Genetic studies of Asiatic Clams, Corbicula, in Thailand - Allozymes of 21 Nominal Species are Identical

10 Pages·1991·3.3 MB·English

by  V Kijviriya

GENETIC STUDIES OF ASIATIC CLAMS, CORBICULA, IN THAILAND:
ALLOZYMES OF NOMINAL SPECIES ARE IDENTICAL
21
VARAPORN KIJVIRIYA
DEPARTMENT OF BIOLOGY, FACULTY OF SCIENCE
RAMKHAMHAENG UNIVERSITY
RAMKHAMHAENG ROAD, BANGKOK 10240, THAILAND
SUCHART UPATHAM
E.
VITHOON VIYANANT
CENTER FOR APPLIED MALACOLOGY AND ENTOMOLOGY
DEPARTMENT OF BIOLOGY, FACULTY OF SCIENCE
MAHIDOL UNIVERSITY
RAMA 6 ROAD, BANGKOK 10400, THAILAND
DAVID S. WOODRUFF
DEPARTMENT OF BIOLOGY AND CENTER FOR MOLECULAR GENETICS
UNIVERSITY OF CALIFORNIA, SAN DIEGO
LA JOLLA, CALIFORNIA 92093-0116, U.S.A.
ABSTRACT
FreshwaterclamsofthegenusCorbicula,collectedfrom40sitesupto 1500kmapartinThailand,
andrepresenting21 nominalspecies,shownosignificantgeographicvariationat24electrophoretically
detectedallozymelociandaremostprobablyall referabletothewidespreadAsianspecies,C. fluminea
(Muller, 1774).
ThaiCorbiculahaveverylittlegeneticvariability: mean numberofallelesperlocus(A)wasvery
low: no variation (A = 1.0) was detected in 30% of the samples, in the remainder, A < 1.1; mean
percentage of loci polymorphic in each sample, P = 4.59% (range: 0.0-12.5%); mean individual
heterozygosity, H = 0.011 (range: 0.000-0.025) with one outlier where H = 0.058). The low level of
populationvariabilityandverylowindividual heterozygositysuggestthatmostoftheCorbiculainThailand
arefacultativeself-fertilizers.Thesmallamountofgeniediversitydetected, andtheobservedgenotype
frequencies,areapparentlymaintainedbylimitedoutcrossing,atrandomwithrespecttoshellphenotype,
internal shell color and allozyme genotype.
Eighty-eight percent of the samples, including one referable to Corbicula fluminea, cluster at
Nei's genetic distance values of D < 0.01. Only five samples from northeast Thailand stand slightly
apartfromtheothers.Theseveryhighgeneticsimilarities,coupledwithalackofsignificantanatomical
variation,providenosupportfortherecognitionofmorethanonespeciesinoursamples.Twentynominal
Thai speciesaresynonymizedaccordinglywith C. fluminea; anotherseven nominal speciesarecandi-
dates for synonymy.
Freshwater clams of the genus Corbicula (Bivalvia: studyandtwo international conferences(Britton, 1979, 1986).
Corbiculidae) occur throughout Asia, eastern Europe and This research hasshown that C. fluminea isquitevariable in
Africa. One species, C. fluminea (Muller, 1774), is the com- shell size, shape, sculptureandcolor: thesameconchological
monest freshwater bivalve in Asiaand has recently been in- characteristicsuponwhich mostCorbiculaspecieshave been
troducedtowestern Europe, North and SouthAmerica(Mor- defined. Morton (1979, 1986) reexamined such conchological
ton, 1986). In the United States it spread rapidly and, as a variation in the numerous nominal speciesofCorbiculafrom
majoreconomic pest, it hasbeen thesubjectofconsiderable China and Japan and found that such characters were
American Malacological Bulletin, Vol. 8(2) (1991):97-106
97
98 AMER. MALAC. BULL. 8(2) (1991)
taxonomically unreliable. Hefoundthat, when morphological studiesonallozymevariation in Corbicula. Smithetal. (1979)
variation was considered together with data on ecology, found nodetectablevariationat 18loci infivepopulationsfrom
physiology, demographyand reproductive behavior, onlytwo across North America. In contrast, theyfoundsomevariation
biologicalspeciescouldbe recognized ineastAsia:freshwater at 12-17loci infourAsian samples: theproportionof locithat
C. fluminea, and estuarine C. fluminalis (Mu'ller, 1774) (Mor- werevariable(P) inthe PhilippineswasP = 0.17-0.23, in Hong
ton, 1986). Hesuggestedthat mostoftheothernominalAsian Kong, P = 0.25, and in Japan, P = 0.76. The Asian clams
taxaaresynonymsofthesetwospecies.Thispaperexamines were thus moderately to highly variable and the mono-
this hypothesis as it applies to the nominal species of Cor- morphismofthe introduced NorthAmericanpopulationswas
bicula in Thailand. attributed to genetic drift associated with the founder effect
Mostoftheoriginal speciesdescriptionsofAsian Cor- duringasinglecolonization. McLeodand Sailstad(1980)then
bicula are due to Prime (1864) and Prashad (1930, and reported consistent (year-round) low levels of genetic varia-
referencestherein). Subsequently, Brandt(1974)catalogedthe tion at seven loci from one population in eastern U.S.A.: P
molluscsofThailand and recognized28speciesofCorbicula, = 0.14, and mean individual heterozygosity, H = 0.005. They
fiveof them new. These systematists worked almost entirely concluded that their genetic observations supported the
with conchological characters in defining their species and hypothesisthat NorthAmerican Corbicula areself-fertilizing.
ignored geographic and ontogenetic variation and other Subsequently, McLeod (1986) reported a study of variation
biological attributes. Theseniorauthor has recentlydescribed in other populations from both eastern and western U.S.A.
variation in Corbicula representing 22 nominal species from andagain found littlevariation withinsamples. Theseearlier
40 localities throughout Thailand (Kijviriya, 1990). In agree- reports, especially that showing high genetic variability
ment with earlier reports, she found considerable conch- elsewhere in Asia, suggested that allozymic variation might
ological variation among clams referable to the different be used in resolving the relationships among the Corbicula
species recognized by Brandt (1974). She alsofound signifi- of Thailand.
