S., Perbius, R.D., Lukas, K.L., 1975. Boring Microorganisms and Microborings in Carbonate Substrates. In: Frey, R.W. (Ed.), The Study of Trace Fossils. New York, Springer-Verlag, pp. 229–259.
Hackett, C.J., 1981. Microscopical focal destruction (tunnels) in exhumed human bones. Medicine, Science and the Law 21, 243–265. Hedges, R.E., Millard, A.P., Pike, A.W.G., 1995. Measurements and relationships of diagenetic alteration of bone from three archaeological sites. Journal of Archaeological Science 22, 201–210. Hernández Pacheco, E., 1924. Noticias sobre el yacimiento Paleontológico de Concud (Teruel). Boletín de la Sociedad Española de Historia Natural 24, 401–404. Jackes, M., 1990. Diagenetic change in prehistoric Portuguese human bone (4000 to 8000 BP). Symposium on Bone Chemistry, Canadian Association for Physical Anthropology, 18th Annual Meeting. Jackes, M., Sherburne, R., Lubell, D., Barker, C., Wayman, M., 2001. Destruction of microstructure in archaeological bone: a case study from Portugal. International Journal of Osteoarchaeology 11, 415–432. Jans, M.M.E., 2005. Histological characterisation of diagenetic alteration of archaeological bone. Geoarchaeological and Bioarchaeological Studies, vol. 4. Institute for Geo and Bioarchaeology, Vrije Universiteit, Amsterdam. Jans, M.M.E., Kars, H., Nielsen-Marsh, C.M., Smith, C.I., Nord, A.G., Arthur, P., Earl, N., 2002. In situ preservation of archaeological bone: a histological study within a multidisciplinary approach. Archaeometry 44 (3), 343–352. Jans, M.M.E., Nielsen-Marsh, C.M., Smith, C.I., Collins, M.J., Kars, H., 2004. Characterisation of microbial attack on archaeological bone. Journal of Archaeological Science 31, 87–95. Knoll, A.H., Golubic, S., Green, J., Sweet, K., 1986. Organically preserved microbial endoliths from the late Proterozoic of East Greenland. Nature 231 (26), 856–857. Liebig, K., 1998. Fossil microorganisms from the Eocene Messel oil shale of Southern Hesse, Germany. Kaupia 7, 1–95. Luque, L., Merino, L., Sanchíz, B., Alcalá, L., 1996. Procesos de fosilización de las ranas miocenas de Libros (Teruel). II Reunión de Tafonomía y Fosilización, Zaragoza, Spain. McNamara, M., Orr, P., Alcalá, L., Anadón, P., Peñalver, E., 2003. Excepcional preservation of frogs from the Spanish Miocene. Abstracts European Palaeontological Association Workshop on Excepcional Preservation. Teruel, Spain. pp. 69–72. McNamara, M.E., Orr, P.J., Kearns, S.L., Alcalá, L., Anadón, P., Peñalver-Mollá, E., 2006. High-fidelity organic preservation of bone marrow in ca. 10 Ma amphibians. Geology 34 (8), 641–644. Mein, P., 1990. Updating of MN zones. In: Lindsay, E.H., Fahlbusch, V., Mein, P. (Eds.), European Neogene Mammal Chronology. Nato ASI Ser, 180. Plenum Press, New York, pp. 73–90. Meyer, D.L., Milsom, C.V., 2001. Microbial Sealing in the biostratinomy of Uintacrinus Lagërstaten in the Upper Cretaceous of Kansas and Colorado, USA. Palaios 16, 535–546. Pesquero, M.D., 2006. Tafonomía del yacimiento de vertebrados miocenos de Cerro de la Garita (Concud, Teruel). Ph.D Thesis, Univ. Complutense Madrid, Spain. Pesquero, M.D., Bessa, V., Sánchez-Chillón, B., Geigl, E.M., Fernández-Jalvo, Y., Alcalá, L., 2005a. Fossil bacteria and morpho-histological modifications from Concud (Teruel, Spain). V International Bone Diagenesis meeting. University of Cape Town, South Africa. p. 34. Pesquero, M.D., Fernández-Jalvo, Y., Alcalá, L., 