ZOOLOGICAL SCIENCE 25: 261–272 (2008)
© 2008 Zoological Society of Japan
Genetic Differentiation of the Fejervarya limnocharis Complex
from Bangladesh and Other Asian Countries Elucidated
by Allozyme Analyses
Mohammed Mafizul Islam1, Md. Mukhlesur Rahman Khan2, Djong Hon Tjong3,
Mohammad Shafiqul Alam1 and Masayuki Sumida1*
1
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University
Higashihiroshima 739-8526, Japan
2
Department of Fisheries Biology and Genetics, Faculty of Fisheries,
Bangladesh Agricultural University, Mymensingh, Bangladesh
3
Department of Biology, Faculty of Science, Andalas University,
Padang 25136, West Sumatra, Indonesia
The present study was conducted to elucidate the genetic divergence and the phylogenetic relationships in the F. limnocharis complex from Bangladesh and other Asian countries such as Sri Lanka,
Thailand, Malaysia, Taiwan and Japan by allozyme analyses. We used a total of 95 frogs of the F.
limnocharis complex from these countries and F. cancrivora from the Philippines as an outgroup.
Based on body size, the F. limnocharis complex from Bangladesh was divided into three distinct
groups: large, medium and small types. Allozyme analyses were carried out with 28 loci encoding 20
enzymes and two blood proteins by horizontal starch-gel electrophoresis. When Nei’s (1972) genetic
distance was calculated, distinct divergence was found among the three types: mean genetic distance
was 0.782 between the small and medium types, 1.458 between the large and medium types, and 1.520
between the large and small types. Phylogenetic trees based on genetic distance showed that all
populations of Bangladesh small type strongly formed a cluster and were found to be most closely
related to the Sri Lanka population; that all populations of Bangladesh large type formed a very strong
cluster and were grouped with several populations from Thailand, Malaysia, Japan, and Taiwan; and
that the medium type was segregated from all other groups. This may imply that each of the three
types is a different species, and that the medium type is possibly an undescribed taxon.
Key words: genetic differentiation, Fejervarya limnocharis complex, allozyme analyses, genetic distance,
Bangladesh
INTRODUCTION
The rice frog Fejervarya limnocharis (Gravenhorst,
1829), previously known as Rana limnocharis (Annandale,
1917) and only recently regarded as belonging to Fejervarya
(Dubois and Ohler, 2000), is one of the most widely distributed frogs in Asia. This species is prevalent throughout
much of South, East, and Southeast Asia and many of the
islands of Indonesia and Malaysia, and is also found in
northern, central, southern, and southwestern China up to
western Japan. Although F. limnocharis was originally
described from Bogor, Java, Indonesia (Gravenhorst, 1829,
and Wiegmann, 1834, in Dubois and Ohler, 2000), many
frogs from other Asian countries were identified as F.
limnocharis, but subsequently were recognized as different
species or subspecies of the genus Fejervarya (henceforth
referred to as the F. limnocharis complex). To date, a total
* Corresponding author. Phone: +81-82-424-7482;
Fax : +81-82-424-0739;
E-mail : [email protected]
doi:10.2108/zsj.25.261
of 31 species in different Asian countries has been
described as being from this complex (Frost, 2007).
Bangladesh is a small riverine country in Southeast Asia
with rich biodiversity. Until now, reliable information on the
number of frog species in Bangladesh was unavailable,
although very limited genetic analysis of Bangladeshi frogs
was performed by Khan et al. (2002). Frost (2007) mentioned four Fejervarya species distributed in Bangladesh as
F. brama, F. frithii, F. syhadrensis, and F. limnocharis, but
was puzzled about whether the distribution of F. brama was
in contemporary India or Bangladesh and considered this as
a synonym of Hoplobatrachus tigerinus. There was little
adequate information for F. frithii, since this species was
only known from the type locality, Jessore in southern
Bangladesh, and the type specimen was lost. According to
recent surveys conducted by IUCN Bangladesh (2000), only
a single species of genus Fejervarya, F. limnocharis, was
reported within the territory of Bangladesh. Among the few
reviews available on Bangladesh F. limnocharis, Sarker
(1999) mentioned that the cricket frog, F. limnocharis (he
used the name Limnonectes limnocharis), was fairly
common in Bangladesh and had a wide distribution mainly
262
M. M. Islam et al.
in irrigated, cultivated fields that created a new habitat for
feeding, sheltering, and breeding.
Bangladesh is surrounded by India, shares a small border with Myanmar, and is adjacent to Nepal, Pakistan, Sri
Lanka, and other South Asian countries with the same geographical and meteorological conditions and practically with
no geographical barriers. Dutta (1997) reported nine species
of the F. limnocharis complex in India. Khan (2004) reported
two Fejervarya species in Pakistan. Dubois (1975, 1984)
declared that the Nepalese F. limnocharis could be separated into four species including F. nepalensis, F. pierrei,
and F. syhadrensis, and also suggested that F. limnocharis
nicobariens was a synonym of F. andamanensis. According
to Frost (2007), in the countries adjacent to Bangladesh,
there were 19 Fejervarya species from India, two from
Myanmar, three from Sri Lanka, and five from Nepal. There
was little research on the F. limnocharis complex in
Bangladesh, and no genetic studies were reported on this type
of frog. During a preliminary survey in Bangladesh, we collected samples of the F. limnocharis complex from different
part of Bangladesh and found conspicuous variation in body
size among this species complex. So, it is imperative to investigate this species complex in Bangladesh. There is a possibility of finding undescribed species from Bangladesh, and it
is also necessary to accurately clarify the taxonomy of this
complex distributed throughout the country after comparisons
with the F. limnocharis complex from other Asian countries.
In the present study, the F. limnocharis complex from
Bangladesh was studied biochemically for the first time. This
study has provided us with the first information regarding the
genetic relationships of the Bangladeshi rice frog with the
possibility of describing distinct species from the complex in
this territory.
