3. Results
3.1. Assignment of indiÕiduals to species types PCA of allelic variation at the Lap, Pgi, Pgm and Mpi loci generated 10 principal
components. Plots of all principal components against one another revealed that PC1 and Ž
PC2 together resolved individuals into three discrete clusters Fig. 3; boundaries
represent 95 confidence intervals which were plotted after assigning individuals to .
species, see below . PC1 explained 21.7 of the total genetic variance and described differences between two groups of oysters that we labelled ‘P’ and ‘B’. Alleles
significantly correlated with PC1 for group ‘P’ possessed positive component loadings
6
Ž .
7
Ž .
3
Ž .
4
Ž .
and were Lap P - 0.001 , Lap
P - 0.001 , Pgi P - 0.001 , Pgi
P - 0.05 ,
7
Ž .
4
Ž . Ž
. Mpi
P - 0.05 and Pgm P - 0.001
Table 3 . Conversely, alleles significantly correlated with PC1 for group ‘B’ possessed negative component loadings, and were
3
Ž .
6
Ž .
7
Ž .
4
Ž .
6
Lap P - 0.001 , Pgi
P - 0.001 , Pgi P - 0.001 , Mpi
P - 0.001 , Pgm Ž
.
7
Ž .
P - 0.001 and Pgm P - 0.001 . PC2 explained 16.9 of the total genetic variance
and split group ‘P’ into two subgroups ‘A’ and ‘C’. Alleles significantly correlated with
6
Ž .
PC2 for group ‘A’ possessed positive component loadings and were Lap P - 0.001 ,
4
Ž .
5
Ž .
4
Ž .
3
Ž .
4
Pgi P - 0.001 , Pgi
P - 0.001 , Mpi P - 0.001 , Pgm
P - 0.05 and Pgm Ž
. Ž .
P - 0.001 Table 3 . Conversely, alleles significantly correlated with PC2 for group
7
Ž .
1
Ž ‘C’ possessed negative component loadings and were Lap
P - 0.001 , Pgi P -
.
3
Ž .
6
Ž .
8
Ž .
7
Ž .
0.01 , Pgi P - 0.001 , Mpi
P - 0.001 , Mpi P - 0.001 and Pgm
P - 0.001 . Comparisons of observed genotype frequencies in our original 12 samples with
Ž .
genotype frequencies expected under Hardy–Weinberg equilibrium HWE identified
Fig. 3. First and second principal components of allozyme data from four marker loci for all Saccostrea spp. collected from Thailand. 95 confidence ellipses are for identification of individuals using diagnostic alleles
at the Lap, Pgi and Mpi loci. Key: Filled triangles — S. cucullata; Filled circles — S. commercialis; Crosses — S. manilai.
Table 3 Ž
. Component loadings correlation coefficients of allelles at four allozyme loci with taxonomically useful
principal components for Saccostrea spp. From Thailand. Locus
Allele PC1
PC2 Lap
1 y0.022
0.004 2
y0.093 0.020
UUU
3 y0.751
0.066 4
0.013 0.094
5 0.087
0.064
UUU UUU
6 0.492
0.267
UUU UUU
7 0.255
y0.496 8
0.019 y0.019
UU
Pgi 1
0.089 y0.158
2 0.005
y0.007
UUU UUU
3 0.515
y0.331
U UUU
4 0.125
0.175
UUU
5 y0.001
0.227
UUU
6 y0.312
0.092
UUU
7 y0.309
0.003 8
y0.095 y0.001
9 y0.016
0.000 Mpi
1 0.018
y0.001 2
0.022 0.019
3 0.065
0.094
UUU UUU
4 y0.355
0.528 5
0.062 y0.008
UUU
6 0.042
y0.248
U
7 0.134
y0.093
UUU
8 0.027
y0.260 9
0.027 y0.030
Pgm 2
0.028 0.051
U
3 0.061
0.130
UUU UUU
4 0.381
0.355 5
0.007 0.001
UUU
6 y0.186
y0.014
UUU UUU
7 y0.269
y0.519 8
y0.023 y0.005
UUU
P - 0.001.
UU
P - 0.01.