cant variation in shell size, shape, sculptureand colorwithin
and between allegedly conspecific populations. Gross
anatomy and dissections of the siphon, digestive and MATERIALS AND METHODS
reproductivesystems, ontheother hand, failedto reveal any
taxonomicallysignificant variation between anyofthepopula- Clams were collected in 1985-1986 at 40 localities
tionssampled (Kijviriya, unpub. data). Inthispaper, wereport (Fig. 1,Appendix)fromvariousprovincesthroughoutThailand.
on detectable genetic variation within and between these Most clamswerecollected from lotic habitats in the riversor
populationsand concludethatamajortaxonomic revision of irrigation canals; exceptionsweretwo lake localities(samples
Thai Corbicula is now required. 1 and 23). Some clams were stored in liquid nitrogen im-
Genetic variation in Corbicula was studied by elec- mediatelyand hand-carriedtothe UniversityofCalifornia, San
trophoreticsurveysof multilocusallozyme patterns. Allozymes Diego(UCSD)forallozymicscreening and protocoldevelop-
haveproven extremely useful in recent systematicstudiesof ment; othersweremaintainedaliveat Mahidol Universityand
molluscs. Intrapopulation allozymicvariation has been used usedfor routineallozymestudies. Notesonclamdensity, shell
to establish mating systems (Selander and Kaufman, 1973; morphology, and habitat type and water pH were recorded.
McCrackenandSelander, 1980; SelanderandWhittam, 1983), Distributional and conchological criteria(Brandt, 1974) were
torevealtheexistenceofsibling species (Davis, 1983; Staub used to identify 39 of the 40 samples to the species level.
ef a/., 1990), to reveal cases of interspecific hybridization Voucherspecimensfromeachsampleweredepositedinthe
(Gould and Woodruff, 1986, 1987, 1990; Woodruffand Gould, Museum at the Center for Applied Malacology and En-
1987) andtostudyenvironmental effectson population struc- tomology, Mahidol University, Bangkok. Voucher specimen
ture (Koehn and Hilbish, 1987; Nevo, 1988). Studiesof inter- lot numbers are given in the Appendix.
population allozymic variation have revealed the extent of
ELECTROPHORESIS
geographic structuring and variation in widespread species
(Gould and Woodruff, 1978; Grant and Utter, 1988) showing Proteins from 30 animals from each sample were
whether disjunct populations are or are not conspecific studied byelectrophoreticanalysisusing horizontal starchgel
(Palmer ef a/., 1990; Woodruff ef a/., 1988), revealed the slabsfollowingthetechniquesofSelanderefal. (1971). Prior
historical pattern of a species dispersal (Mulveyefal. 1988; to electrophoresis, individual clams werethawed and quick-
,
Woodruff ef al., 1986, 1988), and helped define the limits of ly homogenized in 0.1-0.2 ml distilled water. The whole-body
semispecies in syngameons (Woodruff and Gould, 1980; homogenate was centrifuged at 4,500 g for 10 min and the
Woodruff, 1989). The comparison of allozymes among supernatant was absorbed onto Whatman No. 1 filter paper
nominal species has supported numerous taxonomic revi- wicks(3x9mm)and inserted into 12% (w/v) horizontal starch
sionsand phylogeneticdiscussions(Davisefa/., 1981; Ember- gels. Electrostarch Lot 146(Otto Hiller, Madison, Wisconsin)
ton, 1988; Woodruffand Solem, 1990; Woodruffefal. 1987). wasusedthroughoutthisstudy. Clamsfromdifferentsamples
,
Giventhedemonstrable utilityofsuch dataonallozymicvaria- were run on each gel to facilitate comparisons and sample
tion, this approach was included in our study of the Thai 17 from the Loei River (northeast Thailand) was used as a
Corbicula. controlgroup.Threebuffersystemswereemployedto resolve
Three research groups have previously reported the 14 enzyme systems and 24allozymes studied (Table 1).
KIJVIRIYA ET AL GENETIC VARIATION IN THAI CORBICULA 99
:
estimated bydirectcount. Chi-squareand Fisherexacttests
20°N 1 wereusedtocomparethefrequencyofindividual genotypes
with Hardy-Weinbergexpectationsfor a panmicticpopulation.
Population structure was examined using fixation indices or
F - statistics calculated for each locus and sample (Wright,
1978). Nei's(1978) unbiasedgeneticdistancecoefficients(D)
and Rogers' (1972) genetic similarity coefficients (S) were
calculated and clustered using the unweighted pair group
averaging method (UPGMA) for the construction of
phenograms and Wagner trees.
RESULTS
Weobtainedgeneticallyinterpretable resultsfor24loci
persample. Descriptionsofbanding patternsandtheirinter-
pretations are provided by Kijviriya (1990). Allozymic varia-
tion in 40samplesfrom throughoutThailand is summarized
in Table 2.
The mean number of alleles per locus (A ) was very
low: no variation (A = 1.0) was detected in 12 oMhe 40
samples (30%) and in the remaining 28 samples, A < 1.1.
The mean percentage of loci that were polymorphic in each
Table 1. Electrophoretic buffers giving optimal resolution of 24
allozymic loci in Corbicula sp.