MATERIALS AND METHODS
A total of 95 frogs of the Fejervarya limnocharis complex were
used in the present allozyme study. They consisted of both male
and female individuals from 16 different locations in Bangladesh
Table 1.
and other Asian countries such as Sri Lanka, Thailand, Malaysia,
Japan, and Taiwan. Fejervarya cancrivora was used as an outgroup, because it belongs to the genus Fejervarya but is considered
to be remotely related to other Fejervarya species. Among the F.
limnocharis complex from Bangladesh, three different types were
found based on SVL (snout vent length) and external characteristics: the first type has an average SVL of 53.6±6.4 mm, a moderate
snout, a very long dermal ridge, and black-striped grayish coloration
in the thigh region; the second type has an average SVL of 38.0±4.9
mm, a very sharp snout, a moderately long dermal ridge, and blackspotted yellowish coloration in the thigh region; the third type has
an average SVL of 35.6±3.8 mm, a blunt snout, a dot-shaped
dermal ridge, and faint yellowish to grayish coloration in the thigh
region (Islam et. al., unpublished data). The first, second and third
types are referred to as the large, medium, and small types in this
study, respectively. Collecting stations and the number of individuals used in the present study are given in Table 1 and Fig. 1.
Seventeen enzymes extracted from skeletal muscle and two
blood proteins were analyzed by horizontal starch-gel electrophoresis. The procedure followed that described by Nishioka et al. (1980,
1992) with slight modification. The detection of each enzyme was
carried out using the agar overlay method outlined by Harris and
Hopkinson (1976). The amido black staining method was used for
the detection of blood proteins.
The kinds of enzymes and blood proteins analyzed, enzyme
commission numbers (Nomenclature Committee — International
Union of Biochemistry, 1992), tissue samples used, and associated
buffer systems are given in Table 2. Notations of presumptive loci
are made using the abbreviations of enzymes. In cases where more
than two loci control an enzyme system, except for LDH, we added
numerical suffixes, with “1” representing the most anodal locus. In
the case of LDH loci, electromorphs were designated alphabetically
according to their anodal mobility.
To estimate the genetic variability of local populations, the
mean proportion of heterozygous loci per individual (H), mean proportion of polymorphic loci per population (P), and mean number of
alleles per locus (A) were quantified. A locus was considered to be
polymorphic when each of the multiple alleles existed at a frequency exceeding 1%. The fixation index (Fst) was calculated for
all populations of each Bangladesh large or small type using
POPGENE software (Yeh et al., 1997) based on Nei (1987). The
genetic relationship between different types and populations was
Collecting localities and sample sizes of specimens used in the present study.
Collecting station
Species
Ingroup
F. limnocharis complex
Locality
Large
Medium
Bangladesh
Mymensingh, BAU1 Campus
Mymensingh, Shambuganj
Mymensingh, Muktagacha
Mymensingh, Bailor
Kishorganj, Karimganj
Sherpur, Gazni
Dinajpur, Parbotipur
Nawabganj, Nawabganj
Khulna, Kailasganj
Hiroshima
Ishigaki
Chiai
Kuala Lumpur
Kota Kinabalu
Bangkok
Bentota
Manila
Total
14
4
4
2
10
14
3
4
3
5
Japan
Taiwan
Malaysia
Outgroup
1
F. cancrivora
No. of frogs
Country
Thailand
Sri Lanka
Philippines
Bangladesh Agricultural University.
58
Small
Population
Standard
Total
(Abbreviation)
2
2
2
1
1
2
2
2
14
27
6
4
2
11
16
7
4
4
2
2
2
1
1
2
2
2
95
BAU Campus (BAUC)
Shambuganj (Sham)
Muktagacha (Mukt)
Bailor (Bail)
Kishorganj (Kish)
Sherpur (Sher)
Dinajpur (Dina)
Nawabganj (Nawa)
Khulna (Khul)
Hiroshima (Hiro)
Ishigaki (Ishi)
Taiwan (Taiw)
Kuala Lumpur (Kual)
Kota Kinabalu (Kota)
Thailand (Thai)
Sri Lanka (Sril)
F. cancrivora (Canc)
8
2
1
2
4
1
5
18
Fejervarya limnocharis Complex
263
Fig. 1. Map showing the collecting stations for the F. limnocharis complex from Bangladesh and other Asian countries and for F. cancrivora
from the Philippines used in the present study.
Table 2. Enzymes and blood proteins analyzed in the present study.
Enzyme or blood protein
E.C. No. a
No. of locus
Locus
Sample
Buffer systemb
AAT-1
AAT-2
ADA
AK
ALD
CK
FUM
α-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
PEP-A
PEP-B
PEP-C
PEP-D
6-PGD
PGM
SOD
Alb
Hb-1
Hb-2
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Skeletal muscle
Blood serum
Erythrocyte
Erythrocyte
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-E-B pH 8.0
T-E-B pH 8.0
T-C pH 6.0
T-E-B pH 8.0
T-C pH 7.0
T-C pH 7.0
T-C pH 6.0
T-C pH 6.0
T-C pH 6.0
T-C pH 6.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-E-B pH 8.0
T-E-B pH 8.0
T-E-B pH 8.0
T-E-B pH 8.0
T-C pH 7.0
T-E-B pH 8.0
T-E-B pH 8.0
T-E-B pH 8.0
T-E-B pH 8.0
T-E-B pH 8.0
Aspartate aminotransferase
2.6.1.1
2
Adenosine deaminase
Adenylate kinase
Aldolase
Creatin kinase
Fumarase
α-Glycerophosphate dehydrgenase
Glucose-6-phosphate isomerase
Isocitrate dehydrogenase
3.5.4.4
2.7.4.3
4.1.2.13
2.7.3.2
4.2.1.2
1.1.1.8
5.3.1.9
1.1.1.42
1
1
1
1
1
1
1
2
Lactate dehydrogenase
1.1.1.27
2
Malate dehydrogenase
1.1.1.37
2
Malic enzyme
1.1.1.40
2
Mannose-6-phosphate isomerase
Peptidase
5.3.1.8
3.4.3.1
6-Phosphogluconate dehydrogenase
Phosphoglucomutase
Superoxide dismutase
Serum albumin
Hemoglobin
1.1.1.44
5.4.2.2
1.15.1.1
–
–
1
1
1
1
1
1
1
1
1
2
a
b
E.C. No. from the Nomenclature Committee — International Union of Biochemistry (1992).
