VENUS 68 (3–4): 151–163, 2010

©Malacological Society of Japan

Molecular Markers Reveal Genetic Contamination of Endangered
Freshwater Pearl Mussels in Pearl Culture Farms in Japan
Akihisa Shirai1*, Takaki Kondo2 and Tadashi Kajita1
1

Department of Science and Technology, Graduate School of Science,
Chiba University, 1-33, Yayoi, Inage, Chiba 263-8522, Japan
2
Division of Natural Science, Osaka Kyoiku University,
Kashiwara, Osaka 582-8582, Japan

Abstract: Phylogenetic analyses using mitochondrial DNA (Cox2-Cox1) and nuclear DNA(ITS1) were performed on a critically endangered freshwater pearl mussel, Hyriopsis schlegeli,
endemic to Lake Biwa, Japan, and a closely related Chinese species H. cumingii, which wasintroduced to Japan approximately 30 years ago. Using samples of typical H. schlegeli and H.
cumingii, clear genetic differences between the two species were recognized in both molecularmarkers. Surveys of the genetic compositions of freshwater pearl mussels showed evidences ofhybridization between the two species in culture farms in Lakes Kasumigaura and Biwa. Given
that pearl culture farms are not completely isolated from natural water systems in Lake Biwa,
these findings suggest that native H. schlegeli might be threatened by hybridization with H.
cumingii. The wild population in Lake Anenuma, Aomori Prefecture, Japan would be the only
remaining ‘pure’ population of H. schlegeli free from hybridization and therefore be very
important for conservation, although it originated from escaped cultured mussels.
Keywords: conservation genetics, Hyriopsis, endemic species, endangered species, Lake Biwa

Introduction
Exotic species have greatly impacted native ecosystems in a variety of ways. Ecologicalinfluences such as threats to native species from predation and occupation of native niches havebeen well documented (Mack et al., 2000; Mooney & Cleland, 2001). However, hybridizations
between native and exotic species are not easily recognized (Strauss et al., 2006), and may resultin genetic contamination of the native species. The impacts of such hybridization would be great
especially when the native species involved are endangered. Thus, immediate actions are required
to assess the current status of hybridization by determining the genetic composition of both nativeand exotic species.
The freshwater pearl mussel Hyriopsis schlegeli (Martens, 1861) (Bivalvia: Unionidae) is
endemic to Lake Biwa, central Japan (Fig. 1), and is now threatened (Kondo, 1994; Nakanishi &Sekino, 1995). This species has very large shells, and is known to produce freshwater pearls.
Following the development of commercial freshwater pearl culture in Lake Biwa, H. schlegeli
was introduced to other freshwater systems in Japan. Propagation of the species, however, was not
successful in most water systems, with the exception of Lake Kasumigaura in the Kanto district(Kihira et al., 2003). Since the 1930s, Lake Kasumigaura has been stocked several times withmussels from Lake Biwa, and a large wild population of H. schlegeli was established as early as
1963 (Ogiwara, 2002). Freshwater pearl culture farms are currently in operation only in Lakes
Biwa and Kasumigaura (Figs. 1 and 2).
* Corresponding author: aki-shirai@graduate.chiba-u.jp

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Fig. 1. Localities from which samples were obtained.

