5th International Symposium on Eastern Mediterranean Geology Thessaloniki, Greece, 14-20 April 2004

Microbial focal destruction in Late Miocene mammal bone from Kerassia (N. Euboea Island, Greece)
Iliopoulos G.
Department of Geology, University of Leicester, LE1 7RH, Leicester, UK, gi6@le.co.uk Keywords: bioerosion, microorganisms, bacteria, fossil bone, Kerassia, Miocene.

Introduction The taphonomic investigation of Upper Miocene (MN 11-12) fossil mammal bones from Kerassia (Euboea Island, Greece) (Fig. 1) was undertaken. To date, seven fossiliferous sites have been found near the village of Kerassia, where at least two fossiliferous horizons occur, an upper and a lower one (Theodorou et al., in press). The microscopic investigation of the collected material revealed extensive microbial focal destruction (MFD). The term microbial focal destruction was coined by Hackett in 1981 to describe any histomorphological alterations in bone or other vertebrate hard tissues produced by microbes. The different patterns of this damage have been attributed to four types of microorganisms, bacteria (Hackett, 1981), fungi (Wedl, 1864), algae (Davis, 1997) and protozoa (Ascenzi and Silvestrini, 1984). These organisms invade the bone to feed on the collagen (the organic component of vertebrate hard tissues) producing recognisable destructive features in the form of microscopic tunnels or borings. Recent work on archaeological ((Grupe, 2001; Hedges, 2002) and palaeontological bioeroded bone (Trueman and Martill, 2002) has shown that extensively bioeroded bones would have little or no chance to survive as fossils. However, in Kerassia extensively bioeroded bones have survived in the fossil record.

Methodology Polished thin sections of fossil bone and teeth from both horizons of Kerassia and, for comparison, from eight other Late Miocene Greek localities were studied under a Hitachi S 520 scanning electron microscope (SEM) and analysed using a JEOL JXA 8600-S electronmicroprobe where a focused beam at 15kV was used for analyses. Fragments of bone and teeth on stubs only from Kerassia were also used and examined under the SEM. The bone and tooth specimens from Kerassia were collected from five localities representing both fossiliferous horizons, and from different areas in the bone accumulations. The bone specimens from the other Greek localities were randomly selected from material that came from the localities Ravin de la Plui 2, Vathylakkos 1, Ditiko 1 and Ditiko 2 in Macedonia, Samos on Samos Island, whereas the localities of Old Pikermi, New Pikermi and Chalkoutsi are located close to Athens. Stratigraphically, they range from the Late Vallesian (Ravin de la Plui) to the late Turolian (Ditiko) (Fig. 1).

Results and Discussion Almost all Kerassia bones and teeth showed extensive microbial focal destruction (MFD) (Hackett, 1981). MFD was not detected only in two specimens that came from scattered bones above the main bone beds. Thus, the concentration of bones in great numbers and

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5th International Symposium on Eastern Mediterranean Geology Thessaloniki, Greece, 14-20 April 2004 density seems to be a prerequisite for extensive MFD. The MFD was recognised in the bone, the dentine and the cement but not in the enamel where the organic component is less than 1%. MFD can be seen as zones of damaged bone, around the perimeter of the bones, around the marrow cavity and as randomly scattered foci. The MFD foci are ellipsoid nodules with their long axes parallel to the long axis of the bone (Iliopoulos, in review). The rims of these nodules are permineralised (Fig 2a, 2b). Microprobe analyses show that the apatite in the rims is enriched in calcium phosphate relative to the whole bone and calcium phosphate is depleted in the foci. The fluorine content was found to be higher in the undamaged bone than in the rims and lower in the foci. This probably indicates an early recrystallization of the hydroxyapatite crystallites in the foci that followed bacterial action, and impeded the diagenetic fluorine uptake (Iliopoulos, in review).

Figure 1. Sketch map of Greece showing the location of Euboea Island and Kerassia and of the eight other Greek Late Miocene localities used in this study. 1. Samos, 2. Chalkoutsi 3. Old Pikermi, 4. New Pikermi, 5. Ditiko 1, 6. Ditiko 2, 7. Ravin de la Pluie 2 8. Vathylakkos 1. The internal structure of the foci is manifest as a series of parallel microtunnels (Fig 2a, 2b). The diameter of these microtunnels is between 150-600nm (Iliopoulos, 2003). The diameters of the microtunnels and the preservation of fine histological features (lacunae) in the foci (Fig 2a, 2b), indicate that the invading microorganisms were bacteria (Iliopoulos, 2003, in review). The bone material from the other Late Miocene Greek localities (Old Pikermi, New Pikermi, Chalkoutsi, Samos, Vathylakkos 1, Ditiko 1, Ditiko 2, Ravin de la Pluie) revealed the same or similar, extensive bacterial damage. According to Paul and Clark (1989) most soil bacteria are mesophylous, need moderate moisture and grow better in neutrality. Therefore, in order to succeed the maximum development needed to produce such extensive bioerosion bacteria need favourable conditions. Such favourable conditions refer to a temperate to warm and relatively moist climate, and a soil with neutral or near neutral pH. Probably, such favourable conditions prevailed during the Late Miocene of Kerassia and North-Eastern Greece. Also, the presence of foci with and without perminerallized rims that overlap each other, is probably indicative of a seasonal climate that allowed two growth periods for the bacteria per annum (Iliopoulos, in review). According to Schaffer (1973), in Mediterranean type climates today soil microorganisms present two growth cycles per annum, one at the onset of the dry period and a second at the onset of the