2005b. Alteraciones óseas superficiales registradas en los fósiles de vertebrados del yacimiento paleontológico de Cerro de la Garita (Concud, Teruel). Taphos 05. Barcelona, Spain. p. 125. Pesquero, MD, et al. in preparation The site formation of a Miocene vertebrate reference site, Cerro de la Garita (Concud, Teruel, Spain). Roux, W., 1887. Uber eine knocher lebende gruppe von faderpilzen (Mycelites ossifragus). Zeitschr Wiss Zoologic 45, 227–254. Turner-Walker, G., Jans, M.M.E., 2008. Reconstructing taphonomic histories using histological analysis. Palaeogeography, Palaeoclimatology, Palaeoecology 266 (3–4), 207–235. Turner-Walker, G., Nielsen-Marsh, C.M., Syversen, U., Kars, H., Collins, M.J., 2002. Submicron spongiform porosity is the major ultra-structural alteration occurring in archaeological bone. International Journal of Osteoarchaeology 12, 407–414. Underwood, C.J., Mitchell, S.F., 2004. Sharks, bony fishes and endodental borings from the Miocene Montpelier Formation (White Limestone Group) of Jamaica. Cainozoic Research 2, 157–165. Wedl, C., 1864. Ueber einen im zahnbein und knochen keimenden pilz. Sitzungsberichte der Kaiserlichen Academie der Wissenschaften, mathematisch-naturwissenschaftliche Classe 50, 171–193.
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Palaeogeography, Palaeoclimatology, Palaeoecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a l a e o
A new taphonomic bioerosion in a Miocene lakeshore environment M. Dolores Pesquero a,b,⁎, Carmen Ascaso c, Luis Alcalá a, Yolanda Fernández-Jalvo b a b c
Fundación Conjunto Paleontológico de Teruel-Dinópolis, Avda. Sagunto s/n, 44002 Teruel, Spain Museo Nacional de Ciencias Naturales (CSIC), C/José Gutiérrez Abascal 2, 28006 Madrid, Spain Instituto de Recursos Naturales, Centro de Ciencias Medioambientales (CSIC), C/Serrano 115-bis, 28006 Madrid Spain
a r t i c l e
i n f o
Article history: Received 14 October 2009 Received in revised form 7 May 2010 Accepted 28 May 2010 Available online 9 June 2010 Keywords: Miocene Microboring Bacteria Aquatic ecosystems Concud Teruel Spain
a b s t r a c t This study describes a new type of taphonomic alteration of fossil bone that occurred in a continental carbonate palaeolake environment at the reference Spanish Miocene site of Cerro de la Garita (Concud, Teruel). Scanning electron microscopy showed this type of alteration to be characterized by microtunnels that penetrate inward from the bone surface and by a branching-meandering arrangement of microchannels on the bone surface. These microtunnels had a highly electron dense inner wall, seen as a characteristic rim in transverse section. Microspheres were seen inside the microtunnels. Both this electron dense layer and these microspheres were found to be composed of calcium phosphate. These taphonomic modifications bear some similarities to, but also differs from, those caused by bacterial attack on bone and enamel in marine and terrestrial environments, suggesting the present process to be a new type of bioerosion. The microspheres inside the microtunnels were similar in size, shape and composition to the fossilized bacteria covering fossils from Fossil-Lagersttäten palaeolake sites, such as Libros (Teruel, Spain) and Messel (Germany). Under the transmission electron microscope these structures showed an apparent cell wall, suggesting them to be fossilized coccoid bacteria. © 2010 Elsevier B.V. All rights reserved.