Allozyme (E.C. No.) Abbreviation Buffer*
Alkaline phosphatase (3.1.3.1) Alp TC 6.0
Aspartate aminotransferase (2.6.1.1) Aat-1 TC 6.0
Aat-2 TC 6.0
Esterase (alpha-naphthyl acetate) (3.1.1.-) Es-1 LiOH
Es-4 LiOH
Es-5 LiOH
Glucose-6-phosphate dehydrogenase
(1.1.1.49) G6pdh TC 8.0
Glucose-6-phosphate isomerase (5.3.1.9) Gpi TC 8.0
Isocitrate dehydrogenase (1.1.1.42) ldh-1 TC 6.0
ldh-2 TC 6.0
I00°E 05°E Leucine aminopeptidase (3.4.11.-) Lap-1 LiOH
Fig. 1. Location of sample sites in Thailand. Lap-2 LiOH
Malate dehydrogenase (NAD) (1.1.1.37) Mdh-1 LiOH
Mdh-2 LiOH
Electrophoresiswascarried out atconstantvoltagefor 15 hr Malate dehydrogenase (NADP+) (1.1.1.40) Mdhp-1 LiOH
bywhichtimeabromophenol blue markerdye had migrated Mdhp-2 LiOH
100-120 mm anodally. Following electrophoresis, each slab Peptidase (L-leucyl-L-alanine) (3.4.-) Pep-A-1 TC 6.0
was cut intofour orfive slices and stained for a specific en- Pep-A-2 TC 6.0
zyme following standard methods (Murphy ef a/., 1990). Peptidase (L-leucylglycylglycine) (3.4.-) Pep-B-2 LiOH
Pep-B-3 LiOH
Isozymes were numbered, and allozymes were assigned
Phosphoglucomutase (5.4.2.2) Pgm-1 TC 8.0
superscripts (a, borc) in orderofdecreasing anodal mobili- Pgm-2 TC 8.0
ty. Commonlyusedenzymeabbreviationsaretypeset in italics 6-Phosphogluconate dehydrogenase
to indicate the presumed genetic locus. (1.1.1.44) Pgdh TC 6.0
Xanthine oxidase (1.1.1.204) Xdh TC 6.0
STATISTICAL ANALYSES
Dataconsistingof multilocusgenotypesfor individual *gTeCls6.0a:n0d.118:85MfoTrriesl,e0c.t0r6o5deMsc(i1t4rather,, a8d0juvs).teLditOoH.pHso6l.0u;tidoinluAt:ed01.:093fMor
clams scorable at all 24 loci were analyzed using the LiOH, 0.19Mborate, pH8.1;solution B: 0.008Mcitrate, 0.05MTris,
BIOSYS-1 computerprogram (Swofford and Selander, 1981). pH 8.4; 1A:9B for gels, A for electrode. TC 8.0. electrode: 0.678 M
Alocuswasconsidered polymorphic if morethan one allele Tris, 0.157 M citrate, adjusted to pH 8.0; gel: 22.89 mM Tris, 5.22
was detected. Mean heterzygosity per individual (H) was mM citrate, pH 8.0 (14 hr, 100 v).
100 AMER. MALAC. BULL. 8(2) (1991)
sample was P =_4.59% (range: 0.0-12.5%). Mean individual squaretest, theobservationswere inagreementwith Hardy-
heterozygosity (H) was also very low: H = 0.011 (range: Weinbergexpectationsin 40outof43tests(P > 0.05). Inthe
0.000-0.025) with one outlier at Mekong River-5 (sample 15) remaining three cases, two (both involving Lap-2) were also
where H = 0.058. As 24 loci were surveyed in every sample in agreement with Hardy-Weinberg expectation when the
and as mean sample size per locus was 21-28, our results Fisherexact test was used. Onlyone case (involving Es-5 in
are adequate to conclude that the Thai Corbicula have very sample 15) failed both tests: we detected 6 AA, 24 AB and
little genetic variability. noBBindividuals. Thisaccountsfortheelevated heterozygosi-
Minor genetic variation was detected at 28 localities ty noted at this locality.
(70%)aroundThailand; the remaining 12were isogenic. Six- In the Thai Corbicula, we found the simple
teen sampleshad 1 polymorphic locus, 8 had 2 polymorphic morphological dichotomy between whiteand purple internal
loci, and 4had 3polymorphic loci. Thisvariationwasdetected shell color to be generally inapplicable (Kijviriya, 1990); like
in 5 loci, four of which were di-allelic and one was tri-allelic. Morton(1987) in Hong Kong, werecognized moreintermediate
In no case, however, were more than two alleles detected at color types. However, in eight parts of Thailand we found
a locus in a single population. There was no discernible populations with approximately bimodal distributions of col-
geographic pattern tothisvariation although levelsofPwere or morphs(samples: 9, 11, 15, 18-19and 35-37). When these
slightly higher in the northeast (P = 0.065) than elsewhere morph-sorted subsamples were compared they were found
(P = 0.03 - 0.046). to be indistinguishable at the 24 allozymic loci. The genes
Wherea locuswaspolymorphic ataparticularly locali- weexamined includedthreeofthesixlocifoundtoshowfixed
ty, the observed genotype frequencies were compared with differences betweenthecontrasting morphotypesstudied in
the frequencies expected under panmixia. Using the chi- Texas by Hillis and Patton (1982).
Table2. Allelefrequenciesforvariableloci* in40samplesofThaiCorbicula,withsummarystatisticsofgeneticvariability
at 24 loci".
Sample Es-5a Lap-2a Mdh-1a Pep-1a Pgdhc N A p H
1 1.00 0.95 1.00 0.80 0.93 28 1.1 0.13 0.02
2 1.00 1.00 1.00 1.00 1.00 25 1.0 0.00 0.00
3 1.00 1.00 0.83 1.00 1.00 26 1.0 0.04 0.01
6 1.00 0.88 1.00 0.95 1.00 24 1.1 0.08 0.01
7 1.00 0.93 1.00 1.00 1.00 26 1.0 0.04 0.003
9 0.90 0.93 0.92 1.00 1.00 25 1.1 0.13 0.02
10 1.00 1.00 0.88 1.00 1.00 25 1.0 0.04 0.01
11 1.00 1.00 0.97 1.00 0.78 26 1.1 0.08 0.02
12 1.00 1.00 1.00 0.87 1.00 26 1.0 0.04 0.01
13 1.00 1.00 1.00 0.85 1.00 27 1.0 0.04 0.01
14 1.00 1.00 1.00 0.87 1.00 26 1.0 0.04 0.01
15 0.60 0.85 1.00 0.85 1.00 25 1.1 0.13 0.06
16 1.00 0.87 0.93 0.95 1.00 27 1.1 0.13 0.02
18 0.23 1.00 1.00 0.93 1.00 26 1.1 0.08 0.03
19 0.10 0.95 1.00 1.00 1.00 26 1.1 0.08 0.01
20 0.00 0.85 1.00 0.80 1.00 26 1.1 0.08 0.02
21 0.00 1.00 1.00 0.85 1.00 26 1.0 0.04 0.01
22 0.20 1.00 1.00 0.90 1.00 25 1.1 0.08 0.03
24 0.97 1.00 0.85 1.00 1.00 24 1.1 0.08 0.02
25 0.80 1.00 1.00 1.00 1.00 25 1.0 0.04 0.02
26 0.97 1.00 1.00 1.00 1.00 25 1.0 0.04 0.003
28 1.00 0.83 1.00 1.00 1.00 25 1.0 0.04 0.01
31 1.00 1.00 0.78 1.00 1.00 25 1.0 0.04 0.02
32 1.00 1.00 1.00 0.87 1.00 24 1.0 0.04 0.01
33 1.00 0.90 1.00 0.92 1.00 25 1.1 0.08 0.02
34 1.00 0.92 1.00 1.00 1.00 26 1.0 0.04 0.01
35 1.00 0.82 1.00 1.00 1.00 26 1.0 0.04 0.02
36 1.00 0.87 1.00 1.00 1.00 24 1.0 0.04 0.01
39 1.00 0.88 1.00 1.00 1.00 23 1.0 0.04 0.01
'Each variable locus has two alleles except for Pdgh where c/a and c/b occur in samples 1 and 11, respectively. All
other loci (see Table 1) were monomorphic.