Although H. schlegeli was highly abundant in Lakes Biwa and Kasumigaura approximately
five decades ago, a serious decline of wild mussels occurred in both lakes in the mid-1970s (Fig.
2). This might be the results of environmental changes and/or over-collection of seed mussels(Nakanishi & Sekino, 1995; Kondo, 2001; Ogiwara, 2002). Indeed, population crashes were so
extensive that H. schlegeli is now counted as one of the critically endangered species under the
category CR + EN in the Japanese Red Data Book (Ministry of the Environment, 2005).
The decline in wild freshwater pearl mussels induced a shortage of seed mussels forcommercial pearl culture. In Lake Kasumigaura, Hyriopsis mussels, referred to as an “improved
variety” of Hyriopsis species, have been used for pearl culture since 1988. Although the origin of
this improved variety is not clear, the mussel is more tolerant to water contamination than H.
schlegeli (Ogiwara, 2002). The improved variety was transferred from Lake Kasumigaura toculture farms in Lake Biwa in 1992 and was used as seed mussels for pearl culture (Kondo, 2001)(Fig. 2). The present cultured mussels in Lake Biwa are likely dominated by the improved variety,however the taxonomic and genetic identity of this variety has not been clarified. Machii (1995)
suggested that it might be a natural hybrid between H. schlegeli and the related Chinese species
H. cumingii (Lea, 1852), whereas Kondo (2001) suggested that it could be H. cumingii itself
based on morphological features.
Hyriopsis cumingii is endemic to China and is the most important mussel for freshwater pearl
production there (Liu, 1979; Wang et al., 2008). This species was probably introduced to Lake
Kasumigaura from China sometime between the mid-1970s and the mid-1980s, although theintroduction was not clearly documented (Fig. 2). According to Dong et al. (2007), H. schlegeli

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Fig. 2. A brief history of freshwater pearl aquaculture of Hyriopsis species in Japan.

and H. cumingii can be artificially crossed and produce hybrids. If the mussels transferred into
Lake Biwa from Lake Kasumigaura in 1992 included H. cumingii or their hybrids, genetic
contamination of H. schlegeli may have occurred at culture farms in Lake Biwa. This might haveresulted in subsequent genetic contaminations of H. schlegeli in its natural habitat in Lake Biwa,
given that most pearl culture farms are built adjacent to open water. It is necessary to clarify thegenetic composition of native and cultured Hyriopsis mussels in Lakes Kasumigaura and Biwa.
Previous studies using allozyme markers, however, did not provide sufficient evidence todistinguish the two species (Sakai et al., 1997; Kondo, 2001).
Shirai (2008) recently identified a wild population of H. schlegeli in Lake Anenuma, adjacentto Lake Ogawara, in Aomori Prefecture, northern Japan (Fig. 1), and suggested that the wildmussels originated from ones that escaped from a test aquaculture farm established there in 1966.
Given that the test culture in Lake Anenuma used seed mussels from Lake Kasumigaura and took
place before the introduction of H. cumingii, the large wild population of H. schlegeli in Lake
Anenuma might not have been influenced by the introduction of H. cumingii (Shirai, 2008; Fig. 2).
In this study, we used mitochondrial and nuclear DNA markers, which are expected to have

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higher resolutions than allozyme markers. The objectives of this study were to clarify: 1) thegenetic differences between H. schlegeli and H. cumingii, and 2) the genetic composition of wild
and cultured mussels in Japan for the conservation of H. schlegeli, which is an endangered
endemic species in Japan.