1. Introduction 1.1. Microboring and activity of microorganisms Wedl (1864) was the first to describe microboring affecting the bone structure, a phenomenon he attributed to fungal attack. Hackett (1981) distinguished four types of microscopic focal destruction (MFD) of such bone which he divided into three non-Wedl-types (linear longitudinal, lamellate and budded foci) and one Wedl-type. Hackett (1981) attributed the first three types to bacterial activity and described the areas so attacked to be surrounded by zones of remineralization. Hedges et al. (1995) proposed an index of histological destruction (the Oxford Histology Index, OHI) for describing the overall extent to which the histological integrity of post-mortem bone attacked in this way is lost, from stage 0 (with no original histological features identifiable other than Haversian canals) to stage 5 (with less than 5% of bone affected by bacterial attack). The distribution of modifications affecting the bone matrix made by indigenous bacteria during decay is conditioned by the histological microstructure of the bone in question (Bell, 1990; Jans et al., 2002, 2004; Turner-Walker and Jans, 2008). However, the marks left by bacteria in non-Wedl alteration may appear dispersed, independent of the vascular or cell network; such bacterial activity is post-depositional or post-skeleto-
⁎ Corresponding author. Fundación Conjunto Paleontológico de Teruel-Dinópolis, Avda. Sagunto s/n, 44002 Teruel, Spain. Fax: + 34 978 617 638. E-mail address: [email protected] (M.D. Pesquero). 0031-0182/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.palaeo.2010.05.037
nization (i.e., appearing after the soft tissues have disappeared) (Bell and Elkerton, 2008; Fernández-Jalvo et al., 2010). The most commonly observed type of non-Wedl microbial bone deterioration in terrestrial environments involves discrete zones that in backscattered scanning electron microscopy (BSE-SEM) are seen to contain small pores and thin channels 0.1–1.0 μm in diameter (Hackett, 1981; Bell, 1990; Bell et al., 1991, 1996; Turner-Walker et al., 2002; Jans, 2005). These zones are often surrounded by an electron dense region marking the limit of the affected area. Jackes (1990) hypothesized that this was due to coccoid bacteria removing collagen from the solubilized compactum and then reprecipitating bone mineral in a more dense form. Using the scanning electron microscope (SEM), Jackes et al. (2001) showed a rim surrounding the destructive foci to be much more electron dense than unaltered bone. These authors indicated these rims to contain more mineral (a higher percentage of Ca + P) and less organic matter (collagen) than the surrounding unaltered bone. The remaining type of microscopic focal destruction in post-mortem bones (i.e., Wedl microscopic focal destruction) involves microboring similar to that seen by Wedl (1864). Roux (1887) agreed with Wedl that this bioerosion was the product of fungal activity and assigned such microborings to the ichnogenus Mycelites ossifragus. Arnaud et al. (1978) sectioned medieval human bones from a shipwreck and observed microboring similar to this Wedl type alteration. However, these authors were unable to find any microorganism inside the microtunnels that might have given rise to them. Ascenzi and Silvestrini (1984) extended the study to experimental work and analysed both medieval and modern human bones collected from sea water using the
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transmission electron microscope (TEM) and found electron dense spherical aggregates made of calcium phosphate inside the microtunnels. These authors described a thin rim at the edge of the microtunnels stained by methylene blue, indicating that the bone matrix had been attacked (Ascenzi and Silvestrini, 1984, pg. 532). Bell and Elkerton (2008) described a characteristic post-mortem type of microtunnel on skeletal remains from the Mary Rose shipwreck. The distribution of this alteration was always peripheral, reaching some 300 μm into the bone. These tunnels showed quite constant morphology and dimensions (84% of tunnels were 5–8 μm diameter, with a maximum diameter of 19 μm) (Bell and Elkerton, 2008). No remineralization boundary showing increased density was observed on the internal walls of these microtunnels. The lack of such remineralization suggested to these authors that these microtunnels were different to those that had undergone diagenetic alteration in terrestrial contexts where bacteria are considered the main invading organism (Bell et al., 1991, 1996; Bell and Lee-Thorp, 1998; Turner-Walker and Jans, 2008). It was finally concluded that this characteristic microboring was diagnostic of marine exposure and to be the product of endolithic microorganisms of unknown identity. Turner-Walker and Jans (2008) found similar microtunnels to those described by Bell and Elkerton (2008) in modern bone remains from a gravel beach in Cyprus, as well as in fossil specimens from the littoral zone of an estuary (the West Runton Freshwater Bed, UK), and referred to endolithic filamentous cyanobacteria as strong candidates for having caused this bioerosion. Further experimental work and detailed descriptions referring to the bioerosion of different organic and inorganic substrates caused by cyanobacteria, fungi, algae and other organisms can be found in Golubic et al. (1975) (boring microorganisms and microborings in carbonate substrates), Knoll et al. (1986) (detailed description of bioerosion caused by cyanobacteria in ooliths), García-Pichel (2006) (microboring of cyanobacteria in different substrates) and Ehrlich et al. (2008) (bibliographic summary of bioerosion caused by cyanobacteria, fungi and algae etc).
