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Aspartic Acid Racemization in the Dentine of Bears (Ursus etruscus G. Cuvier, Ursus prearctos, Boule, Ursus deningeri von Reichenau and Ursus spelaeus Rosenmüller-Heinroth). Tooth Dentine Amino Acids Versus Mollusca Amino Acids
Advances in BioChirality G. Pfilyi, C. Zucchi and L. Caglioti (Editors) 9 1999 Elsevier Science S.A. All rights reserved.
CHAPTER
247
17
Aspartic Acid Racemization in the Dentine of Bears (Ursus etruscus G. Cuvier, Ursus prearctos, Boule, Ursus deningeri von Reichenau and Ursus spelaeus Rosenmiiller-Heinroth). Tooth Dentine Amino Acids Versus Mollusca Amino Acids T. de Torres, J.F. Llamas, L. Canoira, P. Garcia-Alonso Laboratory of Biomoecular Stratigraphy. Madrid School of Mines, Rios Rosas 21, 28003 Madrid, Spain
Introduction The dating of Quaternary mammal bones and teeth has been considered as a main objective for archaeologists and palaeontologists. U-series based methods seemed do not work accurately because both bone and tooth are open systems. Electro spin resonance dating method offered interesting results but up to now amino acid racemization analysis seems to be the most promising system. There are previous works on amino acid racemization analysis in bones [1-10], teeth enamel, [11-13], and teeth dentine [14-16]. We have obtained interesting results from a wide analytical work of amino acid racemization ratios in the dentine of cave bears of Middle and Upper Pleistocene age. We have also carried out some analysis on the dentine of Lower Pleistocene age bears.
Phylogeny After the unsuccessful attempt of Ursus m i n i m u s Deveze-Debouillet towards more modern bear forms more adaptative and hipocarnivorous with stepped frontal bone appeared Ursus etruscus G. Cuvier; Fig. 1, the common ancestor of the modern european bears. This species was first recorded at the Middle Villafranchian (ca 2 ma) and represents the last successful attempt to evolve from short faced forms to more adaptative long faced: arctoid and speloid species. One of the latest appearance of U. etruscus was in the Venta Micena (Orce Granada, Spain) site, aminoacid racemization dated in circa 1 million years. This bear was a very adaptative species and colonized wide areas of Europe with markedly different environmental characteristics: arid alluvial fan dominated intermontane basins (Puebla de Valverde, Teruel, Spain), rivers (Vald'Arno, Italy), swamps (Tegelen, the Netherlands), hypersaline lacustrine basins (Venta Micena, Granada, Spain) but
248
IH-OL]-;~I~.IT...........................................................................................................................................................................................................................
........! ............. i
U. maritimus
U. arctos
U mediterraneus ~
io~_
|
~
/
U.spelaeus
U.prearctos U.deningeri
_
N
I. i
,,
U.etruscus s
U.minimus
U.ruscinensis
Ursavus
Fig. 1. Pleistocene bears phylogeny.
never colonized caves. Some cave findings in Spain (Almenara, Castell6n) have been interpreted as water-laid fossils. In short: U. etruscus was a very adaptative species which probably never needed to colonize caves during the winter because of favourable palaeoclimatological conditions. U. etruscus 1 million year old A A R dated [17], in an arid basin (C~llar-Baza, Spain) was associated with controversial Homo sp. remains and with uncontroversial lithic artifacts. The arctoid group includes Ursus prearctos Boule, Lower-Middle Pleistocene, and the still living Ursus arctos Linneo and Ursus maritimus Phipps. This group had, and still has, a very noticeable ecological success covering the whole of Europe, de Asiatic an African Mediterranean Border as well as North America, they probably were as opportunistic as U. etruscus was. U. prearctos inhabited the caves of the Atapuerca karst system and ca 800 Ky. was associated with the oldest human remains from Europe (Homo antecessor). The cave bears evolutive line was composed of Ursus deningeri von Reichenau, of Middle Pleistocene age, and Ursus spelaeus Rosenm/iller-Heinroth, of upper Middle Pleistocene-Upper Pleistocene age. Arctoid line representings (U. prearctos, U. arctos and U. maritimus) were oportunistic species, as U. etruscus was. The speloid line representings, the cave bears (U. deningeri and U. spelaeus), became very popular since they have been repeatedly employed as dramatic resources in fictions and films. But in the real life
249 they were only wrongly evolved, too specialized, bears that probably avoided to coincide with contemporary human beings. In spite of, in Europe, the oldest U. deningeri remains appeared in open-air sites as river sands (Sussenborn, Germany), travertines (Bilzinsleben, Germany), in Spain all the U. deningeri sites are in caves. During the upper part of the Middle Pleistocene Ursus deningeri was replaced by the true cave bear U. spelaeus which remains with some rare exceptions were found only in caves. This species reached largest body, bone and tooth sizes. Short and massive postcranial bones and complicated molars are characteristics. The common characteristic on both evolutive lines, arctoid and speloid, of being cave occupants during winter hibernation period minded that all of them had developed liver protective mechanisms (ursodeoxicholic acid) that allowed a winter metabolism on body fat exclusively based.
Geographical distribution of cave bears
The cave bears distribution in Europe, Fig. 2, had a latitudinal control: between 42 ~ and 52 ~, the later roughly coincides with the maximum extent of the polar ice sheet during the last maximum glacial. In Spain, Fig. 3, four occupation areas have been defined: Atlantic Border, Mediterranean Border, Central Part and Pyrenees.
Fig. 2. Cave bears distribution in Europe.