Material and Methods
Samples
Samples were obtained from wild and cultured Hyriopsis mussels in three Japanese lakes(Table 1, Fig. 1). A total of nine wild samples were collected from Lake Anenuma in 2007 andseven cultured samples from Lake Kasumigaura were kindly provided by the Yanase Pearl
Culture Company (Inashiki, Ibaraki Prefecture) in 2007. 40 cultured mussels were purchased fromthe Lake Biwa Freshwater Pearl Cooperative Association (Omihachiman, Shiga Prefecture) in
1994 and 1998 and preserved in a freezer in Baika High School (Toyonaka, Osaka Prefecture). Of
the 40 mussels, 20 were born in 1990–1991 in the culture farm in Lake Biwa, before theintroduction of mussels from Lake Kasumigaura, and were therefore not genetically influenced by
H. cumingii. The remaining 20 mussels were born in 1996–1997 after the introduction, and thus
might have been genetically influenced by H. cumingii or its hybrids with H. schlegeli. Finally,four samples of genuine Hyriopsis cumingii were obtained from Sichuan province, China.
For use as an outgroup in phylogenetic analyses, a sample of Pronodularia japanensis (Lea,
1834) was collected in Sakata City, Yamagata Prefecture in northern Honshu in 2007.
DNA extraction
The CTAB method by Winnepenninckx et al. (1993) was used to extract total DNA from anapproximately 5-mm3 piece of the foot muscle of each specimen. Fresh samples were used if
possible, but in some cases, frozen or ethanol-fixed samples were used. Samples were boiled prior
to DNA extraction according to Ueshima (2002).
Mitochondrial DNA (Cox2-Cox1) and nuclear ribosomal DNA (ITS1 region) sequencing
Sequences of the coding and non-coding regions of the mitochondrial DNA (mtDNA) Cox2Cox1 genes and the internal transcribed spacer region 1 (ITS1) of nuclear ribosomal DNA(nrDNA) were determined using direct sequencing methods. It was expected that male mussels
would have two highly diverged mitochondrial genomes because of doubly uniparental
inheritance (DUI) that is known for some bivalves including Unionidae (Hoeh et al., 1996; Liu etal., 1996). In Unionidae, a unique protein-coding extension is known in the male mitotype of theCox2 gene, which is approximately 600 bp longer than the female one (Curole & Kocher, 2002;
Curole & Kocher, 2005). Based on the different length of amplified fragments, the PCR productsof female mitochondrial Cox2-Cox1 were separated on agarose gels and used for nucleotide
sequencing. The primer pair used to PCR was CoxII.2 (Curole & Kocher, 2002) and HCO2198(Folmer et al., 1994). The ITS1 region of nrDNA was amplified using the universal primers ITS2
and ITS5 (White et al., 1990).
PCR reactions were performed in a mixture containing 18.4 μL of sterile distilled water,
0.125 μL of ExTaq DNA polymerase (5 U/μL; TaKaRa Bio, Shiga, Japan), 2.5 μL of 10 × ExTaq
buffer, 2.0 μL of dNTP solution (2.5 mM each), 0.5 μL of each primer (5 mM), and 1.0 μL of
genomic DNA (10–30 ng) in a final volume of 25 μL. Amplification was performed in a PCR
Thermal Cycler Dice (TaKaRa Bio) under the following conditions: an initial denaturation at
95°C for 3 min followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 45 or 55°C
for 45 s, extension at 72°C for 90 s, and a final extension at 72°C for 10 min. The lower (45°C)
annealing temperature was used for the mitochondrial genes and the higher (55°C) for the nrDNA.

Genetic Contamination of Hyriopsis Species in Japan

155

Table 1. Wild and cultured samples of Hyriopsis used in this study and haplotypes identified bymolecular markers. N designates numbers of samples. Haplotype designations of mtDNA and nrDNA
are shown in Figs. 3 and 4. Biwa* indicates samples collected in Lake Biwa before the introduction
from Lake Kasumigaura in 1992.
Localitycollection
year
2007

mtDNA
haplotype
X1
X1
X2
X7

nrDNA
haplotype
S1
S2
S1
S2

5
1
1
2

Biwa* [n = 20]:
Cultured mussels of Lake Biwa,
before the introduction of mussels from
Lake Kasumigaura (collected samples wereborn in the culture farm before 1992)

1994

X1
X1
X1
X4
X5
X6
X7
X7
X7

S1
S2
S3
S3
S2
S1
S1
S2
S3

2
5
5
2
1
1
1
1
2

Biwa [n = 20]:
Cultured mussels of Lake Biwa,
after the introduction of mussels from Lake
Kasumigaura

1998

X1
X5
X6
X7
X1
X1
X1
X1
X1
X7

S1
S3
S3
S1
C1
C3
S1/C1
S2/C4
S3/C4
S1/C3

8
1
1
1
3
1
1
1
1
2

Kasumigaura [n = 7]:
Cultured mussels of Lake Kasumigaura, after
introduction of H. cumingii from China

2007

X1
X1
X3
Y1
Y1
Y1

C1
C3
C3
S2
C1
C2

2
1
1
1
1
1

China [n = 4]:
Hyriopsis cumingii collected in Sichuan,
China

1999

Y2
Y3
Y4
Y5

C1/C3
C1
C1
C1

1
1
1
1

Anenuma [n = 9]:
Wild mussels of Lake Anenuman

PCR products were purified using the GENECLEAN III Kit (Qbiogene, Inc., Carlsbad, CA,
USA). Cycle sequencing reactions were performed using the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA, USA), using profiles of 28 cycles of 95°C
for 10 s, 48°C for 8 s, and 60°C for 4 min for mtDNA, and 25 cycles of 95°C for 30 s, 50°C for
30 s, and 60°C for 4 min for nuclear ITS. Both strands were sequenced using the same primers