"Samples described in the Appendix. The following 11 samples are not shown but were identical to sample 2: 4, 5,
8, 17, 23, 27, 29, 30, 37, 38, 40 (i.e. N = 22-27, A = 1.0, P = 0, H = 0), where N = mean sample size per locus, A
= mean no. alleles per locus, P = proportion of loci polymorphic, H = mean individual heterozygosity.
KIJVIRIYA ETAL GENETIC VARIATION IN THAI CORBICULA 101
:
Wetestedthe relationshipbetweengenievariability(P)
and local abundance by simple regression analysis. As ex- 21 gguussltaavvllaannaa SS
pected, wefound a significant negative correlation between -4 gustaviana S
abundanceand samplearea(r2 = 0.4). Therewas, however, --58 slpy.diIgnldaenta. CS
nosignificantrelationshipbetweensampleareaorabundance -17 blandlana NE
and P(r2 = 0.13 and 0.09, respectively). Population variabili- -23 lydlgiana NE
ty was not simply a function of population size. -.2279 lfyldulmglinaenaa Cc
We looked for ecological factors that could be -30 lydiglana c
associated with locally elevated levels of P. The four most -37 plngensls N
noelllngi N
variable samples (samples 1,9, 15 and 16) showed no con- 40 lamarckiana N
sistent patternwith respecttowaterdepth, watertemperature, E3286 baudonl c
pH or habitat type. We are unable to distinguish such sam- 41124 lteevnliuusscula NNEE
ple sites ecologically from sites with isogenic clams. 32 noelllngi N
Genetic relationships between the 40 samples were 13 levluscula NE
6 gustavlana S
studied bycalculating various measures ofgenetic distance 33 levluscula N
and clustering these values using various tree building 7 javanica S
algorithms. A phenogram (not shown) based on Nei'sgenetic 34 castanea N
36 Iravadlca N
distance(D)did notdiscriminate between almost 90% ofthe 39 heardl N
samples; one based on Rogers' genetic similarity resolved 28 virescens c
35 messageri N
the trivial differences between some samples (Fig. 2). Both 16 baudonl NE
algorithmsshowthatalltheCorbiculastudiedfromthroughout 3 guslavlana S
Thailand are virtually identical to one another at the 24 10 lamarckiana C
24 lydlgiana C
allozyme loci examined. 31 baudonl N
•9 javanica C
•25 regla C
DISCUSSION •11 tenius NE
-15 arata NE
The interpretations concerning allelic variation were ••2128 mloarmealrectkiiaannaa NNEE
made in averyconservativefashion. Geneticallyinterpretable 19 bocourti NE
variation wasseen in 5 loci: Es-5, Lap-2, Mdh-1, Pep-A-1 and -r 2210 bsollaindduilaana NNEE
Pgdh. In the majority of cases (39 out of 44), where a locus _L
was polymorphic at a particular locality, only one of the two 0.96 0.98 I.00
homozygote genotypes was observed. Both homozygote GENETIC SIMILARITY
genotypes were, however, seen in at least one sample for 4 Fig.2. Dendrogramshowingrelationshipsamong40samplesofThai
of the 5 polymorphic loci (Es-5 being the exception). Thus, CorbiculageneratedbyUPGMAclusteringofRogers' (1972)genetic
ourgenetic interpretationsaresupported bytheobservation similarity(S)estimatesbasedon24loci.Thecopheneticcorrelation
ofall possiblegenotypessegregating inThai Corbicula in the is 0.971.
majority of the variable allozymes.
Another laboratory using different techniques could species of invertebrates. For example, Nevo ef a/. (1984)
find variation where we have found none as single-gel elec- reported that for 361-371 species of invertebrates P = 0.38
trophoretic surveys are known to fail to detect about 20% of and H = 0.10. Our estimates forthe Thai Corbicula are also
true sample variability (Ayala, 1982; Selander and Whittam, lower than those obtained by Smith ef al. (1979) who found
1983). Forthisandotherreasons, thecomparison ofelectro- P = 0.17-0.76 in the Philippines, Hong Kong and Japan. The
phoretic results between different laboratories is not recom- average Thai values are thus closer to those estimated for
mended (Nei, 1987). However, somecommentsonthe results North American populationswhere P = 0.04 and H = 0.002
ofSmithefal. (1979), theonlyotherpublishedsurveyofvaria- (datasummarized by Britton and Morton, 1986). Such mean
tion in Asian Corbicula, areappropriate. Theirstudy involved values can, however, be misleading: the most variable sam-
eight unique loci and ten that were shared with our survey. ple in Thailand (sample 15) had P = 0.125 and H = 0.06and
They too found variation in Mdh-1 and Pgdh in Asian Cor- isthus much morevariablethan mostofthe North American
bicula. In contrast, where they found Gpi-1 was variable in populations.
