(helix aspersa) differing in shell deposition of lead - Journal of ...

J. Moll Stud. (1996), 62,213-223 C 77K Malacological Society of London 1996
GENETIC AND CONCHOLOGICAL COMPARISON OF
SNAILS (HELIX ASPERSA) DIFFERING IN SHELL
DEPOSITION OF LEAD
MARGARET MULVEY 1 , MICHAEL C. NEWMAN 1 and ALAN N. BEEBY 2
'University of Georgia, Savannah River Ecology Laboratory, Aiken, South Carolina, 29802 USA 'School of
Applied Science, South Bank University, London SE1 OAA, UK
(Received 29 December 1994; accepted 15 October 1995)
ABSTRACT
Populations of snails inhabiting areas with different
histories of Pb contamination differed in their deposition
of Pb in shell relative to soft tissues. Genetic
variation, measured using isozymes, was not related
to Pb history nor geographic distance between populations.
Shell characteristic* were significantly different
among sites; shell dry weight was strongly related
to soil calcium levels. Shells of snails from areas with
long histories of Pb contamination were significantly
more robust (greater shell width/shell height ratio)
than snails from other locations. H. aspena adaptation
to Pb contamination may involve significant
changes in shell characteristics but these do not
correlate with genetic traits assessed with allozymes
INTRODUCTION
Populations of animals inhabiting metalcontaminated
environments often display
enhanced tolerances relative to unexposed
populations. However, the evolution of tolerance
and the mechanisms underlying population
adaptation to contaminant stress remain
poorly defined in most situations. Beeby and
Richmond (1988) report reduced soft tissue
assimilation of Pb in the brown garden snail,
Helix aspena MttUer, 1774, apparently associated
with modifications in calcium metabolism.
A population from an urban car park also
deposited a greater proportion of Pb in the
shell relative to soft tissues. Laboratory studies
showed the urban population incorporated
relatively more Pb into their shells and at a
faster rate than snails taken from non-contaminated
habitat (Beeby & Richmond, 1988; Richmond
& Beeby, 1993).
Newman, Mulvey, Beeby, Hurst & Richmond
(1994) extended this work to determine
whether the enhanced incorporation of Pb into
shell occurred in other Pb-contaminated populations
or was characteristic only of the car-
park snails. In a survey of 34 populations from
England and Wales, they report that snails
from areas having long and intense exposure
to Pb had a greater proportion of Pb in their
shells than soft tissues relative to other snails.
Populations from areas experiencing intense but
relatively non-native Pb contamination (largely
derived from automobile petroleum additives)
had lower relative shell Pb levels. Thus, modification
of Ca metabolism and shell deposition
may be a general adaptation in snail populations
experiencing prolonged exposure to lead.
Population adaptation to stressors in the
environment may involve an increase in some
genetic characteristics (e.g., those contributing
to tolerance) and a loss of others. Genetic
variation has been related to growth and reproduction,
and is essential to the long-term
evolutionary potential of populations. Total
genetic variation in populations stressed for
long time periods may differ from populations
subject to more recent stress. Allozyme analysis
has been used to study population genetic
processes in populations experiencing metal
stress. It provides a convenient method to
identify genetic differences among individuals
within populations (Nevo, Beiles & Woo, 1981;
Newman, Diamond, Mulvey, Dixon & Martinson,
1989) and to describe differentiation and
evolutionary divergence among populations
(Heagler, Newman, Mulvey & Dixon, 1992).
Frati, Fanciulli & Posthuma (1992) report a
relationship between allozyme frequencies for
glutamate oxaloacetate transaminase (GOT;
also known as aspartate aminotransferase,
AAT) and Cd pollution in the springtail,
Orchecella cincta. No relationship was detected
for allozymes of phosphoglucomutase or phosphoglucose
isomerase in populations of O. bifasciata
experiencing a gradient of heavy metal
pollution (Tranvik, Bengtssen & Rundgren,
1993, cited in Posthuma & Van Straalen, 1993).
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214 M. MULVEY, M.G NEWMAN & A.N. BEEBY
The snail-Pb system provides an ideal model
to examine the adaptive responses of populations
to environmental stressors. Snails will be
exposed to Pb through soil and foods in contaminated
environments. Snails are relatively
sedentary; a trait that contributes to local
differentiation. Also, genetic differentiation in
H. aspersa populations has been reported on
micro- (Selander & Kaufman, 1975) and
macro-geographic scales (Madec, 1991a;
1991b; Guiller, Madec & Daguzan, 1994).
