Cross-calibration of the racemization rates of leucine and
phenylalanine and epimerization rates of isoleucine
between ostracodes and gastropods over
the Pleistocene in southern Spain
J.E. Ortiz*, T. Torres, F.J. Llamas
Laboratory of Biomolecular Stratigraphy, E. T.S.I. Minas de Madrid, Cj Rios Rosas 21, Madrid 28003, Spain
Abstract
Cross-calibration of the racemization rates of leucine a n d phenylalanine a n d the epimerization rates of isoleucine between Middle a n d Lower Pleistocene ostracodes a n d g a s t r o p o d s from southern Spain is presented. Using these two relationships, along with previously-calculated age estimation algorithms, it is possible to estimate the age of samples from southern a n d central Spain because of the shared thermal history of these regions. They can also be used to establish the a m i n o s t r a t i g r a p h y of Q u a t e r n a r y deposits from the areas. However, for D / L ratios below those in the ostracodes a n d g a s t r o p o d s of the Cortijo del Negro-1 site, the cross-calibration equations m a y n o t be satisfactorily applied. The racemization/epimerization ratios in ostracode shells can be measured precisely because of the excellent preservation of a m i n o acids within a n valves a n d an a b u n d a n c e t h a t m a k e s the s t a n d a r d error or variance smaller. It has been observed t h a t in m o d e r n samples the racemization in ostracodes is slower t h a n in g a s t r o p o d s b u t the D / L ratios become similar in older samples.
1. Introduction
In recent years, the a m i n o acid racemization m e t h o d has become one of the m o s t widely used geochronolo-gical tools for dating b o t h continental a n d marine Q u a ternary deposits. Likewise, it can be used for stratigraphical correlation purposes.
Living beings contain only L - a m i n o acids which gra dually racemize to D - a m i n o acids after death. T h u s , the D / L ratio increases with time after d e a t h until it is equal to 1, t h a t is, when equilibrium is reached. A m i n o acids with two asymmetrical c a r b o n a t o m s (such as isoleucine) u n d e r g o epimerization, which is the t r a n s f o r m a t i o n of an L-diastereomer into a n o t h e r D - d i a s t e r e o m e r . E a c h
a m i n o acid has a unique racemization or epimerization rate.
found differences in the D-alloisoleucine/L-isoleucine ( D - a l l e / L - I l e ) values between Glycymeris sp. a n d Area sp. from the same strata along M e d i t e r r a n e a n coasts. Likewise, Torres et al. (2000) r e p o r t e d differences in the D / L ratios of aspartic acid (Asp), glutamic acid (Glu), isoleucine a n d leucine (Leu) between shells of diverse pelecypoda genera from the east coast of Spain. Finally, Goodfriend a n d Stanley (1996) estimated the age calcu lation e q u a t i o n for the D / L Asp ratio in a pelecypod
(Corbicula sp.) from the Nile delta from c o m p a r i s o n of
the D / L Asp racemization rates with those for a n o t h e r pelecypod (Cerastoderma sp.).
A wide g r o u p of materials is available for dating with this m e t h o d but, in spite of the a b u n d a n c e of ostra-codes, their prevalence in m o s t lacustrine environments a n d their convenience to w o r k with, there are only a few studies based on their D / L ratios (McCoy, 1988; Torres et al., 1995; Oviatt et al., 1999; Ortiz, 2000; Ortiz et al., 2000; K a u f m a n , 2000).
According t o our experience (Ortiz, 2000) ostracodes have t w o m a i n characteristics t h a t m a k e t h e m particu larly useful for a m i n o acid racemization/epimerization dating:
1. T h e excellent preservation of a m i n o acids in their valves allows analysis of a small sample size (10-20 mg) by gas c h r o m a t o g r a p h y (GC), m u c h less t h a n for other organisms, e.g. mol luscs (80 mg). Using reversed phase high per formance liquid c h r o m a t o g r a p h y ( H P L C ) , it is possible t o analyze even a single ostracode valve (cf. K a u f m a n , 2000).
2. I n a single G C analysis, there are typically between 1500 a n d 2000 ostracode valves, so the s t a n d a r d error or variance is low because the sample is statistically significant.
F u r t h e r m o r e , in m o s t cases, ostracodes are a b u n d a n t a n d the only fauna present in outcrops, or along b o t h stratigraphic sections a n d drill cores, so g a s t r o p o d s or bivalves c a n n o t be used t o obtain an entire a n d accurate a m i n o acid chronology.