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used for the PCR as well as the following internal primers: forward UnioFCo1F (5’GATTCGAGCAGAGTTGGGTC-3’) and reverse UNIOFCOIR (Curole & Kocher, 2005) orHyFCo1R (5’-CCCCCAATTATCATAGGTATCAC-3’) primers for the Cox2-Cox1 regions of
mtDNA. Nucleotide sequences were determined using an ABI PRISM 377 automated sequencer(Applied Biosystems) with the standard settings. DNA sequences were edited and aligned using
Sequencing Analysis ver. 3.4.1 (Applied Biosystems) and AutoAssembler ver.2.1.1 (Applied
Biosystems). Multiple alignment was performed with Clustal X version 1.83 (Thompson et al.
1997) using default parameters, and the obtained alignment was manually corrected to maximize

Fig. 3. The strict consensus tree of the six most parsimonious trees obtained from partial sequences of Cox2Cox1 of mtDNA. The numbers above the branches are bootstrap supports obtained by MP and NJ analyses(MP/NJ). Numbers below branches represent branch length. NrDNA haplotypes relevant to the mtDNA
haplotypes are shown after the haplotype names in parentheses. Biwa* indicates samples collected in Lake
Biwa before the introduction from Lake Kasumigaura in 1992.

Genetic Contamination of Hyriopsis Species in Japan

157

sequence similarity using Se-Al Sequence Alignment Editor version 2.0a11 (Rambaut, 2002).
Direct sequencing of nuclear ITS1 detected two different peaks overlapping within a single
sequence profile in some samples. They were presumed to be caused by heterozygotes in ITS1
locus. In order to segregate alleles for these samples, we performed SSCP (single-stranded
conformation polymorphism) analysis mostly following the method of Watano et al. (2004). PCR
products were diluted 1:10 in denaturing formamide buffer, heated to 95°C for 3 min, and then
immediately chilled on ice. They were loaded on nondenatured acrylamide gel containing 2%
glycerol and run for 10 h at 300 V at 20°C in 1 × TBE. After electrophoresis, the DNA bands weredetected by silver staining methods. Each band was cut out from the gel and the DNA of the band
was purified by ethanol precipitation. Using the extracted single-stranded DNA as a template, PCR
reactions were performed using the same PCR primers that were used in the initial PCR reaction.
PCR products were then treated with ExoSAP-IT (USB Corporation, Cleveland, OH, USA) toremove excess primers and dNTPs, and used as a sequencing template as described above.
Phylogenetic analyses
Phylogenetic relationships among the haplotypes of mtDNA and nrDNA sequences wereinferred using maximum parsimony (MP) and neighbor-joining (NJ) methods using PAUP* 4.0 beta
10 (Swofford, 2002). For the MP analysis, all characters were unordered and equally weighted. The
MP tree was constructed using the branch and bound search option with 100 random addition
replicates, the tree bisection reconnection (TBR) branch-swapping algorithm, and the ‘MulTrees’
option. Branch support was assessed by bootstrap analysis (Felsenstein, 1985) based on 1000
replicates using the branch and bound search option with the simple addition of sequence replicates.
Pronodularia japanensis was used as an outgroup species for each analysis. Sequences of Cox2Cox1 genes for P. japanensis were obtained from GenBank (accession no. AB055625, registered as
Inversidens japanensis) and ITS region was sequenced in this study (accession no. AB507769). All
sequences (haplotypes) of Hyriopsis samples were deposited in GenBank (accession nos.
AB507749–AB507761 for mtDNA and AB507762–AB507768 for nrDNA).
Genetic diversity
DnaSP4.5 (Rozas et al., 2003) was used to calculate the mean number of nucleotide
substitutions per site of mtDNA and nrDNA among groups (Dxy) and nucleotide diversity withingroups (π).