250
1313
Li
EF
CUEVA MAYOR (Atapuerr Burgos, Castlllll-l_lXin)
CUEVA LA PASADA (Gurlzo, Contabm)
CUEVA CUETO DE LA LUCIA[,
(ouirlani~, cemix/)
C U E ~ S" mABEL (Ranero, Blzkal, Euskadl)
c.fvA ..6s 14...i (Trlacutela, Lugo, Galicia)
CUEVA DE LEZETXIKI (Mondmg6n, Gulpuzkoll, Eusklldl)
1 ]
] J
CUEVA DE ARRIKRUIZ (Olltl, Gulpuzkoli, Euskadl) CUEtR DE El(Am (l~lba, GuIpuzkoa, Euskadl)
'~ <:
/
0
! :
J
li
~
MADRID
o (i
.,/ 'l ~i{ ,..~
t
,,!__._../L-...."t~
~ .............. ;f" ~ , ~
x,-~""s'{-
[
J
CUEVA DE TROSKAETA (Ataun, Guipuzkoll, Eusklidl) CUEIA CORO TRACrro Feb, Hues~, Aiall~)
]
CUL=VA DE I L 'rOLL (Idols, Baroelona, Cailunya) CUEVA DE EL REGUERILLO (PMones, Mliddd)
Fig. 3. Cave bears distribution in Spain and sampled localities.
In the Atlantic Border area there appear many caves with bear remains: U.
deningeri: La Lucia (Quintanilla, Cantabria), Santa Isabel (Ranero, Vizcaya), Lezetxiki (Mondrag6n, Guipuzcoa). With U. spelaeus they are Eir6s (Triacastela, Lugo), La Lucia, La Pasada (Guriezo, Cantabria), Lezetxiki, Arrikrutz (Ofiate, Guipuzcoa), Ekain (Deba, Guipuzcoa), Troskaeta (Ataun, Guipuzcoa). In the Mediterranean Border there are not many caves with bear remains: Cova Bunica (Olopte, Girona) with U. deningeri and E1 Toll (MoiA, Barcelona) with U. spelaeus. In the Central Part of the Iberian Peninsula, the Sima de los Huesos (Atapuerca, Burgos), delivered many thousands of U. deningeri remains as well as hundreds of Preneandertalian man bones and teeth, being dated (U-series and ESR), (1997), 320 ky old [18]. E1 Reguerillo (Torrelaguna, Madrid) is the only U. spelaeus important locality in the area. In the Pyrenees: Coro Tracito (Tella, Huesca) represents the only high mountain locality. As it is possible to see the Iberian Peninsula represents the species border: that minds that palaeoenvironmental conditions were often near the species stress limits. The cave bears moved southwards only during climatic optimum periods appearing endogamy dominated populations, as a response to minor palaeoenvironmental worsening in the European realm.
The problem
According to our data, there are striking morphological and metrical differences between U. deningeri and U. spelaeus [19,20], being the former more primitive,
251
according to dental and skeletal analysis, and slender, obviously taking into account the strong sex dimorphism always present in bears. When we have compared dental morphologies from U. spelaeus of different localities, it has been possible to observe the persistence of minor morphological features that allowed to identify a certain "tribal" component, a noteworthy of this appeared in the enormous sample of lower fourth premolars from Ekain cave which show an strong cusp in the heel, this robust cusp, that produces a distinguishable wear notch on the opposite fourth upper premolar is absent or it appears as a cusplet in the other cave bear localities. Much more important is the fact that in caves developed in very craggy areas, far enough from wide valleys, plains, flat depressions, the cave bears showed a very peculiar paw morphology: broader and shorter than the normal cave bears used to be. Based in this character a subespecies Ursus spelaeus parvilatipedis Torres was defined [21], being the name based on the most important characteristic: short ~arvus) and broad (latus) feet ~edis). Based on first lower molar morphology Grandal d'Anglade and Vidal Romani [22] concluded that U. s. parvilatipedis, U. spelaeus and U. deningeri were only an expression of politypysm and, in fact, the same species. This assert must be rejected. But it is necessary to take into account that the Iberian Peninsula was the species border and palaeoenvironmental stresses were usually high, the caves were occupied for relative short time periods and population isolation phenomena (endogamy) were not uncommon. To solve these uncertainties we have been working for dating cave bears sites trough aspartic acid racemization analysis.
A b o u t cave bear sites
Cave bear caverns are good sites for amino acid racemization analysis sampling because they are clean and that minds that during the first and further taphonomical stages there are not too many possibilities of foreign protein or amino acids arrival. During the hibernation period adult and young bears do not eliminate faeces or urine and because of the almost mono specific composition of faunal remains proteins, amino acids and other organic origin compounds have the same signature: are the result of cave bear carcasses decay. Hibernation zones were in dark cave places, some tens of meters out of the sun light reach, there algae and fungi do not grow easily. Cave bears caverns were almost exclusively places for bear hibernation, being uncommon findings of other mammal remains, which usually represent less than 0.01%. In an excavation campaign more than 3000 bones and teeth can be recovered. Guano from bats is also infrequent. Man rarely inhabited cave bears sites although it was documented a very short seasonal summer occupation of bear dens by Musterian culture (Middle Paleolithic) man. Scavengers (Crocuta crocuta spelaea) rarely cannibalized cave bears carcasses, in spite of sometimes they did it and phosphatic coprolites appeared scattered in the sediment.
252 Cave bears caverns are the best sites for amino acid racemization dating because the thermal history did not show strong variations and average temperature was moderately low. The Iberian Peninsula was never affected by general glaciation processes. River incision and hillside retreat were very strong: ancient caves accesses are usually closed by rock blocks from cave roof collapses. Local cave hydrogeological reactivations very frequently were the origin of mud beds deposition sealing bear remains. Amino acids (usually free amino acids) reached the cave deposits from soils and rhizospher e in general, some speleothems we have studied contained small amounts of aspartic acid. In short, cave bears cavities are unusually clean sites with stable thermal history.
