Mar. Biotechnol. 3, 407–415, 2001
DOI: 10.1007/s1012601-0060-Z
© 2001 Springer-Verlag New York Inc.
Invasion Without a Bottleneck: Microsatellite Variation in
Natural and Invasive Populations of the Brown Mussel
Perna perna (L)
Brenden S. Holland*
Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street,
Honolulu, HI 96813 U.S.A.
Abstract: Population-level genetic diversity of the brown mussel Perna perna was investigated using nuclear
microsatellite markers in 6 natural and 6 invasive populations. A total of 448 individuals from 12 populations
spanning the natural and introduced ranges of the brown mussel were scored for 2 polymorphic microsatellite
loci. Wright’s hierarchical F statistics (FST), Hardy-Weinberg equilibrium, Nei’s genetic distance, and other
descriptive statistics were used to quantify geographic population subdivision, and to estimate the number of
migrants per generation. FST values (0.007–0.042) revealed that genetic partitioning among populations was
low. Microsatellite data revealed a slight difference in observed heterozygosity and no statistically significant
differences in expected heterozygosity or allelic diversity between natural and introduced populations. Effective
numbers of migrants (Nem) per generation ranged from 6 to 35 individuals. The potential significance of an
invasive species with high genetic variation in terms of the risk of establishment and conservation implications
is discussed.
Key words: Perna perna, population structure, invasive species, DNA markers, microsatellites, F statistics.
I NTRODUCTION
An issue fundamental to the growing field of bioinvasion
science is the role of genetic variation in predisposing introduced species to successful establishment. Results of allozyme-based genetic studies of invasive mollusk populations are mixed, some have demonstrated unexpectedly low
levels of variation due to founder effects (Smith et al., 1979;
McLeod, 1986; Knight et al., 1987; Johnson, 1988), while
others have shown relatively high genetic diversity (WooReceived October 15, 2000; accepted December 29, 2000
*Corresponding author: telephone 808-539-7318; fax 808-599-4817; e-mail:
[email protected]
druff et al., 1985, 1986; Hebert et al., 1989; Duda, 1994).
However, with a few recent exceptions (Bagley and Geller,
1999), studies using molecular techniques to characterize
genetic variation in invasive marine populations during or
immediately following introduction are lacking.
Characterization of genetic structure of invasive populations is important for management as well as scientific
reasons. If genetically variable populations tend to be more
successful as invaders than those that are relatively genetically homogeneous (e.g., Ehrlich, 1986), then genetic data
may provide an important tool to resource managers concerned with invasion risk assessment and prediction. Genetic data may augment management strategy by providing
408 Brenden S. Holland
Figure 1. Sampling locales and mean
genetic diversity for invasive brown
mussel populations from the Gulf of
Mexico. Numbers following each
population code are average number of
alleles for both loci/average direct count
heterozygosity (Hdc) for both loci.
Sample codes correspond to the figure
as follows: (1) PAJ, Port Aransas Jetty;
(2) FPJ, Fish Pass Jetty; (3) MPJ,
Mansfield Pass Jetty; (4) BSP, Brazos
Santiago Pass; (5) TPN, Tuxpan; (6)
TLA, Tecolutla
guidance for focusing control and eradication efforts on
invasive taxa with the highest probability of establishment
(Holland, 2000). Currently molecular methods are proving
helpful in identification of invading taxa (Apte et al., 2000).
Recently developed molecular techniques such as multilocus genotyping may eventually be used to pinpoint geographic sources of invasive taxa (Rannala and Mountain,
1997; Davies et al., 1999). Scientifically, studies of invasion
genetics may eventually contribute to a better understanding of evolutionary processes and impacts associated with
founder effects, genetic drift, gene flow, extinction, inbreeding, and speciation. Detailed knowledge of the genetic structure of invading populations is essential to developing an
understanding of the evolutionary significance of invasive
events.
The brown mussel, Perna perna is considered endemic
to the central and southwestern Atlantic, the southeastern
Atlantic, and the southwestern Indian Oceans (Sidall, 1980).
A single colony of P. perna was discovered on a man-made
jetty at the mouth of Corpus Christi Bay, Texas, in early
1990 (Figure 1) (Hicks and Tunnell, 1993). The taxonomic
identity was verified via cytotaxonomic techniques (Holland et al., 1999). The brown mussel had become established in the northwestern Gulf of Mexico by 1992, and
continued to spread southwards into the Mexican states of
Tampico and Veracruz over the next few years by advection
of veliger larvae via prevailing surface currents (Hicks and
Tunnell, 1995; Holland, 1997).
