Journal of Shellfish Research, Vol. 28, No. 3, 453–458, 2009.
LABORATORY HYBRIDIZATION BETWEEN CRASSOSTREA ARIAKENSIS AND C. SIKAMEA
FEI XU,1,2 GUOFAN ZHANG,1* XIAO LIU,1 SHOUDU ZHANG,1,2 BIN SHI3 AND
XIMING GUO4*
1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 2Graduate University of
Chinese Academy of Sciences, Beijing 100049, China; 3Nantong Ocean and Fisheries Office, Jiangsu
Province 226018, China; 4Haskin Shellfish Research Laboratory, Institute of Marine and Coastal
Sciences, Rutgers University, Port Norris, New Jersey 08349
ABSTRACT To understand possible reproductive interaction between Crassostrea ariakensis (Fujita, 1913) and C. sikamea
(Amemiya, 1928), which coexist in estuaries of China and Japan, we conducted 2 3 2 factorial crosses between the two species.
Asymmetry in fertilization success was observed, where C. sikamea eggs can be fertilized by C. ariakensis sperm, and the reciprocal
cross resulted in no fertilization. Fertilization success in C. sikamea female 3 C. ariakensis male (SA) crosses was lower than that
in the two intraspecific crosses and produced larvae that had similar growth rate as their maternal species during the first nine days
because of maternal effects. After that, genome incompatibility casted negative effects on the growth and survival of the hybrid
larvae. Most hybrid larvae died during metamorphosis, but a small number of spat survived. Genetic analysis revealed that the
survived SA spat contained DNA from both species and were true hybrids. This study demonstrates that hybridization between C.
ariakensis and C. sikamea is possible in one direction.
KEY WORDS: Crassostrea ariakensis, C. sikamea, oyster, hybridization, reproductive isolation, internal transcribed spacer
INTRODUCTION
Species form because of prolonged reproductive isolation.
Hybridization occurs when two species meet and the reproductive isolation is incomplete. Hybridization zones may exist
where two closely related species overlap in distribution.
Studying reproductive isolation and potential interaction
among closely related species is important to our understanding
and management of genetic diversity. It may also shed light on
mechanisms of reproductive isolation and speciation.
There are at least five Crassostrea oyster species naturally
occurring along the coast of China. They include Crassostrea
gigas, C. ariakensis, C. sikamea, C. angulata, and C. hongkongensis (Wang et al. 2006). These species often occur in the same
estuary. In North China, C. ariakensis coexist with C. gigas. In
Central and South China, C. ariakensis, C. hongkongensis, C.
angulata and C. sikamea may be found in the same estuary in
various combinations. In Nantong, for example, two species, C.
ariakensis and C. sikamea, live closely together on the same
oyster reef (Guo et al. 2008). Genetic identification using
multiple genetic markers failed to detect any hybrids among
578 oysters collected from Nantong (Wang et al. 2008). C. gigas,
C. ariakensis, and C. sikamea also coexist in Ariake Sea, Japan,
without any detectable hybrids (Hedgecock et al. 1999).
The absence of naturally occurring hybrids between C.
ariakensis and C. sikamea is interesting and raises the question
whether hybridization between the two species is possible.
Reports on hybridization among Crassostrea oysters appeared
almost every decade since the 1920s. Some species can be easily
hybridized, such as C. gigas and C. angulata (Imai & Sakai 1961,
Menzel 1974, Numachi 1977, Soletchnik et al. 2002, Batista
et al. 2007). The ease to hybridize C. gigas and C. angulata has
led to questions about their status as two independent species
(Huvet et al. 2002). Hybridization has been reported between
some other species without genetic confirmation of the hybrids
*Corresponding authors. E-mail: [email protected], xguo@hsrl.
rutgers.edu
or the parental species (Gaffney & Allen 1993). Zhou et al.
