Zoological Journal of the Linnean Society, 2021, 193, 673–699. With 6 figures.
Revisiting the phylogenetic predicament of the genus
Huia (Amphibia: Ranidae) using molecular data and
tadpole morphology
1
Centrum für Naturkunde-Zoologisches Museum, Universität Hamburg, Edmund-Siemers-Allee 1, 20146
Hamburg, Germany
2
Lee Kong Chian Natural History Museum, National University of Singapore, 2 Conservatory Drive,
117377 Singapore
3
Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie
Boulevard, Fort Worth, TX 76107, USA
4
Amphibian & Reptile Diversity Research Center, Department of Biology, University of Texas at
Arlington, Arlington, TX 76019-0498, USA
5
Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, CH-3005 Bern, Switzerland
6
Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, CH-3012 Bern, Switzerland
7
School of Life Sciences & Technology, Bandung Institute of Technology, Jalan Ganeca 10 Tamansari,
Bandung 40132, Indonesia
Received 3 April 2020; revised 16 October 2020; accepted for publication 27 October 2020
Despite a considerable amount of research, the systematics of the ranid genus Huia have remained unresolved,
mostly owing to insufficient sampling and morphological similarities. As currently circumscribed, Huia consists of
five species, but multiple studies have consistently demonstrated that it is not a monophyletic genus. However, no
study has approached the problem with adequate data and provided a systematically sound solution, leaving the
genus to languish in a classification that is phylogenetically incoherent. We generated the most comprehensive
sampling of Huia to date, based on extensive fieldwork in Java and Sumatra. Using an integrative taxonomy
framework, we analysed four mitochondrial and two nuclear markers and, in conjunction with tadpole morphology,
investigated the phylogenetics of Huia and its congeners. Corroborating previous studies, Huia is recovered as a
paraphyletic group. Huia cavitympanum emerges as the sister taxon to Meristogenys. The remaining members of
Huia form a monophyletic group, sister to the H. cavitympanum + Meristogenys clade. Our extensive geographical
sampling in Sumatra and Java reveals multiple highly divergent lineages that potentially represent undescribed
species. Using our expanded molecular and morphological dataset, we resolve the paraphyly of Huia by restricting
the genus to its type species and propose a new genus to accommodate the strongly supported clade of Sumatran and
Javan populations previously belonging to Huia.
ADDITIONAL KEYWORDS: Anura – cryptic species – genetics – Indonesia – new genera – paraphyly –
phylogenetic systematics – Southeast Asia – species diversity – taxonomy.
INTRODUCTION
*Corresponding author. E-mail: umilaela@gmail.com;
umilaela@uni-hamburg.de
[ Ve r s i o n o f r e c o r d , p u b l i s h e d o n l i n e 1 2 J a n u a r y
2021; http://zoobank.org/ urn:lsid:zoobank.org:pub:
54DD9ED6-0B56-4CA2-837D-A7E05307D81F]
The genus Huia Yang, 1991 currently comprises five
species [Huia cavitympanum (Boulenger, 1893), Huia
masonii (Boulenger, 1884), Huia melasma Stuart &
Chan-ard, 2005, Huia modigliani (Doria, Salvidio, &
Tavano, 1999) and Huia sumatrana Yang, 1991] that
© The Author(s) 2021. Published by Oxford University Press on behalf of The Linnean Society of London.
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673
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UMILAELA ARIFIN1,*, KIN ONN CHAN2, UTPAL SMART3,4, STEFAN T. HERTWIG5,6,
ERIC N. SMITH4, DJOKO T. ISKANDAR7 and ALEXANDER HAAS1
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U. ARIFIN ET AL.
topologies with regard to the other species, albeit with
low support (Wiens et al., 2009; Kurabayashi et al.,
2010; Pyron & Wiens, 2011; Chan & Brown, 2017; Arifin
et al., 2018; Fig. 1). However, the taxonomic sampling
for those studies was limited to only one or two samples
per species, and none incorporated all available species
in the examined genera (Fig. 1).