Selander & Ochman (1983) suggested that
both micro- and macro-geographic population
structure of H. aspersa introduced into the
U.S. resulted from stochastic processes. Crook
(1980) suggested that selection was acting to
determine allelic frequencies for the leucine
aminopeptidase (LAP) locus in H. aspersa
populations in the U.K. Recently, Madec
(1991a) reported low levels of heterozygosity
in a colony of H. aspersa at Fort Bloque and
attributed this to harsh ecological conditions.
In the present report, we describe genetic
and conchological characteristics for the snail
populations examined by Newman et al.
(1994). Since these populations differed with
respect to Pb deposition in the shell, we
wanted to determine if there were also consistent
genetic or conchological patterns relative
to Pb contamination. Two patterns might be
predicted for the population genetic data
based on selection associated with exposure to
heavy metal pollution. First, a decrease in
overall genetic variability might be expected if
there were directional selection for more resistant
genotypes. Second, differential selection
might lead to differences in the distribution of
alleles in tolerant versus nontolerant locations.
If differences in shell deposition between
populations were associated with major genes,
then allozyme loci might serve as 'markers' for
adaptation through chromosomal linkage.
Finally, we examined shell morphology in snail
populations from locations with a long history
of Pb contamination and compared these to
populations from locations with non-native or
no Pb contamination to determine if shell
features, other than relative Pb deposition,
differed for snails from these two habitat
types.
Sample locations
MATERIALS AND METHODS
Helix aspersa populations were collected from 33
locations during August and September 1990 (Table
1, Fig. 1). Locations reflected a range of Pb contamination
from very heavily contaminated sites (e.g.,
sites BI, below a highway interchange in Birmingham
and HA, 3 km downwind of Avonmouth
smelter in Hallen) to locations with insignificant levels
of Pb (e.g., ET and LL, gardens in Ettington and
Llanrhaedr, respectively). Additionally, populations
were chosen that would reflect different histories of
contamination. Five populations were taken from
areas where ratios of Pb with an isotopic signature
characteristic of native Pb were high (BE, FB, HA,
Ml, and SC). Several of these areas had been long
exploited' for metal working. Newman el al. (1994)
report that these populations displayed high Pb in
shells relative to snails from other locations; for the
present study, these populations will be referred to
as 'native Pb'. The remaining populations were in
relatively non-contaminated locations or locations
with non-native, heavy contamination associated
with automotive sources. Pb isotopic ratios for these
locations indicated a relatively greater proportion of
non-native Pb. These populations had relatively low
Pb levels in shells (Newman et al., 1994) and will be
referred to as 'non-native Pb'. Additional details of
sample locations are provided by Newman et al.
(1994). Methods for atomic absorption analyses for
Pb in soil and snails are described in Newman et al.
(1994). Soil Ca levels were determined using standard
methods. Briefly, one g of soil was placed into
glass Kjedahl tubes and 10 mL of 50% (v/v) Analr
grade nitric acidideionized water were added.
Samples were then heated to 150 c C for 2 h, cooled,
filtered through Whatman No. 1 filter paper, and
brought to volume in 25 mL volumetric flasks.
Quality assurance included procedural blanks and
standard materials.
Snails
Snails were placed in plastic containers for 24 hours
to allow clearance of the guL They were washed in
tap water, blotted dry, and frozen at -70 °C. The initial
freezing enabled thawed soft tissues to be easily
removed intact from the shells. A small piece of foot
tissue was removed for electrophoresis and the
remaining soft tissues were used for determination
of Pb contamination (details in Newman et al. 1994).
Protein electrophoresis was used to genetically characterize
populations and describe local differentiation.