(1982) reported success in hybridizing three species, C. gigas, C.
rivularis, and C. plicatula without genetic confirmation. The
identity of these species is now in question because they have
been frequently misidentified. The name C. rivularis was used
for 2 species (C. hongkongensis and C. ariakensis), and C.
plicatula has been rejected as a taxonomic species (Wang et al.
2004, Wang et al. 2007, Wang & Guo 2008a). As far as we can
determine, no hybridization between C. ariakensis and C.
sikamea has been reported. In this study, we conducted
laboratory hybridization between C. ariakensis and C. sikamea,
and used molecular markers to confirm the identity of parental
species and hybrids. Here we report that the two species can
hybridize in one direction.
MATERIALS AND METHODS
Oysters and Gametes
Sexually mature oysters were obtained in mid-June 2007
from Xiaomiaohong oyster reef, Nantong, China. After collection, oysters were transported to Qingdao and cultured in the
laboratory of Institute of Oceanology, Chinese Academy of
Sciences. Single oysters were separated from clusters and
cleaned. Because C. ariakensis and C. sikamea differ considerably in size and shape, initial identification was done based on
shell morphology. After gametes were collected, tissues from all
parents used for hybridization were fixed for subsequent confirmation with genetic markers (Wang & Guo 2008a).
Parental oysters were opened, and gametes were obtained by
dissection. Before working on an individual, all tools and
containers were thoroughly washed with freshwater to avoid
cross-contamination. Gonad of each opened oyster was sampled and examined under a light microscope to determine sex.
Four females and three males from each species were chosen for
gamete collection. Eggs from the four females were rinsed with
sand-filtered seawater and pooled into one beaker. Egg suspension was passed through a 90-mm nylon screen, rinsed on a
453
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XU ET AL.
25-mm nylon screen, and resuspended in seawater. Sperm from
each male were washed into separate beakers and diluted until
suspensions from the three males showed about the same
density or cloudiness. Sperm from the three males were then
pooled in equal volume.
Experimental Design and Larval Culture
Eggs from C. ariakensis (designated as A) were equally
divided into two parts: one inseminated with sperm from C.
ariakensis and the other with sperm from C. sikamea (designated
as S). Similarly, eggs from C. sikamea were divided into two
equal parts and fertilized with sperm from A and S separately.
Thus, 2 3 2 factorial crosses were created producing four
groups: AA, AS, SA, and SS with female species listed first.
Insemination was made within 60 min after the eggs were
dissected. Before insemination, eggs were checked to ensure
there was no uncontrolled fertilization as indicated by the
absence of polar bodies. Sperm were added to the egg suspension, until about 20 sperm surrounded an egg. For interspecific
crosses, about 20% more sperm were added. The experiment
was replicated three times using different sets of parental oysters.
Mixtures of sperm and eggs were sampled and held in beakers to
assess fertilization success and survival to D-stage. The remaining eggs were counted and reared in 40-L bucket with aeration at
a density of 150/mL. Larvae were cultured in filtered-seawater at
23°C, and the salinity was about 30 ppt.
For each bucket, larval density was adjusted to 20 per
milliliter at D-stage or 24 h after fertilization. Excess larvae
from the hybrid SA cross were kept in additional tanks in an
attempt to obtain enough survivors. Seawater was changed
every two days. During the initial period (from Day 1–10),
larvae were fed with Isochrysis galbana. Starting from Day 11,
larvae were fed with a mixture of Platymonas subcordiformis
and I. galbana. Feeding ration was gradually increased with
time from 20,000–90,000 cells/mL. When larvae reached eyedstage and appeared ready to settle around day 35, strings of
scallop shells were hung in the buckets as cultch.
Measurements and Sampling
Fertilization success was determined at 60–90 min postinsemination. Eggs that divided were considered as fertilized,
and the percentage of divided eggs was used as a measure of
fertilization success.
Survival to D-stage was determined as the percentage of
fertilized eggs that developed into D-stage larvae at 24 h
postinsemination. Survival and growth of larvae were monitored during the first 30 days of culturing. Seawater was
completely changed every two days. At each water change,
the number of larvae was determined, and the shell height of 30
larvae was measured. The last larval count and measurement
were taken at Day 35 just before metamorphosis. After
settlement, the number of spat in each group was counted,
and the shell height of 30 spat was measured at Day 93.