samples from Japan, Hong Kong and the Philippines, we Corbiculaflumineahas,atvarioustimes, been regarded
detected novariation inThailand. Theotherthreepolymorphic as dioecious, as monoecious, as a consecutive protandic
loci that we detected in Thailand were not studied by Smith hermaphrodite, and as a simultaneous hermaphrodite
ef al. (1979). (Kraemer, 1979; Brittonand Morton, 1979, 1986). Thelowlevel
The number of individual clams studied per sample of populationvariabilityandverylowindividual heterozygosity
and the number of loci investigated per clam are adequate suggestthat mostoftheCorbicula in Thailand arefacultative
for the estimation of levels of genic_variability. The approx- self-fertilizers. The reduced variability observed is similar to
imate average values of A < 1.1, P = 0.05 and H = 0.01 thatfound in otherself-fertilizing molluscs(Selanderand Kauf-
are all very low relative to those estimated for many other man, 1973; Selanderand Hudson, 1976; Hornbachefal. 1980;
,
102 AMER. MALAC. BULL. 8(2) (1991)
McCracken and Selander, 1980; Stoddard, 1983; Nevoefa/., stand slightlyapartfrom theothers: samples20-22 from the
1984). Morton (1983) recognizedthatdifferent populationsof Mun Riverdrainageandsamples 18-19fromtheMekongRiver
C. fluminea can havedifferent sexual strategies; he noted dif- at Nakhon Phanom. Theirgeneticdifferentiation rests primari-
ferent degrees of self-fertilizatin in lotic and lentic habitats. ly on their having unusually high frequencies of Est-5t> and
The small amount of genie diversity detected in 70% to a lesser extent on their frequencies of Pep-A-1t>. Neither
of the samples must be maintained by either inbreeding oftheseallelesareuniquetotheselocalitiesoreventonorth-
among a few strains with limited outcrossing or by the self- east Thailand, however, so these samples are differentiated
fertilization ofa large numberof heterozygotes. Wefavorthe from the others at a barely significant level (D = 0.03-0.04).
former explanation as we failed to find the alternate Not only were these regionally commoner alleles found
homozygotes in 88% of the situations where we detected elsewhere in Thailand, but we failed to detect them at two
heterozygotes(each situation involved aspecific polymorphic other Mekong River sites (samples 14-15) between Nakhon
locus in aspecific sample). Occasional random outcrossing Phanom and the point 300 km downstream where the Mun
would also account for observations that genotype frequen- River enters the Mekong.
cies conformed to Hardy-Weinberg expectations in 98% of Theoverall level ofgeneticdifferentiation seenamong
the cases and the concomitant lack of a consistent pattern the 40 samples of Corbicula is well within that expected for
of heterozygote deficiencies expected for obligate self- conspecific populations of sexually reproducing species.
fertilizing species. Thorpe's (1983) survey of 1111 published conspecific com-
Linkage disequilibrium, which is common in self- parisonsshowedthat98% ofthegeneticdistanceestimates
fertilizing plants, was notdetected inthevariablesamplesof wereD < 0.10. Self-fertilizing species have received less at-
Corbicula.Therewas nosignificantassociation between alter- tention, but generally show more geographic differentiation
nate alleles at two or more polymorphic loci in any popula- than outcrossing species, i.e. the observed values for Thai
tion.Thus, thevariable populationsinThailandarenotsimply Corbicula are much lower than expected for agroup of self-
composed oftwoor more monogeniccloneswith limited inter- fertilizing congeneric species. The very high genetic
breeding. Instead the pattern we discovered suggests similaritiesestimated here provide nosupportforthe recogni-
amphimixis isoccurringtoday in somepopulationsthat have tion of more than one species of Corbicula in our samples.
a recent historyof automixis. The proportion of progenypro- We hasten toadd that even within acladethere is no
duced by self-fertilization (S) can be estimated from the pro- simple relationship between geneticdistanceandtaxonomic
portion of heterozygous individuals in each subpopulation level. Some most closely related speciesare knownthat are
(Hamrick, 1983; Hillis, 1989). Using Pgdh genotypefrequen- very similar at their allozyme loci (Davis ef a/., 1981; Gould
cies we estimated the frequency of self-fertilization in sam- andWoodruff, 1987). Nevertheless,thepreponderanceofthe
ple 10to be 13%; using Pep-A-1 weestimated the ratesto be evidence on genetic differentiation associated with species
29% in sample 1, 29% in sample20, and 23% in sample 14; of vertebrates (Avise and Aquadro, 1982), invertebrates
using Lap-1 our estimates were 68% in sample 7 and 57% generally (Nei, 1987), and molluscs in particular (Woodruff
in sample 16. These results indicate that multiple reproduc- ef a/., 1988), supports the conclusion that our samples are
tive modes may be used differentially in different localities. conspecific. Furthermore, the degree of differentiation seen
The very low levels of genie variation and small sam- intheThai Corbicula islessthanthatreported inotherspecies
ple sizes make it difficult to study population structure using offreshwaterbivalve(Davis, 1983; Davisefa/., 1981; Kat, 1983;
Wright's (1978) fixation indices or F- statistics. The over-all- Katand Davis, 1984). It issignificantlylessthanthatreported
loci values of F = -0.13 indicates insignificant inbreeding between clonesofotherautomictic molluscs(D = 0.06-0.12)
is
within each sample. However, when the data are examined byMcCrackenand Selander(1980).The lackofgeniedifferen-
on a finer scale, eight significant individual locus F values tiation in an areathe size ofThailand was a little surprising.
were obtained in eight different samples; i.e. Fis > -is0.20 for We failed to detect any evidence for even incipient specia-
Es-5infoursamples, andforLap-2and Mdh-1, intwodifferent tion in clams from different river systems. Had we known, a
samples each. In contrast, the larger F = 0.46 indicates priori, thattheThai Corbiculawereprimarilyself-fertilizingwe
st
significant variation among samples. Thisvalue is similarto would have predicted even more differentiation between
the average fixation index for self-fertilizing plants (F = clones; as was found in North American slugs (McCracken
st
0.44; Hamrick, 1983). Thereduction in individual heterozygosi- and Selander, 1980) and Australian Thaira Stoddard, 1983,
ty in the Thai Corbicula as awhole (dueto both substructur- 1985).