Sampling We have selected five canines or third upper incisors from each locality because their crown conic shaped efficiently protects dentine from contamination. Fifty mg of powdered dentine samples were obtained from the innermost part of the crown via drilling the tooth with a dental diamond drill. Powder from the outer part of the root, up to the limit of 1 mm deep was rejected. This process produced an unavoidable slight sample heating. We have analyzed samples from the following cave bears sites:
U. deningeri: Sima de los Huesos, Santa Isabel, La Lucia. U.spelaeus: La Lucia, Troskaeta, E1 Reguerillo, Eiros, Arrikrutz, Coro Tracito, La Pasada.
Sample preparation and analysis The glassware used for analysis was cleaned by baking in an oven at 500~ for about 2 h. Plastics were new from the factory. All the water used in the analysis was Milli-Q quality from Millipore. All chemicals were HPLC grade or spectroscopy grade. Before hydrolysis and amino acid derivatization, samples were treated to eliminate free amino acids. With this process we aimed to remove foreign amino acids and to obtain a homogeneous protein (from collagen) molecule which racemizates more uniformingly, [13]. Dentine has a 10% of A-type collagen [23]. We have employed a modification of the method proposed by Marzin [24]. The powder sample was dissolved in 1 ml of HC1 2N and sonicated. After 5 ml PBS buffer addition, the sample was dialyzed at 3500 Dalton (Spectra/Por mnco 3500 membrane) during a 20 h period in a buffered solution with magnetic stirring. After lyophilizaton the remaining powder was treated as it has been described elsewhere [17,25] and analyzed. Hydrolysis of ca 50 mg of dentine was carried out in a mixture of 12 N hydrochloric acid (2.9 #l/mg) and 6 N hydrochloric acid (100 #1), in test tubes with an atmosphere of nitrogen, in a heating block at 100~ for 20 h. Desalting was
253 accomplished in conical 1.5 ml Eppendorf plastic micro test tubes with caps, with concentrated hydrofluoric acid added and centrifuged in an Eppendorf centrifuge. The supernatant was frozen in liquid nitrogen, and vacuum dried. Samples were redissolved with 80 #1 distilled water and transferred to 2 ml glass vials. Water was evaporated at vacuum. The first amino acid derivation step consisted of sterification with 250 #1 of 3 M thionyl chloride in isopropanol in a nitrogen atmosphere on the heating block at 100~ for just 1 h and later vacuum dried. The second derivation step consisted of N-trifluoroacetylation with 150 #1 of trifluoroacetic acid anhydride (25% in dichloromethane) in a nitrogen atmosphere and heated at 100~ for just 5 min on the heating block. Dichloromethane solvent and the unreacted trifluoroacetic acid anhydride were evaporated under a flow of nitrogen. Later the samples were dissolved in 125 #1 of n-hexane but most of the n-hexane evaporated in a stream of nitrogen to a final volume of 15-25 #1, then being transferred to injection vials. A sample of 0.2 #1 was injected into a Hewlett-Packard 5890 gas chromatograph. The injection port was kept at 215~ and set for splitless mode for the first 75 s, at the beginning of which the sample was injected, and later set to split mode. We used helium as the carrier gas, at a head column pressure of 5.8 psi, and a Chirasil-Val fused silica column (25 m • 0.39 m m • 0.25 mm) from Chrompack. The gradients we used were as follows: 50~ (1 min), heat at 40~ to 115~ remaining at 115~ for 12 min, heating at 3~ to 190~ remaining at 190~ for 10 min, cooling down to 50~ and remaining at this temperature between runs (80~ if the time between runs is longer, typically overnight). The detector was a N P D set at 300~ Integration of the peak areas was carried out using the PEAK96 integration program from Hewlett-Packard, which runs on a PC computer. Usually DL alanine, uI~ valline, oI~ proline, oI~ leucine, AI isoleuleucine, DL aspartic acid, I~I~ phenilalanine, DL glutamic acid and L hydroxiproline peaks are identified.
Results and conclusions
First at all we want to note that the use of a previous dialysis process allowed a noteworthy accuracy in our analytical results that were not obtained in more than 30 former analysis that we carried out on non dialyzated samples, where we obtained racemization ratios of both free and bonded amino acids. This can be explained according different ways: on one hand we must take into account the effect of hydroxyproline peak which in some analysis could appear superposed to D-Asp peak "rejuvenating" the true sample age and being origin of an inadmissible racemization ratios distribution for statistics calculations. In our opinion the use of all dialysis, new purchased Chirasil L-Val column and N P D GC detector can be interpreted as analytical success key. Up to now this is the first work which deals on a systematic amino acid racemization ratio analysis in the dentine of the same genus teeth and the results seemed to be very encouraging.
254
Fig. 4. Histogram of aspartic acid racemization in cave bear (Ursus deningeriand Ursusspelaeus) teeth dentine samples. Species and localities are identified. For comparison data from a Holocene brown bear (U. arctos) locality has been added.