U
P - 0.05.
samples from three sites which were mixtures of ‘A’, ‘B’ or ‘C’. Significant deviations Ž
from HWE were observed for all four loci in sample five from Chumphon Lap, 0.001; .
Pgi, P - 0.05; Pgm, P - 0.01; Mpi, P - 0.01 , for the Lap locus alone in sample six Ž
. Ž
. from Chumphon P - 0.05 and for sample 10 from Surat Thani P - 0.01 . Significant
tests were in all cases caused by the absence of heterozygous classes between diagnostic alleles identified by PCA. Absent heterozygotes between common alleles in mixed
samples for Lap included 2r6, 2r7, 3r6 and 3r7; for Pgi 3r6 and 3r7; for Mpi 4r9 and 5r6; and for Pgm 3r7.
Examination of genotypes within samples that were in Hardy–Weinberg equilibrium revealed that alleles Lap
1
, Lap
2
and Lap
3
co-occurred within samples 4 and 7, which lacked any of Lap fast alleles 6–8. The absence of ‘P’ alleles at the Pgi locus
Ž
1 3
. Ž .
Pgi –Pgi identified by PCA confirmed that these samples were monomorphic for
species ‘B’. Conversely, only Lap alleles 4–8 were present in ‘P’ samples 2, 8, 9, 11 Ž
7 9
. and 12. Pgi alleles unique to the ‘B’ species Pgi –Pgi
were absent from all these samples. Samples two and three, which were also in Hardy–Weinberg equilibrium,
consisted predominantly of species ‘B’, but included three and two individuals respec- tively of ‘P’ type. Lap
4
and Lap
5
were found at frequencies of 0.1 in both ‘B’ and ‘P’ samples, as were Pgi
4
, Pgi
5
and Pgi
6
, and therefore were regarded as non-diagnos- tic.
We identified individuals in mixed samples as species ‘B’ if they possessed only Lap
1
–Lap
3
, Pgi
7
–Pgi
9
and non-diagnostic alleles at these two loci. Two hybrids were found after sorting for ‘B’ species as described above. One individual from sample one
Ž .
Ž Trat province was heterozygous for ‘B’ and ‘P’ alleles at the Lap locus a 3r6
. Ž
. heterozygote . The other hybrid from sample six Surat Thani province possessed a ‘B’
allele at the Lap locus, being a 2r4 heterozygote, while at the same time possessing a ‘P’ allele at the Pgi locus, being a 3r5 heterozygote. The presence of ‘‘non-diagnostic’’
alleles such as Pgi
4 – 5
, Lap
4 – 5
and Mpi
1 – 5
, that can be found in two or more species prevented the further classification of hybrids. When non-diagnostic alleles occurred at
all marker loci for a given individual, identification of any kind was impossible. In sample ten, there were four individuals that fell into this category.
The next step in classifying remaining individuals was an examination of genotype frequencies within ‘P’ samples that were in HWE. Alleles for the Lap, Mpi, Pgi and
Pgm loci that were identified from PCA analysis as being markers for species ‘C’ were common in samples eight and nine while markers for species ‘A’ were common in
Ž .
samples 2, 11 and 12 refer Table 3 . However, only Mpi alleles were restricted to either ‘A’ or ‘C’ samples. Mpi
4
was present only in ‘A’ samples 2, 11 and 12, and Mpi alleles 7–9 were present only in ‘C’ samples eight and nine. In addition to the unique
alleles listed above, Mpi
6
was found to be common in species ‘C’ samples but rare in Ž
.
1 5
species ‘A’ with frequencies between 0 and 0.05 . Mpi and Mpi was found in similar frequencies in both species. Individuals were therefore classified as species ‘C’ if they
possessed Mpi marker alleles 6–9 and non-diagnostic alleles 1 and 5. Fourteen of the 61 ‘P’ oysters from ‘‘mixed’’ samples fell into this category, thirteen of which came from
Ž .
sample five from Chumphon , with the remaining individual coming from sample six Ž
. also from Chumphon . Examination of variation at the other loci revealed that all but
two of these individuals possessed PCA marker alleles for species ‘C’ at Lap, Pgi and Pgm loci. Both these hybrids came from sample five, and included one individual that
possessed ‘C’ alleles at all but the Pgm locus where it was homozygous for ‘A’ alleles Ž
. 4r4 . The other individual possessed ‘C’ alleles at all but the Pgi locus where it was
Ž .
homozygous for ‘B’ alleles 7r7 . The remaining forty-nine ‘P’ individuals were
identified as species ‘A’. Following identification of individuals to species, our original 12 samples were
resolved into eighteen putative mono-specific samples. Tests of genotype frequencies on Ž
. sixteen of these samples excluding two samples with n - 5 confirmed that these
A.J. Day
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Table 4 Ž .
