The Israeli Journal of Aquaculture – Bamidgeh 60(4), 2008, 237-242.

237

Genetic Diversity and Population Structure of the Peanut

Worm (Sipunculus nudus) in Southern China as Inferred
from Mitochondrial 16S rRNA Sequences
Du Xiaodong*, Chen Zian, Deng Yuewen, Wang Qingheng and Huang Ronglian
Fishery College, Guangdong Ocean University, 524025 Zhanjiang, China

(Received 26.4.06, Accepted 8.7.06)

Key words: peanut worm, Sipunculus nudus, genetic diversity, population structure,
16S rRNA gene

Abstract
Genetic diversity and population structure of the peanut worm (Sipunculus nudus) were investi-
gated by using 536 base-pair fragments of the mitochondrial 16S ribosomal gene. Populations
were collected from three locations along the southern coast of China - Beihai, Sanya, and
Xiamen. Amplified polymerase chain reaction products were sequenced in both directions and
data were analyzed using ClustalX, Arlequin, and MEGA. A total of 69 polymorphic sites and 21
distinct haplotypes were revealed. The mean haplotype and nucleotide diversity of the three pop-
ulations were 0.814% and 0.37%, respectively. The Beihai population had the greatest haplo-
type and nucleotide diversity, followed by the Xiamen and Sanya populations. Analysis of mole-
cular variance (AMOVA) showed significant genetic differentiation among the three populations
(Fst = 0.0619, p<0.05) and distinct population structures among the three sites.

Introduction
Genetic diversity, the fundamental hierarchy markers include nuclear and mitochondrial
of species and ecosystem diversities, is the markers. Mitochondrial DNA (mtDNA) has a
primary subject of biodiversity research (Avise high mutation rate relative to single-copy
and Himrick, 1996). Documentation of genetic nuclear DNA (Brown et al., 1979) and, under
diversity is needed to utilize and manage equilibrium conditions, maternal inheritance
genetic resources. Genetic diversity can be contributes one quarter of nuclear DNA (Birky
assessed by using morphological, cytological, et al., 1983). Thus population differentiation is
physiological, biochemical, and molecular expected to evolve more rapidly within mtDNA
markers. The most frequently used molecular than in allozymes and other coding regions of

* Corresponding author. E-mail: [email protected]