For electrophoretic analysis, foot tissue samples
were ground in approximately equal volumes of cold
grinding solution (0.01 M Tris, 0.001 M EDTA, 0.05
mM NADP, pH 7.0). Samples were centrifuged at
10,000 rpm for 45 sec Hdmogenate fluid was
absorbed onto filter paper wicks, blotted briefly, and
inserted into 12-5% (w/v) horizontal starch gels. Gels
were stained following methods described by
Selander et al. (1971) and Richardson, Baverstock &
Adams (1986). Buffer and enzyme stain combinations
are shown in Table 2. For multilocus systems,
isozymes were numbered in order of decreasing
anodal mobility. The most common allozyme was
arbitrarily designated 3; faster migrating allozymes
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Table 1. Locations and designations of 33 samples of Helix asperse. Also shown are concentrations of Ca and Pb in soil samples and median concentrations
of Pb in snail tissue. Mean shell measurements (HC •= collumellar height, AW - aperature width, Dmax » maximum diameter, Ws = dry weight
of shell). Samples sizes for electrophoretic analysis, percent of loci polymorphic and mean heterozygosity for each of the populations are also given.
Mean
Heterozygosity
Percent
Polymorphic
loci
Mean
Sample
Size
Ws
Dmax
AW
HC
Tissue
Pb
Soil Pb
Soil Ca
Label
Population
o
m
i
o
Z
D
n
oz
0.119
0.143
0.113
0.114
0.180
52.9
82.4
76.5
88.2
64.7
17.1 ±0.5
54.1 ± 1.5
66.0 ± 0.5
78.0 ±1.9
32.4 ±0.9
1.35
1 22
1.44
1.52
0.67
31.5
30.3
31.6
30 8
29.4
19.6
17.9
19.0
18.9
17.8
27.1
27.6
28.0
28.3
24.7
3.8
2.5
150
216
7.8
209
337
455
462
77
2072
1667
2248
35361
676
BE
FB
HA
Ml
SC
o
o
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2
in
O
o
11
0.116
0.113
0.180
0.190
0 164
0.179
0 133
0 158
0.200
0.177
0.100
0.141
0.116
0 129
0.163
0.144
1.138
0.145
0 163
0.146
0.032
0.118
0.157
0.167
0.138
0.147
0.115
0 157
64.7
70.6
70 6
88.2
82.4
76.5
47.1
52.9
82.4
882
88 2
76 5
88.2
82.4
76 5
82.4
64.7
58.8
70.6
94.1
23.5
70.6
82.4
76.5
64.7
70.6
82.4
76 5
28.3 ± 0.4
66.8 ± 2.0
34.8 ± 1.0
42.6 ± 1 2
55.3 ±1.9
44.7 ± 1.0
38.5 ±1.2
22.1 ±0.5
13.5 ±03
68.2 ± 2 8
54.6 ± 1.2
24.7 ±07
67.8 ± 1.1
65 2 ±1.8
37.4 ± 0.9
55.2 ± 1.5
43.8 ± 1.0
44.4 ± 1.2
24.6 ±0.4
57.0 ±09
15.9 ± 0.7
31.8 ±0.6
142.8 ±2.5
32.6 ± 0.7
26.6 ± 0.6
77.9 ±34
42 7 ± 0.6
35.1 ± 1.2
1.13
1.56
1.80
1.20
1.27
1.24
1.14
1.65
0.74
1.13
0.88
1.01
1.91
0.84
1.24
1 55
1 13
1.70
1.79
1.67
0.98
083
0.82
1.16
0.95
0.86
0 85
1 19
33.4
30 9
30.0
29.4
29.5
29.4
28.3
33.1
30.4
29.8
29.7
27.2
28.3
28 3
29.8
30.6
28 8
30.6
32.6
28 5
32.6
26.9
26.8
29.1
27.8
27.6
27.6
28.8
19.0
19.4
17.9
18.0
17.2
17.4
17.2
18.9
17.8
18.6
17.6
17.2
16.4
16.8
179
18.4
17.8
18.0
20.1
168
19.3
16.4
16.3
16.8
16.8
16.7
16.3
17.1
29.0
29.4
27.0
27.4
26.6
25.9
25.7
30.0
27 3
28.5
27.3
26.0
26.2
26 0
26 9
27.0
26.7
26.6
31.4
25.7
31.0
24.2
25.6
24.4
23.9
24.6
26.2
26.0
411.8
1.6
5.3
8.5

216 M. MULVEY, M.C. NEWMAN & A.N BEEBY
Figure 1. Sampling locations for Helix aspena. Locations designated with bold letters had snail populations
identified by Newman el al (1994) as having a high shell ratio of Pb ('native Pb') and locations with standard
letters had relatively lower Pb ratios in shells ('non-native Pb'). See Table 1 for details for locations.
were designated 1 or 2 and slower allozymes were
designated 4, 5 and so on.