The objectives of this study were threefold: (1) to test
for genetic variation within and between invasive and natu-
Microsatellite Variation in Brown Mussel 409
Figure 2. Sampling locales from the natural
range of Perna perna. Sample codes
correspond to the figure as follows: (1) CVN,
Cumana, Venezuela; (2) PDC, Praya de
Cibratel, Brazil; (3) RGB, Rio Grande, Brazil;
(4) CSA, Capetown, South Africa; (5) KOS,
Kenton on Sea, Transkei; (6) DCT, Diaz
Cross, Transkei. Measures of genetic variation
include average number of alleles over both
loci/average direct count heterozygosity (Hdc)
for both loci: (1) 31/0.93; (2) 33/0.90; (3)
20.5/0.90; (4) 19/0.88; (5) 24/0.85; (6)
25.5/0.92.
Table 1. Perna perna Specific Microsatellite Oligonucleotide PCR Primer Sequences
Oligonucleotide primer name and sequence
Perna microsatellite primer 1:
PMS-1f 5⬘-TCA TCT GTT GTT
PMS-1r 5⬘-GAC AAG AAG TTG
Perna microsatellite primer 2:
PMS-2f 5⬘-CGT CTC CAT CTT
PMS-2r 5⬘-GCG CAC TGT CAA
GTC TTT TTG-3⬘
ACT AGA ATA ATG-3⬘
AF236062
TAA TTA CTA-3⬘
TGT T-3⬘
AF236063
ral populations; (2) to test for the presence and extent of a
founder effect in invasive populations; (3) to describe and
compare genetic population structure of P. perna from its
natural range and from its recently expanded range in the
Gulf of Mexico.
M ATERIALS
AND
GenBank accession number of cloned
microsatellite containing insert:
M ETHODS
Sample Collection and DNA Extraction
Mussels were collected between December 1994 and June
1996, from 6 introduced populations in the western Gulf of
Mexico, including Texas and Mexican gulf coast locales
(Figure 1), and 6 natural populations spanning a broad
range of the natural distribution (Figure 2). Samples were
preserved in 80% ethanol, and genomic DNAs were extracted from gill tissue via standard phenol–chloroform–
isoamyl alcohol methods (Hillis et al., 1996).
Isolation of Microsatellites
A small insert (200–700 bp) partial genomic library was
constructed by ligating cohesive ends of Sau3A-digested
mussel DNA fragments into BamHI-linearized pUC18 vector (Invitrogen, Carlsbad, Calif.) and cloned in Escherichia
coli DH5␣ cells (Pharmacia). Recombinant bacterial colonies were identified by standard IPTG X-Gal blue/white
screening, then transferred to nylon membranes, where recombinant colonies were denatured, fixed, and screened via
hybridization to ␥-32P-labeled (GT)12 dinucleotide probes.
Microsatellite-bearing inserts were sequenced and polymerase chain reaction (PCR) primers were designed to regions
flanking the target sequence (Ferraris and Palumbi, 1996;
Hillis et al., 1996; Strassman et al., 1996).
PCR Conditions
Radioactively labeled primers were used in 30-µl PCR reactions with an oil overlay and standard conditions. Pernaspecific microsatellite primer sequences are presented in
Table 1. PCR conditions were optimized, and 32P-labeled
fragments were separated by 6% denaturing polyacrylamide
gel electrophoresis. PCR profiles were run as follows:
PMS-1 94°C, 30 seconds; 48°C, 30 seconds; 72°C, 30 seconds. PMS-2 94°C, 30 seconds; 44°C, 30 seconds; 72°C, 30
410 Brenden S. Holland
Table 2. Direct Count Heterozygosity Values for Introduced
Brown Mussel Populations from the Gulf of Mexico*
Introduced
populations
Observed heterozygote frequencies (SD)†
PAJ
(0.026)
14
FPJ
(0.024)
39
MPJ
(0.020)
59
BSP
(0.012)
32
TPN
(0.035)
29
TLA
(0.024)
49
Mean Values
0.929
(0.082)
14
0.923
(0.0094)
39
0.915
(0.016)
60
0.844
(0.015)
32
0.690
(0.0094)
26
0.816
(0.013)
49
Hi1 = 0.853
(0.024)
Locus PMS1
Table 3. Direct Count Heterozygosity Values for Natural
Populations*
Observed heterozygote
frequencies (SD)†
Locus PMS2
0.571
0.769
0.783
0.781
0.769
0.776
Hi2 = 0.741
(0.024)
*Population acronyms are as follows: MPJ, Mansfield Pass Jetty; BSP,
Brazos Santiago Pass; Mexican Gulf localities are TPN, Tuxpan; TLA,
Tecolutla.