Results
Mitochondrial DNA (Cox2-Cox1)
The 1027 bp nucleotide sequences of the female Cox2-Cox1 mtDNA region of Hyriopsis
Table 2. Haplotypes identified by nucleotide substitutions of ITS1. Substitution sites are denoted in
the aligned sequences of 484 bp excluding indels.
S1
S2
S3
C1
C2
C3
C4

28

72

95

98

100

107

146

191

267

G
A
G
A
A
A
G

C
A
A
A
A
A
A

G
A
G
G
G
G
G

G
G
G
C
C
C
C

G
G
G
A
A
A
A

A
A
A
A
T
T
T

C
C
C
C
G
C
C

G
G
G
G
G
G
A

C
C
C
T
T
T
T

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Fig. 4. The strict consensus tree of the two most parsimonious trees obtained from nucleotide
sequences of the ITS1 region of nrDNA. The numbers above the branches are bootstrap supports
obtained by MP and NJ analyses (MP/NJ). Numbers below branches represent branch length.
MtDNA haplotypes relevant to the nrDNA haplotypes in each individual are shown after the
haplotype names in parentheses. Biwa* indicates samples collected in Lake Biwa before theintroduction from Lake Kasumigaura in 1992.

schlegeli and H. cumingii were determined in this study (269 bp for Cox2 region, 18 bp for noncoding region and 740 bp for Cox1 region). No indels (insertions and deletions) were foundamong sequences. In total, 12 haplotypes (X1–X7 and Y1–Y5) were recognized, containing 96
variable sites and 88 parsimony informative sites. MP and NJ analyses using the 12 haplotypes
with an outgroup species produced trees with almost identical topologies (only the MP tree isshown; Fig. 3).

Genetic Contamination of Hyriopsis Species in Japan

159

The MP analysis produced six most parsimonious trees with a tree length of 367, a consistency
index of 0.924, and a retention index of 0.935. Two major clades were recognized within the strict
consensus tree of the six trees, both of which were supported with very high bootstrap values. The
first clade (hereafter clade X) consisted of haplotypes X1–X7, which were obtained from
Hyriopsis samples from Lakes Biwa (both before and after introduction from Lake Kasumigaura),
Anenuma, and Kasumigaura. The second clade (hereafter clade Y) consisted of haplotypes Y1–
Y5, which were obtained from H. cumingii collected in China and Hyriopsis samples collected in
Lake Kasumigaura. The nucleotide diversity (π) determined by Jukes and Cantor (Jukes andCantor, 1969) was 0.00232 (+ –0.00039) for clade X and 0.01062 (+ –0.00255) for clade Y. The
average number of nucleotide differences between the two clades was 81.543, and the mean
number of nucleotide substitutions per site (Dxy) with Jukes and Cantor between clades was
0.08393 (+ –0.01830).
Nuclear ribosomal DNA (ITS1 region)
The 484–492 bp nucleotide sequences from the ITS1 region of Hyriopsis samples were
determined in this study. The aligned length was 494 bp including 10 bp indels. The direct
sequencing of nuclear ITS1 region exhibited two different peaks overlapping within a single
sequence profile for six samples. This was likely caused by the presence of two allelic sequencesin an individual, and each different allele was separated by PCR-SSCP methods. In total sevenhaplotypes were recognized by nine diagnostic sites (Table 2).
The MP analyses for the seven haplotypes identified the two most parsimonious trees with atree length of 58, a consistency index of 0.983 and a retention index of 0.900 (Fig. 4). The strict
consensus tree derived from the two trees revealed one clade (hereafter group S) comprising
haplotypes S1–S3 supported with a bootstrap value of 84 and by three synapomorphic characters(substitution site 98, 100 and 267; see Table 2). The remaining haplotypes, C1–C4, did not forma clade in the MP analyses. Since they did comprise a clade in the NJ analyses with bootstrap
support of 54, we designated the latter as group C.
Group S consisted of haplotypes obtained from Hyriopsis samples collected in Lakes Biwa
(both before and after the introduction from Lake Kasumigaura), Kasumigaura, and Anenuma.
Group C consisted of haplotypes obtained from H. cumingii collected in China, as well as from
Hyriopsis samples collected in Lake Biwa (after the introduction from Lake Kasumigaura) and
Lake Kasumigaura. The value of π determined using Jukes and Cantor was 0.00415 (+ –0.00146)
for group S and 0.00415 (+ –0.00112) for group C. The average number of nucleotide differences
between the two groups was 5.50, and the Dxy with Jukes and Cantor was 0.0115 (+ –0.00411).
The haplotype compositions of all individuals are shown for each locality (Table 1).