The most recent study (Arifin et al., 2018)
suggested that wider geographical sampling and
integrative taxonomy were needed to assess species
diversity and resolve taxonomic problems related
to Huia. Through this approach, Arifin et al. (2018)
discovered unexpected hidden diversity and a new
genus from Sumatra (Sumaterana). Members of this
genus also possess gastromyzophorous tadpoles and
are ecologically similar to Huia. Sumaterana was
hypothesized to be either the sister lineage to the
Clinotarsus + H. melasma clade (maximum likelihood
tree) or the sister taxon to Clinotarsus (BI tree; Arifin
et al., 2018). Furthermore, significant morphological
variation has been reported in Huia tadpoles from
Sumatra (Manthey & Denzer, 2014). This suggests that
Huia (at least in Sumatra) might harbour undiscovered
species diversity that might not be associated with
obvious phenotypic differentiation in adults. Therefore,
in the light of the prevailing ambiguous and unresolved
systematics of Huia and preliminary data indicating
the potential for hidden diversity in the genus, we
collected additional molecular data from adults and
tadpoles, and morphological data from tadpoles, from
populations never sampled before, across Sumatra and
Java, Indonesia. The aims of the study were as follows:
(1) to infer phylogenetic relationships within Huia
using expanded taxon and geographical sampling; (2)
to assess species-level diversity of Huia, particularly
in Sumatra and Java; and (3) to resolve outstanding
systematic issues in Huia using integrative taxonomy.
MATERIAL AND METHODS
TAXON SAMPLING
Fieldwork in Sumatra and Java was conducted
between 2013 and 2015 to collect adults and larvae of
Huia frogs using rapid biological sampling approaches
(Ribeiro-Junior et al., 2008). Animal handling and
euthanasia were conducted according to the general
legal guidelines of Germany (Tierschutzgesetz;
h t t p s : / / w w w. g e s e t z e - i m - i n t e r n e t . d e / t i e r s c h g /
BJNR012770972.html). Additional tissue samples
that were relevant to this study were obtained from:
the Museum Zoologicum Bogoriense (MZB), Bogor,
Indonesia; California Academy of Sciences (CAS),
CA, USA; Museum of Vertebrate Zoology (MVZ),
Berkeley, CA, USA; Field Museum of Natural History
(FMNH), Chicago, IL, USA; Amphibian and Reptile
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have a disjunct distribution range in western and
northern Thailand, Java, Sumatra and Borneo (notably
absent in the intervening region of Peninsular Malaysia;
Frost, 2019). Huia is one of four Asian ranid genera
with gastromyzophorous tadpoles; the other genera
are Amolops Cope, 1865, Meristogenys Yang, 1991 and
Sumaterana Arifin, Smart, Hertwig, Smith, Iskandar,
and Haas, 2018 . Gastromyzophorous tadpoles are strictly
associated with cascading or torrential stream habitats
(Nodzenski & Inger, 1990; Gan et al., 2016). These
tadpoles possess large abdominal suckers that enable
them to cling onto rocks in fast-flowing torrential zones
of forest streams (Nodzenski & Inger, 1990; Gan et al.,
2016), which provides the high oxygen concentration
required by the tadpoles to survive and which might also
reduce the risk of predation (Nodzenski & Inger, 1990).
The genus Huia was initially distinguished from
other genera with gastromyzophorous tadpoles based
on a single discrete character of its tadpoles in the
scattered glands on the back (see dichotomous key
provided by Yang, 1991: 6). Incidentally, this character
was not mentioned in the description of the type
species, H. cavitympanum, nor was it observed in
subsequent examinations by other authors (Inger,
1985; Stuart, 2008), including us. The lack of definitive
diagnostic characters for the genus Huia has resulted
in numerous species that occupy a similar type of
stream habitat (e.g. genus Odorrana Fei, Ye and
Huang, 1990) being placed for some time under the
genus Huia, until their identities have subsequently
been resolved using molecular approaches (Chen et al.,
2005; Frost et al., 2006; Cai et al., 2007; Stuart, 2008).
Although the five species of Huia (Frost, 2019) are
distributed across a relatively wide range, each species
is endemic to its own region. Huia cavitympanum
is endemic to the island of Borneo, H. melasma to
mainland Asia (Thailand), H. masonii to Java, and
H. modigliani and H. sumatrana to Sumatra (Frost,
2019). These species were described solely based on
morphology, and the first in-depth molecular study that
examined their phylogenetic relationships revealed
that Huia was a paraphyletic group (Stuart, 2008).
The type species, H. cavitympanum, was recovered as
the sister lineage to the genus Meristogenys, and this
H. cavitympanum + Meristogenys clade was sister to a
clade comprising H. masonii and H. sumatrana [albeit
weakly supported on both maximum likelihood (ML)
and Bayesian inference (BI) trees; Stuart, 2008: 55–56].
Huia melasma, in contrast, was weakly supported as
the sister lineage to the genus Clinotarsus Mivart,
1869 (Fig. 1). Subsequent studies confirmed the
paraphyly of Huia and the sister relationship
between H. cavitympanum and Meristogenys
(except for Kurabayasi et al., 2010, who found that
H. cavitympanum was the sister taxon to Huia and both
were sister to Meristogenys), but produced different
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
675
Diversity Research Center (ARDRC) of the University
of Texas at Arlington (UTA), Arlington, TX, USA; and
Naturhistorisches Museum der Burgergemeinde Bern
(NMBE), Bern, Switzerland, where the respective
voucher specimens are deposited.