T-C, Tris-citrate buffer; T-E-B, Tris-borate-EDTA buffer.
264
M. M. Islam et al.
Allele frequencies
The allele frequencies for each population of the F.
limnocharis complex and outgroup F. cancrivora at all 28
loci are represented in Table 4. When only three
Bangladesh frog types were considered, the Bangladesh
large type had diagnostic alleles at 10 loci (ALD, CK, FUM,
α-GDH, IDH-1, LDH-B, MDH-2, ME-2, Hb-1, and Hb-2); the
Bangladesh medium type had diagnostic alleles at six loci
(ALD, FUM, IDH-2, PEP-C, Alb, and Hb-1); and the
Bangladesh small type had diagnostic alleles at eight loci
(AAT-2, ALD, FUM, MDH-1, ME-1, PGM, Alb, and Hb-1).
Among the different presumptive loci, the FUM locus
was one of the most outstanding loci, where the Bangladesh
large, medium, and small types had diagnostic alleles b, a,
and c, respectively (Fig. 2). It is noteworthy here that the
Bangladesh large type shared the same allele, b, as other
Asian populations except for the Sri Lanka one, whereas the
Sri Lanka one shared the same allele, c, as the Bangladesh
small type. At the ADA locus, the Bangladesh large type had
the diagnostic allele g, while the other two Bangladesh types
shared a major allele, c, together with diagnostic alleles a,
b, d, and e found in the Bangladesh small type (Fig. 3). The
evaluated by calculating the genetic identity (I) and genetic distance
(D). The genetic distances among the F. limnocharis complex from
Bangladesh and other Asian countries and outgroup F. cancrivora
were estimated based on gene frequencies at 25 loci using the
method of Nei (1972, 1978). Dendrograms were drawn based on
genetic distances using the neighbor-joining (Saitou and Nei, 1986),
UPGMA, and CONTML methods. After comparing all constructed
trees, we show only the neighbor-joining tree based on Nei’s (1972)
genetic distances. Bootstrap values were calculated with 1000
pseudoreplicates. For data analysis, we used the PHYLIP 3.65
package (Felsenstein, 2005) and the POPGENE program (Yeh et
al., 1997).
RESULTS
Electrophoretic patterns and allelomorphs
The electrophoretic patterns of 17 enzymes and two
blood proteins were presumed to be encoded by genes at
28 loci (Table 3). At 28 loci, two to 14 phenotypes (6.1 on
average) were produced by two to nine alleles (4.8 on
average). Among the 28 loci, the minimal two phenotypes
produced by two alleles (a and b) were found at the AAT-2
and CK loci. The maximum of 14 phenotypes produced by
nine alleles (a–i) was found at the MPI locus.
Table 3. Number of phenotypes and alleles at 28 loci for the Fejervarya limnocharis complex from Bangladesh
and other Asian countries, and for F. cancrivora.
Locus
Alleles
No.
AAT-1
AAT-2
ADA
AK
ALD
CK
FUM
α-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
PEP-A
PEP-B
PEP-C
PEP-D
6-PGD
PGM
SOD
Alb*
Hb-1*
Hb-2*
Average
4
2
9
3
4
2
5
4
5
5
3
4
9
6
7
3
4
9
3
5
5
5
6
3
4
6
6
3
4.8
Phenotypes
Kind
No.
a-d
a-b
a- i
a-c
a-d
a-b
a-e
a-d
a-e
a-e
a-c
a-d
a-i
a-f
a-g
a-c
a-d
a-i
a-c
a-e
a-e
a-e
a-f
a-c
a-d
a-f
a-f
a-c
6
2
10
3
5
2
6
4
7
6
3
4
12
7
7
4
6
14
3
7
6
9
11
3
5
7
6
3
6.1
Kind
AA, BB, CC, DD, AB, AC, BC
AA, BB
CC, EE, FF, GG, AB, AC, AD, CE, EH, GI
AA, BB, BC
AA, BB, CC, DD, AB
AA, BB
AA, BB, CC, AB, BD, CE
AA, CC, DD, BD
BB, CC, DD, AB, AC, BD, CE
CC, DD, EE, AE, BC, BD
AA, BB, CC
BB, CC, DD, AB
BB, CC, DD, EE, FF, GG, HH, II, AD, BG, CD, FH
AA, BB, CC, EE, FF, BC, DF
AA, CC, DD, EE, FF, GG, BD
AA, BB, CC, AB
AA, BB, CC, DD, BC, BD
BB, DD, EE, FF, HH, II, AB, BD, CE, DF, DH, EF, EH, GH
BB, AB, BC
BB, CC, DD, EE, AB, BD, CD
AA, BB, CC, DD, EE, BD
AA, BB, CC, DD, EE, AC, AD, CD, DE
AA, BB, CC, DD, EE, BC, BD, BE, CD, DE, EF
AA, BB, CC
AA, CC, DD, AC, BC
AA, BB, CC, EE, FF, BD, CD
AA, BB, CC, DD, EE, FF
AA, BB, CC
*These loci were not examined in F. limnocharis complex from Taiwan and Kota Kinabalu, and in F. cancrivora.
Fejervarya limnocharis Complex
265
Table 4. Allele frequencies at 28 loci in the F. limnocharis complex from Bangladesh and other Asian countries, and in F. cancrivora.