†Population sample sizes are shown below standard deviations.
seconds. Polyacrylamide gels were vacuum dried and placed
on autoradiographic film at −80°C overnight. F statistics
and genetic distances were calculated using Microsat C
(Minch, 1995).
R ESULTS
Unprecedented levels of genetic variation were revealed in
the present study. Although 6 microsatellites were isolated,
the 2 markers used in this study required an unexpected
amount of time and effort to characterize. Heterozygosity
values of polymorphic loci from introduced versus natural
populations of P. perna are shown in Tables 2 and 3. Levels
of heterozygosity are shown for each population sampled as
well as the mean value and SD for all introduced and all
natural populations for each locus, as well as over all popu-
Natural populations
PMS1
PMS2
CVN
(0.044)
47
RGB
(0.015)
26
PDC
(0.024)
46
CSA
(0.025)
17
KOS
(0.010)
28
DCT
(0.019)
30
Mean values
0.979
(0.014)
41
0.847
(0.021)
22
0.860
(0.009)
46
0.824
(0.010)
15
0.903
(0.048)
29
0.867
(0.025)
29
Hn1 = 0.880
(0.023)
HO = 0.875
(0.024)
0.878
Overall means for
all populations
(natural and introduced)
0.957
0.930
0.933
0.80
0.966
Hn2 = 0.911
(0.021)
HO = 0.829
(0.023)
*Locality acronyms from within the natural range are CVN, Cumana Venezuela; RGB, Rio Grande Brazil; PDC, Praya Del Cibratel Brazil; CSA,
Capetwon, South Aftrica; KOS, Kenton on Sea, Transkei; DCT, Diaz Cross,
Transkei.
†Population sample sizes are shown below standard deviations.
lations. The remainder of the microsatellite data are summarized in Tables 4 through 8. Number of alleles per locus
is shown in Tables 4 and 5 for introduced versus natural
populations. Unbiased heterozygosities were computed and
Hardy-Weinberg equilibrium (HWE) was tested using TFPGA software (Miller, 1997). Nine of the 12 populations
were found to conform to the HWE model for each locus.
Values for numbers of alleles revealed high allelic diversity
both in introduced and in natural populations. Tables 6 and
7 summarize the results in a locus-specific manner, and
include number of samples scored, heterozygosity per
population, and fixation index relative to the Port Aransas
population. Table 8 shows the Microsat C (Minch, 1995)
data summary for both loci and all populations.
Microsatellite Variation in Brown Mussel 411
Table 4. Number of Alleles per Locus from Introduced Populations*
Table 5. Number of Alleles per Locus from Natural Populations*
Number of alleles (SD)†
Number of alleles per locus (SD)†
Natural populations
PMS1
Introduced populations
Locus PMS1
CVN
PAJ
15
(s = 2.61)
14
21
(0.076)
39
25
(1.86)
59
21
(0.52)
32
21
(0.076)
29
22
(0.52)
49
Ni1 = 20.83
(0.94)
30
(3.50)
47
20
(0.97)
26
26
(1.71)
46
14
(3.65)
17
24
(0.82)
28
19
(1.42)
30
Nn1 = 22.17
(s = 2.01)
NO1 = 21.5
(s = 1.48)
14
FPJ
MPJ
BSP
TPN
TLA
Mean values
Locus PMS2
15
(s = 5.59)
RGB
32
(2.01)
39
39
(5.14)
59
27
(0.22)
32
22
(2.46)
26
30
(1.12)
49
Ni2 = 27.5
(2.76)
*For population designation codes see footnote to Table 2.
†Sample sizes are shown below standard deviations.
Table 6 shows FST and Nei’s genetic distance values
produced via Microsat C (Minch, 1995). FST values were
found to be low, and ranged from 0.004 to 0.05 for locus
PMS1, from 0.005 to 0.07 for locus PMS2, and from 0.007
to 0.042 for both loci combined. Individual as well as overall
FST values corresponded to high rates of gene flow among
populations. Number of migrants between populations per
generation was estimated as follows: Nm = 1 − FST/4 FST
(Waples, 1998). The range of migrants per generation was
estimated to be between 6 and 35 individuals exchanged per
generation. This formula provides a rough approximation
and should be used only as a general guideline, particularly
when FST is small (Whitlock and McCauley, 1999).