Until n o w we have developed age calculation algo rithms t o estimate the age of Pleistocene deposits using racemization/epimerization ratios of different a m i n o acids (leucine, isoleucine, phenylalanine, aspartic acid a n d glutamic acid) of fossil g a s t r o p o d s (cf. Torres et al., 1997; Ortiz, 2000). F o r this purpose deposits dated from absolute m e t h o d s were used. However, palaeoenviron-m e n t a l characteristics have resulted in n o t all the gas t r o p o d - b e a r i n g strata being used in the calculation of these algorithms. W h e n ostracodes were present, only in a few cases were we able t o collect the necessary ca. 1500 valves.
Because of these reasons a n d the particularly g o o d characteristics for ostracodes it is obvious t h a t it would
be useful t o determine the equations t h a t relate their a m i n o acid racemization a n d epimerization ratios to those of other groups of fossils such as g a s t r o p o d s in order to o b t a i n or improve either the a m i n o s t r a t i g r a p h y or the a m i n o c h r o n o l o g y of an area.
W e have chosen to w o r k with g a s t r o p o d s a n d ostra codes from the Cullar-Baza Basin, where they are omnipresent. This basin is a " b a s i n - a n d - r a n g e " zone located (Fig. 1) in the southeast of the Iberian Penin sula, in the central p a r t of the Betic R a n g e a n d is filled by alluvial a n d lacustrine-palustrine deposits of Pliocene a n d Pleistocene age. It is one of the few areas of E u r o p e a n d the only one in Spain where almost continuous sedimentation t o o k place over nearly the whole of the Q u a t e r n a r y . A detailed stratigraphical a n d p a l e o n t o -logical description is given in Ortiz (2000).
Samples were recovered from five paleontological localities (Cortijo del N e g r o - 1 , F u e n t e A m a r g a - 1 , Ciillar-Baza-1, Cortes de Baza-182 a n d V e n t a Micena-1), developed u n d e r palustrine conditions, whose geo graphical a n d biostratigraphical details a p p e a r in Table 1.
2. Material and methods
T h e ostracodes from the Cortijo del N e g r o - 1 , F u e n t e A m a r g a - 1 , Cullar-Baza-1 a n d Cortes de Baza-182 paleontological sites were classified as Cyprideis torosa (Jones), while in Venta Micena-1 they belong t o two genera: Ilyocypris a n d Cyprideis because, due t o the n u m b e r s necessary for G C analysis it was impossible at this site to separate a sample comprising only a single genus. I n spite of the racemization process being genus-dependent, these two different ostracode genera were employed t o calculate the cross-calibration racemization a n d epimerization rates. I n fact, in previous studies (McCoy, 1988; Oviatt et al., 1999; K a u f m a n , 2000) only slight differences between D / L ratios from different phylogenetic ostracode groups (Superfamilies Cyprida-cea a n d CytheraCyprida-cea) were reported. In the present w o r k , the ostracodes from Venta Micena-1 belong t o either one of the two Superfamilies: Ilyocypris t o Superfamily Cypridacea a n d Cyprideis t o Superfamily Cytheracea.
Because Torres et al. (1995,1997) found only small differences between racemization ratios in land a n d a q u a t i c snail samples, we decided to combine t h e m , describing t h e m as G a s t r o p o d a b u t including Helix (the m o s t a b u n d a n t ) , Planorbis, Lymnaea, Radix, Bithynia genera representatives.
Malaguide Complex Nevado-Filabride Alpujarride Complex Complex
Fig. 1. G e o g r a p h i c a l setting of the Cullar-Baza Basin a n d location of the palaeontological sites: V e n t a Micena-1 (VM-1), Cortes de Baza-182 (CTB-182), Cullar-Baza-1 (CB-1), F u e n t e A m a r g a - 1 (FA-1) a n d C e r r o del N e g r o - 1 ( C N E - 1 ) .