Locus
Populations
BAUC
(L)
AAT-1
c
Sham
(L)
c
Mukt
(L)
c
Bail
(L)
c
Kish
(L)
c
Sher
(L)
Dina
(L)
a(0.036) c
Nawb
(L)
c
Khul
(L)
c
c(0.964)
BAUC
(M)
BAUC
(S)
a(0.100) c
Sham
(S)
Kish
(S)
Sher
(S)
Dina
(S)
Khul
(S)
Hiro
Ishi
Taiw
Kual
Kota
Thai
Sril
Canc
c
c
c
c
c
c
c
c
c
c
c
c
d
b
a
a
a
b(0.700)
c(0.200)
AAT-2
a
a
a
a
a
a
a
a
a
a
b
b
b
b
b
a
a
a
b
b
ADA
g(0.964) g
g
g
g
g
g
g
g
g
a(0.125) c
c
a(0.500) a(0.375) c
g
g
g(0.750) g(0.500) i
g(0.250) c
f
i (0.250) i (0.500)
h(0.250)
i (0.036)
b(0.063)
b(0.250) c(0.675)
c(0.688)
c(0.250)
i (0.500)
e(0.125)
AK
b(0.929) b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
a
b
b
b
b
b
b
b
c
a
a
a
a
a(0.875) a
a
a
a
a
a
a
a
d
b
c(0.071)
ALD
b
b
b(0.015)
CK
b
b
b
b
b
b
b
b
b
a
a
a
a
a
a
b
b
b
b
b
a
b
FUM
b
b
b
b
b
b
b
b
b
a
c
c
c
c
c(0.750) c(0.500) b(0.750) b
a
b
b
b
a(0.250) c
b
e(0.250) e(0.500) d(0.250)
α-GDH d
d
b(0.125) b(0.250) d
d
a
b(0.750)
d
d
d
a
a
a
a
a
a
d
d
d
d
d
d
a
c
a(0.036) c
c
c
a(0.100) b(0.937) b
b
b
b
b
c(0.750) d
c
c
a(0.500) c
a(0.250) b
c(0.500)
b(0.750)
d(0.875) d(0.750)
GPI
a(0.107) a(0.125) c
c
c
c(0.893) c(0.875)
IDH-1
IDH-2
e
a
e
c(0.964)
e
e
e
e
b(0.900) d(0.063)
e
e(0.250)
a(0.125) e
b(0.100) c
b(0.250) c
e(0.875)
d(0.900)
c(0.750)
c
b(0.250) c
e
e
e
e
e
e
c(0.750)
b(0.250) d
c(0.750)
a
a
a
a
a
a
a
a
c
a
a
a
a
a
a
b
a
a
a
a
a
a
a
LDH-A a(0.071) b
b
b
b
a(0.071) b
b
b
b
b
b
b
b
b
b
d
d
d
d
d
b
b
c
b
b
c(0.900) d
d
d
d
a(0.125) d
e
e
e
f
b(0.929)
LDH-B b
b(0.929)
b
b
b
b
b
b
d(0.100)
MDH-1 c
c
c
c
c
c
c
c
b(0.167) b
d(0.875)
f
f
f
f
c(0.833)
MDH-2 d
d
d
d
d
d
d
d
d
f (0.500) e(0.500) b(0.250) i
h(0.500) h(0.500) g(0.750)
d(0.250) f
a
a
a
b
b
b
f
e
e
a
e
c
c
b(0.250) g
f
f (0.750)
g
g
g
g
g
g
g
d(0.250)
ME-1
ME-2
a(0.893) a(0.875) a(0.750) a(0.750) a(0.900) a(0.964) a
a(0.750) a(0.833) a(0.100) c
b(0.107) b(0.125) b(0.250) b(0.250) b(0.100) b(0.036)
b(0.250) b(0.167) b(0.900)
c
c
c
c
c
c
c
c
c
b
c
c
c
b(0.063) d
d
d(0.938)
MPI
c(0.036) e
e
e
e
c(0.071) e
e
e
a(0.100) d(0.938) d
e(0.893)
e(0.893)
b(0.800) e(0.063)
f (0.071)
f (0.036)
d(0.100)
PEP-A b
b
b
a(0.250) a(0.050) a(0.070) b
b
d
a
b
a
a
a
a
c
a
b(0.250) d
b(0.500) c
c
c
c
c
b(0.250) b
a
d(0.750)
d(0.500)
d
c
c
d(0.875) d
c(0.750)
f
f
f
h
e(0.125)
g(0.500) g(0.250) f (0.250) i
h(0.500) h(0.750) g(0.250)
h(0.500)
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
c(0.050) c(0.071) d
b(0.125) d
b
a(0.060) b
b
b
a(0.125) b
c
c
c
c
c
c
a(0.250) e
d(0.950) d(0.929)
d(0.875)
d
a
a
b
b
b
b
b
e
a(0.750) a(0.750) a
b
e
b
d
d
d
a
e
d(0.200) b(0.250) b(0.250) e(0.500) d(0.250) b(0.250) b(0.500) c
d
c
d
c(0.500) d
a
e
b(0.750) b(0.900) b(0.930)
c(0.050)
PEP-B d
PEP-C b
d
b
PEP-D d(0.964) d
d
b
d
d
b
d
b
d
e(0.036)
b
b
c(0.036) d
b
d
b(0.940)
b
d
c
c
d(0.929)
c
c
c
c
b(0.313) b(0.250) d
b(0.250) d
d(0.688) d(0.750)
d(0.750)
a(0.875) a
a
c(0.063)
e(0.036)
6-PGD b(0.036) c
b(0.875)
b(0.750)
d(0.250) d(0.250)
d(0.063)
c
c
c
c(0.964)
e(0.800) d(0.188) d(0.250) f (0.500) e(0.750) e(0.750) e(0.500)
d(0.500)
e(0.563) e(0.500)
PGM
b
b
b
b
b
b
b
b
b
b
c
c
c
c
c
c
b
b
b
b
b
a
c
c
SOD
c
c
c
c
c
c
b(0.170) c
c
a(0.900) a
a
a
a
a
a
c
c
c
c
c
c
a
d
b(0.833) b(0.500) b
b
b
b(0.500) f
e
–
e
–
e
b
–
c(0.830)
Alb
c(0.962) c
c
c
c
c
c
c(0.100)
c
c
a
d(0.038)
d(0.167) b(0.500)
d(0.500)
Hb-1
c
c
c
c
c
c
c
c
c
a
d
d
d
d
d
d
e
f
–
b
–
b
d
–
Hb-2
b
b
b
b
b
b
b
b
b
a
a
a
a
a
a
a
b
b
–
c
–
c
a
–
ME-1 locus was the most polymorphic in the Bangladesh
large and medium types, whereas it was not polymorphic in
any Asian frogs (Fig. 4). At this locus, the Bangladesh large
type shared the same allele, a, as other Asian populations
except for the Sri Lanka and Ishigaki ones, and the small
type shared the same allele, c, as the Sri Lanka one.
Among the 28 loci, all were found to be polymorphic at
the population level, except for AAT-2, CK, IDH-2, PGM, Hb1, and Hb-2.