These values indicated high gene flow and a lack of
population genetic divergence due to population subdivision. Table 7 summarizes genetic diversity data for all populations grouped by source, as either natural or introduced.
An unweighted pair-group method with arithmetic mean
(UPGMA) tree was constructed using TFPGA (Figure 4).
PDC
CSA
KOS
DCT
Mean values
Mean over all
populations
PMS2
32
(1.42)
41
21
(3.5)
22
40
(5.00)
46
24
(2.16)
15
24
(2.16)
29
32
(1.42)
29
Nn2 = 28.83
(s = 2.61)
NO2 = 28.17
(s = 2.69)
*For population designation codes see footnote to Table 3.
†Sample sizes are shown below standard deviaitons.
Unfortunately, the allelic variation was so high that the
source population could not be resolved for the sample sizes
used in this study.
D ISCUSSION
Population genetic theory predicts that bottlenecks can be
detected by rapid and often drastic decreases in allele frequency (Cornuet and Luikhart, 1996; Hedrick, 2000). Heterozygosity may not be lost at as rapid a rate as allelic
diversity, but if the bottleneck persists over multiple generations, then drift begins to drive allele frequencies toward
fixation (Hartl and Clark, 1997). P. perna populations in the
Gulf of Mexico were characterized by (1) high genetic variation within populations, (2) negligible population subdivision, and (3) lack of heterozygote deficiencies predicted for
bioinvasions and commonly encountered in mussel populations. Mean genetic diversity values for brown mussels are
at the upper extreme for mollusks (Reichow and Smith,
412 Brenden S. Holland
Table 6. Among-population Pairwise Fixation Indices (FST) for Both Loci PMS-1 and PMS-2 (above the diagonal), and Nei’s Genetic
Distance Values Averaged for Each Population (12 populations) for Both Loci PMS-1 and PMS-2 (below the diagonal)*
PAJ
FPJ
MPJ
BSP
TPN
TLA
CVN
RGB
PDC
CSA
KOS
DCT
PAJ
FPJ
MPJ
BSP
TPN
TLA
CVN
RGB
PDC
CSA
KOS
DCT
—
0.252
0.301
0.218
0.375
0.442
0.384
0.338
0.301
0.330
0.297
0.434
0.025
—
0.056
0.149
0.127
0.099
0.255
0.093
0.229
0.177
0.320
0.238
0.028
0.007
—
0.105
0.233
0.145
0.336
0.270
0.134
0.218
0.435
0.101
0.024
0.013
0.010
—
0.180
0.199
0.649
0.273
0.078
0.156
0.388
0.166
0.035
0.013
0.019
0.017
—
0.210
0.426
0.220
0.165
0.201
0.295
0.310
0.037
0.010
0.012
0.017
0.018
—
0.304
0.109
0.172
0.108
0.442
0.306
0.031
0.019
0.028
0.036
0.022
0.026
—
0.202
0.352
0.597
0.329
0.391
0.033
0.011
0.022
0.024
0.021
0.012
0.022
—
0.168
0.220
0.098
0.323
0.028
0.017
0.011
0.008
0.015
0.014
0.020
0.016
—
0.102
0.240
0.164
0.042
0.018
0.019
0.016
0.021
0.013
0.033
0.027
0.011
—
0.310
0.191
0.028
0.023
0.028
0.027
0.023
0.030
0.032
0.010
0.017
0.032
—
0.295
0.036
0.018
0.009
0.014
0.024
0.023
0.038
0.026
0.013
0.019
0.021
—
*Data were generated using Microsat C (Minch, 1995).
Table 7. Comparison of Microsatellite Data from Introduced
and Natural Populations of Perna perna in Terms of Allelic Diversity, Observed and Expected Heterozygosity, and Mean FST
Value*
Population
Introduced
(6 populations)
Natural
(6 populations)
Heterozygote
frequency
(averaged for all
populations,
both loci)
Allelic
diversity
(averaged for all
populations,
both loci)
HE 0.949
HO 0.798
26.67
24.17
0.019
HE 0.945
HO 0.897
25.5
24.84
0.022
Mean
FST
value
*Genetic diversity of natural compared with introduced populations
showed a slight reduction in observed heterozygosity (HO), with no significant difference in ec expected heterozygosity (HE), number of alleles
per locus, or mean FST.