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Fig. 1. Location of the Cerro de la Garita site.
1.2. The Cerro de la Garita site and taphonomic context The site examined in the present work, that of Cerro de la Garita (Concud, Teruel, Spain), is a reference site for the continental Euroasiatic Neogene. Located in northeastern Spain a few kilometres outside Teruel (Fig. 1), the site is around 7 million years old and has yielded many of the mammals that characterise the middle Turolian (Upper Miocene) (MN zone 12 sensu Mein, 1990, or local zone L sensu Dam et al., 2001). A National Heritage site, Cerro de la Garita is of special historic value and has yielded a large number of type and paratype specimens (Hernández Pacheco, 1924; see also Alcalá, 1992 and Alcalá, 1994). The historic collection recovered since the 1920 s and held at the Museo Nacional de Ciencias Naturales (MNCN) consists of 5230 fossils. Detailed taphonomic studies have been possible in more recent systematic excavations undertaken by the MNCN and the Fundación Conjunto Paleontológico de Teruel-Dinópolis between 1997 and 2003. Taphonomic analyses have now also been performed on the historic collection for comparison with the surface alterations found in the modern collection. Unlike in recent excavations, no 3D field coordinates of fossils nor detailed descriptions of finds in the excavation area were recorded during the gathering of the historic collection. The Cerro de la Garita site formed during a transitional stage occurring between sediment levels deposited in alluvial complexes and a sporadic lacustrine system. The site represents a shallow, highly alkaline lake shore. The area was almost certainly a water hole, and therefore a good hunting area for predators and scavengers. Hyena coprolites have been found at the site, and tooth marks have been observed on bone surfaces, the size and shape of some suggesting them to have been made by hyenids. Several bone fragments some 2–5 cm in length show clear evidence of heavy digestion and probable regurgitation; all have a characteristic highly rounded and polished surface. All
these traits coincide with hyenid activity, although other tooth marks may have been made by other carnivores. Abundant trampling marks can be seen on the surface of these bone fossils, congruent with their having been deposited in a damp, lakeshore environment. Most of the remains are broken, and only a few anatomical elements from the same individual have been found close to one another, but not in anatomical connection. No hydrological selection (neither by shape nor size) nor signs of abrasion have been observed in these fossils, although in the ground they showed a certain orientation that suggests the influence of a low energy stream. Despite being an open air site, none of these fossils appears weathered, which is also suggestive that the surrounding environment was a damp lakeshore. Detailed taphonomic analyses and descriptions of the site can be found in Pesquero (2006, in preparation) and Pesquero et al. (2005a,b). 2. Materials and methods A total of 4790 bone fossils were collected during the systematic excavations of the site between 1997 and 2003. All were examined under the binocular light microscope (10× to 60×). However, given the special historic and palaeontological value of the Cerro de la Garita site, only a limited number of fossils were sectioned and processed for further analysis. Twenty-four specimens, 13 of which were sectioned, were examined by environmental SEM (QUANTA 200) at the MNCN. Histological analyses were made of both natural fractures and of the cut sections after embedding in resin. Analyses were performed in secondary electron emission (SE-SEM) and BSE-SEM modes using Oxford energy dispersive spectrometry/wavelength dispersive spectrometry (EDS/WDS) detectors. EDS analyses provided the element composition of specific areas of interest. SEM analysis of the fossil
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Table 1 Samples analysed and techniques used. Label
Surface SEM cut EDS TEM Petrographic X-ray sections microscope Diffraction Fluorescence
CG97-65 CG97-66 CG97-98 CG97-128 CG97-638 CG97 639 CG97-687 CG98 1365 CG98 1809H CG98 1809P CG98-1832 CG98-2014 CG99 2461 CG99-2537 CG99 2601 CG99-2607 CG99-2619 CG99-2728 CG99-2998 CG99-3045 CG99-3081 CG99-3285 CG03-3320 CG03-3436 CG03-3619 CG03-3640 CG03-3676 CG03-3923 CG03-4137a CG03-4137b CG03-4409 CG03-4481 CG03-4538 CG03-4607 CG03-4724 SED
X X X
X
X
X
X
X
X X
X X
X
X
X
X
X X X X X X X X X X X X X X X X X X X X
X X X
X X X
X
X X X
X X
X
X
X
X
X
X
X
X X X X
X X X X X X X
X X
X X
X X
X X
X
X Fig. 2. Scanning electron photomicrographs (backscattered mode) showing a typical terrestrial bacterial attack. (a) Incipient bacterial attack dispersed according to histological traits. (b) Incipient bacterial attack, some signs near Haversian canals. (c) Close-up of previous photomicrograph. (d) Bacterial attack distributed along fissures. Note that cracks are filled with characteristic pores produced by bacteria, indicating bacteria invaded these postdepositional cracks.