Table 1
G e o g r a p h i c a l location a n d geological age of Cullar-Baza basin paleontological sites
Locality L a t i t u d e L o n g i t u d e Elevation (m) Geological age
V e n t a M i c e n a (VM-1) 37°44'8" 2 ° 2 4/2 7/ / 960 ( U p p e r Villafranchian) Lower Pleistocene
M a r t i n e z N a v a r r o (1992), Sese (1994);
Torres et al. (1997) Cortes de Baza-182 (CTB-182) 3 7 ° 3 9 ' 7 2 ° 4 5/2/ / 760 Lower Pleistocene
O m s et al. (1994)
Cullar Baza-1 (CB-1) 37°34/10/ / 2°33/50/ / 940 G a l e r i a n (Middle Pleistocene)
Sese (1994), Torres et al. (1997) F u e n t e A m a r g a - 1 (FA-1) 37°46/7/ / 2 ° 3 5/1 2/ / 880 M i d d l e Pleistocene
Torres et al. (1995) Cortijo del N e g r o - 1 ( C N E - 1 ) 37°46/27/ / 2 ° 3 6/2 1/ / 905 N o t previously d a t e d
have been contained in their valves. Mollusc shells were also washed with running water a n d cleaned by exten sive sonication. Afterwards, we isolated a m o u n t s of 80 mg of gastropods a n d 15-20 m g of ostracodes.
The sample p r e p a r a t i o n protocol is described in Goodfriend (1991) a n d Goodfriend a n d Meyer (1991) a n d involves:
1. Hydrolysis which was performed under a N2
atmosphere in a mixture of 12 N HC1 a n d shell carbonate (2.9 ul/mg) a n d 6 N hydrochloric
acid (100 ul) for 20 h at 100 °C; samples were then desalted in H F a n d the resultant super n a t a n t frozen a n d dried under v a c u u m . 2. Derivatization: a m i n o acids were derivatized in
a two step process, involving first esterification with 250 ul of 3 M thionyl chloride in iso-p r o iso-p a n o l for 1 h at 100 °C under N2; the sam
gentle flow of nitrogen. The sample was t a k e n u p in 125 ul of w-hexane which was vortexed a n d the solvent was reduced in a stream of N2 to a
final volume of 15-25 ul.
Aliquots (1-4 ul) were injected into a H e w l e t t - P a c k a r d 5890 gas c h r o m a t o g r a p h . T h e injection p o r t was kept a t 215 °C a n d set for splitless m o d e for the first 75 s, a t the beginning of which the sample was injected, a n d later set t o split m o d e . W e used helium as the carrier gas, a t a column h e a d pressure of 5.8 psi, a n d a Chirasil-L-Val fused silica c o l u m n (25 m x 0 . 3 8 m m ) from C h r o m p a c k . T h e gradients used were as follows: 50 ° C (1 min), heating at 40 ° C / m i n to 115 °C, 12 m i n a t 115 °C, 3 ° C / m i n to 190 °C, 10 m i n a t 190 °C. The detector was a n N P D set at 300 °C. Integration of the peak areas was carried o u t using the H P P E A K 9 6 inte gration p r o g r a m . As a l a b o r a t o r y routine, D / L - a l a n i n e , D / L - v a l i n e , D-alloisoleucine/L-isoleucine, D / L - p r o l i n e , D / L - a s p a r t i c acid, D / L - l e u c i n e , D / L - p h e n y l a l a n i n e a n d D / L - g l u t a m i c acid peaks were identified.
3. Results and discussion
T h e results for the m e a n values of the D / L ratios of five different a m i n o acids in ostracodes a n d g a s t r o p o d s (Table 2) were plotted o n X Y graphs. M e a n D / L ratios for each a m i n o acid were regressed linearly a n d log arithmically in order to select the best trend. W i t h these correlations, "equivalent r a t i o s " can be obtained from different D / L ratios of a m i n o acids analyzed in ostracodes, which are defined as the D / L ratios t h a t a g a s t r o p o d from a similar horizon w o u l d have. T h e results of the a t t e m p t e d linear a p p r o a c h are:
D - a I l e / L - I l ee q i l I v a l e n t = 0.39133 + 0.67542
x D - a I l e / L - I l eo s t r a c o d e s; r = 0.983, P = 0.003
D / L LEUeq^valent = 0.42581 + 0.50249
x D / L L E Uo s t r a c o d e s; = 0.957, P = 0.011
D / L ASPequivalent = 0.30776 + 0.72441
x D / L A S Po s t r a c o d e s; r = 0.749, P = 0.145
D / L PHEecprivalent = 0.54654 + 0.38697
x D / L P H Eo s t r a c o d e s; r = 0.957, P = 0.011
D / L G L Ue q m v ; 1ie n t = 0.38005 + 0.51751
x D / L G L Uo s t r a c o d e s; r = 0.840, P = 0.075
where r is the correlation coefficient a n d P is the sig nificant level.