Ž .
a Allele frequencies of S. commercialis species ‘A’ in two reference samples from Australia and in samples collected from different sites throughout Thailand Locus
Allele Site 1
Site 2 Site 5
Site 6 Site 10
Site 11 Site 12
Sydney Marnetic
Trat Chon Buri
Chumphon Chumphon
Surat Thani Satun
Trang Australia
Island Australia Aat-1
2 0.091
– 0.031
0.028 0.028
3 1.0
0.976 0.500
– 0.769
0.941 0.889
0.972 0.889
5 0.024
0.409 –
0.200 0.059
0.111 0.083
Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. N
3 21
11 –
15 17
18 18
18 Ap
1 0.026
0.125 0.109
0.028 0.036
2 0.684
0.875 0.667
0.652 0.231
0.095 0.638
0.750 3
0.237 0.083
0.109 0.231
0.309 0.056
0.214 4
0.833 0.052
0.250 0.130
0.538 0.548
0.028 5
0.167 0.048
0.250 Ž
. Ž .
Ž .
Ž . Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 3
19 8
6 23
13 21
18 18
Est-2 3
0.023 –
0.036 0.045
0.306 0.083
5 1.0
0.929 0.955
– 0.892
0.819 0.833
0.639 0.778
7 0.048
– 0.036
0.136 0.167
0.055 0.139
8 0.045
– 0.036
Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. N
3 21
11 –
23 11
21 18
14 Lap
1 0.025
0.030 2
0.030 3
0.350 4
0.214 0.250
0.140 0.024
0.475 0.156
5 0.167
0.119 0.286
0.278 0.160
0.136 0.119
0.345 6
0.833 0.667
0.214 0.611
0.580 0.705
0.666 0.345
7 0.250
0.111 0.120
0.091 0.167
0.150 0.094
8 0.068
0.024 Ž
. Ž .
Ž .
Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. N
3 21
14 9
25 22
21 20
16
A.J. Day
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61 Mpi
1 0.024
0.040 0.023
0.050 2
0.167 0.107
0.060 0.068
0.025 3
0.833 0.262
0.107 0.056
0.360 0.045
0.125 0.263
0.500 4
0.547 0.607
0.721 0.460
0.546 0.650
0.737 0.406
5 0.143
0.143 0.167
0.080 0.273
0.150 0.094
6 0.024
0.036 0.056
0.045 Ž
. Ž .
Ž .
Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. N
3 21
14 9
25 22
20 19
16 Mdh-1
2 0.048
0.022 0.045
0.048 0.028
3 1.000
0.952 0.857
0.667 0.913
0.932 0.952
0.875 0.972
5 0.143
0.333 0.065
0.023 0.125
Ž .
Ž . Ž
. Ž
. Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 3
21 14
9 23
22 21
20 18
Pgi 1
0.036 0.040
0.190 0.265
0.056 2
0.023 3
0.667 0.095
0.214 0.111
0.140 0.931
0.525 0.059
0.167 4
0.333 0.429
0.214 0.278
0.240 0.023
0.190 0.529
0.527 5
0.309 0.429
0.444 0.480
0.023 0.095
0.147 0.250
6 0.167
0.107 0.167
0.100 Ž
. Ž .
Ž .
Ž .
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. N
3 21
14 9
25 22
21 18
18 Pgm
1 0.060
0.050 2
0.024 0.071
0.060 0.023
0.024 0.175
0.194 3
0.333 0.333
0.250 0.056
0.360 0.091
0.048 0.325
0.556 4
0.667 0.619
0.536 0.721
0.480 0.681
0.714 0.375
0.194 5
0.036 0.167
0.020 0.025
0.056 6
0.024 0.107
0.056 0.020
0.182 0.190
0.050 7
0.023 0.024
Ž .