238 Xiaodong et al.

nuclear DNA, making mtDNA a more sensi- 3’ (Zhang and Ryder, 1993). Amplification
tive indicator of population structure. was performed in a 50 µl reaction volume con-
Mitochondrial DNA markers are frequently taining 1 µl DNA dilution, 25 pmol primers, 5 µl
used to evaluate genetic diversity in marine 10x reaction buffer, 25 mM MgCl2, 200 µM
invertebrates (e.g., Boulding et al., 1993; Su dNTP, and 2 U Taq polymerase (Sangon,
et al., 2005; Kumar et al., 2007). Shanghai). The cycling conditions were 2 min
In China, the peanut worm, Sipunculus denaturation at 94°C, 35 cycles of 45 s denat-
nudus, is mainly distributed along the southern uration at 94°C, 1 min annealing at 50°C, 1
coast. The species is widely cultured in south- min extension at 72°C, and a final extension
ern regions of China, especially in Beihai, at 72°C for 5 min. PCR products were sepa-
Guangxi. Sipunculus nudus culture relies rated on 1.5% agarose gels; bands were
entirely on seed collected from the wild, which stained with rhodium bromide and viewed
may result in over-exploitation of wild popula- under a UV light source.
tions. Therefore, it is urgent to obtain informa- A total of 5 µl of each PCR product was
tion on the genetic diversity of wild populations used for 1.5% agarose gel electrophoresis to
so as to manage commercial species. confirm the amplified fragment length with a
Data on population genetics of S. nudus marker. Here, bands were made visible using
have been reported on the basis of random ultraviolet light after an ethidium bromide
amplified polymorphic DNA (Wang et al., bath. The remainder of each PCR product
2006). In the present study, genetic diversity was used as a template for automated
and the population structures of S. nudus col- sequencing reactions performed with a T3
lected from three locations along the coast of Thermocycler and run on an ABI 310 DNA
southern China were investigated using 16S sequencer. The primers used for sequencing
rRNA gene sequence analysis. The objective reactions were the same as those for PCR
was to provide useful information for resource amplification. Sequence data are available
conservation and fishery management of the from GeneBank (accession numbers
species. EU260100-EU260120).
Data analysis. A total of 536 bp sequences
Materials and Methods from 30 individuals were aligned by using
Sample collection. Wild samples of S. nudus ClustalX (Thompson et al., 1997). Data were
were collected from Beihai in Guangxi, Sanya analyzed using Arlequin vers. 3.0 (Excoffier et
in Hainan, and Xiamen in Fujian, all in the al., 2005). The numbers of transition, trans-
South China Sea. Fresh tissues from the mus- version, and haplotype, as well as haplotype
cle and whole individuals were preserved in diversity (h), and nucleotide diversity (pi) val-
70% ethanol. ues (Nei, 1987), were calculated for separate
Total DNA extraction. Total DNA was and combined populations. The total number
extracted from the ethanol-preserved muscle of nucleotide differences, mean sequence
tissues. The tissues were incubated overnight divergence values, and Jukes-Cantor genetic
in lysis buffer with proteinase K at 37°C. DNA distances (Jukes and Cantor, 1969) were cal-
was purified using phenol-chloroform accord- culated for each pair using molecular evolu-
ing to Wang et al. (2006). Isolated DNA was tionary genetics analysis (MEGA vers. 2.1;
dissolved in 50-100 µl distilled water. This Kumar et al., 2004).
solution was further diluted in distilled water to Analysis of molecular variance (AMOVA)
100 ng/µl for polymerase chain reaction was used to partition the total genetic varia-
(PCR). tion into variance components and produce
Amplification and sequencing. A 536-bp fixation indices (Fst). Fst values were calcu-
segment of 16S rRNA was amplified by PCR. lated for the three populations separately and
The primers were: 16SAR (forward) 5’-CGC- together for all pairs of populations on the
CTGTTTATCAAAAACAT3’ and 16SBR basis of information regarding haplotypes and
(reverse) 5’CCGGTTTGAACTCAGATCATG- their frequencies. The statistical significance
 Genetic diversity and population structure of the peanut worm 239

