2. Materials and methods
2.1. Sampling Ž
Samples of Saccostrea oysters as defined by the possession of chomata, Stenzel, .
1971 were collected from 12 sites in six provinces throughout Thailand during
Ž .
September 1995 and April 1996 Table 1 . All samples came from natural populations, where they were collected from a single rock or from within an area of less than 4 m
2
. The exception to this rule was the sample from Ko Samet, where due to low numbers,
Ž .
oysters were collected from approximately 50 m of shoreline. Fig. 1, Table 2 . Within 4 h of collection, the adductor muscle was excised from each oyster and stored in liquid
nitrogen for subsequent allozyme electrophoresis. Shells were retained for morphometry from a subset of seven locations which included two sites from Chumphon and one site
Ž .
from each of Trat, Chon Buri and Surat Thani in the Gulf of Thailand , and Trang and Ž
. Ž .
Satun in the Andaman Sea Table 2 . All shells were cleaned of remaining tissues, and
any that had been damaged by their removal from the substrate discarded. 2.2. Electrophoresis
Tissue samples were homogenised in 75 ml of 0.05 M Tris–HCl pH 7.0 contai- Ž
. ning 20 dimethyl sulphoxide DMSO , and stored at y708C prior to starch-gel
electrophoresis. When two or more zones of activity were visible for a single enzyme, loci were numbered according to mobility such that for a particular enzyme a locus la-
belled — 2 would migrate further towards the anode than a locus labelled — 1. In practise, for each enzyme with multiple loci, one locus consistently stained for more
Table 1 Abundance of three Saccostrea species
a
in samples collected from sites throughout Thailand in 1995 and 1996, which were used for analysis of allozymes
b
Site no Site Name
S. commercialis S. manilai
S. cuccullata Hybrids
Ž . Ž .
Ž . Ž .
n n
n n
1 Ko Chang
3 27
1 2
Ban Si Racha 21
3 Bang Saen 1.
2 42
4 Bang Saen 2.
28 5
Ko Samet 14
12 11
3 6
Ban Sam Saeb 8
15 1
7 Ban Pak Nam
26 8
Ko Jorakae 19
9 Ko Talu
37 10
Ko Prab 25
12 1
11 Ko Patra
21 12
Ban Kantang 22
a
Resolved into species by allozyme variation at three marker loci, Lap, Pgi and Mpi.
b
Possessed diagnostic alleles at marker loci from two different species.
Fig. 1. Locations of sites sampled in Thailand in 1995 and 1996 for species of Saccostrea.
individuals than the others. We include data only for these ‘‘reliable’’ loci. Allelic Ž
. variation was scored at eight enzyme loci, aspartate aminotransferase
Aat-1 , esterase Ž
. Ž
. Est-2, utilising fluorescent stain , leucine aminopeptidase Lap , malate dehydrogenase
Ž .
Ž .
Ž .
Mdh-1 , mannose phosphate isomerase Mpi , peptidase
Ap, substrate gly–leu , Ž
. Ž
. phospho-glucose isomerase Pgi and phospho-glucose mutase Pgm . All enzyme loci
Ž .
were resolved using trisrcitraterEDTA pH 7.0 buffer TCE of Buroker et al., 1975 . Initial scoring of all oysters was on gels that included individuals from at least two
samples and, in addition, a standard individual loaded in every fifth position. Even so the plethora of genotypes observed required that we ran from five to seven line-up gels
per locus as cross-controls. In these gels individuals of the same presumed genotype were located next to one another, and genotypes were loaded in order of increasing
mobility. Mobility’s were compared with two reference samples of S. commercialis
Table 2 Sample sizes of Saccostrea spp collected from sites throughout Thailand in 1995 and 1996, which were used
for analysis of allozymes and morphology Site no
Sea Province
Site Name Allozymes
Morphology Ž .
Ž . n
n 1
Gulf of Thailand Trat
Ko Chang 31
12 2
Chon Buri Ban Si Racha
21 6
3 Chon Buri
Bang Saen 1. 44
– 4
Chon Buri Bang Saen 2.
28 –
5 Chumphon
Ko Samet 40
– 6
Chumphon Ban Sam Saeb
24 –
7 Chumphon
Ban Pak Nam 26
18 8
Chumphon Ko Jorakae
19 –
9 Chumphon
Ko Talu 37
21 10
Surat Thani Ko Prab
42 18
11 Andaman Sea
Satun Ko Patra
22 13
12 Trang
Ban Kantang 21
8
from Australia. One sample was purchased from a commercial hatchery in Sydney the other sample came from a natural population on Magnetic Island in Queensland. We do
not follow the numbering system for loci or alleles that was used for Saccostrea species Ž
. by Buroker et al. 1979b . This study used different buffer systems, including different
substrates and tissues for certain loci. Therefore, we cannot be certain of homology of Ž
. alleles or loci with Buroker et al. 1979b . Alleles were labelled 1, 2, 3 etc. in order of
increasing mobility considering all individuals together. The presence of more than one Saccostrea species in our samples was evident by the association within individuals of
allelic variation at four loci, Lap, Mpi, Pgm and Pgi. Forty-eight tests for deviations of
Ž .
genotypes from Hardy–Weinberg expectations HWE were carried out for these marker loci within the original 12 samples to identify those that were mixtures of different
species. The large number of alleles present at these loci necessitated the pooling of rare Ž
. alleles to avoid low expected numbers of genotypes with df s n n y 1 r2, where n is
Ž .
equal to the number of alleles after pooling Pamilo and Varvio-Aho, 1984 . Signifi- cance levels were adjusted for the number of tests using the sequential Bonferroni
Ž .
method of Hochberg 1988 . Ž
. A principal component analysis PCA was carried out on all alleles at the putative
marker loci, Lap, Mpi, Pgi and Pgm, to identify those most useful for distinguishing between taxa. For all individuals, the presence or absence of each allele at these loci was
Ž .
scored as 0 or 1 McDonald et al., 1991 . 2.3. Morphological analyses
For each individual, the colour of the adductor muscle scar was noted as either black Ž .
Ž . Ž .
Ž . 1 or other colour 2 , and arrangement of chomata as complete 1 or incomplete 2 .
Measurements of 10 quantitative characters were made using a vernier caliper that was Ž
. Ž
. Ž .
accurate to 1 mm Fig. 2 . Dimensions obtained for both right flat valve R and left
Ž Fig. 2. Morphological measurements made on shells of Saccostrea spp from Thailand. Note LTRT, which is
. not illustrated in the total depth of shell with both valves fitted together .
Ž .
Ž . Ž
. Ž
cupped valve L were; anterior–posterior RAPM, LAPM and dorsal–ventral RDVM, .
LDVM measurements of each valve, and the depth of each valve when laid on a flat Ž
. surface RT, LT . Measurements taken on the left valve only included the umbo depth
Ž .
measured as the depth of the shell from the middle of the hinge plate UD , and width of Ž
. Ž
. the adductor muscle scar SW . The length of the hinge plate HL was measured on the
right valve. In addition, the overall depth of both valves fitted together was measured Ž
. LTRT .
The power of morphometrics to distinguish between species was tested for by Ž
. ANOVA and PCA. Stepwise discriminant analysis SDA was used both on the original
raw data and on principal components to determine those morphological measurements, which were most successful at classifying individuals to species. All statistical analyses
Ž .
were performed using SYSTAT V7.0 SPSS .
3. Results
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