It is evident that the bear population remains that have been identified as Ursus deningeri von Reichenau, have homogeneous and higher aspartic acid racemization ratios Fig. 4. We are able from now on to formally define the U. deningeri aminozone in the Iberian Peninsula over 30% ASP racemization ratios and average values near 35%. U. spelaeus samples defined a broad aminozone where two subaminozones could be distinguished: the one with two higher racemization ratio populations (El Reguerillo and Arrikrutz) and the other Troskaeta, Coro Tracito, and La Pasada with very low ASP racemization ratios, in fact they are not very different from Holocene brown bear (U. arctos) sample from Saldarrafiao. La Lucia cave sample is placed into the low ASP racemization ratio group but with higher values but this site would need further considerations because its topographical situation (1600 m a.s.1.) which undoubtedly gave rise to an absolutely different thermal history of the site, and still continues being different. There is an excellent aminostratigraphical differentiation between Ursus deningeri and Ursus spelaeus that proves the former palaeontological analysis rightness. La Lucia cave is an excellent example of this: two very dissimilar species (U. spelaeus and U. deningeri), from different places of the same cave, have strong different ASP racemization ratios. Normal sized cave bears from el Reguerillo and Arrikrutz show similar ASP racemization ratios. It could be interpreted that U. spelaeus reached Madrid area during the most important climatic optimum (Eem ?). Teeth from cave bears with short and broad paws have lowest ASP racemization values: La Pasada, Coro Tracito and Troskaeta, and we interpret it as a expression of a relict subespecies refuged in less favourable areas (craggy). This cave bears were coeval with the still living Iberian brown bear.
255 As has been said in the introduction we have also sampled some tooth dentine of Lower Pleistocene age bears: U. prearctos from the Gran Dolina site in Atapuerca (Burgos) and U. etruscus from Venta Micena (Orce, Granada). In both cases after or before dialysis not amino acids remain. This can be interpreted in an almost total absence of free or bonded amino acids, unprotected from diagenesis in the original collagen fibrils. Amino acid racemization analysis of mollusks and ostracods from Venta Micena site revealed the persistence of considerable amounts of amino acids, free or bonded, which were preserved in their intracristalline position.
Acknowledgements We are very indebted to Dr Veronika. Meyer of the University of Bern. She helped us in an incredible way in the set up of our laboratory. Dr Glenn Goodfriend, now at the Carnegie Institution at Washington, sent us his analysis protocol and GC program. The Biomolecular Stratigraphy Laboratory has been partially founded by E N R E S A (National Company for Radioactive Waste Management).
References [1] J.L. Bada, The dating of fossils using racemization of isoleucine. Ann. Rev. Earth Planet. Sci., 15 (1972) 223-231. [2] J.L. Bada, Racemization of Amino Acids in Bones. Nature, 245 (1973) 308-310. [3] J.L. Bada, Amino acid racemization dating of fossil bones. Ann. Rev. Earth Planet. Sci., 13 (1985) 241-268. [4] G. Dungworth, A.W. Schwartz, L. van de Leemput, Composition and racemization of amino acids in mammoth collagen determined by gas and liquid chromatography. Comp. Biochem. Physiol., 53B (1976) 473-480. [5] K. King, J.L. Bada, Effect of in situ leaching on amino acid racemisation rates in fossil bone. Nature, 28 (1979) 135-137. [6] M.A.S. McMenamin, D.J. Blunt, K.A. Kvenvolden, S.E. Miller, L.F. Marcus, R.R. Pardi, Amino Acid Geochemistry of Fossil Bones from the Rancho La Brea Asphalt Deposit, California. Quat. Res. 18 (1982) 174-183. [7] G. Belluomini, Risultati e prospettive di un nuovo metodo di datazione basato sulla racemizzacione degli aminoacidi. Archeometria, 69 (1985) 135-171. [8] H. Elster, E. Gil-Av, S. Weiner, Amino Acid Racemization of Fossil Bones. Jour. Arch. Sci., 18 (1991) 605-617. [9] R.W.L. Kimber, P.E. Hare, Wide range of amino acids in peptides from human fossil bone and its implications for amino acid racemization dating. Geochim. Cosmochim. Acta, 56 (1992) 739-753. [10] V. Meyer, Amino acid racemization: A tool for dating. Chemtech., (1992) 412-417. [11] J.L. Bada, G. Belluomini, L. Bonfiglio, M. Branca, E. Burgio, L. Delitalia, Isoleucine epimerization ages of Quaternary mammals from Sicily. I1 Quaternario, 4(la) (1991) 49-54. [12] G. Belluomini, J.L. Bada, Isoleucine epimerization age of the dwarf elephants of Sicily. Geology, 13 (1985) 451-452.
256 [13] J.F. Whemilller, Amino Acid Racemization: Applications in Chemical Taxonomy and Chronostratigraphy of Quaternary Fossils. In: Skeletal Biomineralization, J.G. Carter (Ed.), Van Nodstran Ed. NY, 1995, pp. 583-608. [14] B. Blackwell, N.W. Rutter, A. Deb6nath, Amino Acid Racemization in Mammalian Bones and Teeth from La Chaise-de-Vouton (Charente), France. Geoarcheology, 5(2) (1990). [15] M. Sinibaldi, P. Ferrantelli, G.A. Goodfriend, G. Barkay, Aspartic Acid Racemization in Teeth from Late Holocene Burial Caves in Jerusalem. Am. Geophys. Union: Perspectives in Amino Acid and Protein Geochemistry Conference Abstr., (1998) 64. [16] T. Torres, P. Garcia-Alonso, L. Canoira, F.J. Llamas, Aspartic Acid Racemization in the Dentine of Cave Bears (Ursus deningeri von Reichenau and Ursus spelaeus Rosenmiiller-Heinroth). Am. Geophys. Union: Perspectives in Amino Acid and Protein Geochemistry Conference Abstr., (1998) 67. [17] T. Torres, F. Llamas, L. Canoira, P. Garcia-Alonso, A. Garcia-Cortes, H. Mansilla, Amino Chronology of the Lower Pleistocene Deposits of Venta Micena (Orce, Granada, Spain). Org. Geochem., 26 (1997) 85-97. [18] N. Garcia, J.L. Arsuaga, T. Torres, The carnivore remains from the Sima de los Huesos Middle Pleistocene site (Sierra de Atapuerca, Spain). Jour. Human. Evol., 33 2/3 (1997) 130-154. [19] T. Torres, Estudio de la filogenia, distribuci6n estratigr~tfica y geogr~fica, an~ilisis morfol6gico y m6trico de esqueleto y dentici6n de los osos (Mammalia, Carnivora, Ursidae) del Pleistoceno de la Peninsula Ib6rica. Publ. Espec. Instituto Geol6gico y Minero de Espafia, 1989, 314 pp. [20] T. Torres, Excavaci6n en la Cueva de Santa Isabel. Arkeoikuska, 92 (1992) 303-310. [21] T. Torres, R. Cobo, A. Salazar, La poblaci6n de oso de las cavernas (U. s. parvilatipedis n. ssp.) de la Cueva de Troskaeta (Ataun, Guipuzcoa). Munibe, 43 (1991) 3-85. [22] A. Grandal d'Anglade, J.R. Vidal Romani, A population Study on the cave bear (Ursus spelaeus Ros. Hein.) from Cova Eir6s (Triacastela, Galicia, Spain). Geobios, 30(5) (1997) 723-731. [23] J.T. Triffit, The organic matrix of bone tissue, in Fundamentals and Clinic Bone Physiology, M.R. Urist Ed. Lippincott and Co, (1980) 45-82. [24] E. Marzin, Essai de normalisation du protocole d'analyse des taux de rac6misation des acides amines: applications a la datation d'ossements fossiles. Trav. du LAPMO, (1990) 167-178. [25] G.A Goodfriend, V. Meyer, A Comparative study of the kinetics of amino acid racemization/epimerization in fossil and modern mollusc shells. Geochim. Cosmochim. Acta, 55 (1991) 293-302.