Dimensions of all intact shells were determined as
described in Bleakney, Fleming

GENETIC AND CONCHOLOGICAL COMPARISON OF SNAILS
Table 2. Buffer and enzyme stains used to resolve isozymes in Helix aspersa.
Buffer
Amine citrate, pH 6.0*
Tris citrate, pH 8.0"
Tris EDTA borate, pH 8.0*
Poulik"
Enzyme stains
•Clayton StTntiak, 1972
"Selander, Smith, Yang, Johnson & Gentry, 1971.
variance into the following components: among
snath at sampling locations, between locations
within the 'native Pb' and 'non-native Pb' groups
and between groups with different Pb histories.
A 3-way Mantel analysis for matrix correlation
(Sraouse, Long & Solcal, 1986) was done using the
following distance matrices: genetic, geographic and
Pb level. Standard genetic distance was determined
from allozyme data. Geographic distance was determined
as the linear distance between pairs of locations
on an ordinance survey map. A Pb distance
matrix was generated by using the absolute difference
between the mean tissue Pb concentrations
(Newman el a/., 1994) for each pair of locations.
ANOVA (SAS, 1987) was used to examine shell
features and to test for differences between 'native
Pb' and 'non-native Pb' populations. Shell ratio was
defined as the ratio of dry weight of the shell to collumella
height, and shell robustness was defined as
the ratio of aperture width to collumella height Levels
of sod Pb and Ca were examined as factors contributing
to shell size. Data were checked for
deviations from normality and none were found.
RESULTS
Populations of H. aspersa had considerable
intra- and inter-population genetic diversity
(Table 1). Populations had between 23.5 and
882% of loci polymorphic and a range of
heterozygosities between 0.032 and 0.200.
Allozyme frequencies for the 33 populations
are given in Appendix 1. Fifty-seven tests for
fit of data to random mating expectations were
done; eight deviations occurred at P

218 M. MULVEY, M.C. NEWMAN & A.N. BEEBY
Figure 2. Measurements made on intact Helix
aspersa shells. Aperture depth (Ad), aperture width
(Aw), maximum diameter (D,^,), columeUar height
(He), and height of the shell (Hs) (Figure and
measurements as in Bleakney et al., 1989).
between populations. The UPTGMA phenogram
based on genetic distance (Fig. 3) showed
no patterns relative to Pb history, levels of
contamination nor geographical separation.
Analysis of distance matrices (Mantel tests)
indicated no correlation between genetic, Pb,
nor geographical distance (all P > 0.89). The
relationships were: genetic vs. geographic, r 2 =
0.189, genetic vs. Pb, r 2 = 0.110 and geographic
vs. Pb,r 2 =-0.021.
Shell measurements were obtained for 604
intact, mature snails; approximately 20 per
population. There were correlations between
shell measurements within and between populations.
For example, the correlation between
D,^ and Ws was r = 0.19, P = 0.01. There was a
strong positive relationship between soil calcium
levels and dry weight of shell (P = 0.002).
ANOVA results for shell measures are given
in Table 3. Shell ratio (Wj/Hc) varied significantly
among sites but there was no significant
relation with Pb exposure history. However,
shell robustness (D^Hc) was significantly different
among sites and also between the
'native Pb' and 'non-native Pb' snails. 'Native
Pb' snails from the historical Pb locations had
a higher ratio than did snails from other sites.
DISCUSSION
Snails from long-term, highly contaminated
habitats ('native Pb') and those from more
recently or non-contaminated habitats ('nonnative
Pb') did not differ in overall levels of
allozyme variation nor were there any consistent
differences in the occurrence of specific
allozymes. Genetic diversity was high in UK
H. aspersa and was similar to levels reported
elsewhere (Selander & Kaufman, 1975;
Bleakney et a/.1989; Madec, 1991b). Patterns
of genetic variation for H. aspersa in UK were
similar to those reported by Selander & Kaufman
(1975) in a study of microgeographic
population structure, and Madec (1991a, 1991b)
and Guiller et al. (1994) who examined macrogeographic
differentiation. Here, populations
were differentiated on a relatively small scale
but there was no clear large-scale pattern nor
any pattern consistent with duration of Pb
exposure. Previous studies have found Nei
genetic distances of 0.03 to 0.37 for H. aspersa
from France and Algeria (Madec, 1991b) and
0.03 to 0.13 for U.K. and French populations
(Bleakney et al., 1989). These distances are
similar to those reported here for populations
of comparable geographic distance.