Ž . Ž
. Ž
. Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 3
21 14
9 25
22 21
20 18
continued on next page
A.J. Day
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Ž .
Table 4 continued Ž .
Ž .
b Allele frequencies in samples of S. manilai species ‘B’ from different sites throughout Thailand Locus
Allele Site 1
Site 3 Site 4
Site 5 Site 6
Site 7 Site 10
Trat Chon Buri
Chon Buri Chumphon
Chumphon Chumphon
Surat Thani Aat-1
3 1.0
0.696 0.778
0.867 –
0.979 0.9
5 0.304
0.222 0.143
– 0.021
0.1 Ž
. Ž
. Ž
. Ž .
Ž . Ž
. Ž .
N 27
23 9
7 –
25 5
Ap 1
0.059 0.115
0.042 0.042
0.125 2
0.074 0.306
0.354 0.539
0.333 0.167
0.375 3
0.092 0.014
0.059 0.077
0.083 0.125
4 0.352
0.416 0.294
0.192 0.292
0.603 0.292
5 0.389
0.236 0.176
0.077 0.208
0.167 0.083
6 0.093
0.028 0.058
0.042 0.021
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 25
36 17
13 12
24 12
Est-2 3
0.058 0.025
0.056 –
0.120 5
0.923 0.926
0.944 0.857
– 0.880
1.000 7
0.019 0.037
0.143 –
8 0.012
– Ž
. Ž
. Ž
. Ž
. Ž .
Ž .
Ž .
N 27
40 27
7 –
24 10
Lap 1
0.056 0.059
0.019 0.038
0.083 2
0.185 0.095
0.185 0.154
0.100 0.115
0.292 3
0.685 0.703
0.740 0.731
0.833 0.732
0.500 4
0.056 0.119
0.056 0.038
0.067 0.077
0.083 5
0.018 0.024
0.077 0.038
0.042 Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. N
27 42
28 13
15 26
12
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63 Mdh-1
2 0.042
– 0.050
3 0.981
0.861 0.963
0.917 –
0.958 0.950
5 0.019
0.097 0.037
0.083 –
0.042 Ž
. Ž
. Ž
. Ž
. Ž .
Ž .
Ž .
N 27
36 27
6 –
25 12
Mpi 2
0.074 0.020
3 0.093
0.071 0.071
0.038 0.160
0.042 4
0.352 0.584
0.339 0.577
0.500 0.480
0.500 5
0.389 0.345
0.590 0.308
0.467 0.340
0.333 6
0.092 0.077
0.033 0.125
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 27
42 28
13 15
25 12
Pgi 4
0.018 0.012
0.036 0.077
0.083 5
0.093 0.345
0.179 0.154
0.033 0.154
0.125 6
0.315 0.309
0.535 0.346
0.367 0.269
0.250 7
0.370 0.274
0.161 0.231
0.467 0.443
0.250 8
0.167 0.036
0.089 0.154
0.100 0.115
0.250 9
0.037 0.024
0.038 0.033
0.019 0.042
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 27
42 28
13 15
26 12
Pgm 2
0.018 0.033
0.083 3
0.018 0.061
0.036 0.038
0.033 0.096
0.125 4
0.130 0.146
0.089 0.115
0.133 0.135
0.208 5
0.038 6
0.259 0.256
0.393 0.424
0.400 0.404
0.208 7
0.519 0.537
0.446 0.385
0.368 0.327
0.376 8
0.056 0.036
0.033 0.038
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
N 27
41 28
13 15
26 12
continued on next page
A.J. Day
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72 64
Ž .
Table 4 continued Ž .
Ž .
c Allele frequencies in samples of S. cucullata species ‘C’ from different sites throughout Thailand Locus
Allele Site 5
Site 8 Site 9
Chumphon Chumphon
Chumphon Aat-1
2 3
0.950 0.975
0.975 5
0.050 0.025
0.025 Ž
. Ž
. Ž
. Ž
. N
10 20
40 Ap
2 0.167
0.194 0.244
3 0.111
0.083 0.122
4 0.667
0.723 0.594
5 0.055
0.040 Ž
. Ž .