CHAPTER
247
17
Aspartic Acid Racemization in the Dentine of Bears (Ursus etruscus G. Cuvier, Ursus prearctos, Boule, Ursus deningeri von Reichenau and Ursus spelaeus Rosenmiiller-Heinroth). Tooth Dentine Amino Acids Versus Mollusca Amino Acids T. de Torres, J.F. Llamas, L. Canoira, P. Garcia-Alonso Laboratory of Biomoecular Stratigraphy. Madrid School of Mines, Rios Rosas 21, 28003 Madrid, Spain
Introduction The dating of Quaternary mammal bones and teeth has been considered as a main objective for archaeologists and palaeontologists. U-series based methods seemed do not work accurately because both bone and tooth are open systems. Electro spin resonance dating method offered interesting results but up to now amino acid racemization analysis seems to be the most promising system. There are previous works on amino acid racemization analysis in bones [1-10], teeth enamel, [11-13], and teeth dentine [14-16]. We have obtained interesting results from a wide analytical work of amino acid racemization ratios in the dentine of cave bears of Middle and Upper Pleistocene age. We have also carried out some analysis on the dentine of Lower Pleistocene age bears.
Phylogeny After the unsuccessful attempt of Ursus m i n i m u s Deveze-Debouillet towards more modern bear forms more adaptative and hipocarnivorous with stepped frontal bone appeared Ursus etruscus G. Cuvier; Fig. 1, the common ancestor of the modern european bears. This species was first recorded at the Middle Villafranchian (ca 2 ma) and represents the last successful attempt to evolve from short faced forms to more adaptative long faced: arctoid and speloid species. One of the latest appearance of U. etruscus was in the Venta Micena (Orce Granada, Spain) site, aminoacid racemization dated in circa 1 million years. This bear was a very adaptative species and colonized wide areas of Europe with markedly different environmental characteristics: arid alluvial fan dominated intermontane basins (Puebla de Valverde, Teruel, Spain), rivers (Vald'Arno, Italy), swamps (Tegelen, the Netherlands), hypersaline lacustrine basins (Venta Micena, Granada, Spain) but
248
IH-OL]-;~I~.IT...........................................................................................................................................................................................................................
........! ............. i
U. maritimus
U. arctos
U mediterraneus ~
io~_
|
~
/
U.spelaeus
U.prearctos U.deningeri
_
N
I. i
,,
U.etruscus s
U.minimus
U.ruscinensis
Ursavus
Fig. 1. Pleistocene bears phylogeny.
never colonized caves. Some cave findings in Spain (Almenara, Castell6n) have been interpreted as water-laid fossils. In short: U. etruscus was a very adaptative species which probably never needed to colonize caves during the winter because of favourable palaeoclimatological conditions. U. etruscus 1 million year old A A R dated [17], in an arid basin (C~llar-Baza, Spain) was associated with controversial Homo sp. remains and with uncontroversial lithic artifacts. The arctoid group includes Ursus prearctos Boule, Lower-Middle Pleistocene, and the still living Ursus arctos Linneo and Ursus maritimus Phipps. This group had, and still has, a very noticeable ecological success covering the whole of Europe, de Asiatic an African Mediterranean Border as well as North America, they probably were as opportunistic as U. etruscus was. U. prearctos inhabited the caves of the Atapuerca karst system and ca 800 Ky. was associated with the oldest human remains from Europe (Homo antecessor). The cave bears evolutive line was composed of Ursus deningeri von Reichenau, of Middle Pleistocene age, and Ursus spelaeus Rosenm/iller-Heinroth, of upper Middle Pleistocene-Upper Pleistocene age. Arctoid line representings (U. prearctos, U. arctos and U. maritimus) were oportunistic species, as U. etruscus was. The speloid line representings, the cave bears (U. deningeri and U. spelaeus), became very popular since they have been repeatedly employed as dramatic resources in fictions and films. But in the real life
249 they were only wrongly evolved, too specialized, bears that probably avoided to coincide with contemporary human beings. In spite of, in Europe, the oldest U. deningeri remains appeared in open-air sites as river sands (Sussenborn, Germany), travertines (Bilzinsleben, Germany), in Spain all the U. deningeri sites are in caves. During the upper part of the Middle Pleistocene Ursus deningeri was replaced by the true cave bear U. spelaeus which remains with some rare exceptions were found only in caves. This species reached largest body, bone and tooth sizes. Short and massive postcranial bones and complicated molars are characteristics. The common characteristic on both evolutive lines, arctoid and speloid, of being cave occupants during winter hibernation period minded that all of them had developed liver protective mechanisms (ursodeoxicholic acid) that allowed a winter metabolism on body fat exclusively based.