of the Fst values was tested by permutation (Kong et al., 2003) and in 500-bp 16S rRNA
tests (10,000 replicates). AMOVA was con- gene sequences of the oyster, Crassostrea
ducted by using Arlequin vers. 3.0 (Excoffier rivularis, there were 12 haplotypes in 105
et al., 2005). samples at 23 polymorphic sites (Su et al.,
2005). Possible explanations for the high level
Results of variation in S. nudus are higher mutation in
Mitochondrial DNA 16S rRNA polymorphism. the 16S rRNA gene sequence and large effec-
The 536 bp sequences of the 16S rRNA gene tive population sizes of the species.
from 30 individuals produced 69 polymorphic The mean haplotype and nucleotide diver-
sites that defined 21 haplotypes (Table 1). sity of the three populations were 0.814 and
One haplotype (F) was shared by the Beihai 0.37%, respectively. These results are com-
and Sanya populations while the remaining parable with those on other marine inverte-
twenty (95.2%) were unique to one of the brates, for example, 0.57 and 0.14% in
three populations. Forty (58.0%) of the Crassostrea virginica from the Atlantic Ocean
nucleotide substitutions in the 16S rRNA gene (Reeb and Avise, 1990) and 0.07-0.55 and
were synonymous and 29 were non-synony- 0.08%-0.6% for Penaeus monodon from the
mous. Variant substitutions of parsimony Indian Ocean (Kumar et al., 2007). The high
informative sites appeared in the 21 haplo- level of genetic diversity in the present study
types at position 482 (A↔G, A↔T, T↔G, indicates that the 16S rRNA gene sequence
three variants). might be useful as a genetic marker for aqua-
Haplotype, nucleotide diversity, and other culture purposes such as maintaining stock
population-specific diversity indices are diversity and distinguishing hatchery stocks
showed in Table 2. For all indices, the Beihai from wild populations.
population had the greatest values, followed The assessment of genetic diversity and
by the Xiamen and Sanya populations. population structure of S. nudus is important
Population structure. AMOVA from 16S for appropriate conservation and manage-
rRNA gene sequence analysis indicated sig- ment of the species. Commercial culture of S.
nificant genetic structure and population dif- nudus is expanding in southern China (Lan et
ferentiation. It showed that a significant per- al., 2007). With increased culture, a common
centage of the variation was attributable to concern is the loss of genetic diversity in wild
among-population differences (6.19%) and populations. Genetic monitoring and evalua-
population subdivision (Fst = 0.06193, tion can help identify negative effects of aqua-
p<0.01) among all populations (Table 3). culture on genetic diversity. A high level of
Pairwise relationships among the population genetic diversity and significant differentiation
pairs are given in Table 4. between population structures can help buffer
S. nudus from detrimental genetic effects of
Discussion diseases and population decline and facilitate
The present study reports on 16S rRNA gene improvement of stocks of commercially impor-
sequence analysis of S. nudus sampled from tant species through breeding programs.
three locations along the southern coast of In the current study, the significant Fst
China. The data reveal high variation in the value among all the populations shows that S.
16S rRNA gene sequence of S. nudus, nudus populations are genetically structured.
although this region of the mitochondrial This could be a result of insufficient gene flow
genome usually shows low variation. Our among the locations. Gene flow is the
results (21 haplotypes in 30 individuals at 69 exchange of genetic material between popu-
polymorphic sites) were more variable than in lations caused by movement of individuals or
other marine invertebrates. For example, in their successfully fertilizing gametes
592-bp16S rRNA gene sequences of the scal- (Klinbunga et al., 1998). A long planktonic lar-
lop, Chlamys farreri, there were 23 haplotypes vae stage influences the gene flow over large
in 47 specimens at 31 nucleotide positions geographic areas in marine invertebrates
240 Xiaodong et al.

Table 1. Distribution of 536 bp sequences of 16S rRNA gene from 30 peanut worms
(Sipunculus nudus) from three locations produced 69 polymorphic sites that defined 21 haplo-
types.

Haplotype (n = 21)
A B C D E F G H I J K L M N O P Q R S T U
Polymorphic site (n = 69)
30 etc.* T . . . . . . . . . . . . C C C C C C C C
32 A . . . . . . . . . . . . T T T T T T T T
33 etc.* C . . . . . . . . . . . . T T T T T T T T
47 G . . . . . T T T T . . . . . . . . . . .
50 etc.* C . . . . . . . . . . . . A A A A A A A A
59 C . . . . . . . . . . . . T . . . . T . .
66 etc.* A . . . . . . . . . . . . G G G G G G G G
85 T . . . . . . . . . . . . A . . A . . . .
86 A . . . . . . . . . . . . T . . . . . . .
104 T . . . . . C . . . . . . . . . . . . . .
107 C . . . . . T . . . . . . . . . . . . . .
147 G . . . . . C . . . . . . . . . . . . . .
163 etc.* G . . . . . . . . . . . . C C C C C C C C
174 etc.* G . . . . . . . . . . . . A A A A A A A A
215 etc.* T . . . . . . . . . . . . A A A A A A A A
216 T . . . . . . . . . . . . . C . . . . . .
258 T . . . C . . . . . . . . C C C C C C C C
282 etc.* C . . . . . . . . . . . . G G G G G G G G
317 T . . . . . . . . . . . . G G G G G G G G
335 etc.* A . . . . . . . . . . . . C C C C C C C C
373 A . . . . . . . . . . G . . . . . . . . .
412 G . . . . . . . . . . . . A . . . . . . .
462 G . . . . . . . . . . . . T T T T T T T T
474 C . . . . . . . . . A . . . . . . . . . .
482 A . . . . . . . . . . . T G T T T T T T T
522 A . . . . . . . . . . . . C . . . . . . .
523 G . . . . . . . . . . . . . . . . T T T G
525 T C . . . . . . . . . . . . . . . . . . C
526 T . C . C . . . . C . . . . . . . . . . C
534 A G . G . G G G G G G G G G G G G G G G G
Location
Beihai 1 1 2 1 1 1 2 1
Sanya 1 2 1 3 1 2
Xiamen 1 1 1 2 1 1 1 2
* Other occurrences of this haplotype are: for 30 at 35, 179, 181, 197, 264, 279, 324, 326, and 344;
for 33 at 34, 50, 70, 180, 184, 218, 265, 271, 272, 329, 340, 366, and 461; for 50 at 67, 270, 330, 336,
and 481; for 66 at 177, 214, 256, and 261; for 163 at 343; for 174 at 281 and 463; for 215 at 276 and
331; for 282 at 358; and for 335 at 341 and 359.
 Genetic diversity and population structure of the peanut worm 241