Ž .
Ž .
N 9
18 37
Est-2 3
0.200 0.025
5 0.750
0.932 0.925
7 0.050
0.023 0.038
8 0.045
0.012 Ž
. Ž
. Ž
. Ž
. N
10 22
40 Lap
4 0.027
5 0.045
0.026 0.027
6 0.136
0.132 0.203
7 0.727
0.842 0.675
8 0.092
0.068 Ž
. Ž
. Ž
. Ž
. N
11 19
37
A.J. Day
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65 Mpi
1 0.056
5 0.091
0.056 6
0.591 0.250
0.250 7
0.045 0.222
0.167 8
0.091 0.305
0.555 9
0.182 0.111
0.028 Ž
. Ž
. Ž
. Ž
. N
11 18
36 Mdh-1
2 0.053
0.038 3
1.000 0.921
0.900 5
0.026 0.062
Ž .
Ž . Ž
. Ž
. N
7 19
40 Pgi
1 0.227
0.342 0.311
2 0.091
3 0.635
0.632 0.689
4 0.045
0.026 Ž
. Ž
. Ž
. Ž
. N
11 19
37 Pgm
3 0.026
4 0.053
0.135 5
0.079 6
0.318 0.342
0.135 7
0.682 0.500
0.703 8
0.027 Ž
. Ž
. Ž
. Ž
. N
11 19
37
samples were in Hardy–Weinberg equilibrium, and therefore that mixed samples were correctly resolved into species. Numbers of all three species are given in Table 3, and
frequencies calculated for ‘A’, ‘B’ and ‘C’ samples in Table 4a–c.
3.2. Morphology Numbers of Saccostrea from each site that were used for morphological analysis are
given in Table 1. The morphology data set included only those individuals that could be identified with 95 certainty by PCA of allozymes. Total numbers of each species were
Ž .
Ž 39 for species ‘A’ including individuals from five sites , 31 for species ‘B’ including
. individuals from two sites , and 20 for species ‘B’ which came from Ko Talu only.
For qualitative characters, numbers of individuals of each species, in the categories that were scored for scar colour and chomata spacing, are given in Table 5. ‘G’ tests for
Ž .
differences between species Sokal and Rohlf, 1969 were significant for both adductor Ž
. muscle scar colour and chomata spacing independently P - 0.001 , as well as for both
Ž .
characters combined P - 0.001 . The most useful of these two characters for identifica- tion purposes is scar colour. Black adductor muscle scars were almost entirely restricted
to ‘C’ with 75 of individuals having black scars. Scar colour for species ‘B’ was generally brown with pronounced lighter stripes, and in species ‘A’ scar colour was
generally white or brown. However, for species ‘A’, one individual was recorded as having a black scar. Chomata spacing was much more variable. Although 75 of
species ‘C’ were scored as having chomata that did not completely encircle the left valve, a significant number of species ‘A’ and ‘B’ also had ‘‘incomplete’’ chomata. In
both species ‘A’ and ‘B’, chomata spacing and adductor scar colour covaried. For species ‘A’, 84.6 of individuals had adductor scars that were other than black
Table 5 Qualitative morphological characters: numbers of individuals with each character, in samples of Saccostrea
species from Thailand S. commercialis
S. manilai S. cucullata
‘G’ Ž
. Ž
. Ž
. species ‘A’
species ‘B’ species ‘C’
Ž . Ž .
Ž . n
n n
Colour of adductor scar Black
1 15
df s 2; 54.97, P - 0.001 Other
38 37
5 Spacing of chomata
Complete 34
15 5
df s 2; 25.83, P - 0.001 Incomplete
5 16
15 Both characters
Black scar, complete 1
2 df s6; 69.77, P - 0.001
Black scar, incomplete 13
Other colour, complete 33
12 3
Other colour, incomplete 5
16 2
Ž .
generally white , together with chomata that completely encircled the left valve. However, 42.8 of species ‘B’ and 15 of species ‘C’ possessed these same character-
istics. The possession of both black adductor muscle scars and ‘‘Incomplete’’ chomata was entirely confined to species ‘C’, although only 65 of this species fell into this
category.
