Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue8.Oct2000:
Biochemical Systematics and Ecology 28 (2000) 721}736
Evolutionary relationships among 12 species
belonging to three genera of the family
Microhylidae in Papua New Guinea revealed
by allozyme analysis
Masayuki Sumida!,*, Allen Allison", Midori Nishioka!
!Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Higashihiroshima 739-8526,
Japan
"Division of Vertebrate Zoology, Bishop Museum, Honolulu, Hawaii 96817, USA
Received 10 September 1998; received in revised form 14 October 1999; accepted 18 October 1999
Abstract
To elucidate the potential of electrophoretic analysis for understanding relationships among
microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119
individuals from nine species of Cophixalus, two species of Sphenophryne and one species of
Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes
extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These
enzymes were encoded by genes at 20 loci. There were 2}15 phenotypes produced by 2}12
alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion
of polymorphic loci per population, and mean number of alleles per locus in 12 species were
6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's
genetic distances showed that the genus Sphenophryne can be distinguished biochemically from
Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in
external morphology, can be divided biochemically into several species. ( 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Allozyme analysis; Anura; Microhylidae; Sphenophryne; Cophixalus; Barygenys; Papua New
Guinea
* Corresponding author. Tel.: #81-824-24-7482; fax: #81-824-24-0739.
E-mail address: [email protected] (M. Sumida)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 1 5 - 5
722
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
1. Introduction
Microhylid frogs are a diverse group that exhibit considerable ecological and
morphological diversity in the rainforests of New Guinea (Zweifel, 1972), comprising
about 47% of the species of frogs recognized in the region (Zweifel and Tyler, 1982).
The microhylids of New Guinea are arranged in two subfamilies, Asterophryinae and
Genyophryninae ("Sphenophryninae) (Zweifel, 1972). In this region, 39 asterophryine species belonging to seven genera (Asterophrys, Barygenys, Hylophorbus,
Pherophapsis, Callulops, Xenobatrachus and Xenorhina) and 49 named genyophrynine
species belonging to six genera (Choerophryne, Cophixalus, Copiula, Genyophryne,
Oreophryne and Sphenophryne) have been reported (Frost, 1985; Zweifel and Tyler,
1982). The taxonomic diversity is related to the ecological diversity of the family
(Burton and Stocks, 1986). Menzies (1976) conveniently divided the family Microhylidae into four habitat groups; fossorial, terrestrial, scansorial and arboreal.
Two allozyme analyses of New Guinean frogs have resolved the genetic relationships among several species within the genera Litoria (Dessauer et al., 1977) and Rana
(Donnellan et al., 1989). Nishioka and Sumida (1990) examined the genetic relationships between Platymantis papuensis from Papua New Guinea and two Rana species
from East Asia by allozyme analysis. The genetic relationships and phylogeny of New
Guinean hylid frogs were studied by allozyme analysis using 11 species of the family
Hylidae (Sumida et al., 1998). However, allozyme analyses on taxa of New Guinean
microhylid frogs have yet to be conducted using several species belonging to di!erent
genera.
In the present study, the phylogenetic relationships of 12 species belonging to three
genera of the family Microhylidae were investigated by allozyme analysis to assess the
potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea.
2. Materials and methods
A total of 119 specimens from 12 species belonging to three microhylid genera were
used (Table 1, Fig. 1). Fourteen enzymes extracted from muscles and livers were
analyzed by horizontal starth-gel electrophoresis (Nishioka et al., 1980,1992) (Table
2). Enzymes were visualized following the method outlined by Harris and Hopkinson
(1976). Multiple bu!er systems have not been examined for each enzyme, so the
present results probably underestimate the total genetic variation. The genetic distances were calculated following Nei (1972,1975,1987). Two di!erent methods, the NJ
method (Saitou and Nei, 1987) and the maximum-likelihood (ML) method (Felsenstein, 1973), were employed to infer the phylogenetic relationships among taxa on the
basis of genetic distances, using the programs included in version 3.5c of PHYLIP
(Felsenstein, 1993).
Species
Sample size
Body length (mm)
Range
Mean
Date of collection
Individual numbers!
6510}6512, 6583}6586,
6587*}6589*
6576*, 6577*
6501}6503, 6575*, 6578*
6505, 6545}6553, 6666, 6667
6513, 6514, 6516}6519, 6538, 6561,
6565*}6567*, 6595, 6610, 6622
6515, 6594, 6620
6563*, 6564*
6526, 6527, 6554, 6600}6603, 6607,
6609, 6615}6619, 6621, 6623,
6648}6652, 6655, 6659, 6661}6665,
6668, 6671}6675, 6680}6692,
6778}6780
6555}6560, 6579*}6582*
6562, 6571*}6574*, 6591}6593
6570*
6597, 6641
Sphenophryne palmipes
10
30.0}50.4
41.3
Nov. 1982
Sphenophryne rhododactyla
Cophixalus parkeri
Cophixalus kaindiensis
Cophixalus variegatus group sp. 1
2
5
12
14
24.4, 60.2
21.0}29.0
23.6}30.0
15.0}20.6
42.3
26.6
26.2
18.3
Nov.
Nov.
Nov.
Nov.
Cophixalus variegatus group sp. 2
Cophixalus variegatus group sp. 3
Cophixalus cryptotympanum
3
2
50
13.0}18.0
15.6, 17.2
19.6}38.2
15.4
16.4
28.1
Nov. 1982, Jan. 1985
Nov. 1982
Nov. 1982, Jan. 1985
Cophixalus riparius
Cophixalus pansus
Cophixalus sphagnicola
Barygenys yavigularis
10
8
1
2
37.0}53.4
16.5}27.8
19.8
21.8, 22.8
43.3
21.8
19.8
22.3
Nov.
Nov.
Nov.
Jan.
Total
119
1982
1982
1982, Jan. 1985
1982, Jan. 1985
1982
1982
1982
1985
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 1
Specimens of Papua New Guinean microhylid frogs used in the present study
! Asterisked specimens were provided by M. Kuramoto.
723
724
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Fig. 1. Papua New Guinean microhylid frogs. Scale bars equal 2.5 mm. (A,B) Cophixalus variegatus group
sp. 1. (C, D) Cophixalus variegatus group sp. 2. Cophixalus variegatus group sp. 1 and 2 closely resemble each
other in external characters, buta are distinct in abdominal color pattern.
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
725
Table 2
Enzymes analyzed in the present study
Enzyme
Abbreviation
E.C.No.!