Although there is evidence of physiological
adaptation among snail populations inhabiting
locations with high levels of long term exposure
to Pb, differential adaptation was not correlated
with genetic patterns of allozymes. UK
H. aspersa populations displayed genetic diversity
within and between populations; 14% of
the total genetic variance was attributable to
differences between 'native Pb' and 'nonnative
Pb' groups. However, it is unclear what
factors contribute to the 14% since there was
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GENETIC AND CONCHOLOGICAL COMPARISON OF SNAILS 219
•+• -+•
0.060 0.050 0.040 0.030 0.020 0.010 0.00
Standard Distance
Figure 3. UPGMA phenogram illustrating genetic relationships for snail populations. 'Native Pb' populations
(identified in Newman et al., 1994) from long-time Pb contaminated are indicated with *. Bootstrap values
were generated using DISPAN (Ota, 1993).
Table 3. ANCOVA for shell characteristics for garden snails from "native" and "nonnative"
Pb contamination sites. Pb refers to these two characteristics in the model and
site refers to the 33 populations sampled. Covarlates were shell dry weight (dry wgt) or
median soil calcium.
A
B
C
Mean'for
Source of variation df "Native" "Non-native"
Shell dry weight (g)
Pb
Soil calcium (mg/g)
Ratio (dry wgt/Hc)
Pb
Site (Pb)
Robustness [Dm^JHc)
Pb
Site (Pb)
Dry wgt
1
1
1
31
1
31
1
0.21
11.88
1.11
19.72
no correlation with geographic distance nor Pb
differences between sites.
Genetic patterns were surprising because a
correlation between geographic distance and
22.60
2.58
0.17
0.16
0.002
0.29
0.0001
0.0001
0.0001
0.68
1.2J
0.04'
0.69
1.21
0.045
0.66
genetic distance were expected based on an
isolation-by-distance model (Crow & Kimura,
1970). Snails are not continuously distributed
across the landscape and especially for widely
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220 M. MULVEY, M.C. NEWMAN & AJi. BEEBY
distributed populations, gene flow should be
limited. One factor contributing to the
observed pattern might be dispersal of snails
associated with human activities. H. aspersa a
common in domestic and commercial gardens
and might be transported with shipments of
plants and supplies. Lack of correlation
between genetic distance and geographic distance
may reflect unanticipated high levels of
gene flow among populations associated with
human activities.
Like Frati et al. (1992), who reported metal
pollution and tolerance in the springtail, O.
cincta, but did not find an effect on measures
of overall genetic variability, we found no difference
in overall genetic variation between
'native Pb' and 'non-native Pb' populations of
snails. For O. cincta, a correlation was found
with frequency of allozymes of Got (Aat) and
Cd contamination. The Got locus was polymorphic
in the H. aspersa populations but no
allozyme was associated with Pb exposure history.
Lack of relationship with allozyme loci
suggests that these enzymes are not important
to tolerance per se or are not closely linked to
loci responsible for tolerance and so do not
serve as effective 'markers'.
Newman et al. (1994) reported differences in
shell deposition of Pb for snails from the
'native Pb' and 'non-native Pb' locations. Consistent
with their findings, robustness of the
shell (Dn^/Hc) was significantly different
between these populations, in the present
study. Adaptation to heavy metal stressors
may involve modification of existing metabolic
pathways and sequestering of toxic substances
in shell or exoskeleton, as is often observed in
invertebrates. Thus, H. aspersa adaptation to
Pb contamination apparently involves significant
changes in shell characteristics but these
do not correlate with genetic traits assessed
with the allozymes examined here.
ACKNOWLEDGMENTS
Travel for the collections was partially funded
by the NATO Collaborative Research Grants
program (CRG900469). Support for the
research also came from contract
DE/AC09/76SROO/819 between the U.S.
Department of Energy and the University of
Georgia's Savannah River Ecology Laboratory.
We thank M.M. Kelak and J.M.
McCloskey for laboratory assistance. Carol
Ercolano prepared the tables. James Novak
provided comments on an earlier draft of the
manuscript.
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Appendix 1. Allozyme frequencies for 34 populations of Helix aspersa. Population abbreviations are given in Table 1. Deviation from Hardy-Weinberg expectations:
*, p < 0.05; •*, p < 0.01.