For PCA, data were first log-transformed to standardise variances. Shell size was highly variable. Therefore, PCA was carried out on correlation matrices, as these isolate
size variation to the first principal component to a greater extent than covariance Ž
. Ž
. Ž
. matrices Somers, 1986 . Principal components one PC1 and two PC2 accounted for
52 and 20 of the total variance, and individuals classified as species ‘A’, ‘B’ or ‘C’ Ž
. cluster according to species when these two PCs are plotted
Fig. 4 . Stepwise discriminant analysis of the first 10 principal components revealed that PC4 also varied
between species, although this principal component explained less than 7 of the total variance. These three PCs, together successfully classified 100 of species ‘C’ and 85
of each of the other two species. F-values for PC1, PC2, and PC4 were 43.6, 46.3 and
Ž .
4.1, respectively Wilks-Lambda 0.188, P - 0.00005 . Ž
. Correlation coefficients component loadings for each of the 10 measured characters
with PC1, PC2 and PC4 are given in Table 6. Because species varied in size, PC1 Ž
. possessed significant component loadings
P - 0.001 for all characters except umbo depth. ANOVA of all raw measurements revealed that length and width of both shells
Ž .
Ž .
Ž .
LDVM, RDVM,LAPM and RAPM , hinge length HL , adductor scar width SW , and Ž
. thickness of the right valve RT were significantly smaller in species ‘A’ compared to
Ž
2
the other two species R varied from 27 to 49, with P - 0.0005 for all compar-
. Ž .
isons means and standard deviations are given in Table 7 . PC2 reflected the degree of
Fig. 4. First and second principal components of shell morphology data for all individuals of Saccostrea spp. collected from Thailand. Key: Filled triangles — S. cucullata; Filled circles — S. commercialis; Crosses —
S. manilai.
Table 6 Ž
. Component loadings correlation coefficients of morphological characters with taxonomically useful principal
components, for Saccostrea spp. from Thailand Character
PC1 ‘‘Size’’ PC2 ‘‘Cupping’’
PC4 ‘‘Umbo cavity size’’ External measurements
UUU
LDVM 0.684
0.185 0.171
UUU
RDVM 0.832
0.196 0.039
UUU U
LAPM 0.872
0.183 0.243
UUU U
RAPM 0.889
0.235 0.124
UUU UUU
U
LTRT 0.654
y0.655 y0.242
Measurements
UUU UU
UD 0.001
y0.857 0.298
UUU U
HL 0.789
y0.198 0.258
UUU UUU
SW 0.727
0.446 y0.118
UUU UUU
LT 0.570
y0.678 y0.118
UUU
RT 0.767
y0.015 y0.475
Eigenvalue 5.20
2.02 0.57
UUU
P - 0.001.
UU
P - 0.01.
U
P - 0.05.
‘‘cupping’’ of the left valve with the highest component loadings being for the depth of Ž
. Ž
. the left valve LT , the depth of left and right valves together LTRT and the umbo
Ž .
depth UD . The left valve was deepest in species ‘C’ with measurements for LT and LTRT that were roughly one and a half times bigger than those of species ‘A’ and ‘B’
Ž
2
. R of 48 for characters, with P - 0.0005 . Umbo depth varied significantly between
Table 7 Ž
. Means of 10 morphological measurements cm made on the shells of Saccostrea species from Thailand
Ž .
two standard errors Variable
S. commercialis S. manilai
S. cucullata ANOVA
2
Ž .
Ž .
Ž .