Tissue
Aspartate aminotransferase
Adenylate kinase
Creatine kinase
a-Glycerophosphate dehydrogenase
Glucose phosphate isomerase
Isocitrate dehydrogenase
Lactate dehydrogenase
Malate dehydrogenase
Malic enzymes
Mannose phosphate isomerase
Peptidase
Phosphogluconate dehydrogenase
Phosphoglucomutase
Superoxide dismutase
AAT
AK
CK
a-GDH
GPI
IDH
LDH
MDH
ME
MPI
PEP
PGD
PGM
SOD
2.6.1.1
2.7.4.3
2.7.3.2
1.1.1.8
5.3.1.9
1.1.1.42
1.1.1.27
1.1.1.37
1.1.1.40
5.3.1.8
3.4.3.1
1.1.1.44
2.7.5.1
1.15.1.1
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Liver
Skeletal
Skeletal
Skeletal
Bu!er system"
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
T}C pH 7.0
T}C pH 7.0
T}B}E pH 8.0
T}C pH 6.0
T}B}E pH 8.0
T}C pH 7.0
T}C pH 6.0
T}C pH 6.0
T}C pH 7.0
T}C pH 7.0
T}B}E pH 8.0
T}C pH 7.0
T}B}E pH 8.0
T}B}E pH 8.0
!Enzyme Commission numbers (Nomenclature Committee of Biochemistry, 1992).
"T}C, Tris-citrate bu!er. T}B}E, Tris-borate-EDTA bu!er.
3. Results
3.1. Electrophoretic patterns and allelomorphs
The electrophoretic patterns of 14 enzymes analyzed are encoded by genes at 20
loci. Electrophoretic bands corresponding to multiple alleles at each locus are named
A, B, C, etc., in the order of mobility from fast to slow, and alleles are indicated by a, b,
c, etc. The CK and MDH-2 loci are the least variable: two phenotypes are produced
by two alleles. The MPI locus is the most polymorphic; 15 phenotypes are produced
by 12 alleles. At these 20 loci, there are an average of 8.7 phenotypes produced by an
average of 7.0 alleles (Table 3).
3.2. Allele frequencies
Table 4 presents the gene frequencies for each species at 20 loci. At three loci,
a-GDH, LDH-A and PEP-D, each genus has one or more alleles not found in either
of the other genera. At seven other loci, AK, CK, IDH-1, LDH-B, MDH-1, MDH-2
and PEP-A, alleles in Sphenophryne are not found in Cophixalus and Barygenys
allowing alleles at these loci to identify Sphenophryne. Thus, a total of 10 loci can be
interpreted as diagnostic for Sphenophryne.
3.3. Genetic variation
The mean proportion of heterozygous loci per individual, the mean proportion of
polymorphic loci per population, and the mean number of alleles per locus in 12
726
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 3
The numbers and kinds of alleles and phenotypes at 20 enzyme loci in 12 species belonging to three genera
of the family Microhylidae
Locus
Alleles
No.
Phenotypes
Kind
No.
AAT-1
AAT-2
AK
CK
a-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
8
3
6
2
7
8
9
7
6
11
8
2
8
5
12
a}h
a}c
a}f
a, b
a}g
a}h
a}i
a}g
a}f
a}k
a}h
a, b
a}h
a}e
a}l
11
3
6
2
8
12
11
9
7
13
9
2
9
6
15
PEP-A
PEP-D
PGD
PGM
7
7
8
9
a}g
a}g
a}h
a}i
8
9
11
14
SOD
6
a}f
8
Average
7.0
Kind
AA, BB, CC, DD, EE, FF, GG, HH, AD, CG, FH
AA, BB, CC
AA, BB, CC, DD, EE, FF
AA, BB
AA, BB, CC, DD, EE, FF, GG, CD
AA, BB, CC, DD, EE, FF, GG, HH, BC, DH, EH, FG
AA, BB, CC, DD, EE, FF, GG, HH, II, BF, FI
AA, BB, CC, DD, EE, FF, GG, AB, BD
AA, BB, CC, DD, EE, FF, BD
AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, BC, GI
AA, BB, CC, DD, EE, FF, GG, HH, DG
AA, BB
AA, BB, CC, DD, EE, FF, GG, HH, BC
AA, BB, CC, DD, EE, BD
AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, LL, AC,
HJ, IJ
AA, BB, CC, DD, EE, FF, GG, AC
AA, BB, CC, DD, EE, FF, GG, CD, DE
AA, BB, DD, EE, FF, GG, HH, AD, BD, CD, EG
AA, BB, CC, DD, EE, FF, GG, HH, II, AD, CE, EG,
GH, GI
AA, BB, CC, DD, EE, FF, BD, CF
8.7
species are 0}12.5% (x6 "6.1%), 0}35.0% (x6 "17.1%), and 1.00}1.35 (x6 "1.17),
respectively (Table 5). There are no distinct di!erences between the actual and
expected values, expect for species represented only by one or two specimens.
3.4. Genetic distances
The genetic distances among the 12 species examined are shown in Table 6. The
distance is 1.501 between two Sphenophryne species. Among nine species of genus
Cophixalus, the genetic distances are 0.553}2.069 (x6 "1.284), with the smallest value
obtained between C. variegatus group sp. 1 and C. variegatus group sp. 2 (0.553), and
then between C. pansus and C. sphagnicola (0.732). Cophixalus cryptotympanum and C.
parkeri show the largest distance value (2.069) within the genus. The intergeneric
genetic distances are 0.803}1.860 (x6 "1.321) between Cophixalus and Barygenys,
Table 4
Gene frequencies at 20 enzyme loci in 12 species belonging to three genera of the family Microhylidae. If single letter, frequency"1.00
(Sample
size)
Sphenophryne palmipes
(10)
Sphenophryne rhododactyla
AAT-1
AAT-2
AK
CK
e
b
d
b
( 2)
b
a
f
b
Cophixalus parkeri
( 5)
d
b
b
a
Cophixalus kaindiensis
(12)
b
a
e
a
b
Cophixalus variegatus group sp. 1
(14)
d
b
c
a
a
Cophixalus variegatus group sp. 2
( 3)
b
c
a
f
Cophixalus variegatus group sp. 3
( 2)
c
g
(0.83) (0.17)
c
c
a
a
b
Cophixalus cryptotympanum
(50)
a
a
a
a
Cophixalus riparius
(10)
a
b
a
g
Cophixalus pansus
( 8)
f
(0.57)
a
(0.65)
f
b
c
a
b
Cophixalus sphagnicola
( 1)
f
b
c
a
a
Barygenys yavigularis
( 2)
f
a
a
a
e
h
(0.43)
d
(0.35)
a-GDH
GPI
IDH-1
c
b
(0.90)
c
d
d
(0.25) (0.75) (0.50)
b
e
c
(0.10)
g
(0.50)
d
g
a
f
c
f
g
f
i
(0.39) (0.61) (0.32) (0.68)
f
h
g
a
d
(0.03) (0.97)
e
e
(0.31)
d
(0.50)
f
h
(0.69)
h
(0.50)
IDH-2
a
b
(0.55) (0.45)
b
a
b
d
(0.96) (0.04)
g
f
h
c
e
c
b
b
a
b
i
e
b
f
(0.25) (0.75)
b
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Species
2continued
727
728
Species
(Sample
size)
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
S. palmipes
(10)
c
e
a
a
d
d
g
S. rhododactyla
( 2)
e
a
c
a
b
d
h
C. parkeri
( 5)
a
b
f
b
f
C. kaindiensis
