AMER. ZOOL., 29:863-871 (1989)
Allozyme Variation in a Natural Population
of the Nile Crocodile1
ROBIN LAWSON
Osher Foundation Laboratory for Molecular Systematics and
Department of Herpetology, California Academy of Sciences,
San Francisco, California 94118 and
Department of Biological Sciences, Stanford University,
Stanford, California 94305
CHRISTOPHER P. KOFRON2
Department of Biological Sciences, University of Zimbabwe,
Harare, Zimbabwe
HERBERT C. DESSAUER
Department of Biochemistry and Molecular Biology, Louisiana State
University Medical Center, New Orleans, Louisiana 70112
SYNOPSIS. Blood samples were collected for allozyme studies from 92 Crocodylus niloticus
from the Runde River in Gonarezhou National Park, southern Zimbabwe. Two (glucose
phosphate isomerase and erythrocyte acid phosphatase) of 27 protein coding loci were
polymorphic when examined by starch-gel electrophoresis. This amount of variability is
similar to that found in another crocodilian, Alligator mississippiensis and is not unusually
low as has been found in a number of large vertebrates. In a single semi-isolated population,
allele frequencies at both polymorphic loci were in Hardy-Weinberg equilibrium suggesting a random mating pattern with no severe bottleneck effect in the founding of this
population. Population F-statistics suggest that panmixia exists within and among the
three main breeding sites studied.
by Mitton and Grant, 1984). In the absence
of directional selection, the loss of genetic
variability within natural populations of
normally outbreeding species can most
often be attributed to bottlenecking effects
or genetic drift.
A recent increase in the tempo of extinctions of animal species, most noticeable
among larger animals, has been attributed
mainly to the loss of resources brought
about by rapidly expanding human populations. Concern for the preservation of
endangered species has resulted in the
setting aside of natural reserves, with
the intent that these provide the habitat
requirements for the continued natural
propagation of indigenous faunas. These
well intentioned efforts in some cases may
fail in their purpose due to fragmentation
of habitats with resultant population bottlenecks and possible extinctions brought
1
From the Symposium on Biology of the Crocodilia about by community-level effects (Wilcox
presented at the Annual Meeting of the American and Murphy, 1985). Because of the possiSociety of Zoologists, 27-30 December 1987, at New ble deleterious effects of severe inbreedOrleans, Louisiana.
2
Present address of Christopher P. Kofron is Divi- ing, a measure of genetic variability within
sion of Science, Cuttington University College, P.O. reserve populations may help in manageBox 277, Monrovia, Liberia.
ment strategy. At present no method exists
INTRODUCTION
Genie heterozygosity within natural
populations has been shown to be correlated with Darwinian fitness (Mitton and
Grant, 1984; Endler, 1986; and references
therein). The cheetah, Acinonyx jubatus, a
species with greatly reduced allelic variability at a high proportion of loci, may
suffer diminished fertility with reduced
sperm counts and relatively high mortality
of young when compared to other felines
(O'Brien et al., 1983, 1985; Wildt et al,
1983). This species exemplifies the possible consequences of severe heterozygosity
loss. On the other hand, association of
increase in heterozygosity with increased
population fecundity and individual vigor
and survivability has been demonstrated
for a number of animal species (reviewed
863
864
R. LAWSON ET AL.
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132
FIG. 1. Location of Gonarezhou National Park in southern Zimbabwe.
which will allow examination of variability
of the genome as a whole. However, allozyme data, though not reflecting an
unbiased sampling of the genome, can,
when used comparatively, serve as an estimate of variability among structural loci
coding for soluble proteins.
The purpose of this study is to estimate
genie variability in Nile crocodile populations of Gonarezhou National Park, Zimbabwe. This is undertaken as part of an
overall investigation of the reproductive
biology of these populations (Kofron, in
preparation). The results of this study will
be compared with other surveys of genie
variability in crocodilians (Gartside et al.,
1977; Menzies et al, 1979; Adams et al,
1980). Based on these comparisons we will
attempt to predict the effect of genetic factors on the continued survival of the Nile
crocodile in Gonarezhou National Park.