X
X
X
X
X
X
X
X
X
surface was complemented by thin section observations made under a petrographic microscope (Nikon YM-EPI) using parallel and crossed nichols. Imaging analyses were completed by transmission electron microscopic (TEM) (Zeiss EM 910) examination of ultra-thin preparations at the Electron Microscopy Unit of the Institute of Environmental Sciences of the CSIC. Some samples were analysed by X-ray diffraction (Philips PW-1830) and fluorescence measurements (Philips PW-1404) to obtain their mineral and element compositions (Table 1).
for analysis under the petrographic microscope (Fig. 4a). Both techniques revealed microtunnels that penetrated 200–300 μm into the bone; all contained highly electronic dense spherical aggregates or ‘microspheres’. The distribution of this microboring in the bone sections was peripheral (OHI 0, according to Hedges et al., 1995), with no histologically-orientated organization (Figs. 3 and 4a). The diameters of these tightly clustered microtunnels ranged from 7 to 18 μm (N= 73; mean = 9.84 ± 2.6), their dimensions and morphology remaining constant (Fig. 4b). The microtunnel walls showed a rim of highly electron dense material with a width of 0.1–1.5 μm (N = 45;
3. Results Some of the Cerro de la Garita fossils showed typical signs of terrestrial bacterial attack (Fig. 2). The signs of this bacterial attack in the histologically-studied samples showed three types of distribution, although all were very incipient (OHI 4, according to Hedges et al., 1995). Some specimens showed bacterial alteration around the osteons (Fig. 2a), others showed dispersed attacks near the Haversian canals (Figs. 2b and c) and others still showed signs of attack that occurred post-depositionally following the cracking of the specimens due to sediment compaction (Fig. 2d). Well according to other authors this alteration show small pores with diameters of some 0.9 μm. The precise number of fossils affected by microorganisms remains unknown since histological sections were not prepared of all 4790 specimens. About a dozen of the examined fossils showed a characteristic form of macroscopic damage undescribed until now (Fig. 3a). In order to identify the origin of this alteration and to determine its intensity, the surface and natural fractures of one of these fossils (CG03-4538) were analysed by SEM (Fig. 3b) after transverse sectioning of the specimen and embedding in resin (Figs. 3c and d). Thin sections were also made
Fig. 3. New type of taphonomic alteration. (a) Fossil fragment from the Cerro de la Garita site (CG03-4538), the surface of which has been intensely affected by bacteria. (b) SEM photomicrograph of a natural fracture in fossil CG03-4538. (c) SEM micrograph of the same specimen, cut and embedded in resin; note the signs of intense attack in the cortical layer contrasting with the deeper bone. (b) Close-up of previous picture showing distinctive microboring.
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grooves (Fig. 5c white arrows) or branching-meandering arrangements (Fig. 5c black arrows) were distinguished in less altered areas. Microspheres with a diameter of 1–2.8 μm (N = 49; mean = 2.01 ± 0.44) were observed inside the tunnels (Fig. 6). EDS analysis of these microspheres showed them to have a calcium phosphate composition (Fig. 7) in similar proportion to non-affected bone. Fig. 8 shows ultrathin sections of bone observed under the TEM. Fig. 8a shows dark circles (black arrows) representing the microspheres shown in Fig. 6a under SEM at a similar magnification. Fig. 8b and d show further microtunnels containing these microspheres that appear mineralized at the interior (asterisks). Fig. 8b, d, e and f show the cell walls of what appear to be mineralized microorganism (white arrows). 4. Interpretation and discussion Some of the components of the Cerro de la Garita fossil association showed typical signs of incipient terrestrial bacterial attack characterized by microscopic focal destruction involving nanometric-sized pores within the bone. Some of these alterations followed a Fig. 4. New type of taphonomic alteration. Alteration in specimen CG03-4538. (a) Thin section under the petrographic microscope showing invasive microtunnels penetrating into the bone from the outer cortical lamellae (bottom of the picture). (b) SEM photo micrograph of invasive microtunnels containing microspheres. The walls of these tunnels show a higher electron density than the unaltered bone. (c) EDS analyses of the highly electron dense microtunnel wall (transverse section; spectrum 3, right side top diagram) and non-affected bone (spectrum 4, right side bottom diagram).