(12)
d
b
b
a
C. variegatus group sp. 1
(14)
b
b
(0.83)
f
b
h
c
(0.80)
b
(0.88)
a
C. variegatus group sp. 2
( 3)
b
f
b
g
a
C. variegatus group sp. 3
( 2)
C. cryptotympanum
(50)
C. riparius
(10)
C. pansus
( 8)
C. sphagnicola
B. yavigularis
b
c
(0.17)
d
g
(0.07) (0.93)
d
e
(0.20)
d
(0.13)
g
k
a
c
(0.43) (0.57)
b
j
h
b
c
d
e
d
e
b
e
d
h
g
b
b
k
e
b
c
(0.50)
c
(0.56)
c
( 1)
b
i
e
b
b
(0.50)
b
(0.44)
a
i
(0.96)
h
(0.90)
l
b
d
( 2)
f
g
(0.75)
e
b
b
(0.25)
c
(0.75)
d
f
b
d
(0.97) (0.03)
b
i
(0.25)
b
j
(0.04)
j
(0.10)
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 4*continued
Table 4*continued
(Sample
size)
PEP-A
S. palmipes
(10)
d
a
S. rhododactyla
( 2)
f
b
C. parkeri
( 5)
e
c
d
e
C. kaindiensis
(12)
d
d
C. variegatus group sp. 1
(14)
C. variegatus group sp. 2
( 3)
c
c
b
d
(0.08) (0.92)
c
d
e
(0.71)
a
(0.14)
f
C. variegatus group sp. 3
( 2)
b
f
(0.50) (0.50)
h
b
C. cryptotympanum
(50)
c
C. riparius
(10)
e
C. pansus
( 8)
c
a
c
(0.58) (0.42)
g
C. sphagnicola
( 1)
c
B. yavigularis
( 2)
e
PEP-D
c
PGD
f
a
d
(0.25) (0.75)
PGM
SOD
g
h
(0.60) (0.40)
c
g
i
(0.75) (0.25)
a
e
g
(0.29)
d
(0.86)
c
d
e
g
(0.17) (0.83) (0.21) (0.79)
d
d
e
g
(0.23) (0.77)
g
d
e
(0.57) (0.43)
e
c
e
(0.71) (0.29)
e
g
h
b
a
g
f
c
(0.07)
f
f
(0.93)
f
f
b
d
(0.65) (0.35)
c
e
f
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Species
729
730
Species
Sample
size
Mean proportion of
heterozygous loci
per individual! (%)
Mean proportion of
polymorphic loci
per population (%)
Mean number of
alleles per locus
Sphenophryne palmipes
Sphenophryne rhododactyla
Cophixalus parkeri
Cophixalus kaindiensis
Cophixalus variegatus group sp. 1
Cophixalus variegatus group sp. 2
Cophixalus variegatus group sp. 3
Cophixalus cryptotympanum
Cophixalus riparius
Cophixalus pansus
Cophixalus sphagnicola
Barygenys yavigularis
10
2
5
12
14
3
2
50
10
8
1
2
7.5 ( 5.8)
12.5 ( 8.1)
0 ( 1.6)
5.4 ( 7.4)
8.9 (10.3)
3.3 ( 3.9)
0 ( 0)
8.2 ( 8.0)
6.0 ( 8.0)
8.8 ( 9.1)
5.0 ( 2.5)
7.5 ( 5.6)
15.0
20.0
5.0
25.0
35.0
10.0
0
35.0
20.0
20.0
5.0
15.0
1.15
1.20
1.05
1.25
1.35
1.10
1.00
1.35
1.20
1.20
1.05
1.15
6.1 ( 5.9)
17.1
1.17
Average
!Parentheses show an expected value.
9.9
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 5
Genetic variabilities at 20 enzyme loci in 12 species belonging to three genera of the family Microhylidae
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
731
Table 6
Genetic distance (D) among 12 species belonging to three genera of the family Microhylidae
Species
S. pal
S. rho C. par
S. rho
C. par
C. kai
C. var sp. 1
C. var sp. 2
C. var sp. 3
C. cry
C. rip
C. pan
C. sph
B. ya
1.501
2.022
3.241
2.843
2.946
2.966
2.543
2.876
2.022
2.953
2.219
3.233
1.516
2.908
3.914
2.548
1.791
1.323
2.542
4.327
1.478
1.237
1.155
1.294
1.889
2.069
1.098
1.084
1.366
1.860
C. kai C. var
sp. 1
C. var
sp. 2
C. var
sp. 3
1.459
1.345
1.540
1.158
0.952
1.271
1.335
1.076
1.366
1.296
1.602
1.136
1.171
1.561
1.011
1.072
1.232
1.884
1.218
0.553
1.429
1.275
1.252
1.270
1.017
1.642
C. cry C. rip C. pan C. sph
1.193
1.100 1.223
0.958 1.555 0.732
0.803 1.016 1.209
1.507
1.478}2.219 (x6 "1.849) between Sphenophryne and Barygenys, and 1.323}4.327
(x6 "2.695) between Sphenophryne and Cophixalus.
3.5. Phylogentic relationships
The NJ tree show that the genera examined are largely divided into two groups, one
containing the genus Sphenophryne and the other containing the genera Barygenys and
Cophixalus (Fig. 2A). The ML tree also show that the genera examined are largely
divided into two groups, one containing two species of the genus Sphenophryne and
the other containing 10 species belonging to the genera Barygenys and Cophixalus
(Fig. 2B). In contrast to the ML tree, the NJ tree show the genus Barygenys to have
a sister-group relationship with the genus Cophixalus.
4. Discussion
Microhylid genera have been diagnosed largely on the basis of skeletal morphology. Two genera, Cophixalus and Sphenophryne, are distinguished by features of the
pectoral girdle: Cophixalus lacks the clavicles and procoracoid cartilages exhibited by
Sphenophryne (Zweifel, 1962). Tyler (1971, 1972) showed that supplementary slips of
the M. intermandibularis are relatively consistent within taxonomic groups and thus
make useful taxonomic characters. Burton (1984) found that the supplementary slips
of the M. intermandibularis in Cophixalus and Sphenophryne exhibit intrageneric
similarity and intergeneric di!erence, making the two genera readily distinguishable.
The present study showed that the two genera, Cophixalus and Sphenophryne, of the
subfamily Genyophryninae could be clearly distinguished by diagnostic alleles at only
the CK and MDH-2 loci. The genetic distances between the two genera were very
732
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
large (1.323}4.327, x6 "2.695). These values roughly correspond to the intergeneric
genetic distances between two genera, Rana and Platymantis, of the subfamily Raninae
obtained by Nishioka and Sumida (1990) (3.128}3.200, x6 "3.164). On the other hand,
the genetic distances between the genus Barygenys of the subfamily Asterophryinae
and the genera Cophixalus and Sphenophryne of the subfamily Genyophryninae are
smaller (0.803}2.219, x6 "1.337) than those between the latter two genera
(1.323}4.327, x6 "2.695). These results may imply the inappropriate assignments of the
genera to the two subfamilies, or may suggest that allozyme analysis is not a preferable technique for elucidating the relationships between these genera or subfamilies.