MATERIALS AND METHODS
Description of study site
The study site was that part of the Runde
River (also called the Lundi River) flowing
through the Gonarezhou National Park in
the lowveld of southeastern Zimbabwe (Fig.
1) at an elevation of about 350 m above
sea level. The Runde is a seasonal river that
flows only during the rainy season, from
around October through April. During the
dry season it consists of a series of pools in
the dry river bed. The source of the Runde
is a number of smaller seasonal rivers.
The main three collecting localities used
in this study are shown in Figure 2. Most
of the crocodiles were captured at Chipinda Pools at the northern boundary of
the park. Directly below Chipinda Pools
continuous bedrock is exposed which forms
rapids and the Chiviriga Falls when the
river flows. Movement of crocodiles down
river from Chipinda Pools is somewhat
impeded by the rapids and falls, but is not
totally prevented. However, there are several species offish below the Chiviriga Falls
that do not occur above it. There is no
restriction to movement up river from Chipinda Pools. About 65 km below Chipinda
Pools the Runde joins the Sabi River, which
then flows through Mozambique to empty
ALLOZYME VARIATION IN CROCODYLUS NILOTICUS
865
2. Chinguli
3. Pokwe
FIG. 2. Location of the three main study sites on the Runde River in Gonarezhou National Park, Zimbabwe.
into the Indian Ocean. Additional study
site information is provided by Kofron (in
preparation).
At the termination of sample collection the
frozen specimens were snipped by air to
the United States.
Sample collection
Electrophoresis and data analysis
On thawing, the cellular fractions, due
Adult and subadult Nile crocodiles were
captured by baited mouth snares (Kofron, to their high concentration of DNA, conin preparation). The traps were tended, sisted mainly of a stringy clot. These clots
and the snared undrugged crocodiles were were broken down mechanically by
pulled ashore and the jaws secured as homogenization with a "Tekmar tissuequickly as possible. At the time of first cap- mizer" following the addition of an equal
ture each crocodile was fitted with a plastic volume of homogenizing buffer consisting
tag, thus allowing the accumulation of of 0.25 M sucrose containing 10 mM merextensive capture-recapture data for the captoethanol, 0.001 mM EDTA, and 0.01
Chipinda Pools population. A 15 ml sample M Tris adjusted to neutrality with hydroof blood was drawn by heparinized syringe chloric acid. After centrifugation at 48,000
from the inferior jugular vein of each croc- x g for 50 minutes, the particle-free superodile (Olson et at, 1975). Each crocodile natants were stored at — 55°C until needed
was measured and the sex determined by for electrophoretic assay.
palpation (excepting two individuals from
A total of 27 protein coding loci were
Chipinda Pools). The blood samples were assayed for electrophoretic variability. The
centrifuged and the plasma and cellular loci scored, with buffer system, blood fracfractions pipetted into separate labelled tion and electrophoretic system used for
cryotubes, frozen and stored in liquid air. each locus are given in Table 1, which also
866
R. LAWSON ET AL.
TABLE 1. Loci examined with protein source, buffer and electrophoresis systems used, and the total number of animals
examined at each locus.