Surviving spat were sampled at Day 93 and fixed in 95%
ethanol for genetic confirmation.
Genetic Confirmation
DNA was extracted from ethanol-fixed samples using the
TIANamp Marine Animals DNA kit (Tiangen). Genetic iden-
tification of parents was conducted using a multiplex speciesspecific PCR targeting the mitochondrial cytochrome oxidase I
(COI) gene (Wang & Guo 2008a). A common forward primer
(5#-GGTCAACAAATCATAAAGATATTGG) was used with
two species-specific reverse primers: 5#-AAGTAACCTTAAT
AGATCAGGGAACC for C. sikamea and 5#-AAAAAAGAT
TATAACTAATGCATGTCGG for C. ariakensis, generating
fragments of different sizes. PCR amplification was performed
in 25-mL volume containing 2.0 mM MgCl2, 0.15 mM dNTP,
0.2 mM of each primer, 20 ng of template DNA, 1 U Taq
polymerase, 2.5 mL 10 3 PCR buffer. The thermal cycler
protocol consisted of an initial denature at 95°C for 2 min, 30
cycles of 95°C for 1 min, 51°C for 1 min and 72°C for 1 min, with
a final extension at 72°C for 5 min. A negative control with no
template and two positive controls with DNA template from
previously identified C. sikamea and C. ariakensis were included
in the experiment.
Genetic confirmation of hybrids was conducted using the
ITS1 (internal transcribed spacer 1) markers (Wang & Guo
2008b). Primer sequences for ITS1 were 5#-GTTTCCGTAGG
TGAACCTGC (28S forward) and 5#-ACACGAGCCGAGTG
ATCCAC (5.8S reverse). PCR was performed in 25 mL volume
containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 mM of each
primer, 20 ng of template DNA, 1 U Taq polymerase, 2.5 mL
10 3 PCR buffer, and 0.4 mg/mL BSA. The thermal cycler
protocol consisted of an initial denature at 95°C for 5 min, 30
cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min,
with a final extension at 72°C for 5 min. Three controls were
included in the experiment: one with DNA from an identified C.
sikamea parent, one with DNA from a C. ariakensis parent, and
the other with mixed DNA of these two parents.
All amplified fragments were separated on agarose gels,
1.5% for COI and 3% for ITS1, containing 0.2 mg/mL ethidium
bromide, and visualized under a UV transilluminator (BIORAD)
for species identification based on fragment length.
Data Analyses
One-way ANOVA was used to test the effects of crosses.
Fertilization success and survival to D-stage were arcsinetransformed prior to analysis, and cumulative larval survival
to Day 35 and 93 was square root-transformed. Fisher LSD ttest was used to compare differences among groups.
RESULTS
Fertilization
Fertilization in two intraspecific crosses was good and comparable. The mean fertilization success of C. ariakensis 3 C.
ariakensis (AA) crosses was 84.5%, and that of C. sikamea 3
C. sikamea (SS) crosses was 75.4% (Table 1). The difference was
not statistically significant (Table 2). Eggs of C. sikamea can be
fertilized by sperm from C. ariakensis without obvious delays or
signs of abnormality, although the fertilization success, 12.5%,
was significantly lower than the two intraspecific crosses. No
fertilization occurred in the C. ariakensis $ 3 C. sikamea #
(AS) hybrid crosses, despite the addition of excess sperm. No
polar body release or cell division was observed, and the eggs
decomposed within 24 h. In SA crosses, the eggs of C. sikamea
that were not fertilized by C. ariakensis sperm, because of low
HYBRIDIZATION BETWEEN C. ARIAKENSIS AND C. SIKAMEA
455
TABLE 1.
