„
2005 Zoological Society of JapanZOOLOGICAL SCIENCE

22

: 157–168 (2005)

Phylogeography of Loaches of the Genus

Lefua

(Balitoridae,
Cypriniformes) Inferred from Mitochondrial DNA Sequences

Motohiro Mihara

1

, Takehiko Sakai

1

, Koushin Nakao

1

, Leonardo de Oliveira Martins

2

,
Kazumi Hosoya

3

and Jun-Ichi Miyazaki

1

*

1

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

2

Laboratory of Biometry and Bioinformatics, Graduate School of Agriculture and
Life Sciences, University of Tokyo, Yayoi 1-1-1,
Bunkyo-ku, Tokyo 113-8657, Japan

3

Department of Fisheries, Faculty of Agriculture, Kinki University,
Nakamachi 3327-204, Nara 631-8505, Japan

ABSTRACT

—In order to elucidate phylogenetic relationships and intraspecific variations and to infer the
evolutionary process of loaches of the genus

Lefua

, we analyzed nucleotide sequences of the mitochon-
drial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus

Lefua

includes three described species,

L. nikkonis

,

L. echigonia

, and

L. costata

and an undescribed species,

Lefua

sp. Our results showed that each species of

Lefua

formed a monophyletic group, indicating clearly
that

Lefua

species can be genetically distinguished from one another.

Lefua nikkonis

was the most closely
related to

L. costata

, while

L.

sp. was the most closely related to

L. echigonia

. Specimens of

L.

sp. were
grouped into two intraspecific populations and specimens of

L. echigonia

were grouped into six popula-
tions. These populations were well separated geographically from one another by mountain ranges and
highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and
speculated on the evolutionary process of the genus

Lefua

. Species of

Lefua

are severely threatened. Fun-
damental genetic information is indispensable for conservation. We presented genetic background in order
to protect these threatened loaches.

Key words:

intraspecific variation, D-loop, mitochondria, conservation, speciation, dispersal, vicariance

INTRODUCTION

In the genus

Lefua

(Balitoridae, Cypriniformes), three
species are formally described,

L. costata

(Kessler),

L.
nikkonis

(Jordan and Fowler), and

L. echigonia

Jordan and
Richardson, and

Lefua

sp. was recently separated from

L.
echigonia

by morphological (Hosoya, 1994, 2000), ecologi-
cal (Yamashina

et al

., 1994), and genetical (Sakai

et al

.,
2003) studies. It is conceivable that loaches of

Lefua

can be
readily isolated and accumulate genetic variations because
of their specific and restricted habitats. They inhabit rela-
tively fast-flowing streams with gravelly beds on mountains
or relatively slow-flowing streams with muddy beds between
hills.

Lefua costata

is distributed widely in East Asia includ-
ing Korea, China, and Russia, but not in Japan.

Lefua nikko-
nis

is endemic to the Hokkaido Island of Japan.

Lefua echig-
onia

inhabits the Honshu Island from the Tohoku region to
the Kinki region.

Lefua

sp. is distributed from the Chubu
region to the Chugoku region in the Honshu Island and also
in the eastern Shikoku Island. We previously reported that

L. echigonia

was more closely related to

L.

sp. than to

L.
nikkonis

and

L. costata

, and that neither

L. nikkonis

nor

L.
costata

was monophyletic and these species together com-
prised a clade (Sakai

et al

., 2003).

Lefua echigonia

is known
to have large intraspecific variations (Fujita and Okawa,
1975) and four local populations were distinguished geneti-
cally (Sakai

et al

., 2003). These populations may reflect the
evolutionary process of the genus

Lefua

and form part of the
evolutionary history of East Asian freshwater fish fauna;
thus, they could be considered to be evolutionarily signifi-
cant units (ESU; Morits, 1994).
The nucleotide sequence of mtDNA is informative to
examine genetic divergence and gene flow among popula-
tions because of the maternal inheritance of mitochondria.
Advanced statistics can be applied to analyze sequence
data. Sequencing of mtDNA has been thus used to study
geographic variations of genotypes to reconstruct the pro-

* Corresponding author. Phone: +81-298-53-4665;
Fax : +81-298-53-6614;
E-mail: junichi@biol.tsukuba.ac.jp

M. Mihara

et al

.158

cess of intraspecific diversification of freshwater fishes (Brito

et al

., 1997; Kitagawa

et al

., 2001; Miyake

et al

., 2001; Oka-
zaki

et al

., 2002; Takehana

et al

., 2004; Yamamoto

et al

.,
2004). The mitochondrial D-loop (displacement-loop), which
is a non-protein-coding control region, is useful for analysis
of intraspecific genetic relationships because of its high evo-
lutional rate (Shedlock

et al

., 1992; Koskinen

et al

., 2002;
Salzburger

et al

., 2003).
Phylogenetic studies using molecular information are
now considered to be the most powerful approach to inves-
tigate genetic backgrounds in order to protect freshwater
fishes facing extinction. Species of

Lefua

are severely
threatened owing to rapid destruction of their habitats
through human activities.

Lefua echigonia

and

L.

sp. were
assigned to endangered species, and

L. nikkonis

to vulner-
able species in the Red Data Book by the Ministry of the
Environment of Japan (Hosoya, 2003; Goto, 2003). Funda-
mental genetic information is indispensable for conservation
of these threatened loaches.
In this study, we investigate interspecific relationships
and intraspecific variations of the genus

Lefua

by sequenc-
ing mtDNA of the D-loop region with more specimens from
new localities. We infer the evolutionary process of the
genus

Lefua

by estimating the evolutionary time of species
and intraspecific populations under consideration of rate
variation in molecular evolution. Our data also present
genetic background for protecting the threatened loaches,
and establish six and two intraspecific populations of

L.
echigonia

and

L.

sp., respectively, to be conserved.

MATERIALS AND METHODS

Materials

Collecting localities of

Lefua nikkonis

,

L. costata

,

L

. sp., and

L.
echigonia

are shown in Fig. 1 and sample numbers are listed in
Table 1.

Noemacheilus barbatulus toni

of the family Balitoridae was
used as an outgroup species. Two of the 8 specimens of

L. costata

were obtained from commercial sources, and both were collected
in Korea, but the exact sampling location of one of the specimens
(sample number 14) is unknown.

Lefua

sp. and

L.



echigonia

were
sympatric in Nishikata, Kyoto Prefecture (sample numbers 23 and
62) and Kasuga, Hyogo Prefecture (sample numbers 24 and 63).

DNA sequencing of the mitochondrial D-loop region

The back muscle or caudal fin from each individual was
homogenized in 250

m

l of a proteinase solution (0.1 mg/ml protein-
ase K, 50 mM KCl, 1.5 mM MgCl

2

, 0.1% gelatin, 0.45% NP-40,
0.45% Tween 20, and 10 mM Tris-HCl·pH 8.0). The homogenate
was incubated at 37

∞

C overnight or at 55

∞

C for 2 hours, and
extracted with phenol, phenol/chloroform, and chloroform. The
supernatant was mixed with the same volume of 4 M ammonium

Fig. 1.

Collection sites of loaches.

*

,

Noemacheilus



barbatulus toni

; #,

Lefua nikkonis

; △ , L. costata ; ☆ , L . sp.; ○ , L. echigonia . Refer to
Table 1 for details of the collection sites and sample numbers.