T h e results of the logarithmic a p p r o a c h are:
D - a I l e / L - I l ee q i u v a k ; n t = 1.0716 + 0.40618
x L n ( D - a I l e / L - I l eo s t r a c o d e s) ; r = 0.997, P = 0.000
D / L LEUecprivalent = 0.84298 + 0.1990
x L n ( D / L L E Uo s t r a c o d e s) ; r = 0.869, P = 0.056
D / L A S PE q m v a l e n t = 0.98219 + 0.45856
x L n ( D / L A S Po s t r a c o d e s) ; r = 0.759, P = 0.137
Table 2
D-alle/L-Ile a n d D / L m e a n values for a m i n o acids in g a s t r o p o d a a n d ostracodes from Venta Micena-1 (VM-1), Cortes d e Baza-1 (CTB-1), Cullar-Baza-1 (CB-1), F u e n t e A m a r g a - 1 (FA-1) a n d Cortijo del Negro-1 (CNE-1) sites
Sample Material n D-alle/L-Ile D / L L e u D / L A s p D / L P h e D / L G l u Sample Material n
M e a n S.D. M e a n S.D. M e a n S.D. M e a n S.D. M e a n S.D. V M - 1 G a s t r o p o d a 7 1.160 0.031 0.893 0.017 0.922 0.012 0.896 0.013 0.835 0.033
O s t r a c o d a 3 1.156 0.057 0.848 0.004 0.756 0.011 0.850 0.062 0.720 0.010 CTB-182 G a s t r o p o d a 3 1.045 0.032 0.760 0.011 0.755 0.009 0.818 0.029 0.690 0.031 O s t r a c o d a 3 0.998 0.019 0.710 0.026 0.742 0.035 0.804 0.005 0.750 0.083 CB-1 G a s t r o p o d a 12 0.828 0.030 0.639 0.054 0.809 0.016 0.799 0.027 0.633 0.019 O s t r a c o d a 8 0.559 0.040 0.479 0.065 0.575 0.005 0.485 0.074 0.372 0.010 F A - 1 G a s t r o p o d a 13 0.713 0.056 0.577 0.052 0.711 0.023 0.711 0.043 0.531 0.038 O s t r a c o d a 1 0.419 0.000 0.376 0.000 0.587 0.000 0.373 0.000 0.364 0.000 C N E - 1 G a s t r o p o d a 2 0.480 0.105 0.563 0.040 0.624 0.016 0.616 0.026 0.534 0.015 O s t r a c o d a 5 0.228 0.019 0.181 0.021 0.494 0.010 0.240 0.024 0.349 0.005
D / L P H Ee q u l v a l e n t = 0.89643 + 0.19391
x L n ( D / L P H Eo s t r a c o d e s) ; r = 0.972, P = 0.006
D / L G L Ue q u i v aie n t = 0.84675 + 0.27574
X L n ( D / L G L Uo stra c o d e s ) ; r = 0.850, P = 0.068
Only the linear correlations obtained for isoleucine, leucine a n d phenylalanine were significant ( P < 0 . 0 5 ) , with high correlation coefficients. Based o n the corre lation coefficients, the logarithmic trend is the best fit for isoleucine (Fig. 2) a n d phenylalanine (Fig. 3) whereas for leucine (Fig. 4) the best fit is a linear trend. These results c a n n o t be extrapolated to lower g a s t r o p o d a n d ostracode racemization ratios t h a n those obtained in the
Cortijo del Negro-1 sample (see Table 2), in o u r opinion, because the racemization/epimerization is a non-linear process (Goodfriend, 1991) in which the racemization rate eventually decreases with time. In fact, some a u t h o r s have found different behaviour b e y o n d D / L ratios of 0.3 (Masters a n d Bada, 1977; K r i a u s a k u l a n d Mitterer, 1980) or 0.5 (Wehmiller a n d H a r e , 1971; B a d a a n d Schroeder, 1972), depending o n the material.