Discussion
Genetic differences between H. schlegeli and H. cumingii based on mtDNA and nrDNA
sequences
The phylogenetic analyses of partial nucleotide sequences of Cox2-Cox1 of female mtDNA
and the ITS1 region of nrDNA revealed the presence of two distinct groups (clades X and Y in
the mtDNA tree; groups C and S in the nrDNA tree). In both phylogenetic trees, H. schlegeli bornin Lake Biwa before 1992 (i.e., before the introduction of mussels from Lake Kasumigaura) andH. cumingii collected in China were assigned to different groups (Figs. 3 and 4). This suggeststhat the two species can be clearly distinguished by species-specific markers of mtDNA andnrDNA. The differences between the two species (Dxy) were 8.4% in the Cox genes and 1.2% in
the ITS region, which were slightly higher than the values (5.6% in Cox and 0.9% in ITS)
reported between closely related freshwater mussels in North America (Campbell et al., 2008).

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Our study provides new molecular markers that can be used to assess the genetic composition of
the two species.
Molecular evidence of hybridization between H. schlegeli and H. cumingii in pearl culture farms
in JapanOur results suggest that hybridizations between H. schlegeli and H. cumingii have occurred inJapanese culture farms. Four samples from Lake Kasumigaura and four samples from Lake Biwa
exhibited the mtDNA haplotype of H. schlegeli (X1 or X3) and nrDNA haplotypes of H. cumingii(C1 or C3). On the other hand, one sample from Lake Kasumigaura exhibited the mtDNA
haplotype of H. cumingii (Y1) and nrDNA haplotype of H. schlegeli (S2) (Table 1). These results
are likely caused by hybridization between the two species. Given that only female lineage of
mtDNA were surveyed in this study, our results suggest that crossings between the two species
have occurred in both directions in the cultured mussels of Lake Kasumigaura, which is consistent
with the recent report of artificial reciprocal crosses between the two species (Dong et al., 2007).
Five samples from Lake Biwa exhibited the mtDNA haplotypes of H. schlegeli and both nrDNA
haplotypes of H. schlegeli and H. cumingii (S1/C1 in Table 1, for example). These results would
represent heterozygotes produced by crossings between H. schlegeli and H. cumingii or theirhybrids.
If we assessed the extent of genetic influence of H. cumingii using the proportion of its
haplotypes in Hyriopsis mussels sampled in Japan, 43% of mtDNA and 86% of nrDNA were of
H. cumingii origin in Lake Kasumigaura, on the other hand, 0% of mtDNA and 45% of nrDNA
were of H. cumingii origin in Lake Biwa (after the introduction from Lake Kasumigaura).
Haplotypes of H. cumingii origin were found more in Lake Kasumigaura than in Lake Biwa. This
result might reflect the history of the introductions: H. cumingii was introduced to Lake
Kasumigaura from China sometime between the mid-1970s and the mid-1980, and then Hyriopsis
mussels were transferred to Lake Biwa from Lake Kasumigaura in 1992 (Fig. 2). Although somesamples from Lakes Biwa and Kasumigaura may represent pure H. schlegeli and H. cumingii
based on the two molecular markers (X1, 5, 6, or 7 with S 1 or 3 for H. schlegeli, and Y1 with
C1 or C2 for H. cumingii in Table 1), these might have been caused by backcross involving theirhybrids. Future studies with a larger number of molecular markers with higher resolution will benecessary to clarify their identity.
Conservation of H. schlegeli
Hyriopsis schlegeli is one of the representative relict endemic species in Lake Biwa
(Takahashi, 1989), and its fossils are found in the Pleistocene Kobiwako (Palaeo-Lake Biwa)
Group (Matsuoka, 1987; Nishino and Watanabe, 2000). On the other hand, the Chinese species H.
cumingii has a wide distribution range throughout mainland China (Liu, 1979). The two species
are considered closely related, given that they are morphologically similar and able to produce
artificial hybrids (Dong et al., 2007). Although previous studies did not provide molecularmarkers to distinguish them (Sakai et al., 1997; Kondo, 2001; Wu et al., 2008), our molecular
data, in contrast, have shown clear differences between the two species in both mtDNA andnrDNA markers (Figs. 3 and 4). H. schlegeli has accumulated original genetic differences from its
sister species since isolation of the population to Lake Biwa.
Hyriopsis schlegeli is now considered a critically endangered species, and conservation of thespecies is very important. Our findings of crossings between H. schlegeli and H. cumingii in
culture farms in Lake Biwa, however, raise an alarm for the conservation of H. schlegeli. Given
that culture farms are built adjacent to natural water systems, mussels with non-native genes thatescape from the farms may cause genetic contamination of wild H. schlegeli in Lake Biwa.
Indeed, H. cumingii-like individuals have been collected outside farms in Lake Biwa, which