Genetic variation
The most differentiated locus was AAT-1 for the
Bangladesh large type, with an Fst value of 0.201 (Table 5).
No differentiation was found at the AAT-2, ALD, CK, FUM,
IDH-2, LDH-B, MDH-2, ME-2, PEP-C, PGM, Hb-1, and Hb2 loci, whereas the other 15 loci showed some degree of
differentiation, with Fst values of 0.028–0.174. For the
Bangladesh small type, the most differentiated locus was
Alb, with an Fst value of 0.395, followed by FUM (0.333),
ADA (0.325), and ME-2 (0.294), whereas no differentiation
was found at the AAT-1, AAT-2, AK, CK, α-GDH, IDH-2,
LDH-A, MDH-2, ME-1, PEP-A, PGM, SOD, Hb-1, or Hb-2,
and the other 10 loci showed some degree of differentiation
with Fst values of 0.053–0.217.
The mean proportion of heterozygous loci per individual
(H) is shown in Table 6. The values of heterozygous loci per
individual ranged from 1.2% to 5.4% (mean, 3.1%) in the
Bangladesh large type, was 8.6% in the Bangladesh
266
M. M. Islam et al.
Fig. 2. Geographic distribution of ADA alleles in the F. limnocharis complex from Bangladesh and other Asian countries.
Fig. 3.
Geographic distribution of FUM alleles in the F. limnocharis complex from Bangladesh and other Asian countries.
Fejervarya limnocharis Complex
Fig. 4.
Geographic distribution of ME-1 alleles in the F. limnocharis complex from Bangladesh and other Asian countries.
Table 5. F-statistics for 28 loci in the large and small
types of the F. limnocharis complex from Bangladesh.
Locus
AAT-1
AAT-2
ADA
AK
ALD
CK
FUM
α-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
PEP-A
PEP-B
PEP-C
PEP-D
6-PGD
PGM
SOD
Alb
Hb-1
Hb-2
Average
267
Large type
Small type
0.201
0
0.028
0.064
0
0
0
0.174
0.079
0.113
0
0.057
0
0.151
0
0.064
0
0.067
0.142
0.075
0
0.039
0.032
0
0.151
0.032
0
0
0.086
0
0
0.325
0
0.106
0
0.333
0
0.053
0.156
0
0
0.106
0.217
0
0
0.294
0.075
0
0.075
0.16
0.137
0.155
0
0
0.395
0
0
0.206
medium type, and ranged from 3.6% to 14.3% (mean, 9.5%)
in the Bangladesh small type. There were no remarkable differences between the foregoing actual frequencies and the
expected values (Table 6).
The mean proportion of polymorphic loci per population
(P) was calculated for 28 loci in each of the 24 populations
(Table 6). The mean proportion of polymorphic loci per population ranged from 3.6% to 32.1% (mean, 13.9%) in the
Bangladesh large type, was 28.6% in the Bangladesh
medium type, and ranged from 3.6% to 35.7% (mean,
20.3%) in the Bangladesh small type.
The mean number of alleles per locus (A) ranged from
1.04 to 1.39 (mean, 1.15) in the Bangladesh large type, was
1.36 in the Bangladesh medium type, and ranged from 1.04
to 1.50 (mean, 1.24) in the Bangladesh small type.
Genetic distance
Table 7 shows Nei’s genetic distance (below diagonal)
and genetic identity (above diagonal) among different types
and populations of the F. limnocharis complex and the outgroup, F. cancrivora. Within the Bangladesh large type,
genetic distances were very minute, ranging from 0.001 to
0.009 (mean, 0.004). Within the Bangladesh small type,
genetic distances were also comparatively low, ranging from
0.007 to 0.047 (mean, 0.025). The genetic distances
between the Bangladesh large and medium types ranged
from 1.386 to 1.488 (mean, 1.458). The genetic distances
between the Bangladesh large and small types ranged from
1.437 to 1.628 (mean, 1.520). The genetic distances between
the Bangladesh medium and small types ranged from 0.739 to
268
M. M. Islam et al.
Table 6. Genetic variation at 28 loci for 24 populations of the F. limnocharis complex, and F. cancrivora. Expected values are
in parentheses.
Population
Sample size
Mean proportion of heterozygous
loci per individual (%)
14
4
4
2
10
14
3
4
3
5
8
2
1
2
4
1
2
2
2
1
1
2
2
2
3.8 ( 4.2)
1.8 ( 1.8)
2.7 ( 2.4)
5.4 ( 5.4)
1.8 ( 1.7)
3.8 ( 3.7)
1.2 ( 1.2)
5.4 ( 4.9)
2.4 ( 2.4)
8.6 ( 8.0)
8.9 ( 9.7)
7.1 ( 8.3)
3.6 ( 3.6)
10.7 (10.1)
12.5 (12.5)
14.3 (14.3)
3.6 ( 3.6)
0 ( 0 )
2.0*( 2.0)
3.6 ( 3.6)
16.0*(16.0)
7.1 ( 9.5)
10.7 (10.1)
0* ( 0 )
BAU Campus (L)
Shambuganj (L)
Muktagacha (L)
Bailor (L)
Kishorganj (L)
Sherpur (L)
Dinajpur (L)
Nawabganj (L)
Khulna (L)
BAU Campus (M)
BAU Campus (S)
Shambuganj (S)
Kishorganj (S)
Sherpur (S)
Dinajpur (S)
Khulna (S)
Hiroshima
Ishigaki
Taiwan
Kuala Lumpur
Kota Kinabalu
Thailand
Sri Lanka
F. cancrivora
Mean proportion of polymorphic
loci per population (%)
Mean number of
alleles per locus
32.1
7.1
7.1
10.7
10.7
32.1
3.6
14.3
7.1
28.6
35.7
14.3
3.6
17.9
35.7
14.3
7.1
3.6
4.0*
0
16.0*
17.9
17.9
0*
1.36
1.07
1.07
1.11
1.14
1.39
1.04
1.14
1.07
1.36
1.50
1.18
1.04
1.21
1.36
1.14
1.07
1.00
1.04*
1.04
1.16*
1.18
1.21
1.00*
*Except the Alb, Hb-1 and Hb-2 loci.