The results for aspartic acid a n d glutamic acid are n o t satisfactory. A m o n g the five a m i n o acids used, aspartic acid a n d glutamic acid racemize faster t h a n phenyl alanine, leucine a n d isoleucine. In o u r opinion this could explain the p o o r correlation between aspartic acid a n d glutamic acid D / L ratios of ostracodes a n d g a s t r o p o d s . This is confirmed by the results obtained by Torres et al.
1.2
Fig. 2. M e a n D-alloisoleucine/L-isoleucine values logarithmic regression p l o t of ostracodes a n d g a s t r o p o d s from different p a l e o n t o logical sites of the CullarBaza Basin. V e n t a Micena1 (VM1), C o r t e s de Baza182 (CTB182), CullarBaza1 (CB1), F u e n t e A m a r g a -1 (FA--1) a n d C e r r o del N e g r o - -1 ( C N E - -1 ) .
D / L L e uo s t r a c o d e s
Fig. 4. M e a n leucine D / L values linear regression plot of ostracodes a n d g a s t r o p o d s from different paleontological sites of the Cullar-Baza Basin. V e n t a Micena-1 (VM-1), Cortes de Cullar-Baza-182 (CTB-182), Cullar-Cullar-Baza-1 (CB-1), F u e n t e A m a r g a - 1 (FA-1) a n d C e r r o del N e g r o - 1 ( C N E - 1 ) .
(2000) w h o analyzed the reliability of diverse amino acids from different genera of marine pelecypoda in old samples, noting t h a t isoleucine a n d leucine results are the most reliable. Some authors, e.g. H e a r t y et al. (1986) calculated for marine pelecypoda shells from Mallorca (Spain) the "equivalent r a t i o s " only by the division of the D / L values of the " s t a n d a r d g e n u s " by those in the other genus using only a single sample, applying this relationship directly as a non-proportionality factor. It is obvious t h a t the correlation analysis using m o r e samples t h a n in the present w o r k will provide m o r e accurate results.
The leucine, phenylalanine a n d isoleucine D / L ratios of the gastropods a n d ostracodes, which are the only ones t h a t are well correlated, were plotted in c o m p a r a tive histogram plots; it can be seen t h a t in the m o d e r n samples these a m i n o acids racemize faster in gastropods t h a n in ostracodes (Fig. 5). However, ostracode D / L ratios become closer to those of gastropods with age, being similar in old samples. This is a n o t h e r reason why we think t h a t for D / L ratios below those for the ostra codes a n d gastropods in the Cortijo del Negro-1 sample, these relationships c a n n o t be applied, especially for the leucine linear a p p r o a c h .
O u r tentative interpretation is t h a t the differences in the racemization rates of ostracodes a n d gastropods are mainly due to the taxa-effect, t h a t is the different geo-chemical composition of their shells since they belong to different groups of fossils. While ostracode valves are mainly m a d e of low-magnesium-calcite (Sohn, 1958; C a d o t a n d Kaesler, 1977; Bordegat, 1979, 1985), g a s t r o p o d shells are m a d e of aragonite ( M o o r e 1969). The lower stability of aragonite, which changes to cal-cite, can explain why gastropods initially racemize faster. This could explain why the differences in the racemization rates are in some cases described by linear regression and in others by a logarithmic approach.
The intra-genera effect (Murray-Wallace, 1995) has also to be t a k e n into account, although according to o u r
experience the differences in D / L ratios tend to diminish with time. The relationships have been calculated using gastropods a n d ostracodes from different genera a n d in spite of the small differences t h a t have been reported, some error has to be assumed.
T a b l e 3
A m i n o acid racemization ratios of o s t r a c o d e s from samples F R A - 1 a n d N O R - 1 a n d c o m p a r i s o n of the ages o b t a i n e d using (1) the a m i n o acid racemization m e t h o d a n d (2) p r e v i o u s d a t i n g
Sample n D-alle/L-Ile D / L L e u D / L P h e A g e 1 (ka)
Age 2 (ka) M e a n S.D. M e a n S.D. M e a n S.D.
A g e 1 (ka)
Age 2 (ka)
F R A - 1 N O R - 1
3 5 0.725 0.504 0.046 0.055 0.570 0.318 0.061
0.048 0.429 0.085
746 ± 4 6 445 ± 3 0
ca. 780 ca. 419^112
n, N u m b e r of analysis; S.D., s t a n d a r d deviation.
W i t h these correlations, equivalent ratios can be obtained from the leucine, phenylalanine a n d isoleucine D / L a n d D - a l l e / L - I l e ratios analyzed either for ostra codes or in g a s t r o p o d s .