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5th International Symposium on Eastern Mediterranean Geology Thessaloniki, Greece, 14-20 April 2004 wet period. Thus, the foci with the permineralised rims were possibly formed during the warm and dry season, and similarly the foci without the permineralised rims were possibly formed during the cold and wet season (Iliopoulos, in review). This climatic interpretation is in accord with the recent results of Ivanov et al. (2002) for the climate of the Late Miocene of Bulgaria. Their palynological analysis showed that during the Late Miocene in Bulgaria there was a general alternation of a dry and hot season with a humid and cool season, indicating a seasonal climate. F

r HC L

m F Figure 2: a. Backscattered electron image (BEI) of extensively damaged bone tissue. Haversian systems are still clear and the foci (F) of bacterially eroded bone can be easily identified around the Haversian canals (HC). Portions of undamaged bone are also evident (black arrow), (scale bar 150 m). b. At higher magnifications the structure of the foci is evident, the distinctive rims (r) of the foci and the microtunnel (m) network of the invading bacteria. Interestingly, some lacunae (L) are situated in the foci, BEI (scale bar= 30m). Furthermore, the prevailed geochemical conditions (redox potential, pH, hydrological regime, etc.) in the surrounding sediments and the burial environment, in general, of Kerassia and the other studied localities were such that favoured the preservation of extensively bioeroded bones and teeth. Under these conditions the apatite (carbonated hydroxyapatite) crystallites in the bones and teeth recrystallized and allowed the fossilization of the bones and teeth.

Conclusions Contrary to the established ideas of bone preservation (Grupe, 2001; Trueman and Martill, 2002; Hedges, 2002), in Kerassia and eight other Late Miocene Greek localities, bones and teeth with extensive bacterial damage have survived in the fossil record with quite good preservation of their histology and bioerosion features. Consequently, such extensive bioerosion possibly indicates that, a warm enough and moist enough seasonal climate that favoured the development of soil bacteria did prevail during the Late Miocene of Kerassia and probably during the Late Miocene of the whole North-Eastern Greece.

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5th International Symposium on Eastern Mediterranean Geology Thessaloniki, Greece, 14-20 April 2004 Medicine, Science and the Law, 21(4), 243-265. Hedges R.E.M. (2002). Bone diagenesis: An overview of processes, Archaeometry, 44, 319328. Ivanov D., Ashraf A.R., Mosbrugger V. & Palamarev E. (2002). Palynological evidence for Miocene climate change in the Forecarpathian Basin (Central Paratethys, NW Bulgaria), Palaeogeography, Palaeoclimatology, Palaeoecology, 178, 19-37. Iliopoulos G. (2003). Invading the bone: Microbial focal destruction in Late Miocene mammal bone, Scanning, 25, 94-95. Iliopoulos G. (in review). Extensive microbial destruction in Late Miocene mammal bone, Journal of the Geological Society, London. Paul E.A. & Clark F.E. (1989). Soil microbiology and biochemistry, San Diego, Academic Press, Inc. Schaefer R. (1973). Microbial activity under seasonal conditions of drought in Mediterranean climates, In: Castri F.D. & Mooney H.A. (eds.), Mediterranean type ecosystems: Origin and structure, London, Chapman and Hall Limited, 191-198. Theodorou G., Athanassiou A., Roussiakis S. & Iliopoulos G. (in press). Remarks on the Late Miocene vertebrates of Kerassia (Northern Euboea, Greece), Deinsea, 10. Trueman C.N. & Martill D.M. (2002). The long term survival of bone: The role of bioerosion, Archaeometry, 44(3), 371-382. Wedl C. (1864). Uber einen im Zahnbein und Knochen keimenden Pilz. Akademi der Wissenschafen in Wien, Fitzungberichte Naturwissenschaftliche Klasse ABI. Mineralogi biologi erdkunde, 50, 171-193.

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