Species ‘A’ Species ‘B’
Species ‘C’ R
ns 36–39 ns 29–31
ns17–20 External measurements
LDVM 3.920.30
5.060.38 5.220.48
0.27 RDVM
3.280.23 4.590.38
4.570.49 0.34
LAPM 2.760.17
3.630.26 3.680.19
0.38 RAPM
2.350.17 3.340.26
3.280.18 0.42
LTRT 1.670.12
1.770.13 2.630.22
0.48 Measurements
UD 0.700.10
0.420.11 1.490.12
0.66 HL
0.960.06 1.210.10
1.620.15 0.49
SW 0.840.08
1.300.12 1.050.07
0.36 LT
1.390.13 1.500.13
2.350.24 0.48
RT 0.510.05
0.820.10 0.980.16
0.38
Ž
2
. all species R
of 66, P - 0.0005 . Species ‘C’ had an umbo depth that was more than double the size of the other two species. However, species ‘A’ also had a
Ž .
comparatively large umbo depth for its small size. The relative thicknesses depths of both left and right valves compared to the size of umbo cavity was reflected in PC4 with
significant negative loadings for RT and LTRT and positive loadings for hinge length Ž
. Ž
. Ž
. HL , umbo depth UD and width of the left valve LAPM .
From stepwise discriminant analysis, the ‘‘best’’ subset of ‘‘raw’’ characters to discriminate between species comprised UD, RAPM and RT, with F-values of 68.5,
Ž .
17.3 and 5.8, respectively Wilks-Lambda 0.205, P - 0.00005 . This combination of characters correctly classified 100 of species ‘C’, 88 of species ‘A’ and 85 of
species ‘B’. RAPM is the only one of these measurements that does not require oysters to be killed. Therefore, we also tested subsets of measurements that can be made
externally for their ability to classify living oysters. The first subset comprised RAPM, LTRT and RDVM, which had the highest F-values of the five ‘‘external’’ characters.
Classification using these three characters was equally successful for S. commercialis, with again 85 of individuals successfully classified to species. However, successful
Ž classifications fell to 90 for species ‘C’ and only 68 for species ‘B’ Wilks-Lamda
. 0.314, P - 0.00005 . The F-value for LDVM was substantially lower at 0.8 than the
Ž .
other two characters F s 2.6 for LTRT and 5.8 for RAPM . Repetition of the analysis excluding LDVM reduced successful identification by only three to five percent for each
species with the highest percentage being 85 for species ‘C’.
3.3. Identification of species Mobility’s of common alleles at all eight scored loci of type ‘A’ were identical to
those of reference S. commercialis from Australia, and allowed identification of species ‘A’ as S. commercialis. Species ‘B’ and ‘C’ were identified by conchological characters
and relative mobilities of alleles at marker loci. Shell morphology of species ‘C’ Ž
. matched that of S. cucullata from India Awati and Rai, 1931 . Similar characters
included a deeply cupped left valve with a wide hinge-line, well-developed cavity at the umbo, chomata that did not completely encircle the right valve, and an adductor muscle
scar that was generally black. Shell morphology of species Species ‘B’ was similar to
Ž .
that described for S. malabonensis from the Philippines Carreon, 1968 . Key features included a small hinge-line, a striped brown and white adductor scar that was displaced
dorsally, and large patches of greenish-brown coloration on interior surfaces. Buroker et Ž
. al.
1979a,b published allele frequencies for four species of Saccostrea from the indo-pacific, S. commercialis, S. cucullata, S. malabonensis and S. manilai. Compari-
Ž .
son with the data sets of Buroker et al. 1979a,b was only possible for Lap and Pgi. Ž
. Buroker et al. 1979a,b scored two loci for each of our other six enzymes and in spite
of using the same buffer system for our gels we could not be certain which of these loci Ž
. was homologous to those that we scored. Buroker et al.
1979a,b found that S.
commercialis and S. cucullata had similar mobility’s for common alleles at both Lap and Pgi, and this was also the case for S. commercialis from Thailand and our species
‘C’. Allozyme frequencies are therefore consistent with our morphological identification Ž
. Ž
. of species ‘C’ as S. cucullata Born . Buroker et al. 1979b identified a cryptic sibling
species to S. malabonensis that they named S. manilai. For this species, the zone of activity for the Lap locus was slowest of all scored Saccostrea species while the zone of
activity for the Pgi locus was fastest. This was the case for our species ‘B’, suggesting Ž
. that this may be the cryptic species S. manilai Buroker . For S. malabonensis itself,
although mobility’s of common Lap alleles were slower than S. commercialis and S. cucullata, the zone of activity for the Pgi locus was of similar mobility to all species
Ž .
except S. manilai. We therefore identify species ‘B’ as S. manilai Buroker .
4. Discussion
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