Table 7. Nei’s (1972) genetic distance (D) and genetic identity (I) among the F. limnocharis complex from Bangladesh and other Asian
countries, and F. cancrivora.
BAUC
(L)
Sham
(L)
Mukt
(L)
Bail
(L)
Kish
(L)
Sher
(L)
Dina
(L)
Nawa
(L)
Khul
(L)
BAUC
(M)
BAUC
(S)
Sham
(S)
Kish
(S)
Sher
(S)
Dina
(S)
Khul
(S)
Hiro
Ishi
Taiw
Kual
Kota
Thai
Sril
Canc
BAUC (L)
–
0.999
0.997
0.993
0.998
0.999
0.997
0.994
0.997
0.228
0.227
0.215
0.201
0.229
0.233
0.206
0.591
0.536
0.679
0.668
0.673
0.698
0.272
0.201
Sham (L)
0.001
–
0.998
0.994
0.999
0.998
0.998
0.995
0.998
0.232
0.230
0.218
0.204
0.232
0.237
0.208
0.585
0.530
0.671
0.662
0.668
0.695
0.276
0.197
Mukt (L)
0.003
0.002
–
0.997
0.998
0.996
0.996
0.996
0.998
0.237
0.231
0.219
0.205
0.233
0.238
0.209
0.581
0.531
0.668
0.659
0.660
0.692
0.275
0.192
Bail (L)
0.008
0.006
0.003
–
0.995
0.993
0.991
0.991
0.994
0.228
0.222
0.211
0.196
0.225
0.229
0.201
0.571
0.522
0.660
0.651
0.651
0.683
0.267
0.184
Kish (L)
0.002
0.001
0.002
0.005
–
0.999
0.998
0.995
0.998
0.227
0.226
0.214
0.200
0.228
0.233
0.204
0.588
0.527
0.675
0.667
0.667
0.700
0.270
0.194
Sher (L)
0.001
0.002
0.004
0.007
0.001
–
0.998
0.994
0.997
0.227
0.225
0.213
0.199
0.226
0.231
0.203
0.597
0.532
0.685
0.673
0.677
0.703
0.269
0.201
Dina (L)
0.003
0.002
0.004
0.009
0.002
0.002
–
0.993
0.997
0.226
0.229
0.217
0.203
0.231
0.236
0.207
0.585
0.516
0.672
0.664
0.664
0.696
0.273
0.201
Nawa (L)
0.006
0.005
0.005
0.009
0.005
0.006
0.007
–
0.995
0.250
0.228
0.216
0.202
0.230
0.235
0.206
0.577
0.528
0.666
0.657
0.657
0.690
0.272
0.195
Khul (L)
0.003
0.002
0.002
0.007
0.002
0.003
0.003
0.005
–
0.241
0.230
0.219
0.204
0.233
0.238
0.209
0.589
0.533
0.676
0.674
0.675
0.708
0.275
0.196
BAUC (M) 1.478
1.461
1.442
1.477
1.485
1.485
1.488
1.386
1.425
–
0.453
0.458
0.447
0.451
0.458
0.478
0.186
0.225
0.185
0.236
0.241
0.270
0.462
0.238
BAUC (S)
1.485
1.471
1.467
1.504
1.489
1.494
1.474
1.480
1.468
0.791
–
0.993
0.985
0.985
0.983
0.971
0.170
0.220
0.224
0.235
0.239
0.294
0.848
0.230
Sham (S)
1.537
1.524
1.520
1.558
1.542
1.547
1.527
1.532
1.521
0.781
0.007
–
0.987
0.971
0.977
0.971
0.167
0.216
0.218
0.229
0.231
0.284
0.844
0.226
Kish (S)
1.607
1.591
1.587
1.628
1.610
1.615
1.594
1.600
1.588
0.806
0.015
0.013
–
0.966
0.976
0.969
0.164
0.202
0.204
0.204
0.211
0.257
0.824
0.222
Sher (S)
1.476
1.461
1.458
1.493
1.479
1.486
1.464
1.468
1.459
0.795
0.015
0.030
0.034
–
0.978
0.954
0.170
0.220
0.221
0.243
0.245
0.302
0.831
0.240
Dina (S)
1.456
1.440
1.437
1.472
1.458
1.467
1.443
1.450
1.438
0.781
0.017
0.023
0.024
0.022
–
0.969
0.168
0.208
0.210
0.221
0.228
0.277
0.809
0.246
Khul (S)
1.582
1.570
1.567
1.607
1.589
1.594
1.573
1.580
1.567
0.739
0.030
0.030
0.032
0.047
0.032
–
0.168
0.206
0.208
0.208
0.215
0.268
0.841
0.227
Hiro
0.526
0.536
0.544
0.560
0.531
0.516
0.536
0.549
0.530
1.680
1.771
1.789
1.807
1.773
1.784
1.786
–
0.762
0.911
0.656
0.662
0.595
0.170
0.152
Ishi
0.624
0.636
0.632
0.650
0.641
0.631
0.661
0.639
0.630
1.490
1.515
1.532
1.599
1.516
1.569
1.579
0.273
–
0.756
0.667
0.646
0.604
0.209
0.200
Taiw
0.388
0.399
0.404
0.416
0.393
0.378
0.397
0.407
0.392
1.688
1.495
1.525
1.592
1.509
1.562
1.571
0.094
0.280
–
0.753
0.767
0.694
0.253
0.202
Kual
0.404
0.412
0.416
0.429
0.405
0.396
0.410
0.420
0.394
1.444
1.447
1.476
1.589
1.415
1.509
1.568
0.421
0.405
0.283
–
0.948
0.835
0.275
0.202
Kota
0.396
0.404
0.416
0.430
0.405
0.391
0.410
0.420
0.393
1.421
1.432
1.467
1.558
1.406
1.478
1.537
0.413
0.436
0.266
0.054
–
0.818
0.278
0.209
Thai
0.360
0.364
0.368
0.381
0.357
0.352
0.362
0.372
0.346
1.310
1.226
1.261
1.359
1.197
1.283
1.317
0.520
0.504
0.365
0.180
0.201
–
0.338
0.201
Sril
1.303
1.289
1.291
1.319
1.309
1.313
1.297
1.303
1.291
0.772
0.165
0.169
0.194
0.186
0.212
0.173
1.773
1.565
1.375
1.293
1.280
1.085
–
0.199
Canc
1.602
1.626
1.649
1.692
1.641
1.603
1.604
1.636
1.632
1.436
1.468
1.486
1.504
1.425
1.404
1.483
1.882
1.609
1.602
1.599
1.568
1.603
1.616
–
Genetic distance (D) is given below the diagonal and genetic identity (I) is given above.