A total of 44 adult Huia samples from Borneo (N = 3),
Java (N = 4), Sumatra (N = 35) and mainland Asia
(Thailand, N = 1; Laos, N = 1) were included in this
study. For taxonomic assurance and clarity, we included
samples from the type localities or nearby whenever
possible. The samples UTA A-64939 and UTA A-64940
were collected from Bukit Lawang, Provinsi Sumatera
Utara, the type locality of H. sumatrana; MZB.
AMPH.23526 was collected from Panyabungan, Provinsi
Sumatera Utara, ~170 km south of Si Rambé, Provinsi
Sumatera Utara, the type locality of H. modigliani;
MZB.AMPH.22371 was collected from Cibodas, Provinsi
Jawa Barat, the type locality of Huia javana Yang, 1991.
Unidentified tadpoles of Huia from Sumatra (N = 2)
and Java (N = 1) were also included, in addition to
other genera possessing gastromyzophorous tadpoles:
Sumaterana (N = 6), Meristogenys (N = 12) and Amolops
(N = 4). We also included Odorrana hosii (Boulenger,
1891) (N = 1), which does not have gastromyzophorous
tadpoles, but occurs in syntopy with Huia on the island
of Sumatra and has previously been confused with it.
Likewise, the genus Clinotarsus (N = 2) does not possess
gastromyzophorous larvae, but has been inferred to be
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Figure 1. Several studies showing phylogenetic relationship of frogs previously assigned as Huia to other closely related
species within the family Ranidae.
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U. ARIFIN ET AL.
LABORATORY PROTOCOLS AND PHYLOGENETIC
ANALYSES
All tissue samples (muscle or liver), which were preserved
in ethanol (96%), RNAlater (Sigma-Aldrich, USA) or
Lysis buffer (0.5 M Tris, 0.25% EDTA and 2.5% sodium
dodecyl sulphate, pH 8.2), were extracted and sequenced
following the protocols described by Arifin et al. (2018).
Four mitochondrial [12S, 16S+tRNAval, cytochrome c
oxidase subunit I (COI); NADH dehydrogenase 2
(ND2)] and two nuclear markers [recombinationactivating gene 1 (RAG) and tyrosinase exon 1 (TYR)]
were sequenced. For tadpoles, we sequenced only
the 16S+tRNA val genes as a molecular barcode for
identification. Primer information and polymerase chain
reaction (PCR) annealing temperatures are provided in
the Supporting Information (Table S1). The sequence
quality of both strands was checked with their respective
chromatograms, then assembled and edited (if necessary)
using GENEIOUS v.8.0 (Kearse et al., 2012).
Subsequently, sequences for each locus were aligned
separately using the MAFFT v.7.017 module (Katoh &
Standley, 2013) implemented in GENEIOUS v.8.0. The
program GBLOCK v.0.91b (Castresana, 2000; Talavera
& Castresana, 2007), implemented on an online
server (phylogeny.fr; Dereeper et al., 2008), was used
to eliminate poorly aligned positions and divergent
regions of an alignment for each locus, using a less
stringent selection setting that allowed smaller final
blocks and gap positions within the final block. Aligned
Table 1. Summary of the species, sample locality, and sample size
Species name
Sample locality
Sample size
Amolops afghanus
Amolops indoburmanensis
Amolops marmoratus
Amolops panhai
Chalcorana chalconota
Clinotarsus alticola
Clinotarsus penelope
Huia Borneo
Huia Java
Huia Sumatra
Huia Mainland Asia
Huia Tadpoles
Hylarana erythraea
Meristogenys amoropalamus
Meristogenys jerboa
Meristogenys kinabaluensis
Meristogenys orphocnemis
Meristogenys poecilus
Meristogenys sp.
Odorrana hosii
Pulchrana picturata
Sumaterana crassiovis
Sumaterana dabulescens
Sumaterana montana
Staurois guttatus
Total specimens
Myanmar, Kachin State
Myanmar, Chin State
Myanmar, Shan State
Myanmar, Tanintharyi Division
Indonesia, Provinsi Jawa Tengah
Thailand, Prachuap Kirikhan
Thailand, Ranong Prov.