1999) (Table 4). Perna perna populations sampled from the
natural range are also characterized by (1) high genetic
variation within populations, (2) negligible geographic subdivision among populations, and (3) lack of heterozygote
deficiencies commonly encountered in bivalve mollusks
(e.g., Zouros and Foltz, 1984).
The high levels of genetic diversity shown by P. perna in
the Gulf of Mexico result from the introduction of a genetically diverse assortment of larvae. Several possible explanations include more than one introduction event, with
larvae originating from numerous source populations; a
single introduction of larvae picked up at several genetically
distinct source populations; and a single introduction of a
large number of larvae from a genetically diverse source
population. These hypotheses were tested by comparing the
genetic composition and population structure of introduced versus natural populations. The result of this comparison revealed a lack of genetic structure in natural and
nonindigenous P. perna populations, as evidenced by the
low FST values. These values indicated little or no inbreeding due to subdivision of populations and reflected regular
exchange of alleles among populations, with little or no
population-level differentiation detectable. This lack of
population subdivision was likely brought about by extremely high levels of gene flow. If the founding population
had originated from multiple source populations, a Wahlund effect would be expected, characterized by higher heterozygosity in introduced populations than in each natural
population (Ayala, 1982; Hartl and Clark, 1997; Hedrick,
2000). This was not observed.
The pattern of genetic variability observed provided
support for the hypothesis that the genetic structure of
source populations of P. perna is reflected in the structure of
introduced populations. The high levels of allelic diversity
and heterozygosity exhibited in invasive populations were
characteristic of the source population, and would not have
Microsatellite Variation in Brown Mussel 413
Table 8. Exact Tests, ␹2 Tests, and Calculation of Expected Heterozygosities (Miller, 1997)*
Population
Introduced
PAJ
FPJ
MPJ
BSP
TPN
TLA
Natural
CVN
PDC
RGB
CSA
DCT
KOS
Expected heterozygote frequency
(unbiased heterozygosity
averaged for both loci)
HWE locus 1
exact test
HWE locus 2
exact test
HWE locus
1 ␹2
0.9431
0.9559
0.9471
0.9521
0.9487
0.9490
+
+
+
+
−
+
+
+
−
+
+
−
+
+
+
+
−
+
+
+
−
+
+
−
0.9497
0.9378
0.9618
0.9388
0.9370
0.9468
+
−
+
+
+
+
−
+
+
+
+
+
+
−
+
+
+
+
−
+
+
+
+
+
HWE locus
2 ␹2
*Results of exact tests and ␹2 were identical, i.e., the 2 tests identified the same loci or populations in violation of Hardy-Weinberg equilibrium. The plus
symbol is used to indicate conformation with the HWE model, while the minus symbol indications violation of the model. One introduced population
was not at HWE for locus 1, and 2 introduced populations violated HWE at locus 2. For natural populations, one population violated HWE at locus 1,
and one population violated HWE at locus 2. In summary, 38 of 48 tests conformed to HWE.
been detected in the case of a severe bottleneck event. Although a slight decrease in observed heterozygosity was seen
in introduced populations, no significant differences in allelic diversity were found. Since only minor differences were
detected in population structure (FST) and in genetic variation of natural versus introduced populations, we can conclude that the effective population size of introduced mussel
larvae was large, likely consisting of many thousands of
successful recruits. Although only a single colony of P. perna
was discovered in 1990 (Hicks and Tunnell, 1993), genetic
evidence indicated that many thousands of undetected
mussels were present in the Gulf at that time.
Perna perna had been in the Gulf for between 4 and 6
years when tissue samples were collected. A conservative
estimate is that brown mussels in the Gulf may spawn 3
times per year (D. Hicks, University of Texas, personal
communication). Therefore, P. perna had been in the Gulf
for at least 12 to 18 generations. Theoretical models of the
effects of genetic drift on allele frequency have shown that
allele fixation and loss are observed as founding populations
grow (e.g., Hartl and Clark, 1997). The exact number of
generations required for the effects of drift to become apparent depends on the effective population size and genetic
composition of the founders. The fact that introduced
populations of P. perna have not exhibited significant impacts due to genetic drift to date, coupled with the high
heterozygosity shown by invasive populations, eliminates
the possibility that genetic drift simply has not yet been
observed.