Zweifel and Allison (1982) revealed that Cophixalus pansus di!ered more from
typical scansorial Cophixalus chie#y in ways associated with a ground-dwelling as
opposed to an arboreal way of life: the complete absence of toe discs and terminal
grooves, relatively small eyes, and short legs. Cophixalus pansus may represent the
extreme of adaptation to terrestrial environments seen in the Cophixalus evolutionary
line. The only other species of Cophixalus that lacks expanded terminal discs on both
"ngers and toes is C. sphagnicola (Green and Simon, 1986; Zweifel and Allison, 1982).
Large size, longer "rst "nger and absence of terminal grooves on digits distinguish
C. pansus from C. sphagnicola. Zweifel and Allison (1982) argued that the similarity of
the two species does not necessarily re#ect their close a$nity, although C. sphagnicola
has been compared with C. pansus in determining generic status. They suggested that
their common and presumably derived states of characters may represent parallel
adaptations to a terrestrial life style. However, the present study showed that the
genetic distance between the two species was relatively small (0.732) in comparison
with those between Cophixalus species examined in the present study, and that C.
pansus is closely related to C. sphagnicola. On the other hand, Kuramoto and Allison
(1989) reported that C. pansus is a tetraploid with 2N"52 chromosomes, and that its
haploid set is very similar to that of C. riparius based on a statistical examination of
karyotypes. The present study showed that the genetic distance between C. pansus and
C. riparius is 1.223, and that these two species are not closely related genetically.
The present study revealed that the C. variegatus group contains three species (sp.
1}3), of which sp. 1 and 2 closely resemble each other in external characters
(Fig. 1A,C), but are distinct in abdominal color pattern (Fig. 1B,D). The genetic
distance between C. variegatus group sp. 1 and 2 (0.553) was the smallest one as the
interspeci"c value for the genus obtained in this study. Nevertheless, they were
distinguished by diagnostic alleles at a-GDH, IDH-2, ME-1, MPI, PEP-A and PGM
loci. Thus, they were considered to be independent sibling species. According to
karyological observations by Kuramoto and Allison (1989), C. variegatus group `sp.
1a is unique in that it has many small pairs with a high arm ratio. Karyotypes of
C. variegatus group `sp. 1a and C. variegatus group `sp. 2a (C. variegatus group sp. 3 in
the present study) di!er remarkably (Kuramoto and Allison, 1989), and they have
di!erent acoustic features (Allison, unpublished). The present study showed that
C. variaegatus group sp. 3 is distinct form C. variegatus group sp. 1 and 2 by alleles at
AAT-2, AK, LDH-B, ME-2 and PEP-D loci. The generic distances between C.
variegatus group sp. 3 and the latter two were comparatively large, being 1.429 and
1.366, respectively. Thus, it is evident that C. variegatus group sp. 3 should be
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
733
separated from the other two into a di!erent species group. There are many undescribed species that very closely resemble C. variegatus (Allison, unpublished data),
and the taxonomic revision of this complex requires further study. Burton and Zweifel
(1995) thought it appropriate to formalize the recognition of the variegatus group as
a genus distinct from Cophixalus, and created a new genus, Albericus, to accommodate
three species, including the variegatus group, removed from the genus Cophixalus.
However, the present study showed that the variegatus group could not be distinguished from other species of the genus Cophixalus.
Zweifel (1979) described one new species, Cophixalus kaindiensis, found on Mt.
Kaindi, Morobe District, Papua New Guinea. This species is virtually identical with
its sympatric congener C. parkeri in size and proportions, although the two taxa di!er
slightly in color patterns and greatly in mating calls. The present study showed that
the genetic distance between C. kaindiensis and C. parkeri (1.237) is larger than that
between C. riparius and C. kaindiensis (0.952). Both NJ and ML trees showed that C.
kaindiensis is closest to C. riparius. These two taxa di!er greatly in size; C. riparius may
grow up to 50 mm in body length, which makes it the largest known species in the
genus, whereas C. kaindiensis is a small species not growing much over 30 mm in body
length.
According to Zweifel and Tyler (1982), two subfamilies, Asterophryinae and
Genyophryninae, share direct embryonic development, and thus are considered
monophyletic. Kuramoto and Allison (1989), after examining the karyotypes of 15
species of New Guinean microhylid frogs belonging to "ve of these genera from both
subfamilies, suggested that the Asterophryinae and Genyophryninae are closely
related to each other and are karyologically conservative. Results of the present
allozyme analysis, which was the "rst attempt at a molecular genetic approach to
Papuan microhylid systematics, do not support the monophyly of the two subfamilies,
Genyophryninae and Asterophryinae, due to the relatively close a$nity of Barygenys
with Cophixalus. Monophyly of the genera Cophixalus and Sphenophryne is supported
by our results. The present study also revealed that nine species of the genus
Cophixalus seem to be remotely related to each other (Fig. 2). This result could suggest
that a common ancestor diverged into several species during a comparatively short
period to adapt to the ecologically variable environments, and thereafter considerable
parallel evolution of adaptive types may have taken place, leading to great diversity in
external appearance as suggested by Zweifel and Allison (1982) and Zweifel and Tyler
(1982).
The genetic distances between taxa of various levels has been reviewed mainly in
vertebrates (Avise and Aquadro, 1982; Thorpe, 1982; Nei, 1987). According to these
studies, the interspeci"c genetic distances vary from 0.22 to 1.60 in the majority of
cases of congeneric species. If birds and some mammals are excluded, the interspeci"c
genetic distances are about 0.05 or larger and can be as large as 3.00 or more, and
intergeneric distances are larger than interspeci"c distances (Nei, 1987). The present
study showed that the genetic distances between congeneric species of genus
Cophixalus were 0.553}2.069 (x6 "1.284), and those between three confamilial genera
of the family Microhylidae were 0.803}4.327 (x6 "2.210). These values almost correspond to the range of variation stated above, but clearly show by far greater genetic
734
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Fig. 2. NJ tree (A) and ML tree (B) for 12 species of Papua New Guinean microhylid frogs belonging to
three genera of the family Microhylidae based on Nei's genetic distances. Scale bars represent branch length
in terms of Nei's distances for the NJ and ML trees.
distances than those of any non-amphibian vertebrates shown by Nei (1987) and Avise
and Aquadro (1982). These results may imply that genera and families in the di!erent
classes of vertebrates are not equivalent in levels of structural gene divergences as
suggested by Avise and Aquadro (1982).
Acknowledgements
The authors are especially indebted to T. Nakajima, who was the Field Research
Project Representative in Papua New Guinea, for this invaluable support in numerous ways throughout the "eld work. The authors are also grateful to M. Kuramoto for
his kindness in providing us with valuable specimens. The authors also thank the
participants of this project; Y. Komiya, T. Yasuhara, Y. Kuroda, H. Koda and E.
Sugiyama, and the sta! of Wau Ecology Institute for their cooperation and aid in
collecting specimens in Papua New Guinea. Field work in Papua New Guinea was
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
735
supported by a Grant-in-Aid for Overseas Scienti"c Survey from the Ministry of
Education, Science and Culture, Japan (No. 59041025).
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Lawrence, Kansas.
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Nei, M., 1972. Genetic distance between populations. Amer. Natur. 106, 283}292.
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Nishioka, M., Sumida, M., Ohtani, H., 1992. Di!erentiation of 70 populations in the Rana nigromaculata
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1}70.