mean= 0.81± 0.25). EDS analysis showed the chemical composition of the unaltered bone and these rims to be the same (Fig. 4c). i.e., calcium phosphate. BSE-SEM images showed this rim to have a bright white colour, indicating it to be denser than unaltered bone. The distribution of the surface alteration of the fossil bones was heterogeneous (see Fig. 5a in contrast with Fig. 5b). Individual
Fig. 5. New type of taphonomic alteration. SEM photomicrographs of fossil bone surfaces showing distribution of alteration produced by invasive microboring (CG034538). (a) Some areas appear affected by an intense pitting, increasing the bone porosity. (b) In contrast, some areas are less affected. (c) In areas where surface microboring is less intense, individual grooves (white arrows) can be distinguished in contrast to areas where these channels form a ramified arrangement (black arrow). (d) Longitudinal grooves on bone surface (black arrows) that may be related to the incipient stage of this alteration; photographed at a higher magnification than in the previous picture.
Fig. 6. (a), (b), (c) New type of taphonomic alteration. SEM image of fossilized microspheres (black arrows) inside a microtunnel. These photomicrographs represent a naturally, transversally broken surface of the same specimen at different magnifications. (d) SEM photomicrograph of a fossil from the Fossil-Lagersttäten site of Libros (Teruel, Luque et al., 1996). The picture, taken at a magnification similar to that used in (c), represents the preserved soft tissues of an amphibian and shows a diatom and abundant fossil bacteria covering the fossil surface. (e) SEM photomicrograph of a section of a modern bone collected from Neuadd (Wales, UK), showing a typical terrestrial bacterial attack with mineral redeposition rims around individual colonies. EDS analysis returned a calcium phosphate chemical composition for the bone and the outer ring. (f) New type of taphonomic alteration. Section of a fossil showing mineral redeposition on the internal wall of microtunnels, seen as a rim in transverse section (high electron density). The EDS mineral composition is shown in Fig. 3 (spectrum 3).
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experimental work by Davis (1997). The latter author reported strong similarities with the microboring described by Wedl (1864) and Roux (1887), but assigned this type of alteration to endolithic cyanobacteria or algae based on the microorganisms found in the examined bone. Underwood and Mitchell (2004) reported branching-meandriform microtunnels affecting the surface of the tooth roots of Mesozoic fossil sharks identical to that observed by Ascenzi and Silvestrini (1984) and Davis (1997) on human and bird bones respectively. Underwood and Mitchell (2004) concluded that the causative agent was Mycelites ossifragus. Table 2 lists the similarities and differences between these various types of alteration, suggesting that the second type of microboring seen in the present work represents a formerly unknown type of bioerosion. Chemical analysis of the inner wall of the new type of microboring showed it to be similar in chemical composition (calcium phosphate) to unaffected bone, suggesting this high density structure represents bone remineralization. The highly electron dense microspheres inside the tunnels have identical calcium phosphate composition. These microspheres are similar in shape, size and chemical composition to those found covering amphibian fossils with preserved soft tissues (Luque et al., 1996; Meyer and Milsom, 2001; McNamara et al., 2003, 2006) from the Fossil-Lagerstätten site of Libros (Teruel). The latter authors interpret the microspheres they observed to be bacteria preserved in calcium phosphate. Liebig (1998) also documented that a large percentage of the bacteria fossilized in the Messel Oil Shale (Germany) were preserved in amorphous calcium phosphate or that cryptocrystalline apatite had replaced them. When observed under TEM the present microspheres showed strong similarities in size and structure to those reported by Ascenzi and Silvestrini (1984). Some of the present microspheres show a preserved cell wall; this, plus their size and shape suggests them to be coccoidal forms in the range of bacteria. TEM-SEM and EDS analyses ruled out any possibility of modern bacterial contamination; the present microspheres were clearly mineralized and preserved in calcium-phosphate, and found inside tunnels located around 300 μm into the bone, ruling out secondary infilling from the external environment (see Figs. 3d and 4b). Fig. 7. Above: SEM picture of the microspheres inside microtunnels (specimen CG03538), showing the specific area analysed (+) using EDS detectors. Below: Element composition of the microspheres as determined by EDS.