Genetic Contamination of Hyriopsis Species in Japan161

suggests that the wild H. schlegeli may be threatened by H. cumingii or their hybrids. Further
genetic assessments of wild population of H. schlegeli in Lake Biwa are necessary, althoughcollecting wild H. schlegeli samples is extremely difficult.
Our results also revealed that mussels of Lake Anenuma found by Shirai (2008) exhibit only
native haplotypes of H. schlegeli in both mtDNA and nrDNA markers. The composition of
haplotypes was similar to that of the Lake Biwa mussels born before 1992 (Table 1). These results
suggest that the mussels from Lake Anenuma are free from the genetic influence by H. cumingii.
Although it originated with escaped animals from the test culture farm in the 1960s, the wild
population in Lake Anenuma would be the only remaining ‘pure’ and abundant population of H.
schlegeli free from hybridization, and would therefore be very important for conservation.
Acknowledgments
We are grateful to Drs. Y. Watano, T. Asakawa, K. Takayama, N. Wakita, and other members of ourlaboratory at Chiba University for their valuable advice, and Mr. M. Matsuda and Dr. K. Nakai of Lake Biwa
Museum for their useful information. Many thanks are also given to those who provided samples, especially
Dr. S. Fukuhara of Baika High School, Osaka, Mr. K. Yanase of the Yanase Company, Ibaraki, Japan and
Dr. A. Shi of Sichuan University, Chengdu, Sichuan, China. We also thank anonymous reviewers for theiruseful comments. This study represents a portion of the doctoral dissertation of A. Shirai prepared under thesupervision of T. Kajita, which was partly supported by Research Assistant Programs of the Graduate Schoolof Science, Chiba University.

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Genetic Contamination of Hyriopsis Species in Japan163

分子データから示された琵琶湖固有の絶滅危惧種イケチョウガイの移入種
ヒレイケチョウガイによる遺伝的撹乱白井亮久・近藤高貴・梶田

要

忠

約

琵琶湖固有の絶滅危惧種イケチョウガイと中国からの移入種ヒレイケチョウガイの交雑の実態を探る
RNA遺伝子の I
TS1領ため，分子マーカーを用いて解析を行った。ミトコンドリアの Cox遺伝子と核 r域の塩基配列を用いた系統解析の結果，両種はいずれの分子マーカーでも区別されることが分かった。
遺伝的組成を調べたところ，琵琶湖と霞ヶ浦の淡水真珠養殖場では両種の交雑に由来すると考えられる個体がみつかった。琵琶湖の養殖場は自然環境とは完全には隔離されていないため，逸出した養殖個体とイケチョウガイの野生個体との交雑が懸念される。姉沼の野生集団は移植された養殖個体の逸出に由来するものの，唯一交雑の影響を受けていない純粋なイケチョウガイの集団であると考えられるため，保全上非常に重要である。