Saitou, N., Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic
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Sumida, M., Allison, A., Nishioka, M., 1988. Genetic relationships and phylogeny of Papua New Guinean
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Tyler, M.J., 1972. Super"cial mandibular musculature, vocal sacs and the phylogeny of Australo-Papuan
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Evolutionary relationships among 12 species
belonging to three genera of the family
Microhylidae in Papua New Guinea revealed
by allozyme analysis
Masayuki Sumida!,*, Allen Allison", Midori Nishioka!
!Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Higashihiroshima 739-8526,
Japan
"Division of Vertebrate Zoology, Bishop Museum, Honolulu, Hawaii 96817, USA
Received 10 September 1998; received in revised form 14 October 1999; accepted 18 October 1999
Abstract
To elucidate the potential of electrophoretic analysis for understanding relationships among
microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119
individuals from nine species of Cophixalus, two species of Sphenophryne and one species of
Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes
extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These
enzymes were encoded by genes at 20 loci. There were 2}15 phenotypes produced by 2}12
alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion
of polymorphic loci per population, and mean number of alleles per locus in 12 species were
6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's
genetic distances showed that the genus Sphenophryne can be distinguished biochemically from
Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in
external morphology, can be divided biochemically into several species. ( 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Allozyme analysis; Anura; Microhylidae; Sphenophryne; Cophixalus; Barygenys; Papua New
Guinea
* Corresponding author. Tel.: #81-824-24-7482; fax: #81-824-24-0739.
E-mail address: [email protected] (M. Sumida)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 1 5 - 5
722
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
1. Introduction
Microhylid frogs are a diverse group that exhibit considerable ecological and
morphological diversity in the rainforests of New Guinea (Zweifel, 1972), comprising
about 47% of the species of frogs recognized in the region (Zweifel and Tyler, 1982).
The microhylids of New Guinea are arranged in two subfamilies, Asterophryinae and
Genyophryninae ("Sphenophryninae) (Zweifel, 1972). In this region, 39 asterophryine species belonging to seven genera (Asterophrys, Barygenys, Hylophorbus,
Pherophapsis, Callulops, Xenobatrachus and Xenorhina) and 49 named genyophrynine
species belonging to six genera (Choerophryne, Cophixalus, Copiula, Genyophryne,
Oreophryne and Sphenophryne) have been reported (Frost, 1985; Zweifel and Tyler,
1982). The taxonomic diversity is related to the ecological diversity of the family
(Burton and Stocks, 1986). Menzies (1976) conveniently divided the family Microhylidae into four habitat groups; fossorial, terrestrial, scansorial and arboreal.
Two allozyme analyses of New Guinean frogs have resolved the genetic relationships among several species within the genera Litoria (Dessauer et al., 1977) and Rana
(Donnellan et al., 1989). Nishioka and Sumida (1990) examined the genetic relationships between Platymantis papuensis from Papua New Guinea and two Rana species
from East Asia by allozyme analysis. The genetic relationships and phylogeny of New
Guinean hylid frogs were studied by allozyme analysis using 11 species of the family
Hylidae (Sumida et al., 1998). However, allozyme analyses on taxa of New Guinean
microhylid frogs have yet to be conducted using several species belonging to di!erent
genera.
In the present study, the phylogenetic relationships of 12 species belonging to three
genera of the family Microhylidae were investigated by allozyme analysis to assess the
potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea.
2. Materials and methods
A total of 119 specimens from 12 species belonging to three microhylid genera were
used (Table 1, Fig. 1). Fourteen enzymes extracted from muscles and livers were
analyzed by horizontal starth-gel electrophoresis (Nishioka et al., 1980,1992) (Table
2). Enzymes were visualized following the method outlined by Harris and Hopkinson
(1976). Multiple bu!er systems have not been examined for each enzyme, so the
present results probably underestimate the total genetic variation. The genetic distances were calculated following Nei (1972,1975,1987). Two di!erent methods, the NJ
method (Saitou and Nei, 1987) and the maximum-likelihood (ML) method (Felsenstein, 1973), were employed to infer the phylogenetic relationships among taxa on the
basis of genetic distances, using the programs included in version 3.5c of PHYLIP
(Felsenstein, 1993).
Species
Sample size
Body length (mm)
Range
Mean
Date of collection
Individual numbers!
6510}6512, 6583}6586,
6587*}6589*
6576*, 6577*
6501}6503, 6575*, 6578*
6505, 6545}6553, 6666, 6667
6513, 6514, 6516}6519, 6538, 6561,
6565*}6567*, 6595, 6610, 6622
6515, 6594, 6620
6563*, 6564*
6526, 6527, 6554, 6600}6603, 6607,
6609, 6615}6619, 6621, 6623,
6648}6652, 6655, 6659, 6661}6665,
6668, 6671}6675, 6680}6692,
6778}6780
6555}6560, 6579*}6582*
6562, 6571*}6574*, 6591}6593
6570*
6597, 6641
Sphenophryne palmipes
10
30.0}50.4
41.3
Nov. 1982
Sphenophryne rhododactyla
Cophixalus parkeri
Cophixalus kaindiensis
Cophixalus variegatus group sp. 1
2
5
12
14
24.4, 60.2
21.0}29.0
23.6}30.0
15.0}20.6
42.3
26.6
26.2
18.3
Nov.
Nov.
Nov.
Nov.
Cophixalus variegatus group sp. 2
Cophixalus variegatus group sp. 3
Cophixalus cryptotympanum
3
2
50
13.0}18.0
15.6, 17.2
19.6}38.2
15.4
16.4
28.1
Nov. 1982, Jan. 1985
Nov. 1982
Nov. 1982, Jan. 1985
Cophixalus riparius
Cophixalus pansus
Cophixalus sphagnicola
Barygenys yavigularis
10
8
1
2
37.0}53.4
16.5}27.8
19.8
21.8, 22.8
43.3
21.8
19.8
22.3
Nov.
Nov.
Nov.
Jan.
Total
119
1982
1982
1982, Jan. 1985
1982, Jan. 1985
1982
1982
1982
1985
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 1
Specimens of Papua New Guinean microhylid frogs used in the present study
! Asterisked specimens were provided by M. Kuramoto.
723
724
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Fig. 1. Papua New Guinean microhylid frogs. Scale bars equal 2.5 mm. (A,B) Cophixalus variegatus group
sp. 1. (C, D) Cophixalus variegatus group sp. 2. Cophixalus variegatus group sp. 1 and 2 closely resemble each
other in external characters, buta are distinct in abdominal color pattern.
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
725
Table 2
Enzymes analyzed in the present study
Enzyme
Abbreviation
E.C.No.!