Protein type and protein
Oxidoreductases
Lactate dehydrogenase-2
Malate dehydrogenase-1
Malic enzyme-1
Isocitrate dehydrogenase-1
Phosphogluconate dehydrogenase
Glucose-6-phosphate dehydrogenase
Hexose-6-phosphate dehydrogenase
Glutathione reductase
Catalase
Superoxide dismutase-1
Transferases
Hexokinase
Adenylate kinase
Hydrolases
Esterase (a-naphthyl acetate)*
Esterase (4-methyl umbelliferyl acetate)
Acid phosphatase
Tripeptidase-1 (leucyl-glycyl-glycine)
Dipeptidase (valyl-leucine)
Proline dipeptidase (phenylalanyl-proline)
Lyases
Lactoylglutathione lyase
Isomerases
Mannose phosphate isomerase
Glucose phosphate isomerase
Phosphoglucomutase-1
Non-enzyme proteins
Albumin
Transferrin
a2-macroglobulin
Haptoglobin
Hemoglobin
E C number
Blood
fraction b
Buffer
system1
1.1.1.27
1.1.1.37
1.1.1.40
1.1.1.42
.1.1.44
.1.1.49
.1.1.47
.6.4.2
.11.1.6
.15.1.1
c
c
c
c
c
c
c
c
c
c
1
2,3
2
3
' 2.7.1.1
!2.7.4.3
c
c
[ 5.1.1.2
[ 5.1.1.1
'5.1.3.2
'5.4.11.1
I 5.4.13.11
I 5.4.13.9
c
c
c
c
c
3
3
3
3
3
1.4.1.5
c
5.3.1.8
5.3.1.9
5.4.2.2
C
C
l
Electrophoresis
system11
Total
number
of animals
examined
3
3
3
1
H
H
H
H
H, V
H, V
H
H
H
H
85
89
89
91
91
92
3
3
H
H
83
89
V
H
H
H
H
H
84
92
84
78
83
91
4
H
86
3
1
3
H
H
H
92
92
91
P
P
P
V
V
V
P+C
H, V
c
H
85
85
85
85
92
3
3
p
C
90
92
92
87
* Reagent in parenthesis following enzymes indicates substrate used.
b
C = cellular fraction, P = plasma.
' Buffer 1: TRIS-citrate, pH 8.6 (Poulik, 1957). 2: TRIS-EDTA-maleate, pH 8.0 (Selander et al., 1971), 3:
TRIS-EDTA-maleate, pH 7.4 (Selander et al., 1971). 4: 0.2 M phosphate, pH 6.7 (Parr et al., 1977).
d
H = horizontal system (Ayala et al., 1972). V = vertical system (Brewer, 1970).
lists total number of animals examined at
each locus. Starch-gels were composed of
11.8% electrostarch (Otto Hiller, Madison,
Wisconsin, USA. Batch number 307).
Enzyme electromorphs were localized by
using specific substrate and cofactor linked
methods (Harris and Hopkinson, 1976) and
haptoglobin by the method of McCombs
and Bowman (1969). Albumin, transferrin
and a2-macroglobulin were identified by
their relative mobilities using a general
protein stain.
From only three of the five collecting
localities (Chipinda Pools, Chinguli, and
Pokwe) were sample sizes large enough to
make inter-population comparisons. For
the large Chipinda Pools population a comparison of presumed genotype frequencies
in the sexes was possible.
With electromorphs interpreted as allelic products, presumed genotypes were
scored at all loci for most individual crocodiles (see Table 1). For polymorphic loci,
genotype classes were tested for confor-
TABLE 2. Genotype and gene frequencies at two polymorphic enzyme coding loci in Crocodylus niloticus.
Population
Chipinda-males
Freq. obs.
Freq. exp.
Chipinda-females
Freq. obs.
Freq. exp.
Chipinda-total
Freq. obs.
Freq. exp.
Chinguli
Freq. obs.
Freq. exp.
okwe
Freq. obs.
Freq. exp.
All animals
Freq. obs.
Freq. exp.
fA
(23)
(41)
0
6
0.000
0.017
0.261
0.227
0
0.000
0.015
(66)
0
0.000
0.017
(10)
(9)
(92)
10
0.244
0.214
17
0.742
0.759
0
1
9
0.000
0.003
0.100
0.095
0.900
0.903
0
0
9
0.000
0.000
0.000
0.000
1.000
1.000
0
18
0.196
0.177
0.130
0.870
0.122
0.878
0.129
0.871
0.050
0.950
0.000
1.000
(36)
(59)
49
0.258
0.224
0.000
0.010
(21)
31
0.756
0.711
(10)
(8)
(84)
74
0.804
0.814
AB
fB
17
0.739
0.756
>
Erythrocyte acid phosphatase
Glucose phosphate isomeras*
0.098
0.902
BB
0
1
0.000
0.001
0.048
0.047
0
3
0.000
0.002
0.083
0.080
0
4
0.000
0.001
0.068
0.066
0.932
0.933
0
1
9
0.000
0.003
0.100
0.095
0.900
0.903
fA
fB
H
0.024
0.976
0.011
0.952
0.953
0.042
0.958
0.012
0.034
0.966
0.012
0.050
0.950
0.007
0.125
0.875
0.009
0.048
0.952
0.011
33
0.917
0.918
0
2
6
0.250
0.219
0.750
0.766
0
8
0.000
0.002
0.095
0.091
S
w
<
s
>
z
§
76
0.905
0.907
K
O
55
0.000
0.016
O
N
20
S
I
1
o
868
TABLE 3.