ing and inbreeding) is also quite substantial (F = 0.39). IntheabsenceofcomparableallozymestudiesofCor-
lt
Thephenetictreebasedon Rogers' geneticsimilarity bicula from elsewhere in Southeast Asia, we are unable to
(Fig. 2)shows howall 40samplesofThai Corbiculaarealmost discusstheevolutionorphylogeneticrelationshipsoftheThai
identical at the loci studied. In terms of the more widely clams. Their close similarity probably reflects recency of
reported Nei'sgeneticdistances,88%ofthesamples(35/40) divergence. Time for the accumulation of genetic variation
cluster at averaged genetic distancevaluesofD < 0.01; such has been limited and self-fertilization hasworked constantly
valuesaresmallerthan their associated standard erros (Nei toeliminateheterozygotesandrarealleles.Thedegreeofdif-
efa/. 1986). Most of the Thai Corbicula are thus genetically ferentiation observed among the Thai Corbicula could have
,
identical or indistinguishable at a reasonably large sample evolved in less than 200,000 years if Nei's (1987) moderate
of allozyme loci. Only five samples from northeast Thailand calibrationoftheprotein-electrophoreticclock(withaDvalue
KIJVIRIYA ETAL. GENETIC VARIATION IN THAI CORBICULA 103
:
of 1.0 equivalent to about 5 million years) is applicable. This, then, isourprincipal result:thefreshwaterclams
Theallozymic similarityofthe40samplesofThai Cor- of the genus Corbicula, collected from sites up to 1500 km
bicula, together with their lack of taxonomically significant apart in Thailand, show no significant geographic variation
anatomical or conchological variation (Kijviriya, 1990), in- at 24genetic loci and are referable tothe widespread Asian
dicates that all are referable to a single species. Two speciesC. fluminea. This resultwould beconfirmed if itwere
argumentscan bemadethatthisspecies is C. fluminea. First, shown that the Thai Corbicula are closely related to better
Morton(1986)and Brittonand Morton(1986) havearguedthat known C. fluminea from Hong Kong and North America. We
most, ifnotall, oftheAsianfreshwaterCorbiculaaresynonyms predictthattheChineseclamswill beverysimilargenetical-
of C. fluminea. Second, Brandt (1974) identified C. fluminea ly to those in Thailand (D < 0.10). Those of the Philippine
as occurring at several localities in Thailand and we have islands could show greater differentiation associated with
studied variation in clams from one of these localities (sam- foundereffectsandperhapsreducedvariabilityasin thecase
ple 27) and found that morphologically they aretypical of C. ofthe introduced NorthAmericanclams.Thereported higher
fluminea. Furthermore, as we have discovered that these levelsofvariation in Japan (P = 0.76, H = 0.23; Smith efal.
,
clams are genetically indistinguishable from all other Cor- 1979) are paradoxical and requireconfirmation. Perhapsthe
bicula studied in Thailand, we proposethefollowing taxabe radically different level of variation in this peripheral popula-
synonymized with C. fluminea (Muller, 1774): tion indicates that the Japanese clams have adopted a dif-
Corbicula arata (Sowerby, 1877) (Brandt, 1974:313; pi. 27, ferent mating strategy to those used elsewhere. Alternative-
fig. 73) ly, theislandcould havebeencolonized repeatedlybyclams
C. baudoni Morelet, 1866 (Brandt, 1974:323; pi. 29, fig. of diverse geographically origin.
102) In partsofthe United States, two morphotypesof Cor-
C. blandiana Prime, 1864 (Brandt, 1974:313; pi. 27, fig. 72) bicula fluminea have been identified based on differences in
C. bocourti (Morelet, 1865) (Brandt, 1974:314; pi. 27, fig. 80) colorofthe nacre: whiteordark purple. Thetypical form has
C. castanea (Morelet, 1865) (Brandt, 1974:317; pi. 27, fig. 79) colored shellsandthedarkform haspurpleshells. Hillisand
C. gustaviana Martens, 1900(Brandt, 1974:320; pi. 28, fig. 87) Patton (1982) found that in Texas the two morphs had fixed
C. heard! Brandt, 1974:328; pi. 29, fig. 104 differencesatsixof26allozyme loci. McLeod(1986)reported
C. iravadica Hanley and Theobald, 1876 (Brandt, 1974:323; asimilarfinding. Although Hillisand Patton (1982)andothers
pi. 28, fig. 91) have used these genetic differences as evidence for the
C.javanica(Mousson, 1849)(Brandt, 1974:315; pi. 27, fig. 82) presence of two species in North America, the analyses of
C. lamarckiana Prime, 1864(Brandt, 1974:316; pi. 27, fig. 76-77) Britton and Morton (1986)favoraone-species model. Britton
C. leviuscula Prime, 1864 (Brandt, 1974:326; pi. 28, fig. 95) and Morton argued that otherconchological, ecological and
C. lydigiana Prime, 1861 (Brandt, 1974:316; pi. 27, fig. 74-75) behavioral traitsareassociated with thisdimorphism in both
C. messageri Bavay and Dautzenberg, 1901 (Brandt, 1974: North America and Hong Kong. These suites of associated
327; pi. 29, fig. 100) characters are apparently local adaptive responses to en-
C. moreletiana Prime, 1867(Brandt, 1974:321; pi. 28, fig. 89-90) vironmental variables and the contrasting morphotypes are
C. noetlingi Martens, 1899 (Brandt, 1974:319; pi. 28, fig. 88) thusbetterregarded asecophenotypes. Accordingto Britton
C. pingensis Brandt, 1974:324; pi. 28, fig. 93 and Morton (1986), allozymic variation has now been de-
C. regia Clessin, 1879 (Brandt, 1974:320; pi. 28, fig. 86) scribed in 22 North American populations of the white
C. solidula Prime, 1861 (Brandt, 1974:326; pi. 28, fig. 96 morphotype; P =_ 0.04 (range = 0-19%) and H = 0 in 17
C. tenuis Clessin, 1887 (Brandt, 1974:318; pi. 28, fig. 85) populations and H = 0.0013-0.0049 in 5 populations. The 3
C. virescens Brandt, 1974:324; pi. 29, fig. 101 samplesofthepurple morphotypethat have beendescribed
As Brandt (1974) has provided fully referenced synonyms, were all monomorphic (P = 0, H = 0). No other genetic
notes on conchology, types, localities and distribution, and studies have been published, although work in progress on
excellentphotographsof representativeshells, on the pages rRNAvariationwill reopenthequestionofwhethermorethan
and plates cited above, such data are not repeated here. one species is present in North America (D. Hillis, pers.