The lack of evidence of a severe population bottleneck
provides, in a sense, additional cause for concern in the face
of the increasing incidence of global ballast water introductions. Data presented in this investigation provide the first
evidence of the ability of a single ballast water introduction
to capture and translocate a gene pool virtually in its natural
state, a phenomenon that might be termed a “gene pool
capture” event. As the bottleneck model illustrates, founder
events result in genetically limited and therefore usually
suboptimal assortments of genotypes. On the one hand,
when faced with heterogeneous foreign habitats and “unfamiliar” ecological conditions, a genetically homogeneous
founder population has a limited gene pool from which to
reproductively “draw” successful allelic combinations. On
the other hand, the fitness of a highly variable assortment of
genotypes is predicted to be adaptively and evolutionarily
superior under a heterogeneous, or changing set of selective
414 Brenden S. Holland
Figure 3. Representative 32P-labeled microsatellite PCR amplification products from P. perna are shown. Following separation via
denaturing 6% polyacrylamide gel electrophoresis, microsatellites
were visualized via exposure to autoradiographic film. Both dinucleotide microsatellite markers were highly polymorphic for all
12 populations surveyed. Individuals shown were from a single
population, amplified using primer set PMS-1.
conditions. The result is a theoretical increase in the probability of successful establishment of introduced taxa with
high genetic diversity.
Although the delivery mechanism of P. perna in the
Gulf of Mexico is unknown, it is most likely due to ballast
water discharge (Holland, 1997; Hicks and Tunnell, 1995).
Ballast water transport provides a potential mechanism for
introduction of hundreds of millions of planktonic individuals in a single discharge event, owing to the massive
volumes of ballast water routinely conveyed by commercial
bulk carriers (Schormann et al., 1990) and the rapid transit
times typical of modern commercial shipping practices.
This study reveals yet another insidious and devastating
potential effect of the ballast water delivery mechanism—
that is, its ability to transport larvae in sufficient numbers to
avoid bottleneck effects and reduction of genetic diversity.
The brown mussel has maintained high levels of genetic
variation in introduced populations, reflecting panmixia
within populations, high gene flow among populations, and
possibly balancing selection in natural populations. This
emphasizes the serious nature of the global environmental
threat posed by ballast water introductions. Several authors
have suggested that high genetic variability is an important
Figure 4. UPGMA tree showing relationships among 12 Perna
perna populations based on Nei’s genetic distance (1978) generated via TFPGA (Miller, 1997). Populations 1–6 were introduced
to the Gulf of Mexico, 7–12 are from the natural range of this
species. OTU’s are as follows: (1) PAJ, Port Aransas Jetty, Texas;
(2) FPJ, Fish Pass Jetty, Texas; (3) MPJ, Mansfield Pass Jetty,
Texas; (4) BSP, Brazos Santiago Pass, Texas; (5) TPN, Tuxpan,
Mexico; (6) TLA, Tecolutla, Mexico; (7) CVN, Cumana Venezuela; (8) PDC, Praya de Cibratel, Brazil; (9) RGB, Rio Grande,
Brazil; (10) CSA, Capetown, South Africa; (11) KOS, Kenton on
Sea, Transkei; (12) DCT, Diaz Cross, Transkei.
characteristic of successful invasive populations (e.g.,
Ehrlich, 1986). The levels of genetic variation in P. perna
from the Gulf of Mexico observed in this study and the
corresponding success of establishment by the brown mussel support this theory. The high genetic diversity found in
introduced P. perna populations also underscores the serious nature of the threat to natural marine communities
posed by the transport and discharge of nonindigenous marine taxa by commercial shipping. The application of molecular techniques to biological invasions may prove a crucial tool in the efforts to better understand, predict, and
ultimately prevent future introduction events.
A CKNOWLEDGMENTS
This study was supported by the Texas A&M University
Research Foundation, and was conducted in partial fulfillment of the requirements for a Doctor of Philosophy degree
in the Department of Oceanography at Texas A&M University. I thank S.R. Gittings, S. Davis, E. Arrevalo, and D.
Gallagher, and my committee for guidance during this
Microsatellite Variation in Brown Mussel 415
study. I also thank M. Jackson for field assistance in Texas,
Y. Barrios for field assistance in Mexico, and J. Lindsay and
D.W. Hicks for providing mussel samples from southern
Africa, and S.K. Davis, E.C. Rios, D.W. Hicks, and V. Fagundes for providing samples from Brazil and Venezuela.
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