Geographical distribution of cave bears
The cave bears distribution in Europe, Fig. 2, had a latitudinal control: between 42 ~ and 52 ~, the later roughly coincides with the maximum extent of the polar ice sheet during the last maximum glacial. In Spain, Fig. 3, four occupation areas have been defined: Atlantic Border, Mediterranean Border, Central Part and Pyrenees.
Fig. 2. Cave bears distribution in Europe.
250
1313
Li
EF
CUEVA MAYOR (Atapuerr Burgos, Castlllll-l_lXin)
CUEVA LA PASADA (Gurlzo, Contabm)
CUEVA CUETO DE LA LUCIA[,
(ouirlani~, cemix/)
C U E ~ S" mABEL (Ranero, Blzkal, Euskadl)
c.fvA ..6s 14...i (Trlacutela, Lugo, Galicia)
CUEVA DE LEZETXIKI (Mondmg6n, Gulpuzkoll, Eusklldl)
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Fig. 3. Cave bears distribution in Spain and sampled localities.
In the Atlantic Border area there appear many caves with bear remains: U.
deningeri: La Lucia (Quintanilla, Cantabria), Santa Isabel (Ranero, Vizcaya), Lezetxiki (Mondrag6n, Guipuzcoa). With U. spelaeus they are Eir6s (Triacastela, Lugo), La Lucia, La Pasada (Guriezo, Cantabria), Lezetxiki, Arrikrutz (Ofiate, Guipuzcoa), Ekain (Deba, Guipuzcoa), Troskaeta (Ataun, Guipuzcoa). In the Mediterranean Border there are not many caves with bear remains: Cova Bunica (Olopte, Girona) with U. deningeri and E1 Toll (MoiA, Barcelona) with U. spelaeus. In the Central Part of the Iberian Peninsula, the Sima de los Huesos (Atapuerca, Burgos), delivered many thousands of U. deningeri remains as well as hundreds of Preneandertalian man bones and teeth, being dated (U-series and ESR), (1997), 320 ky old [18]. E1 Reguerillo (Torrelaguna, Madrid) is the only U. spelaeus important locality in the area. In the Pyrenees: Coro Tracito (Tella, Huesca) represents the only high mountain locality. As it is possible to see the Iberian Peninsula represents the species border: that minds that palaeoenvironmental conditions were often near the species stress limits. The cave bears moved southwards only during climatic optimum periods appearing endogamy dominated populations, as a response to minor palaeoenvironmental worsening in the European realm.
The problem
According to our data, there are striking morphological and metrical differences between U. deningeri and U. spelaeus [19,20], being the former more primitive,
251
according to dental and skeletal analysis, and slender, obviously taking into account the strong sex dimorphism always present in bears. When we have compared dental morphologies from U. spelaeus of different localities, it has been possible to observe the persistence of minor morphological features that allowed to identify a certain "tribal" component, a noteworthy of this appeared in the enormous sample of lower fourth premolars from Ekain cave which show an strong cusp in the heel, this robust cusp, that produces a distinguishable wear notch on the opposite fourth upper premolar is absent or it appears as a cusplet in the other cave bear localities. Much more important is the fact that in caves developed in very craggy areas, far enough from wide valleys, plains, flat depressions, the cave bears showed a very peculiar paw morphology: broader and shorter than the normal cave bears used to be. Based in this character a subespecies Ursus spelaeus parvilatipedis Torres was defined [21], being the name based on the most important characteristic: short ~arvus) and broad (latus) feet ~edis). Based on first lower molar morphology Grandal d'Anglade and Vidal Romani [22] concluded that U. s. parvilatipedis, U. spelaeus and U. deningeri were only an expression of politypysm and, in fact, the same species. This assert must be rejected. But it is necessary to take into account that the Iberian Peninsula was the species border and palaeoenvironmental stresses were usually high, the caves were occupied for relative short time periods and population isolation phenomena (endogamy) were not uncommon. To solve these uncertainties we have been working for dating cave bears sites trough aspartic acid racemization analysis.
A b o u t cave bear sites
Cave bear caverns are good sites for amino acid racemization analysis sampling because they are clean and that minds that during the first and further taphonomical stages there are not too many possibilities of foreign protein or amino acids arrival. During the hibernation period adult and young bears do not eliminate faeces or urine and because of the almost mono specific composition of faunal remains proteins, amino acids and other organic origin compounds have the same signature: are the result of cave bear carcasses decay. Hibernation zones were in dark cave places, some tens of meters out of the sun light reach, there algae and fungi do not grow easily. Cave bears caverns were almost exclusively places for bear hibernation, being uncommon findings of other mammal remains, which usually represent less than 0.01%. In an excavation campaign more than 3000 bones and teeth can be recovered. Guano from bats is also infrequent. Man rarely inhabited cave bears sites although it was documented a very short seasonal summer occupation of bear dens by Musterian culture (Middle Paleolithic) man. Scavengers (Crocuta crocuta spelaea) rarely cannibalized cave bears carcasses, in spite of sometimes they did it and phosphatic coprolites appeared scattered in the sediment.