BE BJ CA CN Cfl ET FB FR GS HA HS ID IF 1R KB LU Lt LE UC
LL
LW
Ml
NC
NS
RO
SA
SB
SC
SF
SH
SJ ST SU TO
10 10 10 10 10 10 1 10 0 10
1 10 0 1.0
098
1.0
to 10 10
1 10098 0 098 10
10
1 10 0 10 10 099 10
0 02
0 02
0 01
1.0
1 0
1 0
10
10
10
1.0
1.0
1.0
0 14 0 02
0 01 0 01 0 01
002
10 0 79 79 098 099 10 10 039 0 99 99 1 10 0 0.99 10 1.0 1.0 0.98 099 10 10 10 10098 098 10 10 1.0 10 10
0 07 0 01
0 01
0 01
0 01
0.03
007 008 0 11 006 0.18 0 01 002 0.19 003 0 16 0 08 0 13 0 05 0 04 0.19 0.28 0 20 0 17 0 07 001
1.0 0 93 0 92 0 89 0 94 0 84 0 99 038 0 81 10 0 97 0.86 0 92 0 87 10 0 96 0.96 031 0 79 030 030 0 93 0 99
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002
098
1.0
10
0 02
0.97
0 01
10
0 01
099
099
0.01
039
0 01
10
1 0
10
006
0.95
0.03
034
0 19
031
0.02
038
0.04
096
0.24
0.78
0.02
098
004
1 0 036 098 099 10 096 0 97 10 10 096099 10 0 98 0 93 10 10 10 099 035 0 98 0 87 0 99 0.99
0 10 0 02 0.01 0 04 0 03 0 04 0 01 0 02 0 07 0 01 0 18 0 02 0 13 0 01 0 01
10
1.0
1.0
036
005
039
001
0.96
004
10
10
10
3 1.0
4
G8pd
2
3 10
4
6
Gpl
1
2 an
3 039
Pgm 1
2
3 10
4
3 10 093 099 082 036 083 10 0 99 0.98 091 0 88 032 0 90 0 94 038 0 98 0 99 0 98 0 97 10 0 98 0.97 1.0 0 99 10 0 99 0 64 095 0 93 10 10 10 0 84
4 0.07 0.01 0.18 0 15 0 17 0 01 0 04 0.09 0 02 0 18 010 0 06 0 02 0 02 0 01 0 02 0 03 0 02 0 03 0 01 0 01 0 36 0 05 0 07 0 18
Mdhi
2 0.18 011 003 0 17 0.05 0 07 0 18 0.18 0 07 0 07 0 02 0 08 0.16 0 03 0 12 0 10 0 04 004 0 06 002 0 11 0 02 0 13 0 09 0 03 0 08 0 05 001 0 10 0.03* 0 08
3 0.38 0 41 031 0.17 0 79 0 24 0 34 0 62 036 0 86 038 0.54 0 53 0 06 0 58 0 43 0 71 0 67 0 63 0 93 0 66 0 72 0 74 0 51 0 94 0 42 0 44 0 47 0 63 0 62 0 34 0 51* 0 49
4 0 44 048 006068 0 16 069 0480300 67 008 038 0 31 0 91 030 0 47 0 24 0 29 0 42 0 05 0 23 0 26 0 13 0 40 0 03 0 60 0 61 0 52 0.27 0 48 0 58 0 49 0 43
6 0 01
Mdh2
1 0 01
2 0 03 0 01 0 03 0 02 0 02 0 02 0 04 0 02 0 02 0 21 0 02
3 10 1.0 1.0 1.0 10 0.97 10 1.0 10 0 99 1.0 1.0 0 96 0 98 038 038 10 10 098 10 10 10 10 098 10 10 098 1.0 10 10 0 78 098 10
4 001
Gpd
2 005 002 0.06 036 002 0 04 0 09 001 0 02 011 0 17 0 02 0 02 0 04 0 01 001 001 0.01 001 001 0 04 0 03 0 03 0.03 0 02 0 01 001
3 0 97 093 098 0 94 0 96 098 038 0 91 0.99 0 98 0 89 0.79 0 97 0.98 038 0 99 0 99 0 89 0 89 1.0 0 99 1.0 0 90 036 10 0 98 0.97 0 97 0 97 1.0 0 99 10 0 99
4003 0040 01 0 02

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