distribution around the Haversian canals, while others showed a more dispersed distribution or occurred in postdepositional cracks (Fig. 2). A second type of alteration was also observed (Figs. 3–8), characterized by a peripheral microboring intensively affecting the outer compact bone layer. These microtunnels and their internal microspheres differed from the typical damage caused by terrestrial bacterial attack. In transverse section, the rim at the edge of these microtunnels was highly electron dense, similar to the hypermineralized zones surrounding the microscopic focal destruction described in terrestrial environments. This second type of alteration also shows similarities and differences to the bacterial microboring of bones that occurs in marine environments. Certainly, the present microtunnels are similar in size and distribution to those described in bones collected in marine environments (Bell and Elkerton, 2008; Turner-Walker and Jans, 2008), but the latter authors observed no distinct, highly electron dense rim at the edge of the microtunnels they examined. The presence or absence of an outer rim appears to differentiate between lacustrine and marine bacterial microboring. On the bone surface, this microboring showed a meandriformbranching arrangement of channels similar to that reported in marine
5. Conclusions The microboring-modified fossils of the Cerro de la Garita show similarities and differences to taphonomic modifications described by other authors in skeletal remains collected from marine and terrestrial environments. As seen in terrestrial bacterial attacks, the new type of microboring in the present specimens showed characteristic remineralizations, forming highly electron dense microtunnel walls of calcium phosphate (appearing as rims in transverse section). Unlike in terrestrial bacterial attack, however, this highly electron dense layer covers the entire microtunnel inner wall; in terrestrial microscopic focal destruction it appears in discrete zones around nanometric pores and channels. Further, the present modification is characterized by microspheres some 1–2.8 μm in diameter made of calcium phosphate inside the microtunnels. The new type of microboring also showed a meanderingbranching arrangement at the bone surface, similar to that seen in bones collected form marine environments. TEM analyses of the microspheres inside the tunnels showed them to be the same as those described by Ascenzi and Silvestrini (1984). However similar bioerosion in marine environments is not associated with the remineralized, highly electron dense calcium phosphate microtunnel walls seen in the present fossils. The cocci-like microspheres detected in this work had shape, size and a distribution similar to the spherical structures described in fossils from the lacustrine Fossil-Lagersttäten deposits of Libros
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Fig. 8. TEM images of ultrathin bone sections. (a) The dark grey circles (black arrows) are the microspheres shown in the previous figures. (b), (c), (d) (e), (f) Closer views of these microspheres; a mineralized cell wall (white arrows) can be distinguished. Asterisks indicate mineralized microspheres.
(Spain) and Messel (Germany) (Luque et al., 1996; Liebig, 1998; McNamara et al., 2003, 2006). The latter microspheres are composed of calcium phosphate, as are those described in the present work. The latter authors considered these structures to be fossilized bacteria.
The present microspheres observed in Cerro de la Garita may also be fossilized bacteria, or at least, coccoidal forms in the range of bacteria. The present work provides evidence that a type of bone alteration, different to that seen in marine and terrestrial environments, may
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Table 2 Main features of the taphonomic structures discussed in the present work.