252 Cave bears caverns are the best sites for amino acid racemization dating because the thermal history did not show strong variations and average temperature was moderately low. The Iberian Peninsula was never affected by general glaciation processes. River incision and hillside retreat were very strong: ancient caves accesses are usually closed by rock blocks from cave roof collapses. Local cave hydrogeological reactivations very frequently were the origin of mud beds deposition sealing bear remains. Amino acids (usually free amino acids) reached the cave deposits from soils and rhizospher e in general, some speleothems we have studied contained small amounts of aspartic acid. In short, cave bears cavities are unusually clean sites with stable thermal history.
Sampling We have selected five canines or third upper incisors from each locality because their crown conic shaped efficiently protects dentine from contamination. Fifty mg of powdered dentine samples were obtained from the innermost part of the crown via drilling the tooth with a dental diamond drill. Powder from the outer part of the root, up to the limit of 1 mm deep was rejected. This process produced an unavoidable slight sample heating. We have analyzed samples from the following cave bears sites:
U. deningeri: Sima de los Huesos, Santa Isabel, La Lucia. U.spelaeus: La Lucia, Troskaeta, E1 Reguerillo, Eiros, Arrikrutz, Coro Tracito, La Pasada.
Sample preparation and analysis The glassware used for analysis was cleaned by baking in an oven at 500~ for about 2 h. Plastics were new from the factory. All the water used in the analysis was Milli-Q quality from Millipore. All chemicals were HPLC grade or spectroscopy grade. Before hydrolysis and amino acid derivatization, samples were treated to eliminate free amino acids. With this process we aimed to remove foreign amino acids and to obtain a homogeneous protein (from collagen) molecule which racemizates more uniformingly, [13]. Dentine has a 10% of A-type collagen [23]. We have employed a modification of the method proposed by Marzin [24]. The powder sample was dissolved in 1 ml of HC1 2N and sonicated. After 5 ml PBS buffer addition, the sample was dialyzed at 3500 Dalton (Spectra/Por mnco 3500 membrane) during a 20 h period in a buffered solution with magnetic stirring. After lyophilizaton the remaining powder was treated as it has been described elsewhere [17,25] and analyzed. Hydrolysis of ca 50 mg of dentine was carried out in a mixture of 12 N hydrochloric acid (2.9 #l/mg) and 6 N hydrochloric acid (100 #1), in test tubes with an atmosphere of nitrogen, in a heating block at 100~ for 20 h. Desalting was
253 accomplished in conical 1.5 ml Eppendorf plastic micro test tubes with caps, with concentrated hydrofluoric acid added and centrifuged in an Eppendorf centrifuge. The supernatant was frozen in liquid nitrogen, and vacuum dried. Samples were redissolved with 80 #1 distilled water and transferred to 2 ml glass vials. Water was evaporated at vacuum. The first amino acid derivation step consisted of sterification with 250 #1 of 3 M thionyl chloride in isopropanol in a nitrogen atmosphere on the heating block at 100~ for just 1 h and later vacuum dried. The second derivation step consisted of N-trifluoroacetylation with 150 #1 of trifluoroacetic acid anhydride (25% in dichloromethane) in a nitrogen atmosphere and heated at 100~ for just 5 min on the heating block. Dichloromethane solvent and the unreacted trifluoroacetic acid anhydride were evaporated under a flow of nitrogen. Later the samples were dissolved in 125 #1 of n-hexane but most of the n-hexane evaporated in a stream of nitrogen to a final volume of 15-25 #1, then being transferred to injection vials. A sample of 0.2 #1 was injected into a Hewlett-Packard 5890 gas chromatograph. The injection port was kept at 215~ and set for splitless mode for the first 75 s, at the beginning of which the sample was injected, and later set to split mode. We used helium as the carrier gas, at a head column pressure of 5.8 psi, and a Chirasil-Val fused silica column (25 m • 0.39 m m • 0.25 mm) from Chrompack. The gradients we used were as follows: 50~ (1 min), heat at 40~ to 115~ remaining at 115~ for 12 min, heating at 3~ to 190~ remaining at 190~ for 10 min, cooling down to 50~ and remaining at this temperature between runs (80~ if the time between runs is longer, typically overnight). The detector was a N P D set at 300~ Integration of the peak areas was carried out using the PEAK96 integration program from Hewlett-Packard, which runs on a PC computer. Usually DL alanine, uI~ valline, oI~ proline, oI~ leucine, AI isoleuleucine, DL aspartic acid, I~I~ phenilalanine, DL glutamic acid and L hydroxiproline peaks are identified.
Results and conclusions
First at all we want to note that the use of a previous dialysis process allowed a noteworthy accuracy in our analytical results that were not obtained in more than 30 former analysis that we carried out on non dialyzated samples, where we obtained racemization ratios of both free and bonded amino acids. This can be explained according different ways: on one hand we must take into account the effect of hydroxyproline peak which in some analysis could appear superposed to D-Asp peak "rejuvenating" the true sample age and being origin of an inadmissible racemization ratios distribution for statistics calculations. In our opinion the use of all dialysis, new purchased Chirasil L-Val column and N P D GC detector can be interpreted as analytical success key. Up to now this is the first work which deals on a systematic amino acid racemization ratio analysis in the dentine of the same genus teeth and the results seemed to be very encouraging.
254
Fig. 4. Histogram of aspartic acid racemization in cave bear (Ursus deningeriand Ursusspelaeus) teeth dentine samples. Species and localities are identified. For comparison data from a Holocene brown bear (U. arctos) locality has been added.