I n order to check the relability of these equations we t o o k some ostracode samples (Cyprideis torosa) from two previously-dated horizons from the Cullar-Baza Basin. According to the m a g n e t o s t r a t i g r a p h i c studies of the geological record of this Basin (Oms et al., 1994; Ortiz, 2000), two i m p o r t a n t palaeomagnetic events, the M a t u y a m a / B r u n h e s b o u n d a r y (780 ka; C a n d e a n d K e n t , 1995) a n d a short reverse polarity event which can be correlated to either E m p e r o r or Lake Biwa III excursions, d a t e d as ca. 419 ka a n d ca. 412 k a (Cande a n d K e n t , 1995), have been reported. These horizons were n a m e d F R A - 1 (lati tude: 37°38'57'; longitude: 2°44'50"; elevation: 782 m ) a n d N O R - 1 (latitude: 37°47'4"; longitude: 2°29'0"; elevation: = 1004 m) respectively, D / L ratios are in Table 3. Unfortunately, for F R A - 1 samples, the phe nylalanine peaks were n o t satisfactorially isolated in the c h r o m a t o g r a m .
I n order t o o b t a i n the ages of the horizons we first calculated the equivalent D / L ratios using the equations obtained in this work; then, they were introduced in the age calculaton algorithms.
Isoleucine :
sft= 1.0484+ 14.088 L n
Leucine:
v ^ = 0.41668 + 13.857Ln
Phenylalanine:
Jt = 0.79019 + 11. HOLnl
D — a l l e / L — He
0.565- D / L
1 + D - a l l e / L - He
1 + D / L '
1 - D / L
1 + D / L '
1 - D / L
T h e ages resulting after calculating the D / L equivalent ratios of ostracodes from these three localities a n d using the age calculation algorithms are shown in Table 3, where it can be seen t h a t the results are reliable.
4. Conclusions
T h e cross-calibration of the racemization a n d epi merization rates of leucine a n d isoleucine, respectively, between ostracodes a n d g a s t r o p o d s over the M i d d l e a n d Lower Pleistocene in southern Spain have been calcu lated, resulting in:
D - a l l e / L - I l e ^ v a k n t = 1.0716 + 0.40618 x ( D - a l l e / L - i l eo s t r a c o d e s)
D / L L E Ue q m v a l m t = 0.42581 + 0.50249
X D / L L E Uo st r a c o d e s
D / L P H Ee q m v a l e n t = 0.89643 + 0.19391
x ( D / L P H Eo s t r a c ode s)
W i t h these three relationships it should be possible to either estimate the aminostratigraphy of south a n d central Spain, or to calculate accurately the ages of ostracode samples for a time range between ca. 1 M y a n d 200 k a because of the special characteristics of this taxa (excellent preservation of amino acids a n d abundance that makes the variance smaller) by using the age calculation algorithms for g a s t r o p o d D / L ratios defined by Torres et al. (1997) a n d Ortiz (2000). F o r values lower t h a n those in the ostracode a n d g a s t r o p o d D / L ratios in the Cortijo del Negro-1 sample, the cross-calibration equations c a n n o t be satisfactorily applied, especially for the leucine linear a p p r o a c h .
has been observed t h a t the a m i n o acids in ostracodes racemize m o r e slowly t h a n those in gastropods in the early stages b u t the D / L ratios become similar in old samples.
Acknowledgements
F u n d i n g was obtained t h r o u g h the project "Evolu tion Paleoclimatica de la M i t a d Sur de la Peninsula Iberica" of E N R E S A (National C o m p a n y for R a d i o active W a s t e M a n a g e m e n t ) . R o b e r t Rhew from Scripps Institution of O c e a n o g r a p h y m a d e helpful comments on a n earlier draft. W e are indebted to D r . Veronika Meyer of the University of Bern w h o helped in the setting u p of our laboratory. D r . Glenn Goodfriend from the Carne gie Institution in W a s h i n g t o n sent us the analysis p r o tocol a n d G C p r o g r a m . The Biomolecular Stratigraphy L a b o r a t o r y has been partially funded by E N R E S A . W e w a n t t o t h a n k Professor Michael Engel a n d Professor M a r k Teece for reviewing the manuscript.
Associate Editor—J.R. Maxwell
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