Tissue
Aspartate aminotransferase
Adenylate kinase
Creatine kinase
a-Glycerophosphate dehydrogenase
Glucose phosphate isomerase
Isocitrate dehydrogenase
Lactate dehydrogenase
Malate dehydrogenase
Malic enzymes
Mannose phosphate isomerase
Peptidase
Phosphogluconate dehydrogenase
Phosphoglucomutase
Superoxide dismutase
AAT
AK
CK
a-GDH
GPI
IDH
LDH
MDH
ME
MPI
PEP
PGD
PGM
SOD
2.6.1.1
2.7.4.3
2.7.3.2
1.1.1.8
5.3.1.9
1.1.1.42
1.1.1.27
1.1.1.37
1.1.1.40
5.3.1.8
3.4.3.1
1.1.1.44
2.7.5.1
1.15.1.1
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Liver
Skeletal
Skeletal
Skeletal
Bu!er system"
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
muscle
T}C pH 7.0
T}C pH 7.0
T}B}E pH 8.0
T}C pH 6.0
T}B}E pH 8.0
T}C pH 7.0
T}C pH 6.0
T}C pH 6.0
T}C pH 7.0
T}C pH 7.0
T}B}E pH 8.0
T}C pH 7.0
T}B}E pH 8.0
T}B}E pH 8.0
!Enzyme Commission numbers (Nomenclature Committee of Biochemistry, 1992).
"T}C, Tris-citrate bu!er. T}B}E, Tris-borate-EDTA bu!er.
3. Results
3.1. Electrophoretic patterns and allelomorphs
The electrophoretic patterns of 14 enzymes analyzed are encoded by genes at 20
loci. Electrophoretic bands corresponding to multiple alleles at each locus are named
A, B, C, etc., in the order of mobility from fast to slow, and alleles are indicated by a, b,
c, etc. The CK and MDH-2 loci are the least variable: two phenotypes are produced
by two alleles. The MPI locus is the most polymorphic; 15 phenotypes are produced
by 12 alleles. At these 20 loci, there are an average of 8.7 phenotypes produced by an
average of 7.0 alleles (Table 3).
3.2. Allele frequencies
Table 4 presents the gene frequencies for each species at 20 loci. At three loci,
a-GDH, LDH-A and PEP-D, each genus has one or more alleles not found in either
of the other genera. At seven other loci, AK, CK, IDH-1, LDH-B, MDH-1, MDH-2
and PEP-A, alleles in Sphenophryne are not found in Cophixalus and Barygenys
allowing alleles at these loci to identify Sphenophryne. Thus, a total of 10 loci can be
interpreted as diagnostic for Sphenophryne.
3.3. Genetic variation
The mean proportion of heterozygous loci per individual, the mean proportion of
polymorphic loci per population, and the mean number of alleles per locus in 12
726
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 3
The numbers and kinds of alleles and phenotypes at 20 enzyme loci in 12 species belonging to three genera
of the family Microhylidae
Locus
Alleles
No.
Phenotypes
Kind
No.
AAT-1
AAT-2
AK
CK
a-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
8
3
6
2
7
8
9
7
6
11
8
2
8
5
12
a}h
a}c
a}f
a, b
a}g
a}h
a}i
a}g
a}f
a}k
a}h
a, b
a}h
a}e
a}l
11
3
6
2
8
12
11
9
7
13
9
2
9
6
15
PEP-A
PEP-D
PGD
PGM
7
7
8
9
a}g
a}g
a}h
a}i
8
9
11
14
SOD
6
a}f
8
Average
7.0
Kind
AA, BB, CC, DD, EE, FF, GG, HH, AD, CG, FH
AA, BB, CC
AA, BB, CC, DD, EE, FF
AA, BB
AA, BB, CC, DD, EE, FF, GG, CD
AA, BB, CC, DD, EE, FF, GG, HH, BC, DH, EH, FG
AA, BB, CC, DD, EE, FF, GG, HH, II, BF, FI
AA, BB, CC, DD, EE, FF, GG, AB, BD
AA, BB, CC, DD, EE, FF, BD
AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, BC, GI
AA, BB, CC, DD, EE, FF, GG, HH, DG
AA, BB
AA, BB, CC, DD, EE, FF, GG, HH, BC
AA, BB, CC, DD, EE, BD
AA, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, LL, AC,
HJ, IJ
AA, BB, CC, DD, EE, FF, GG, AC
AA, BB, CC, DD, EE, FF, GG, CD, DE
AA, BB, DD, EE, FF, GG, HH, AD, BD, CD, EG
AA, BB, CC, DD, EE, FF, GG, HH, II, AD, CE, EG,
GH, GI
AA, BB, CC, DD, EE, FF, BD, CF
8.7
species are 0}12.5% (x6 "6.1%), 0}35.0% (x6 "17.1%), and 1.00}1.35 (x6 "1.17),
respectively (Table 5). There are no distinct di!erences between the actual and
expected values, expect for species represented only by one or two specimens.
3.4. Genetic distances
The genetic distances among the 12 species examined are shown in Table 6. The
distance is 1.501 between two Sphenophryne species. Among nine species of genus
Cophixalus, the genetic distances are 0.553}2.069 (x6 "1.284), with the smallest value
obtained between C. variegatus group sp. 1 and C. variegatus group sp. 2 (0.553), and
then between C. pansus and C. sphagnicola (0.732). Cophixalus cryptotympanum and C.
parkeri show the largest distance value (2.069) within the genus. The intergeneric
genetic distances are 0.803}1.860 (x6 "1.321) between Cophixalus and Barygenys,
Table 4
Gene frequencies at 20 enzyme loci in 12 species belonging to three genera of the family Microhylidae. If single letter, frequency"1.00
(Sample
size)
Sphenophryne palmipes
(10)
Sphenophryne rhododactyla
AAT-1
AAT-2
AK
CK
e
b
d
b
( 2)
b
a
f
b
Cophixalus parkeri
( 5)
d
b
b
a
Cophixalus kaindiensis
(12)
b
a
e
a
b
Cophixalus variegatus group sp. 1
(14)
d
b
c
a
a
Cophixalus variegatus group sp. 2
( 3)
b
c
a
f
Cophixalus variegatus group sp. 3
( 2)
c
g
(0.83) (0.17)
c
c
a
a
b
Cophixalus cryptotympanum
(50)
a
a
a
a
Cophixalus riparius
(10)
a
b
a
g
Cophixalus pansus
( 8)
f
(0.57)
a
(0.65)
f
b
c
a
b
Cophixalus sphagnicola
( 1)
f
b
c
a
a
Barygenys yavigularis
( 2)
f
a
a
a
e
h
(0.43)
d
(0.35)
a-GDH
GPI
IDH-1
c
b
(0.90)
c
d
d
(0.25) (0.75) (0.50)
b
e
c
(0.10)
g
(0.50)
d
g
a
f
c
f
g
f
i
(0.39) (0.61) (0.32) (0.68)
f
h
g
a
d
(0.03) (0.97)
e
e
(0.31)
d
(0.50)
f
h
(0.69)
h
(0.50)
IDH-2
a
b
(0.55) (0.45)
b
a
b
d
(0.96) (0.04)
g
f
h
c
e
c
b
b
a
b
i
e
b
f
(0.25) (0.75)
b
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Species
2continued
727
728
Species
(Sample
size)
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
S. palmipes
(10)
c
e
a
a
d
d
g
S. rhododactyla
( 2)
e
a
c
a
b
d
h
C. parkeri
( 5)
a
b
f
b
f
C. kaindiensis