R. LAWSON ET AL.
Population F statistics* for River Runde Croc-
odylus niloticus.
Glucose phosphate isomerase
Erythrocyte acid phosphatase
Average sample
size = 28.34
Total sample size = 85
FST = 0.03
F1T = -0.09
Fls=-0.12
Average sample
size = 25.67
Total sample size = 77
FST = 0.01
F1T = -0.03
FIS = -0.05
* Wright (1965) and Weir and Cockerham (1984).
mation with Hardy-Weinberg expectations
by using the chi-square test and observed
genotype classes were used to calculate
allele frequencies. In analyzing the electrophoretic data we used several measures
of genie variation (see Hedrick e< a/., 1986).
These are average number of alleles per
locus, A, proportion of polymorphic loci,
%P, and population heterozygosity, H
averaged over all loci. As a measure of possible inter-population differentiation, we
calculated the genetic distances of Rogers
(1972) and Nei (1972). For the latter we
used the method of Sattler and Hilburn
(1985) which incorporates the modification of Nei (1978) for small sample size,
the redefinition of genetic identity to
reduce any bias which results from unequal
rates of amino acid substitution at all loci
(Hillis, 1984), and the jackknife approach
(Mueller and Ayala, 1982) to reduce the
bias of sampling a small number of loci.
Allele frequencies at all loci were used in
the calculation of the genetic distance measures. As an additional test for population differentiation we have calculated the
F-statistics, FST, F IT and FIS (Wright, 1965,
1978) at each of the polymorphic loci. In
calculating F-statistics we used the formulae of Weir and Cockerham (1984)
which take into account differences in sample sizes. Additionally, we used the jackknife approach recommended by Weir and
Cockerham (1984) to produce less biased
statistics.
RESULTS
Of the 27 loci examined, two, glucose
phosphate isomerase (GPI) and erythrocyte acid phosphatase (EAP), showed allelic
variation. As just two alleles were found at
each of these loci the average number of
alleles per locus, A, is 1.07 and the proportion of polymorphic loci, %P, is 7.41.
Observed genotype frequencies and those
expected under Hardy-Weinberg equilibrium are given in Table 2, in addition to
allele frequencies and average heterozygosities for Chipinda males and females
separately, for the three main populations
sampled and for the total of all animals
sampled which includes animals captured
outside of the three main collecting sites.
The observed and expected heterozygosities averaged over the three main
populations sampled were both 0.009. Chisquare tests showed no significant departures of observed genotype frequencies
from those expected under Hardy-Weinberg equilibrium (a table of these chi-square
values and their exact probabilities is available from the senior author). Table 3 lists
the population F-statistics for the two polymorphic loci and Table 4 compares several
population statistics for Alligator mississippiensis and Crocodylus niloticus. T h e genetic
distance measures of Nei (1978) and Rogers (1972) among the three main population samples were zero or close to zero.
DISCUSSION
The population statistic FST, the fixation
index, measures the amount of inbreeding
due solely to population subdivision or the
reduction in heterozygosity of a subdivision due to genetic drift and as such has
been used as a measure of differentiation
between subpopulations (Hartl, 1981;
Hedrick, 1983). Wright (1978) has suggested that the range of FST values from
0.15 to 0.25 indicates moderately great differentiation, but guidelines for the interpretation of lower values are few. Hedrick
(1983, p. 295) interprets a value of 0.072
as being relatively small and indicative of
only minor allelic heterogeneity among
samples. Barrowclough (1980) used chisquare tests of FST = 0 for the significance
of FST estimates, a method suggested by Li
and Horvitz (1953). By this method, FST =
0.071 (his highest estimated value) was not
significantly different from zero. We interpret the FST estimates of 0.01 and 0.03 for
869
ALLOZYME VARIATION IN CROCODYLUS NILOTICUS
TABLE 4.