Fertilization success, percent survival of fertilized eggs to D-stage, and cumulative survival of D-stage larvae to different days
postfertilization in 2 3 2 factorial crosses between C. ariakensis (A) and C. sikamea (S). Female species are listed first in group names.
Group
Replicate
AA
1
2
3
Mean
1
2
3
Mean
AS
SA
SS
1
2
3
Mean
1
2
3
Mean
No. of Eggs
( 3 1,000)s
Fertilization
(%)
Survival to
D-stage (%)
Survival to
Day 3 (%)
Survival to
Day 11 (%)
Survival to
Day 35 (%)
Survival to
Day 93 (%)
14,667
8,667
5,000
81.60
86.70
85.30
84.53
0
0
0
0
95.80
60.80
59.40
72.00
—
—
—
95.80
60.80
58.09
71.56
—
—
—
27.59
9.79
7.13
14.84
—
—
—
2.40
0.06
1.71
1.39
0.0575
0.0006
0.0653
0.0411
2.70
14.90
19.90
12.50
74.00
82.00
70.30
75.43
45.00
50.50
70.80
55.43
62.60
45.50
68.50
58.87
45.00
50.50
59.76
51.75
52.52
34.49
35.83
40.95
11.25
4.70
9.42
8.45
13.40
9.65
3.90
8.98
0.29
0.03
0.09
0.14
0.21
0.04
0.04
0.10
0.0009
0.0002
0.0014
0.0008
0.0313
0.0023
0.0274
0.0203
14,667
8,667
5,000
20,000
22,667
11,500
20,000
22,667
11,500
fertilization success, remained intact for two to three days in
seawater.
Survival
Although fertilization success differed among crosses, survival of fertilized eggs to D-stage larvae was similar in the three
crosses where fertilization occurred. The survival of fertilized
eggs to D-stage was 72.0% for AA, 58.9% for SS, and 55.4%
for SA (Table 1). The differences were not statistically significant (Table 2). Considering differences in fertilization, the
percentage of eggs that were fertilized and developed to
D-stage was 66.1% for AA, 45.5% for SS, 7.6% for SA and
0% for AS.
Survival of D-stage larvae within the first 13 days was about
the same in all three groups (Fig. 1). Larvae in SA and SS suffered
heavier mortalities than AA after Day 13. At Day 35, cumulative
survival of D-stage larvae was 1.39% for AA, 0.10% for SS, and
0.14% for SA (Table 1). Severe mortality occurred in SA crosses
during metamorphosis, and only 16 spat were obtained at Day 93
from three replicates, corresponding to a cumulative survival of
0.008%. Another 152 SA spat were obtained from 3.8 million
excess D-larvae that were maintained separately, producing a
similar survival of 0.004%. In comparison, AA and SS crosses
produced 1,000 (0.04% of D-larvae) and 580 (0.02% of D-larvae)
spat, respectively, at Day 93. The cumulative survival to Day 93
was about three to seven times higher in the intraspecific crosses
than in the SA hybrid cross (Table 1).
Larval and Juvenile Growth
The growth of early larvae was influenced by the maternal
species more than the paternal species. Larvae produced from
eggs of C. sikamea (SS and SA) grew slower than that produced
from C. ariakensis (AA) under our conditions. At Day 1, the
average shell height of D-stage larvae was 73.6 mm for SS,
72.3 mm for SA, and 82.4 mm for AA (Table 3). There was no
significant difference between SS and SA larvae, but both were
significantly (P < 0.01) smaller than AA larvae (Table 2).
During the first nine days, SA and SS larvae grew at similar
TABLE 2.
One-way ANOVA analysis of several parameters with crosses as a factor.
Traits
DF
MS
P
Multiple Comparison
(LSD)*
Fertilization success
Hatching success
Larval survival at day 35
Juvenile yield at day 93
Larval shell height at day 1
Larval shell height at day 11
Larval shell height at day 35
Juvenile shell height at day 93
2
2
2
2
2
2
2
2
0.6700
0.0600
0.0050
0.0002
787
6,368
109,442
201
<0.01
0.431
0.131
0.176
<0.01
<0.01
<0.01
<0.01
SAa < SSb < AAb
SA < SS < AA
SS < SA < AA
SA < SS < AA
SAa < SSa < AAb
SAa < SSb < AAc
SAa < SSb < AAc
SAa < AAb < SSc
* Different letters within each row indicate the means are different significantly (P < 0.05).