(12)
d
b
b
a
C. variegatus group sp. 1
(14)
b
b
(0.83)
f
b
h
c
(0.80)
b
(0.88)
a
C. variegatus group sp. 2
( 3)
b
f
b
g
a
C. variegatus group sp. 3
( 2)
C. cryptotympanum
(50)
C. riparius
(10)
C. pansus
( 8)
C. sphagnicola
B. yavigularis
b
c
(0.17)
d
g
(0.07) (0.93)
d
e
(0.20)
d
(0.13)
g
k
a
c
(0.43) (0.57)
b
j
h
b
c
d
e
d
e
b
e
d
h
g
b
b
k
e
b
c
(0.50)
c
(0.56)
c
( 1)
b
i
e
b
b
(0.50)
b
(0.44)
a
i
(0.96)
h
(0.90)
l
b
d
( 2)
f
g
(0.75)
e
b
b
(0.25)
c
(0.75)
d
f
b
d
(0.97) (0.03)
b
i
(0.25)
b
j
(0.04)
j
(0.10)
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 4*continued
Table 4*continued
(Sample
size)
PEP-A
S. palmipes
(10)
d
a
S. rhododactyla
( 2)
f
b
C. parkeri
( 5)
e
c
d
e
C. kaindiensis
(12)
d
d
C. variegatus group sp. 1
(14)
C. variegatus group sp. 2
( 3)
c
c
b
d
(0.08) (0.92)
c
d
e
(0.71)
a
(0.14)
f
C. variegatus group sp. 3
( 2)
b
f
(0.50) (0.50)
h
b
C. cryptotympanum
(50)
c
C. riparius
(10)
e
C. pansus
( 8)
c
a
c
(0.58) (0.42)
g
C. sphagnicola
( 1)
c
B. yavigularis
( 2)
e
PEP-D
c
PGD
f
a
d
(0.25) (0.75)
PGM
SOD
g
h
(0.60) (0.40)
c
g
i
(0.75) (0.25)
a
e
g
(0.29)
d
(0.86)
c
d
e
g
(0.17) (0.83) (0.21) (0.79)
d
d
e
g
(0.23) (0.77)
g
d
e
(0.57) (0.43)
e
c
e
(0.71) (0.29)
e
g
h
b
a
g
f
c
(0.07)
f
f
(0.93)
f
f
b
d
(0.65) (0.35)
c
e
f
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Species
729
730
Species
Sample
size
Mean proportion of
heterozygous loci
per individual! (%)
Mean proportion of
polymorphic loci
per population (%)
Mean number of
alleles per locus
Sphenophryne palmipes
Sphenophryne rhododactyla
Cophixalus parkeri
Cophixalus kaindiensis
Cophixalus variegatus group sp. 1
Cophixalus variegatus group sp. 2
Cophixalus variegatus group sp. 3
Cophixalus cryptotympanum
Cophixalus riparius
Cophixalus pansus
Cophixalus sphagnicola
Barygenys yavigularis
10
2
5
12
14
3
2
50
10
8
1
2
7.5 ( 5.8)
12.5 ( 8.1)
0 ( 1.6)
5.4 ( 7.4)
8.9 (10.3)
3.3 ( 3.9)
0 ( 0)
8.2 ( 8.0)
6.0 ( 8.0)
8.8 ( 9.1)
5.0 ( 2.5)
7.5 ( 5.6)
15.0
20.0
5.0
25.0
35.0
10.0
0
35.0
20.0
20.0
5.0
15.0
1.15
1.20
1.05
1.25
1.35
1.10
1.00
1.35
1.20
1.20
1.05
1.15
6.1 ( 5.9)
17.1
1.17
Average
!Parentheses show an expected value.
9.9
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Table 5
Genetic variabilities at 20 enzyme loci in 12 species belonging to three genera of the family Microhylidae
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
731
Table 6
Genetic distance (D) among 12 species belonging to three genera of the family Microhylidae
Species
S. pal
S. rho C. par
S. rho
C. par
C. kai
C. var sp. 1
C. var sp. 2
C. var sp. 3
C. cry
C. rip
C. pan
C. sph
B. ya
1.501
2.022
3.241
2.843
2.946
2.966
2.543
2.876
2.022
2.953
2.219
3.233
1.516
2.908
3.914
2.548
1.791
1.323
2.542
4.327
1.478
1.237
1.155
1.294
1.889
2.069
1.098
1.084
1.366
1.860
C. kai C. var
sp. 1
C. var
sp. 2
C. var
sp. 3
1.459
1.345
1.540
1.158
0.952
1.271
1.335
1.076
1.366
1.296
1.602
1.136
1.171
1.561
1.011
1.072
1.232
1.884
1.218
0.553
1.429
1.275
1.252
1.270
1.017
1.642
C. cry C. rip C. pan C. sph
1.193
1.100 1.223
0.958 1.555 0.732
0.803 1.016 1.209
1.507
1.478}2.219 (x6 "1.849) between Sphenophryne and Barygenys, and 1.323}4.327
(x6 "2.695) between Sphenophryne and Cophixalus.
3.5. Phylogentic relationships
The NJ tree show that the genera examined are largely divided into two groups, one
containing the genus Sphenophryne and the other containing the genera Barygenys and
Cophixalus (Fig. 2A). The ML tree also show that the genera examined are largely
divided into two groups, one containing two species of the genus Sphenophryne and
the other containing 10 species belonging to the genera Barygenys and Cophixalus
(Fig. 2B). In contrast to the ML tree, the NJ tree show the genus Barygenys to have
a sister-group relationship with the genus Cophixalus.
4. Discussion
Microhylid genera have been diagnosed largely on the basis of skeletal morphology. Two genera, Cophixalus and Sphenophryne, are distinguished by features of the
pectoral girdle: Cophixalus lacks the clavicles and procoracoid cartilages exhibited by
Sphenophryne (Zweifel, 1962). Tyler (1971, 1972) showed that supplementary slips of
the M. intermandibularis are relatively consistent within taxonomic groups and thus
make useful taxonomic characters. Burton (1984) found that the supplementary slips
of the M. intermandibularis in Cophixalus and Sphenophryne exhibit intrageneric
similarity and intergeneric di!erence, making the two genera readily distinguishable.
The present study showed that the two genera, Cophixalus and Sphenophryne, of the
subfamily Genyophryninae could be clearly distinguished by diagnostic alleles at only
the CK and MDH-2 loci. The genetic distances between the two genera were very
732
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
large (1.323}4.327, x6 "2.695). These values roughly correspond to the intergeneric
genetic distances between two genera, Rana and Platymantis, of the subfamily Raninae
obtained by Nishioka and Sumida (1990) (3.128}3.200, x6 "3.164). On the other hand,
the genetic distances between the genus Barygenys of the subfamily Asterophryinae
and the genera Cophixalus and Sphenophryne of the subfamily Genyophryninae are
smaller (0.803}2.219, x6 "1.337) than those between the latter two genera
(1.323}4.327, x6 "2.695). These results may imply the inappropriate assignments of the
genera to the two subfamilies, or may suggest that allozyme analysis is not a preferable technique for elucidating the relationships between these genera or subfamilies.