Kijviriya(1990) also providescolor photographs of represen- comm.).
tative shells (see Appendix for specific figure numbers). A paradox to emerge from our study concerns Cor-
These taxonomic conclusions are based on the Thai bicula fluminea's great ecological success despite its innate
material examined. We cannot assess the validity of any of lackofgeneticvariability. We havefoundthatoneofthecom-
these 21 taxa from elsewhere in Asia, e.g. Corbicula gusta- monest bivalves is as genetically depauperate in parts of its
vianafrom SumatraorC.javanicafromJava. In addition,the native range as it is in areas it hassuccessfullycolonized in
following seven nominal Thai species were not collected (C. the last 50 years. Although some workers have maintained
cyreniformis Prime, 1860; C. gubernatoria Prime, 1869; C. oc- that genie variability is positively related to ecological and
cidentiformis Brandt, 1974; C. pisidiformis Prime, 1866; C. evolutionaryadaptability, it isclearthat C. fluminea'ssuccess
siamensis Prashad, 1929; C. vokesi Brandt, 1974) or were isbasedon anarrowgeneticbase. McCrackenand Selander
found in inadequatenumbersforelectrophoreticanalysis(C. (1980) found a similar pattern in some European slugs intro-
erosa Prime, 1861). We therefore have no opinion as to the duced into North America. Homozygousself-fertilizingspecies
validity of these taxa but suspect that they too are junior ofslugsoccupied awiderrangeof habitatsand had superior
synonyms of C. fluminea. colonizing abilities than some more heterozygous outcross-
104 AMER. MALAC. BULL. 8(2) (1991)
ing species. Corbicula appears to have such a less variable Davis, G. M., W. H. Heard, S. L. H. Fuller and C. Hesterman. 1981.
general purposegenome. Clearly,the largerissuespresented Molecular geneticsandspeciation in E///pf/oand its relation-
by Corbicula cannot be resolved from within Thailand; a ships to other taxa of North American Unionidae (Bivalvia).
broader geographic surveyof genie variabilityand breeding BiologicalJournal ofthe Linnean Society 5:131-150.
systems in this widespread species is now required. Only Emberton, K. C. 1988. The genitalic, allozymic, and conchological
when this is done will we begin to understand the relation- evolutionoftheeastern Triodopsinae(Gastropoda: Pulmonata:
Polygyridae). Malacologia 28:159-273.
smhairpkebdetcwoenecnhogleongeitciacls,,etchoeloegnivciarlonamnedntb,ehaavnidorCa.lfvlaurmiianteiao'n.s Gould,LSi.ttJl.eaBnadhaD.maS.BWaonodkr—uffa.r1e9v7i8s.ioNnabtuarsaeldHoinstgoernyeotficCse,rimoonr.pVhIIoI-.
metries and geographic distribution. Bulletin ofthe Museum
of Comparative Zoology 148:371-415.
ACKNOWLEDGMENTS Gould, S. J. and D. S. Woodruff. 1986. E—volutionand systematicsof
Cerionon NewProvidenceIsland Aradicalrevision.Bulletin
WethankCaptainVinyooKijviriya, RTN, Dr. UsaKlinhom, Miss of the American Museum ofNatural History. 182:389-490.
Duangduen Ratanaponglakha, Mr. Thongleum Saesonthi and Mr. Gould, S. J. and D. S. Woodruff. 1987. Systematics and levels of
SomyosHatsanatefortheirassistancewithcollectingclams. Vimonsri covariation inCerionintheTurksandCaicosIslands. Bulletin
Manasarn and M. Patricia Carpenter provided excellent technical of the Museum of Comparative Zoology 151(6):321-363.
assistancein thelaboratory in Bangkokand San Diego, respective- Gould, S. J. and D. S. Woodruff. 1990. History as a cause of Area
ly. The seniorauthorwassupported by Ramkhamhaeng University Effects:An illustrationfromCeriononGreatInagua, Bahamas.
and the National Research Council ofThailand and acknowledges BiologicalJournal ofthe Linnean Society 40:67-98.
theadviceofDrs. MaleeyaKruatrachueandRojanaS.Keawjam.Work Grant,W. S. and F. M. Utter. 1988.Geneticheterogeneityondifferent
in San Diegowassupported bytheAcademic Senateofthe Univer- geographic scales in Nucella lamellosa (Prosobranchia,
Thaididae). Malacologia 28:275-287.
sity of California and, indirectly, by grants from the U.S. National
Science Foundation, to DSW. We thank David M. Hillis and an Hamrick, J. L. 1983. Thedistribution ofgeneticvariation within and
anonymous reviewer for their comments on the manuscript. amongnatural plantpopulations: In:GeneticsandConserva-
tion, C. M. Schonewald-Cox, C. M. Chambers, B. MacBryde
and L.Thomas,eds. pp. 333-348. Benjamin/Cummings, Menlo
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106 AMER. MALAC. BULL. 8(2) (1991)
APPENDIX
The40samplesarelistedbynumber(1-40, in boldfacetype); 18. Phocanal-1, Huai Pho, Nakhon Phanom province,(5), C. morele-
locality; (relativeabundance); andspeciesnameaccordingtocriteria tiana (MUFS-TH00-179, pi. 3, fig. 16A/B).
of Brandt (1974). These sample numbers are placed by locality on 19. Phocanal-2, Huai Pho, NakhonPhanomprovince,(5),C. bocourti
themapofThailand(Fig. 1). Clamabundancewasscoredofascale (MUFS-TH00-150, pi. 1, fig. 4).
from 1 (rare) to 5 (superabundant), where absolutes densities per 20. Mun River, Phibun Mangsahan district, Ubon Ratchathani pro-
square meter are approximately 10, 100, 500, 1000, and >1000. In vince, (4), C. blandiana (MUFS-TH00-149, pi. 1, fig. 3B).
brackets following each species name, we give the voucher 21. Pan River, Yang Talat district, Kalasin province, (3), C. solidula
specimen's lot number together with a reference to the color (MUFS-TH00-186, pi. 4, fig. 2).
photograph (plate and figure number) of representative shells in 22. Chi River, Muang district, Mahsarakham province, (3), C.