XU ET AL.
456
AA larvae being the largest and SA larvae the smallest. Eyed
larvae in SA appeared three days later than the two intraspecific
controls. After metamorphosis, the growth of SS spat accelerated. At Day 93, spat from SS became the largest among
the three crosses, whereas spat of SA remained the smallest
(Table 3).
Genetics Confirmation
Figure 1. Growth (A) and survival (B) curves of hybrid and control larvae
from Day 1–29. Survival data were standardized by setting Day 1 as
100%, multiplied by 1,000 and then Log10-transformed.
rates (Fig. 1). After that, the growth of hybrid SA larvae slowed,
and significant (P < 0.01) difference in shell height between SA
and SS larvae appeared on Day 11 (Table 2). When larvae
reached eyed stage at Day 35, larvae from all three groups
showed significantly different shell height (P < 0.01), with
PCR was successful with COI and ITS1 primer sets for all
samples analyzed. All parents used in this study were unambiguously identified as C. ariakensis or C. sikamea with both COI
(photo not shown here) and ITS1 (Fig. 2) markers. Spat of C.
ariakensis and C. sikamea produced single bands with ITS1
primers at about 515 and 510 bp, respectively. All hybrid spat
from SA produced two bands: one between 510 and 515 bp, and
the other at about 530 bp. The same two bands were obtained in
the positive control where mixed DNA from C. sikamea and C.
ariakensis was used as template (Fig. 2), suggesting that spat
from SA are true hybrids. We noticed, however, neither of the
two bands matched the fragment size of the two pure species
and were both slightly higher than the expected sizes. Therefore,
we sequenced all four fragments, ‘‘a’’, ‘‘s’’, ‘‘ab’’, and ‘‘sb’’ (Fig.
2), for confirmation.
Fragment ‘‘s’’ produced a 530 bp sequence containing a 465
bp ITS1, which matched to the ITS1 sequence of C. sikamea
(FJ222344.1, e-value ¼ 0.0, identities ¼ 98%). The ITS1
sequence (470 bp) from fragment ‘‘sb’’ also matched to that of
C. sikamea (FJ222345.1, e-value ¼ 0.0, identities ¼ 98%), but
was 5 bp longer than fragment ‘‘s’’ because of six insertions and
one deletion (Fig. 3). Most of the changes in sequence involved
tandem repeats. For example, between 209 and 217 bp, sequence
‘‘s’’ had (CCT)3, but sequence ‘‘sb’’ had (CCT)4. Fragment ‘‘a’’
produced a sequence of 536 bp containing a 471 bp ITS1 that
matched to the ITS1 sequence of C. ariakensis (EU073288.1, evalue ¼ 0.0, identities ¼ 100%). The ITS1 sequence (479 bp)
from fragment ‘‘ab’’ also matched to the ITS1 of C. ariakensis
(EU073286.1, e-value ¼ 0.0, identities ¼ 98%), but was 8 bp
longer than sequence ‘‘a.’’ The increased length was mostly
caused by insertions that involve poly-T or poly-A repeats.
TABLE 3.
DISCUSSION
Size of larvae and spat from C. ariakensis 3 C. ariakensis (AA),
C. sikamea 3 C. sikamea (SS) and C. sikamea 3 C. ariakensis
(SA) crosses at different days postfertilization.