Zweifel and Allison (1982) revealed that Cophixalus pansus di!ered more from
typical scansorial Cophixalus chie#y in ways associated with a ground-dwelling as
opposed to an arboreal way of life: the complete absence of toe discs and terminal
grooves, relatively small eyes, and short legs. Cophixalus pansus may represent the
extreme of adaptation to terrestrial environments seen in the Cophixalus evolutionary
line. The only other species of Cophixalus that lacks expanded terminal discs on both
"ngers and toes is C. sphagnicola (Green and Simon, 1986; Zweifel and Allison, 1982).
Large size, longer "rst "nger and absence of terminal grooves on digits distinguish
C. pansus from C. sphagnicola. Zweifel and Allison (1982) argued that the similarity of
the two species does not necessarily re#ect their close a$nity, although C. sphagnicola
has been compared with C. pansus in determining generic status. They suggested that
their common and presumably derived states of characters may represent parallel
adaptations to a terrestrial life style. However, the present study showed that the
genetic distance between the two species was relatively small (0.732) in comparison
with those between Cophixalus species examined in the present study, and that C.
pansus is closely related to C. sphagnicola. On the other hand, Kuramoto and Allison
(1989) reported that C. pansus is a tetraploid with 2N"52 chromosomes, and that its
haploid set is very similar to that of C. riparius based on a statistical examination of
karyotypes. The present study showed that the genetic distance between C. pansus and
C. riparius is 1.223, and that these two species are not closely related genetically.
The present study revealed that the C. variegatus group contains three species (sp.
1}3), of which sp. 1 and 2 closely resemble each other in external characters
(Fig. 1A,C), but are distinct in abdominal color pattern (Fig. 1B,D). The genetic
distance between C. variegatus group sp. 1 and 2 (0.553) was the smallest one as the
interspeci"c value for the genus obtained in this study. Nevertheless, they were
distinguished by diagnostic alleles at a-GDH, IDH-2, ME-1, MPI, PEP-A and PGM
loci. Thus, they were considered to be independent sibling species. According to
karyological observations by Kuramoto and Allison (1989), C. variegatus group `sp.
1a is unique in that it has many small pairs with a high arm ratio. Karyotypes of
C. variegatus group `sp. 1a and C. variegatus group `sp. 2a (C. variegatus group sp. 3 in
the present study) di!er remarkably (Kuramoto and Allison, 1989), and they have
di!erent acoustic features (Allison, unpublished). The present study showed that
C. variaegatus group sp. 3 is distinct form C. variegatus group sp. 1 and 2 by alleles at
AAT-2, AK, LDH-B, ME-2 and PEP-D loci. The generic distances between C.
variegatus group sp. 3 and the latter two were comparatively large, being 1.429 and
1.366, respectively. Thus, it is evident that C. variegatus group sp. 3 should be
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
733
separated from the other two into a di!erent species group. There are many undescribed species that very closely resemble C. variegatus (Allison, unpublished data),
and the taxonomic revision of this complex requires further study. Burton and Zweifel
(1995) thought it appropriate to formalize the recognition of the variegatus group as
a genus distinct from Cophixalus, and created a new genus, Albericus, to accommodate
three species, including the variegatus group, removed from the genus Cophixalus.
However, the present study showed that the variegatus group could not be distinguished from other species of the genus Cophixalus.
Zweifel (1979) described one new species, Cophixalus kaindiensis, found on Mt.
Kaindi, Morobe District, Papua New Guinea. This species is virtually identical with
its sympatric congener C. parkeri in size and proportions, although the two taxa di!er
slightly in color patterns and greatly in mating calls. The present study showed that
the genetic distance between C. kaindiensis and C. parkeri (1.237) is larger than that
between C. riparius and C. kaindiensis (0.952). Both NJ and ML trees showed that C.
kaindiensis is closest to C. riparius. These two taxa di!er greatly in size; C. riparius may
grow up to 50 mm in body length, which makes it the largest known species in the
genus, whereas C. kaindiensis is a small species not growing much over 30 mm in body
length.
According to Zweifel and Tyler (1982), two subfamilies, Asterophryinae and
Genyophryninae, share direct embryonic development, and thus are considered
monophyletic. Kuramoto and Allison (1989), after examining the karyotypes of 15
species of New Guinean microhylid frogs belonging to "ve of these genera from both
subfamilies, suggested that the Asterophryinae and Genyophryninae are closely
related to each other and are karyologically conservative. Results of the present
allozyme analysis, which was the "rst attempt at a molecular genetic approach to
Papuan microhylid systematics, do not support the monophyly of the two subfamilies,
Genyophryninae and Asterophryinae, due to the relatively close a$nity of Barygenys
with Cophixalus. Monophyly of the genera Cophixalus and Sphenophryne is supported
by our results. The present study also revealed that nine species of the genus
Cophixalus seem to be remotely related to each other (Fig. 2). This result could suggest
that a common ancestor diverged into several species during a comparatively short
period to adapt to the ecologically variable environments, and thereafter considerable
parallel evolution of adaptive types may have taken place, leading to great diversity in
external appearance as suggested by Zweifel and Allison (1982) and Zweifel and Tyler
(1982).
The genetic distances between taxa of various levels has been reviewed mainly in
vertebrates (Avise and Aquadro, 1982; Thorpe, 1982; Nei, 1987). According to these
studies, the interspeci"c genetic distances vary from 0.22 to 1.60 in the majority of
cases of congeneric species. If birds and some mammals are excluded, the interspeci"c
genetic distances are about 0.05 or larger and can be as large as 3.00 or more, and
intergeneric distances are larger than interspeci"c distances (Nei, 1987). The present
study showed that the genetic distances between congeneric species of genus
Cophixalus were 0.553}2.069 (x6 "1.284), and those between three confamilial genera
of the family Microhylidae were 0.803}4.327 (x6 "2.210). These values almost correspond to the range of variation stated above, but clearly show by far greater genetic
734
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
Fig. 2. NJ tree (A) and ML tree (B) for 12 species of Papua New Guinean microhylid frogs belonging to
three genera of the family Microhylidae based on Nei's genetic distances. Scale bars represent branch length
in terms of Nei's distances for the NJ and ML trees.
distances than those of any non-amphibian vertebrates shown by Nei (1987) and Avise
and Aquadro (1982). These results may imply that genera and families in the di!erent
classes of vertebrates are not equivalent in levels of structural gene divergences as
suggested by Avise and Aquadro (1982).
Acknowledgements
The authors are especially indebted to T. Nakajima, who was the Field Research
Project Representative in Papua New Guinea, for this invaluable support in numerous ways throughout the "eld work. The authors are also grateful to M. Kuramoto for
his kindness in providing us with valuable specimens. The authors also thank the
participants of this project; Y. Komiya, T. Yasuhara, Y. Kuroda, H. Koda and E.
Sugiyama, and the sta! of Wau Ecology Institute for their cooperation and aid in
collecting specimens in Papua New Guinea. Field work in Papua New Guinea was
M. Sumida et al. / Biochemical Systematics and Ecology 28 (2000) 721}736
735
supported by a Grant-in-Aid for Overseas Scienti"c Survey from the Ministry of
Education, Science and Culture, Japan (No. 59041025).
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