Source
Gartside et al.
Menzies et al.
Adams et al.
Adams et al.
Adams et al.
This study
Comparative population statistics for two species of crocodilians.*
Species
Locality
No. of loci
A.m.
A.m.
A.m.
A.m.
A.m.
C.n.
USA, LA
USA,FL
USA,SC
USA, LA
USA,FL
49
44
GNP
27
27
27
21
.08
.08
.15
.07
.19
.07
0.060
0.045
0.186
0.074
0.191
0.074
0.021
0.009
0.034
0.012
0.022
0.011
* Species, A.m. = Alligator mississippiensis, C.n. = Crocodylus niloticus. Locality, LA = Louisiana, FL = Florida,
SC = South Carolina, GNP = Gonarezhou National Park, Zimbabwe, A = average number of alleles per
locus. P = proportion of loci polymorphic. H = heterozygosity averaged over all loci.
the polymorphic loci in Crocodylus niloticus,
in conjunction with the zero or near zero
genetic distances between populations as
indicative of no differentiation among the
three populations tested.
FIS and F IT are inbreeding coefficients
which indicate departures from random
mating in subpopulations and their total
respectively. Lack of positive values for
these coefficients indicate that neither
within any of the three populations sampled nor in their total is there any inbreeding. These coefficients can also be used as
measures of departure from Hardy-Weinberg equilibrium with negative values indicating heterozygote excess. Because the
calculated numbers of expected homozygotes for the less frequent allele are less
than one for population samples Chinguli
and Pokwe at both polymorphic loci, and
for Chipinda at the EAP locus and only
one for the Chipinda sample at the PGI
locus, we consider the small negative values
for FIS and F IT (Table 3) due to sampling
error. Taken overall, the population
F-statistics indicate that no departure from
random mating within the populations
sampled is taking place, population differentiation due to genetic drift is not occurring and there are no effective barriers to
gene flow among the populations sampled.
Comparison of population genie variability in the Nile crocodile with that of
three American alligator populations shows
them to be remarkably similar (Table 4).
It is known that some of these alligator
populations may have undergone severe
genetic bottlenecks due to overhunting
during several decades prior to 1960, but
with rapid recovery to at least former den-
sities following protection (Joanen and
McNease, 1987). In other alligator populations, notably those of Florida, no such
genetic bottleneck has occurred within historic times. Thus, the hypothesis that
genetic bottlenecking is the cause of the
generally low levels of heterozygosity in
the American alligator has been rejected
(Gartside^ al., 1977; Menzies et al, 1979).
In the absence of evidence for bottlenecking in the Nile crocodile we also reject it
as a probable cause for the similar low level
of heterozygosity in this species.
Another hypothesis presented as a possible cause of low heterozygosity in the
American alligator is that of directional
selection in response to environmental factors (Gartside et al, 1977). This hypothesis
has been discussed in detail by Gartside et
al. (1977) and by Adams et al. (1980) and
will be only briefly reviewed here. According to this ecological theory, large, longlived, and vagile animals with wide geographic ranges are largely unaffected by
microheterogeneity of their environment;
they experience their environment as finegrained (Levins, 1968). The model of
genetic strategy in adaptation to an environment experienced as fine-grained is that
such species will have less genetic variability than those experiencing their environment as coarse-grained (Valentine, 1976).
Gartside et al (1977) suggest that the
American alligator experiences its environment as fine-grained and that the low
level of genetic variability in this species
reflects adaptation to a narrow niche within
a stable environment. Nevo (1978) in his
survey of genetic variation in natural populations of a large number of animal and
870
R. LAWSON ET AL.
niques. Academic Press, New York, San Franplant species, concluded that the data taken
cisco, and London.
overall seem to support the hypothesis that
R. H. 1967. The American alligator—
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land Publishing Company, Amsterdam.
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Hard, D. L. 1981. A primer of population genetics. Sinconcordance between these measures of
auer Associates, Inc., Sunderland, Mass.
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Hedrick, P. W., P. F. Brussard, F. W. Allendorf, J.
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