Kijviriya (1990). Shells were given Mahidol University, Faculty of lamarckiana (MUFS-TH00-173, pi. 3, fig. 13D).
Science(MUFS)codenumbersanddeposited inthe Museumofthe 23. Ubonrat reservoir, Khon Kaenprovince, (3), C. lydigiana(MUFS-
CenterforApplied Malacologyand Entomology, Mahidol University. TH00-167, pi. 2, fig. 12B).
24. Panthong canal, Panthong village, Chonburi province, (4), C.
1. Lake at Lampam, Phattalung province, (5), Corbicula gustaviana lydigiana (MUFS-TH00-170, pi. 2, fig. 12E).
(MUFS-TH00-155, pi. 2, fig. 8B). 25. PraSaeBonRiver,Glaengdistrict, Rayongprovince,(3),C. regia
2. Ta Pi River-1, Chawang district, Nakhon Si Thammarat province, (MUFS-TH00-185, pi. 4, fig. 19A/B).
(2), C. gustaviana (MUFS-TH00-157, pi. 2, fig. 8D). 26. Chao Phraya River-1, Pamoke district, Angthong province, (3),
3. Mincanal,Chawangdistrict, NakhonSiThammaratprovince,(2), C. baudoni (MUFS-TH00-146, pi. 1, fig. 2D).
C. gustaviana (MUFS-TH00-156, pi. 2, fig. 8C). 27.ChaoPhrayaRiver-2,KrungThonbridge,Sanghaearea, Bangkok,
4.TaPi River-2, Lampooisland, Muangdistrict,SuratThaniprovince (3), C. fluminea (MUFS-TH00-153, pi. 1, fig. 7A/B).
(3), C. gustavania (MUFS-TH00-158, pi. 2, fig. 8E). 28. Chao Phraya River-3, Muang district, Nakhon Sawan province,
5. Tachin River, Muang district, Trang province, (4), C. sp. indet. (4), C. virescens (MUFS-TH00-190, pi. 4, fig. 22).
(MUFS-TH00-191, pi. 4, fig. 23). 29. Chao Phraya River-4, Kwae Noi River, Sena district, Ayutthaya
6. Ngae canal, Khlong Ngae Village, Sadao district, Songkhla pro- province, (3), C. lydigiana (MUFS-TH00-169, pi. 3, fig. 12D).
vince, (4), C. gustaviana. (MUFS-TH00-154, pi. 2, fig. 8A). 30. Irrigationcanal, Lopburitown, Lopburiprovince,(3),C. lydigiana
7. Saiburi River, Saiburi district, Pattani province, (5), C. javanica (MUFS-TH00-166, pi. 2, fig. 12A).
(MUFS-TH00-163, pi. 2, fig. 11A/B). 31. PingRiver-1,Wuttikulbridge,Takprovince,(3),C.oaudon/(MUFS-
8. Maeklong River, Muang district, Ratchaburi province, (3), C. TH00-147, pi. 1, fig. 2E).
lydigiana (MUFS-TH00-168, pi. 2, fig. 12C). 32. Ping River-2, Naowaraj bridge, ChiangMaiprovince, (3), C. noet-
9. Phetchaburi River, Muang district, Phetchaburi province, (2), C. lingi (MUFS-TH00-180, pi. 4, fig. 17D).
javanica (MUFS-TH00-164, pi. 2, fig. 11C). 33. Ping River-3, Naowaraj bridge, Chiang Mai province, (3), C.
10. Phetchaburi irrigationcanal, Muangdistrict, Phetchaburi province, leviuscula (MUFS-TH00-175, pi. 3, fig. 14B).
(2), C. lamarckiana (MUFS-TH00-172, pi. 3, fig. 13C). 34. Yom River, Muangdistrict, Praeprovince,(4),C. castanea(MUFS-
11. MekongRiver-1, Khemmaratdistrict, Ubon Ratchathani province, TH00-151, pi. 1, fig. 5).
(2), C. tenius (MUFS-TH00-189, pi. 4, fig. 21C). 35. Kuang River, Muangdistrict, Lamphun province,(3), C. messageri
12. Mekong River-2, Huai Klor, Nong Khai province, (3), C. leviuscula (MUFS-TH00-178, pi. 3, fig. 15).
(MUFS-TH00-176, pi. 3, fig. 14C). 36. MoeiRiver-1, BanTakham, Takprovince,(2),C. iravadica(MUFS-
13. Mekong River-3, Phaengvillage, Nakhon Phanom province, (3), TH00-162, pi. 2, fig. 10B).
C. leviuscula (MUFS-TH00-177, pi. 3, fig. 14D). 37. Moei River-2, Mae Sot district, Tak province, (1), C. pingensis
14. MekongRiver-4, Muangdistrict, Mukdahanprovince,(4), C. tenius (MUFS-TH00-183, pi. 4, fig. 18A).
(MUFS-TH00-188, pi. 4, fig. 21B). 38. Fang River, Fang district, Chiang Mai province, (3), C. noetlingi
15. Mekong River-5, Pakchom district, Nong Khai province, (3), C. (MUFS-TH00-181, pi. 4, fig. 17C).
arata (MUFS-TH00-142, pi. 1, fig. 1). 39. MaeLaoRiver, Muangdistrict,ChiangRaiprovince,(1),C. heard!
16. Suei River, near Nong Khai town, Nong Khai province, (5), C. (MUFS-TH00-160, pi. 2, fig. 9B).
baudoni (MUFS-TH00-144, pi. 1, fig. 2B). 40. Kaek River, Wang Thong district, Phitsanuloke province, (2), C.
17. Loei River, Muangdistrict, Loei province,(1), C. blandiana(MUFS- lamrackiana (MUFS-TH00-171, pi. 3, fig. 13A/B).
TH00-148, pi. 1, fig. 3A).