Results of this study clearly demonstrate that hybridization
between C. ariakensis and C. sikamea is possible in one
Group
Replicate
Day 3
(mm)
Day 11
(mm)
Day 35
(mm)
Day 93
(mm)
AA
1
2
3
Mean
78.2
81.5
87.5
82.4
93.4
91.8
112.0
99.0
243.8
184.7
264.9
231.1
3.6
3.6
3.2
3.5
SA
1
2
3
Mean
72.0
72.4
72.4
72.3
83.9
83.0
79.9
82.3
180.0
150.5
160.3
163.6
1.4
1.6
2.2
1.7
1
2
3
Mean
72.3
71.9
76.7
73.6
98.2
84.8
85.1
89.4
247.8
197.5
192.4
212.6
5.9
7.2
5.8
6.3
SS
Figure 2. ITS1 PCR fragments from parental species and their offspring.
Sample codes are: M, marker DL2000; 1, C. sikamea parent; 2, C.
ariakensis parent; 3, mixed DNA of 1 and 2; 4–11, hybrid spat from C.
sikamea female 3 C. ariakensis male cross; 12–14, spat of C. sikamea;
15–17, spat of C. ariakensis. Fragments marked with ‘‘s’’, ‘‘a’’, ‘‘sb’’, and
‘‘ab’’ are purified and sequenced.
HYBRIDIZATION BETWEEN C. ARIAKENSIS AND C. SIKAMEA
Figure 3. Alignment of four ITS1 sequences corresponding PCR fragments ‘‘a’’, ‘‘ab’’, ‘‘s’’, and ‘‘sb’’ in Figure 2. Different sites of the four
sequences are shown in bold letters. Interspecific differences are indicated
by shaded letters. Difference sites between ‘‘a’’ and ‘‘ab’’, ‘‘s’’, and ‘‘sb’’
are shown in boxed letters.
direction. C. sikamea eggs can be fertilized by C. ariakensis
sperm, but fertilization does not occur in the other direction.
Although fertilization was possible in SA crosses, the success
level was lower in SA than in the intraspecific crosses. In
addition to the three replicates reported here, four other
replicates of fertilization experiments were made on July 5,
2006 and June 28, 2007, and all showed the same results (i.e.,
fertilization in SA but none in AS crosses). The asymmetry in
cross-fertilization is clear and consistent. Asymmetry in crossfertilization success is common and has been reported between
C. gigas and C. sikamea (Numachi 1977, Banks et al. 1994).
Interestingly, similar to what we observed here, C. sikamea eggs
can be fertilized by C. gigas sperm, but C. sikamea sperm cannot
fertilize C. gigas eggs. If we assume that all Crassostrea species
evolved from a common ancestor and shared the same set of
sperm-egg recognition molecules prior to speciation, the fact
that eggs of C. sikamea can be fertilized by sperm of two other
species suggests receptors on C. sikamea eggs have not changed
457
much. On the other hand, C. sikamea sperm cannot fertilize eggs
of either C. ariakensis or C. gigas indicates that their recognition
molecules might have gone through considerable changes. It
would be interesting to identify sequence and compare genes
involved in sperm-eggs recognition in all Crassostrea species, in
conjunction with hybridization experiments.
Although SA hybrid crosses had lower fertilization success,
the hybrid zygotes were capable of normal development as
indicated by their close to normal survival to D-stage compared
with that of the two intraspecific crosses. Hybridization had no
effects on early growth of hybrid larvae during the first nine
days. Actually, the early growth of hybrid SA larvae was similar
to that of SS crosses, suggesting a maternal effect. Maternal
effects are common in intraspecific and interspecific hybridization in marine bivalves (Hedgecock et al. 1995, Zhang et al.
2007, Beaumont et al. 2004, Zhou et al. 1982, Soletchnik et al.
2002). Maternal effects are usually detected in viability and
growth rate at early developmental stages (Schwabl 1996, Eising
et al. 2001). This is not surprising as early development is
programmed in the eggs and supported by yolk materials
(Garamszegi et al. 2007).
