ISSN 10227954, Russian Journal of Genetics, 2010, Vol. 46, No. 1, pp. 69–80. © Pleiades Publishing, Inc., 2010.
Original Russian Text © L.A. Skurikhina, A.D. Kukhlevsky, A.G. Oleinik, N.E. Kovpak, 2010, published in Genetika, 2010, Vol. 46, No. 1, pp. 79–91.
Recently, numerous works succeeded in clarifica
tion of the intraspecific variation and phylogenetic
relationships between and within smelts (family
Osmeridae) by using various methods [1–11]. On the
other hand, the data on the relationships between the
genera of this family are confined to only classic
approaches of comparative morphology [12–16]. The
only exclusion is the work by Fu et al. , who studied
the phylogenetic position of salangid fishes (subfamily
Salanginae) and phylogenetic relationships between
the genera of this subfamily using partially sequenced
mitochondrial genes of cytochrome
and 16S rRNA.
The fact that clarification of many taxonomic and
phylogenetic issues must be supported by analysis of
primary DNA sequences is now doubtless. Due to its
properties, mitochondrial DNA (mtDNA) is most fre
quently used for these purposes . The gene encod
) [18–23] is widely used for
phylogenetic analysis and verification of the taxo
nomic status of vertebrates, fish included; this gene
can be used for assessing the divergence of taxa to the
level of families .
We have analyzed the variation of
RFLP (restriction fragment length polymorphism)
and direct sequencing. The evolution of this gene has
been studied in all the smelt species inhabiting Russian
waters that are most abundant representatives of the
were compared with the phylogenetic schemes based
on classic comparative morphology approaches.
MATERIALS AND METHODS
The specimens collected over 2001–2007 were
studied. The sample sizes and sampling sites are listed
in the table.
Total DNA was isolated from fixed heart and liver
tissues according to a standard protocol .
The mtDNA variation was studied by
RFLP analysis of the region with a length of 2599 base
pairs (bp) containing the coding region of
and noncoding Dloop regulatory region (
) amplified by polymerase chain reaction (PCR).
The primer sequences, localization of the region in
mtDNA map, and amplification conditions are given
in . The following 17 restriction endonucleases
were used for analysis:
. The reaction was conducted
according to the manufacturer’s protocols (MBI Fer
mentas, Lithuania; SibEnzim, Russia). The restric
tion fragments were separated in 1.5–2.0% agarose gel
containing Ultra Pure
agarose (BRL Gibco,
United States) and Synergel
Inc., United States) at a ratio of 1 : 2 in 0.5
borate buffer . The DNA fragments in gel were
stained with ethidium bromide and photographed in
incident UV light. The DNA of
Phylogenetic Analysis of Smelts (Osmeridae) Based
on the Variation of Cytochrome
L. A. Skurikhina, A. D. Kukhlevsky, A. G. Oleinik, and N. E. Kovpak
Zhirmunsky Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok, 690041 Russia;
Received January 14, 2009
—Putative phylogenetic relationships between all smelt species inhabiting Russian waters were stud
ied using RFLP and the data on divergence between nucleotide sequences of the mitochondrial gene encod
. All types of phylogenetic trees (NJ, MP, and Bayesian) displayed stable clustering into iso
lated groups corresponding to the division of the subfamily Osmerinae into three genera,
It was demonstrated that the
phylum was the first to diverge from a hypothetical
common ancestor of the smelts. Later, it divided into two clusters corresponding to the modern subspecies
Mallotus villosus villosus
M. v. catervarius.
The phylum of the genus
diverged later than the genus
and also divided into two clusters, one including
of the Pacific (Sea of Japan) and Arctic
(Kara and Barents Seas) basins and the other, anadromous and resident ecotypes of the
Baltic Sea basin. The smelts of the genus
is the youngest phylogenetic group among the taxa stud
ied; it forms three individual clusters matching the species
H. olidus, H. nipponensis
proposed phylogenetic hypothesis is discussed from the standpoint of its compliance with the phylogenetic
constructions based on the external morphological and osteological traits.