0.806 (mean, 0.782). It is noteworthy that the Bangladesh
small type showed small genetic distances with the Sri Lanka
population, ranging from 0.165 to 0.212 (mean, 0.183). The
Bangladesh large type showed the smallest genetic distances
with the Thailand population, ranging from 0.352 to 0.381 with
a mean of 0.362. The outgroup F. cancrivora constantly
showed large genetic distances from all types of the F.
limnocharis complex, ranging from 1.404 to 1.882 (mean,
1.581).
Phylogenetic relationships
The phylogenetic relationships were inferred from the
Fejervarya limnocharis Complex
dendrogram drawn using the neighbor-joining method
(Saitou and Nei, 1987) based on Nei’s (1972) genetic distances (Fig. 5). In this dendrogram, F. cancrivora was used
as the outgroup to root the tree. The dendogram shows that
all of the frogs of the F. limnocharis complex included in the
present study are divided into two main clusters. The first
includes the Bangladesh large type and other Asian populations (supported by a very high bootstrap value of 96.1%),
and the second includes the Bangladesh medium and small
types and the Sri Lanka population (supported by a
bootstrap value of 72.8%). The first main cluster comprises
several subclusters. Among them, the Bangladesh large
type forms a very strong subcluster (supported by a boot-
269
strap value of 99.7%) with considerably less population
divergence, but the other Asian populations form several
subclusters, including 1) Hiroshima, Ishigaki, and Taiwan,
and 2) Kuala Lumpur, Kota Kinabalu, and Thailand (Fig. 5).
The second main cluster comprises two subclusters, the
Bangladesh medium type and other South Asian populations, with the latter subdivided into the Sri Lanka population
and the Bangladesh small type (supported by a bootstrap
value of 82.3%). The Bangladesh small type forms a very
strong subcluster (supported by a bootstrap value of 89.4%).
The Bangladesh medium type is greatly diverged from all
other types, and is also remotely related to the Bangladesh
small type and the Sri Lanka population (Fig. 5).
Fig. 5. Neighbor-joining tree for the F. limnocharis complex from Bangladesh and other Asian countries based on Nei’s (1972) genetic
distances. Values above branches are bootstrap values>50% from 1000 pseudoreplicates. The scale bar represents branch length in terms of
Nei’s genetic distance.
270
M. M. Islam et al.
DISCUSSION
The genetic distance between taxa at various levels has
been reviewed mainly in vertebrates (Avise and Aquadro,
1982; Thorpe, 1982; Nei, 1987). Nishioka and Sumida
(1990) reported that the genetic distances between populations, between subspecies, and between species of 15 anuran species were 0.030–0.241, 0.145–0.521, and 0.301–
1.715, respectively.
Several studies have been conducted to determine
genetic distances in the F. limnocharis complex from Asia
(Nishioka and Sumida 1990; Toda et al., 1997, 1998a,
1998b; Veith et al., 2001; Djong et al., 2007; Sumida et al.,
2007). Djong et al. (2007) found that Indonesian specimens
of F. limnocharis from the type locality and other Asian
Fejervarya species were largely divided into two groups,
South Asian and East-Southeast Asian, and that the genetic
distance was 1.160–1.408 between these two groups.
These authors also mentioned that the East-Southeast
Asian group was subdivided into two subgroups, the F.
limnocharis subgroup consisting of populations from Indonesia, Malaysia, Japan, and Taiwan, and the F. iskandari subgroup consisting of populations from Indonesia, Thailand,
and Bangladesh. Djong et al. (2007) also found genetic distances of 0.271 and 0.534 between the Bangladesh large
type and F. iskandari, and between the Bangladesh large
type and F. limnocharis, respectively. They found that the
Bangladesh large type clustered in the F. iskandari subgroup and was distinctly different from the F. limnocharis
subgroup.
In the present study, the Bangladesh large-type frog
showed mean genetic distances of 0.408 from the Malaysia
population and 0.524 from the Japan/Taiwan population;
these distances are sufficiently large to consider the
Bangladesh population as a distinct species, according to
the genetic delimitation of species reviewed by Highton
(1989), Rafinsky and Arntzen (1987), and Nishioka and
Sumida (1990). Nishioka and Sumida (1990) reported that
the mean genetic distance among seven populations of F.
limnocharis in western Japan was 0.077. The Iriomote population was differentiated from the Hiroshima, Okinawa and
Taiwan populations with a mean genetic distance of 0.320.
Toda et al. (1997) furthermore demonstrated that the mean
genetic distance between the southern Ryukyu and East
Asian populations was 0.59. Based on allozyme data, Toda
et al. (1998a) found a cryptic species from Java with a
genetic distance of 0.458 from the syntopic F. limnocharis,
and Veith et al. (2001) described this as a distinct species,
F. iskandari, with a genetic distance of 0.316 from the
syntopic F. limnocharis.
In the present study, the genetic distance between the
Bangladesh large and small types was 1.520, whereas it
was 0.782 between the medium and small types and 1.458
between the large and medium types. These values clearly
show that each of the three Bangladesh types could be considered as different species. It is remarkable that even
though the Bangladesh large type of frogs was collected
from different parts of the country, the genetic distance
among populations was negligible (0.001–0.009), which also
somehow applied to the Bangladesh small type (0.007–
0.047). These values show that there was only a slight dif-
ferentiation or none at all among the populations of each
type. In contrast, the Bangladesh large type showed a
higher genetic distance from the relevant types of frog from
Japan, Taiwan, Malaysia, and Thailand (0.378–0.661).