Cerro de la Garita Terrestrial change Turner Walker et al. (2002) Mary Rose (Bell and Elkerton, 2008) Hackett tunnels (Davis, 1997)
Distribution of this alteration
Depth into the cortical layer
Diameter
Rim
Peripheral Discrete zones Peripheral Parallel to the periosteal surface
200–300 μm Variable N 300 μm 0.2 mm
7–18 μm 0.1–1.0 μm 5–8 μm 0.25 mm
Yes Yes No No
occur in aquatic continental environments. Experimental work, especially in lacustrine environments, is needed to identify the origin of this type of alteration and differentiate it from others environments. Acknowledgements The excavations at the Cerro de la Garita site, the preparation of the present fossils and their examination was made possible by support from the Departamento de Educación Cultura y Deporte, the Dirección General de Patrimonio Cultural (projects 022/97, 133/98, 142/99 and 197/00) and the Departamento de Ciencia, Tecnología y Universidad (Grupo de Investigación Emergente E-62) of the Regional Government of Aragón (Spain), and the Spanish Ministry of Science and Innovation (Project CGL2007-66231). M.D.P was the recipient of a Juan de la Cierva contract. (Ref. JCI-2007-132-565). We are grateful to the Laboratories of Electron Microscopy at the Museo Nacional de Ciencias Naturales (MNCN) and the Centro de Ciencias Medioambientales (CCMA), as well as the Laboratory of Fluorescence and X-ray diffraction at the MNCN. We also thank microbiologists Blanca Pérez Uz and Lynne Bell for their comments and suggestions. References Alcalá, L., 1992. Macromamíferos neógenos de la fosa de Alfambra-Teruel. Ph.D Thesis, Univ. Complutense Madrid, Spain. Alcalá, L., 1994. Macromamíferos neógenos de la fosa de Alfambra-Teruel. Instituto de Estudios Turolenses, Teruel. Arnaud, G., Arnaud, S., Ascenzi, A., Bonucci, E., Graziani, G., 1978. On the problem of the preservation of human bone in sea-water. Journal of Human Evolution 7, 409–420. Ascenzi, A., Silvestrini, G., 1984. Bone-boring Marine Micro-organisms: an experimental investigation. Journal of Human Evolution 13, 531–536. Bell, L.S., 1990. Palaeopathology and diagenesis: an SEM evaluation of structural changes using backscattered electron imaging. Journal of Archaeological Science 17, 85–102. Bell, L.S., Elkerton, A., 2008. Unique marine taphonomy in human skeletal material recovered from the Medieval warship Mary Rose. International Journal of Osteoarchaeology 18 (5), 523–535. Bell, L.S., Lee-Thorp, J.L., 1998. Advances and constraints in the study of human skeletal remains: a joint perspective. In: Cox, M. (Ed.), Grave Concerns. York, CBA, pp. 238–246. Bell, L.S., Boyde, A., Jones, S.J., 1991. Diagenetic alteration to teeth in situ illustrated by backscattered electron imaging. Scanning 13, 173–183. Bell, L.S., Skinner, M.F., Jones, S.J., 1996. The speed of post-mortem change to the human skeleton and its taphonomic significance. Forensic Science International 82, 129–140. Dam, J.A., Alcalá, L., Alonso Zarza, A.M., Calvo, J.P., Garcés, M., Krijgsman, W., 2001. The upper Miocene mammal record from the Teruel-Alfambra region (Spain): biostratigraphy and chronology. Journal Vertebrate Paleontology 21 (2), 367–385. Davis, P.G., 1997. The bioerosion of bird bones. International Journal of Osteoarchaeology 7, 388–401. Ehrlich, H., Koutsoukos, P.G., Demadis, K.D., Pokrovsky, O.S., 2008. Principles of demineralization: modern strategies for the isolation of organic frameworks. Part. 1. Common definitions and history. Micron 39, 1062–1091. Fernández-Jalvo, Y., Andrews, P., Pesquero, D., Smith, C., Marín-Monfort, D., Sánchez, B., Geigl, E.M., Alonso, A., 2010. Early bone diagenesis in temperate environments Part I: Surface features and histology. Palaeogeography, Palaeoclimatology, Palaeoecology 288, 62–81. García-Pichel, F., 2006. Plausible mechanisms for the boring in carbonates by microbial phototrophs. Sedimentary Geology 185, 205–213. Golubic, S., Perbius, R.D., Lukas, K.L., 1975. Boring Microorganisms and Microborings in Carbonate Substrates. In: Frey, R.W. (Ed.), The Study of Trace Fossils. New York, Springer-Verlag, pp. 229–259.
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