Table 2. 16S rRNA gene polymorphism of Sipunculus nudus collected from three locations
along the southern Chinese coast.

Population No. sequences Haplotype Nucleotide Avg no.

analyzed diversity diversity (%) nucleotide
differences

Beihai 10 0.871 0.43 2.29

Sanya 10 0.756 0.32 1.43
Xiamen 10 0.816 0.37 1.96
Average 10 0.814 0.37 1.89

Table 3. Analysis of molecular variance (AMOVA) of 16S rRNA sequences for three popula-
tions of Sipunculus nudus.

Source of variation Degree of Sum of Variance Percentage

freedom squares components of variation

Among populations 2 1.557 0.03095Va 6.19

Within populations 27 12.658 0.46880Vb 93.81
Total 29 14.215 0.49975 -
Fst: 0.06193 - - - -

Table 4. Pair Fst values among the popu- study was a mere 5.9-13.4 individuals per
lations based on Jukes-Cantor genetic dis- generation. The reason why S. nudus larvae
tances of 16S rRNA. have not dispersed in the South China Sea,
resulting in genetic homogeneity, may thus be
Beihai Sanya Xiamen explained by the existence of some yet-undis-
covered natural barrier between the locations.
Beihai 0.00000 - - In summary, 16S rRNA gene sequences
Sanya 0.07007* 0.00000 - revealed a relatively high level of genetic
diversity and significant genetic differentiation
Xiamen 0.03622* 0.07778* 0.00000
among three populations of S. nudus collect-
* p<0.05 ed along the coast of southern China. Our
results will provide useful information for
genetic resource conservation and fishery
such as Strongylocentrotus purpuratus and S. management of this species.
droebachiensis (Palumbi and Wilson, 1990)
and C. farrei (Yu et al., 2007). The long pelag- Acknowledgements
ic period in Crassostrea virginica larvae could This project was supported by the Natural
result in a moderate gene flow among wild Science Foundation of Guangdong Province
populations (Hare and Avise, 1996). The (no. 30371026) and the Foundation of
planktonic larvae stage in S. nudus is 7.5-27 Guangdong Ocean University (no. 06031).
days at 21.5-34.0°C (Lan et al., 2007). Yet, We thank the reviewers for their critical com-
the estimated inter-population migration in our ments on the manuscript.
242 Xiaodong et al.