In this study, maternal effects began to fade away after the
first nine days, after which the growth of SA larvae started to
slow down. The slow growth rate is probably caused by genome
incompatibility between the two species. Because AA larvae
grow faster than SS larvae, the SA hybrids should show
intermediate growth if growth is additive. The fact that hybrid
larvae grow slower than either parental species after Day 9
suggests that the hybrid genome has negative effects on growth
in the absence of maternal effects. These results are similar to
observations on C. virginica 3 C. gigas hybrid larvae, which
are apparently normal but would stop growing after the first
week (Allen et al. 1993). These findings suggest that maternal
effects may last for about 1–2 wk in oysters, after which genome
compatibility becomes more important. Clearly, there is no
heterosis between C. ariakensis and C. sikamea.
The incompatibility of the two genomes is also reflected in
the delay of SA larvae reaching eyed stage and their poor
survival through metamorphosis. The survival of SA larvae was
similar to that of SS larvae before metamorphosis, both were
lower than that of AA larvae. However, the SA hybrids clearly
had problems surviving metamorphosis as only 16 spat were
obtained from SA compared with hundreds from SS and AA
crosses. It should be pointed out that the culture condition in
this study may not be optimal for one or both species because
survival in the two intraspecific crosses was low, and the larvae
took over four weeks to settle. The survival data may not be
representative, but it is clear that some SA hybrids can survive
through metamorphosis to 93 days or a juvenile size of 0.62 cm
at 10 months age. Three of the SA spat survived to one year of
age. Genetic analysis clearly shows that the SA spat and
juveniles contain DNA from both parental species. Therefore,
we conclude that some SA hybrids are viable.
While there is no question that SA spat are hybrids, PCR
amplification of the hybrids generated two fragments that are
similar but not identical to these of the two parental species.
Sequence analysis shows that the new ITS1 sequences arise from
insertions at sites with simple sequence repeats. The insertion
apparently occurred during PCR, because mixing DNA from
the two species just before PCR also generate these new
sequence variants (Fig. 2, lane 3). This is interesting as DNA
458
XU ET AL.
from each pure species gives one species-specific ITS1 sequence
without variation, but when mixed, produces new sequence
variants with duplications or insertions. We speculate that
ITS1 DNA from the two species may form heteroduplexes
during PCR, and errors in the form of duplications and insertions are generated when DNA polymerase passing through
regions of simple sequence repeats. Whatever the mechanism
may be, there is no question that ITS1 sequences of both
species were present in SA spat, and these SA spat are true
hybrids.
Our data suggest that prezygotic and postzygotic barriers
exist between C. sikamea and C. ariakensis, but none of them
are complete. If C. sikamea eggs can be fertilized by C.
ariakensis sperm and the hybrids are viable, how do the two
species coexist in the same environment? We know that C.
ariakensis was genetically distinct with C. sikamea in COI, ITS,
and 28S rRNA sequences (Wang et al. 2004). Genetic analysis
of hundreds of wild oysters from the same reef found no hybrids
(Wang et al. 2008). One AS hybrid was found from hundreds of
samples collected from southern China, which is at odds with
finding of this study. This study indicates fertilization does not
occur in AS crosses. There are two possible explanations for this
discrepancy: either fertilization in AS can occur under some
conditions or the AS hybrid observed is an artifact. Either way,
the zero or extremely low frequency of naturally occurring
hybrids suggests that other forms of reproductive isolation may
exist. Further studies are needed to understand how C. ariakensis and C. sikamea avoid hybridization and maintain their
species identity in sympatry.
ACKNOWLEDGMENTS
The authors thank the Fisheries Technical Extension Station
of Nantong, for their kind support; Haixiang Ge, Jianzhong Ni,
and Xihui Guo provided help during sampling; Yi Xin and
Runshan Du, for their assistance in the hatchery and farm
operation; Qi Wu, for kind assistance with molecular identification. This research was supported by a grant from National
Natural Science Foundation of China (NO. 40730845 to Zhang,
Guo and Liu). Guo’s participation is supported by OTP of
Chinese Academy of Sciences and a grant (NA04NMF4570424)
from the US NOAA CBO Non-native Oyster Research Program.
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