Among them, the minimum distance was that with the
Thailand population, which belongs to the F. iskandari subgroup according to Djong et al. (2007). These authors also
found that although the Bangladesh large type belonged to the
F. iskandari subgroup, it possessed high genetic distance
from the F. iskandari subgroup, indicating a distinct species
for this subgroup. The Bangladesh small type was found to
closely resemble F. limnocharis from Sri Lanka. Djong et al.
(2007) also showed that the Bangladesh small type was
more closely related to the Sri Lanka population than to the
India and Thailand populations. According to them, the
genetic distance was 0.181 between the Bangladesh small
type and the Sri Lanka population, 0.588 between the Indian
and Sri Lanka populations, 0.526 between the Bangladesh
small type and the Indian population, and 1.104–1.310
between the Thailand population and the populations of
India, Sri Lanka, and the Bangladesh small type. In their
molecular phylogenies, Sumida et al. (2007) found that the
Sri Lanka F. limnocharis included in their study was closely
related to F. syhadrensis from Sri Lanka included by
Meegaskumbura et al. (2002) and F. sp (hp A) from India
reported by Kurabayashi et al. (2005), which was
subsequently described as F. granosa by Kuramoto et al.
(2007). In the present study, we used the same samples
from Sri Lanka as those used by Sumida et al. (2007), which
indicated that Bangladesh small-type frogs are the same
species as, or closely-related to, F. syhadrensis.
A crossing experiment among the three types of
Bangladesh frogs (Islam et al., 2006) supported the present
allozyme data. The Bangladesh large type was found to be
reproductively isolated from the Bangladesh small type by
complete hybrid inviability at the tadpole stage, indicating
that these two types represent two different species according to the biological species concept. However, the
Bangladesh large-type frog was found to produce viable and
fertile reciprocal hybrids with the Thailand population (Islam
et al., 2006), which also supports the conclusion that the
Bangladesh large type is closely related to the Thailand population. The Bangladesh medium type was found to produce
viable hybrids with the Bangladesh small-type frog, but all of
the hybrids were found to be unisexual, i.e., male and sterile. These hybrids showed remarkably abnormal spermatogenesis. These findings are also supported by our large
genetic distances between the Bangladesh small- and
medium-type frogs, and imply that the Bangladesh mediumand small-type frogs may represent different species. Additionally, the Bangladesh small type produced viable, bisexual, fertile hybrids with the Sri Lanka population, which also
supports the close relationship between the Bangladesh
small type and the Sri Lanka population.
Mitochondrial DNA sequence data also showed that the
Asian F. limnocharis complex was divided into two groups,
South Asian and East-Southeast Asian, and that the latter
was further divided into two subgroups, F. limnocharis and
F. iskandari (Sumida et al., 2007). In a preliminary report of
mtDNA data (Islam et al., 2007), the Bangladesh large type
was found to be included in the East-Southeast Asian group,
Fejervarya limnocharis Complex
making a cluster with the F. iskandari subgroup, which
included F. orissaensis from India and F. limnocharis from
Thailand. The Bangladesh medium and small types were
included in the South Asian group, in which the Bangladesh
medium type formed a cluster with F. limnocharis from
Myanmar, and the Bangladesh small type formed a cluster
with several Fejervarya species, including F. syhadrensis
(Islam et al., 2007). These molecular data also support our
present findings that each of the three types may be a distinct species
In the present allozyme study, the mean proportion of
heterozygous loci per individual, mean proportion of polymorphic loci per population and mean number of alleles per
locus were found to be 3.1%, 13.9%, and 1.15, respectively,
for all populations of the Bangladesh large type and 9.5%,
20.3%, and 1.24 for all populations of the Bangladesh small
type, respectively. Nishioka and Sumida (1990) found these
three values in six populations of F. limnocharis to be 8.5%,
23.1%, and 1.23, respectively. These values for the three
parameters of F. limnocharis were in distinct contrast to
those found in several anurans: 16.1%, 56.0%, and 1.97 in
Rana togoi (Nishioka et al., 1987a); 12.6%, 48.0%, and 1.56
in Buergeria buergeri; and 20.7%, 64.0%, and 2.06 in
Rhacophorus schlegelii (Nishioka et al., 1987b). Dessauer
et al. (1975) reported high heterozygosity in organisms living
in an ecologically variable environment and suggested that
the high degree of genetic variability operates as an
adaptive strategy. The comparatively low degree of genetic
variability found in F. limnocharis could possibly be an
adaptation to an ecologically stabilized environment such as
cultivated rice fields and their periphery, in which the geographical and meteorological conditions are almost constant
throughout Bangladesh.
The present study showed that within both the
Bangladesh large type and the small type, the fixation index
value was very small. In contrast to our work, Toda et al.
(1998b) checked 12 Taiwanese populations of F.
limnocharis and found an Fst value of 0.433, noting that
remarkable heterogeneity existed among them. Sumida and
Nishioka (1996) studied the Fst value for 16 populations of
Rana ornativentris from Honshu, Japan, and found Fst values ranging from 0.00 to 0.876, with an average of 0.306
excluding five invariant loci, which were much larger than
the present values. Our results indicate that even though
these populations were from different geographical locations
in Bangladesh, they are of the same species, with little differentiation among populations within the same type.
From the present data together with those from Djong et
al. (2007) and Sumida et al. (2007), it is clear that the three
Bangladesh types represent three distinct taxa. All three
types of Bangladesh frogs occurred syntopically in the same
locality of the BAU campus, but no hybrids have ever been
found among them. To understand their taxonomic position
and assign appropriate names, further study will be necessary by comparing the external morphology and mating calls
of the three types with those of previously described species
of the F. limnocharis complex.
ACKNOWLEDGMENTS
We are grateful to Professor M. Matsui of Kyoto University,
Emiratus Professor M. Kuramoto of the Fukuoka University of
271
Education, Emeritus Professor M. Nishioka and Professor A.
Kashiwagi of Hiroshima University, Japan, and Md. Abul Hasanat of
Bangladesh Agricultural University, Bangladesh for their kindness in
collecting and providing us valuable specimens from several localities. We also thank to Professor H. Ota of the University of the
Ryukyus for his suggestions in interpreting data. This work was
supported by a Grant-in-Aid for Scientific Research (C) (No.
17570082) to M. Sumida from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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(Received March 31, 2007 / Accepted November 8, 2007)