1063-0740/04/3004- © 2004
Russian Journal of Marine Biology, Vol. 30, No. 4, 2004, pp. 259–265.
Original Russian Text Copyright © 2004 by Biologiya Morya, Skurikhina, Oleinik, Pan’kova.
The marine, anadromous, and native species of the
most common genera of the family Osmeridae
) are encountered
in the waters of the Asian Paciﬁc coast and in the
marine coastal waters of the Northeast Paciﬁc Ocean
[1, 6, 9–13, 30, 31, 35]. The systematics of the genera
is now generally accepted. In
contrast, the taxonomical relationships and biogeogra-
phy of the genus
have been extensively
debated and revised [2–5, 14–16, 27–29, 31, 38].
In recent years, molecular biology techniques have
proven most useful in solving a wide range of taxonomical
problems and testing hypotheses. The mitochondrial
genome has received a great deal of attention for being
passed on to offspring solely by the mother without
recombination. Moreover, the nucleotide substitution rate
of the mitochondrial DNA is believed to be faster than the
substitution rate of the nuclear genome , therefore
allowing closely related taxa to be clearly differentiated.
This paper deals with mitochondrial DNA (mtDNA)
diversity in ﬁve smelt species that belong to the genera
of the family Osmeridae mentioned above.
MATERIALS AND METHODS
DNA was extracted from a total of 120 individuals,
which were sampled in spring and summer 2001 (Fig. 1).
All species were identiﬁed by I.A. Chereshnev using
The total genomic DNA was extracted from ﬁxed
liver and heart tissues according to the routine protocol
. For an analysis of mtDNA diversity, a segment the
mitochondrial DNA coding for NADH-dehydrogenase
subunits ND5/ND6 was ampliﬁed with the polymerase
chain reaction (PCR) and screened for restriction frag-
ment length polymorphism (RFLP). This segment is
2488 base pairs in length; its location on the mtDNA
map, primer sequences, and ampliﬁcation conditions
have been reported by Gharrett
The ampliﬁed fragment was digested with the fol-
lowing eleven restriction enzymes: AsuI (G’GNCC),
AvaII (G’GWCC), BstNI (CC’WGG), BstUI
(CG’CG), EcoRV (GAT’ATC), HinfI (G’ANTC),
MboI ('GATC), MspI (C’CGG), RsaI (GT’AC), StyI
(C’CWWGG), and TagI (TC’GA). The digestion was
performed according to the manufacturer’s protocol
(MBI Fermentas, Lithuania, and SibEnzim, Russia).
Restriction digests were separated in a 1.5–2% agarose
gel composed of part of Ultra Pure
Gibco, Grand, N.Y.) and two parts of Synergel
(Diversiﬁed Biotech Inc., Boston, MA) in 0.5
borate buffer . The gel was stained with ethidium
bromide and photographed in transmitted ultraviolet
light. DNA fragments of phages
with PstI and HinfI, respectively, as well as a 100-base
pair ladder (BRL Gibco, Grand, N.Y.) were used as
molecular length standards.
The heterogeneity among the samples was assessed
by the Monte Carlo method using the REAP software
package . This software was also used to calculate
the number of nucleotide substitutions per site (
dard deviation, divergence among samples (
otide diversity (
), and haplotype diversity (
). We also
performed a phenetic analysis with the NTSYS soft-
ware using the unweighted pair-group method of arith-
metic averages (UPGMA) .
Comparative Analysis of Mitochondrial DNA Diversity in Smelts
L. A. Skurikhina, A. G. Oleinik, and M. V. Pan’kova
Institute of Marine Biology, Far East Division, Russian Academy of Sciences,
Vladivostok, 690041 Russia
Received November 26, 2003
—The polymerase chain reaction (PCR) was used to amplify a segment of the mitochondrial DNA
coding for NADH-dehydrogenase subunits ND5/ND6 in ﬁve smelt species (family Osmeridae). Ampliﬁed
DNA was screened for restriction fragment length polymorphism (RFLP). Nucleotide sequence divergence of
mitochondrial DNA between species ranges from 11.9 (between
Osmerus mordax dentex
Mallotus villosus catervarius
). The genetic divergence between
Osmerus mordax dentex
was 0.32, 0.08 to 0.15, and 0.025%,
respectively. The absence of common haplotypes enables differentiation of closely related smelt species and,
therefore, can be used for solving current problems in the taxonomy and biogeography of this family.
Osmeridae, mitochondrial DNA, restriction fragment length polymorphism analysis (RFLP), poly-
merase chain reaction (PCR), genetic divergence.