References
Avise J.C. and J.L. Hamrick, 1996. evolutionary genetics analysis and sequence
Conservation Genetics: Case Histories from alignment. Briefings in Bioinformatics, 5:150-
Nature. Chapman & Hall, New York. 163.
Birky C.W. Jr., Maruyama T. and P. Fuerst, Kumar N., Lakra W.S., Majumdar K.C.,
1983. An approach to population and evolu- Goswami M. and K. Ravinder, 2007.
tionary genetic theory for genes in mitochon- Genetic diversity in the Indian population of
dria and chloroplasts, and some results. Penaeus monodon (Fabricius, 1978) as
Genetics, 103:513-527. revealed by mtDNA sequence analysis.
Boulding E.G., Boom J.D.G. and A.T. Aquac. Res., 38:862-869.
Beckenbach, 1993. Genetic variation in one Lan G.B., Liao S.M. and B. Yan, 2007. Effect
bottlenecked and two wild populations of the of water temperature on larval development
Japanese scallops (Patinopecten yessoen- and metamorphosis of Sipunculus nudus. J.
sis): empirical parameter estimates from cod- Fish. China, 31(5):633-638.
ing regions of mitochondrial DNA. Can. J. Nei M., 1987. Molecular Evolutionary
Fish. Aquat. Sci., 50:1147-1157. Genetics. Columbia Univ. Press, New York.
Brown W.M., George M. Jr. and A.C. Palumbi S.R. and A.C. Wilson, 1990.
Wilson, 1979. Rapid evolution of animal mito- Mitochondrial DNA diversity in the sea urchins
chondrial DNA. Proc. Natl. Acad. Sci. USA, Strongylocentrotus purpuratus and S. droe-
76:1967-1971. bachiensis. Evolution, 44:403-415.
Excoffier L.G., Laval G. and S. Schneider, Reeb C.A. and J.C. Avise, 1990. A genetic
2005. Arlequin ver. 3.0: an integrated soft- discontinuity in a continuously distributed
ware package for population genetics data species: mitochondrial DNA in the American
analysis. Evolutionary Bioinformatics Online, oyster, Crassostrea virginica. Genetics,
1:47-50. 124:397-406.
Hare M.P. and J.C. Avise, 1996. Molecular Su T.F., Jiang S.G., Zhou F.L., Zhu C.Y. and
genetic analysis of a stepped multilocus cline P.M. Chen, 2005. Mitochondrial 16SrRNA
in the American oyster (Crassostrea virgini- gene fragment sequence analysis in popula-
ca). Evolution, 50:2305-2315. tions of Crassostrea rivularis. High Technol.
Jukes T.H. and C.R. Cantor, 1969. Evolution Letter, 15(2):100-103.
of protein molecules. In: H.N. Munro (ed.). Thompson J.D., Gibson T.J., Plewniak F.,
Mammalian Protein Metabolism. Academic Jeanmougin F. and D.G. Higgins, 1997. The
Press, New York. ClustalX Windows interface: flexible strate-
Klinbunga S., Penman D.J., McAndrew gies for multiple sequence alignment aided by
B.J., Tassanakajon A. and P. Jarayabhand, quality analysis tools. Nucleic Acids Res.,
1998. Genetic variation, population differenti- 24:4876-4882.
ation and gene flow of the giant tiger shrimp Wang Q.H., Du X.D. and K. Li, 2006. Genetic
(Penaeus monodon) inferred from mtDNA- diversity of Sipunculus nudus as revealed by
RFLP data. In: T.W. Flegel (ed.). Advances in RAPD. Mar. Fish. Res., 27(3):57-61.
Shrimp Biotechnology. Natl. Center Genet. Yu Z.N., Wei X.H., Kong X.Y. and S.S. Yu,
Eng. Biotechnol., Bangkok. 2007. Application of the first internal tran-
Kong X.Y., Yu Z.N., Liu Y.J. and L.L. Chen, scribed spacer (ITS-1) of ribosomal DNA as a
2003. Intraspecific genetic variation in mito- molecular marker to population analysis in far-
chondrial 16S ribosomal gene of Zhikong rer’s scallop Chlamys farreri. Acta
scallop Chlamys farreri. J. Shellfish Res., Oceanologica Sinica, 26(1):93-100.
22:655-660. Zhang Y.P. and O.A. Ryder, 1993.
Kumar S., Tamura K. and M. Nei, 2004. Mitochondrial DNA evolution in the Artoidea.
MEGA 3.0: integrated software for molecular Proc. Natl. Acad. Sci. USA, 90:9557-9561.