It is evident that the bear population remains that have been identified as Ursus deningeri von Reichenau, have homogeneous and higher aspartic acid racemization ratios Fig. 4. We are able from now on to formally define the U. deningeri aminozone in the Iberian Peninsula over 30% ASP racemization ratios and average values near 35%. U. spelaeus samples defined a broad aminozone where two subaminozones could be distinguished: the one with two higher racemization ratio populations (El Reguerillo and Arrikrutz) and the other Troskaeta, Coro Tracito, and La Pasada with very low ASP racemization ratios, in fact they are not very different from Holocene brown bear (U. arctos) sample from Saldarrafiao. La Lucia cave sample is placed into the low ASP racemization ratio group but with higher values but this site would need further considerations because its topographical situation (1600 m a.s.1.) which undoubtedly gave rise to an absolutely different thermal history of the site, and still continues being different. There is an excellent aminostratigraphical differentiation between Ursus deningeri and Ursus spelaeus that proves the former palaeontological analysis rightness. La Lucia cave is an excellent example of this: two very dissimilar species (U. spelaeus and U. deningeri), from different places of the same cave, have strong different ASP racemization ratios. Normal sized cave bears from el Reguerillo and Arrikrutz show similar ASP racemization ratios. It could be interpreted that U. spelaeus reached Madrid area during the most important climatic optimum (Eem ?). Teeth from cave bears with short and broad paws have lowest ASP racemization values: La Pasada, Coro Tracito and Troskaeta, and we interpret it as a expression of a relict subespecies refuged in less favourable areas (craggy). This cave bears were coeval with the still living Iberian brown bear.
255 As has been said in the introduction we have also sampled some tooth dentine of Lower Pleistocene age bears: U. prearctos from the Gran Dolina site in Atapuerca (Burgos) and U. etruscus from Venta Micena (Orce, Granada). In both cases after or before dialysis not amino acids remain. This can be interpreted in an almost total absence of free or bonded amino acids, unprotected from diagenesis in the original collagen fibrils. Amino acid racemization analysis of mollusks and ostracods from Venta Micena site revealed the persistence of considerable amounts of amino acids, free or bonded, which were preserved in their intracristalline position.
Acknowledgements We are very indebted to Dr Veronika. Meyer of the University of Bern. She helped us in an incredible way in the set up of our laboratory. Dr Glenn Goodfriend, now at the Carnegie Institution at Washington, sent us his analysis protocol and GC program. The Biomolecular Stratigraphy Laboratory has been partially founded by E N R E S A (National Company for Radioactive Waste Management).
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256 [13] J.F. Whemilller, Amino Acid Racemization: Applications in Chemical Taxonomy and Chronostratigraphy of Quaternary Fossils. In: Skeletal Biomineralization, J.G. Carter (Ed.), Van Nodstran Ed. NY, 1995, pp. 583-608. [14] B. Blackwell, N.W. Rutter, A. Deb6nath, Amino Acid Racemization in Mammalian Bones and Teeth from La Chaise-de-Vouton (Charente), France. Geoarcheology, 5(2) (1990). [15] M. Sinibaldi, P. Ferrantelli, G.A. Goodfriend, G. Barkay, Aspartic Acid Racemization in Teeth from Late Holocene Burial Caves in Jerusalem. Am. Geophys. Union: Perspectives in Amino Acid and Protein Geochemistry Conference Abstr., (1998) 64. [16] T. Torres, P. Garcia-Alonso, L. Canoira, F.J. Llamas, Aspartic Acid Racemization in the Dentine of Cave Bears (Ursus deningeri von Reichenau and Ursus spelaeus Rosenmiiller-Heinroth). Am. Geophys. Union: Perspectives in Amino Acid and Protein Geochemistry Conference Abstr., (1998) 67. [17] T. Torres, F. Llamas, L. Canoira, P. Garcia-Alonso, A. Garcia-Cortes, H. Mansilla, Amino Chronology of the Lower Pleistocene Deposits of Venta Micena (Orce, Granada, Spain). Org. Geochem., 26 (1997) 85-97. [18] N. Garcia, J.L. Arsuaga, T. Torres, The carnivore remains from the Sima de los Huesos Middle Pleistocene site (Sierra de Atapuerca, Spain). Jour. Human. Evol., 33 2/3 (1997) 130-154. [19] T. Torres, Estudio de la filogenia, distribuci6n estratigr~tfica y geogr~fica, an~ilisis morfol6gico y m6trico de esqueleto y dentici6n de los osos (Mammalia, Carnivora, Ursidae) del Pleistoceno de la Peninsula Ib6rica. Publ. Espec. Instituto Geol6gico y Minero de Espafia, 1989, 314 pp. [20] T. Torres, Excavaci6n en la Cueva de Santa Isabel. Arkeoikuska, 92 (1992) 303-310. [21] T. Torres, R. Cobo, A. Salazar, La poblaci6n de oso de las cavernas (U. s. parvilatipedis n. ssp.) de la Cueva de Troskaeta (Ataun, Guipuzcoa). Munibe, 43 (1991) 3-85. [22] A. Grandal d'Anglade, J.R. Vidal Romani, A population Study on the cave bear (Ursus spelaeus Ros. Hein.) from Cova Eir6s (Triacastela, Galicia, Spain). Geobios, 30(5) (1997) 723-731. [23] J.T. Triffit, The organic matrix of bone tissue, in Fundamentals and Clinic Bone Physiology, M.R. Urist Ed. Lippincott and Co, (1980) 45-82. [24] E. Marzin, Essai de normalisation du protocole d'analyse des taux de rac6misation des acides amines: applications a la datation d'ossements fossiles. Trav. du LAPMO, (1990) 167-178. [25] G.A Goodfriend, V. Meyer, A Comparative study of the kinetics of amino acid racemization/epimerization in fossil and modern mollusc shells. Geochim. Cosmochim. Acta, 55 (1991) 293-302.