Borneo
Indonesia, Java
Indonesia, Sumatra
Laos and Thailand
Indonesia, Java and Sumatra
Indonesia, Provinsi Aceh
Malaysia, Borneo
Malaysia, Borneo
Malaysia, Borneo
Malaysia, Borneo
Malaysia, Borneo
Malaysia, Borneo
Malaysia, Borneo
Indonesia, Provinsi Aceh
Indonesia, Sumatra
Indonesia, Sumatra
Indonesia, Sumatra
Malaysia, Borneo
1
1
1
1
1
1
1
3
4
35
2
3
1
3
1
4
1
1
2
1
1
2
2
2
1
76
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nested within the clade of ranid frogs with this type of
tadpole and as the sister group to certain Huia species in
previous studies (Stuart, 2008; Wiens et al., 2009; Pyron
& Wiens, 2011; Arifin et al., 2018). Other closely related
Southeast Asian ranids were represented by Chalcorana
chalconota (Schlegel, 1837), Hylarana erythraea
(Schlegel, 1837) and Pulchrana picturata (Boulenger,
1920). Staurois guttatus (Günther, 1858) was used to root
the tree, following Pyron & Wiens (2011). We generated
all sequences used in this study, except for FMNH 237299
and KUHE 12055, which were obtained from GenBank.
Information regarding voucher specimens (N = 76),
localities, collection numbers and GenBank accession
numbers is provided in Appendix 1 (for a summary of
the species, sample locality and sample size, see Table 1).
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
& Bull, 1993) and posterior probabilities (PPs) ≥ 0.95 for
Bayesian analyses (Huelsenbeck & Ronquist, 2001) were
considered as highly supported (Huelsenbeck & Ranala,
2004; Mulcahy et al., 2011). Pairwise uncorrected genetic
distances (p) for 16S+tRNAval mitochondrial genes were
calculated using MEGA v.7.0.25 (Kumar et al., 2016),
and the distribution of genetic distances between
reciprocally monophyletic clades was compared and
visualized using the R package ggridges (Wilke, 2018).
TADPOLE MORPHOLOGY
Manthey & Denzer (2014) detected morphological
variations [denticle formula or labial tooth row formula
(LTRF) and maximal length] in tadpoles of Huia from
Sumatra, Java and Borneo. Additional data for these
characters were scored and assessed by U.A. from
the holdings of the Lee Kong Chian Natural History
Museum, Singapore (LKCNHM) and the Zoologisches
Museum Hamburg, Germany (ZMH). From those
collections, a total of 122 larval specimens of Huia
(see APPENDIX 2) from numerous localities across
Sumatra (N = 74), Java (N = 23) and Borneo (N = 25)
were examined with a focus on LTRF and the shape of
upper and lower jaw sheaths, which have been shown
to be diagnostic for this group. Representative tadpoles
were photographed using an EOS 6D Canon camera
(50 mm, 1:2) of the Passport II Imaging System (Dun,
Inc.).
RESULTS
PHYLOGENETIC ANALYSES AND GENETIC DISTANCE
In general, phylogenies produced in this study
(mtDNA only, 3859 bp, 2233 distinct patterns; nuDNA
only, 1355 bp, 362 distinct patterns; concatenated
mtDNA + nuDNA, 5214 bp, 2595 distinct patterns;
phased nuDNA only, 1355 bp, 321 and 326 distinct
patterns for individual 1 and 2, respectively; and
concatenated mtDNA + phased nuDNA, 5214 bp,
2559 and 2554 distinct patterns for individual 1 and
2, respectively) show that the genera Clinotarsus,
Huia, Meristogenys and Sumaterana form a highly
supported clade. In agreement with previous studies,
Huia (coloured branches labelled A–H in Fig. 2) is
inferred as a paraphyletic group in relationship to the
genus Meristogenys. Except for the mtDNA-only BI
tree (Supporting Information, Fig. S1B), all trees infer
H. cavitympanum (type species; clade H; Fig. 2) as
the sister lineage to the genus Meristogenys, together
forming a clade comprising Bornean endemics.
Huia from mainland Asia, Huia from Borneo, Huia
from Java and Sumatra, in addition to the genera
Clinotarsus, Meristogenys and Sumaterana, are all
recovered as monophyletic with high support across all
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sequences for each gene were then concatenated for
phylogenetic analyses.
We ran P ARTITION F INDER v.1.1 (Lanfear et al.,
2012) on the concatenated dataset (including the
three tadpole sequences; N = 76), applying the
Bayesian information criterion (BIC) to find the bestfit substitution models and partitioning strategies
for each locus. This resulted in eight partitions
(Supporting Information, Table S2). We then used
ML and BI to construct phylogenetic trees. The ML
analysis was performed using RAXML v.8 (Stamatakis,
2014), and branch support was assessed using 1000
bootstrap replicates. We tested the topology of this
tree using the Shimodaira–Hasegawa test (SH test;
Shimodaira & Hasegawa, 1999) implemented through
the IQ-TREE v.1.6.12 webserver (Trifinopoulous et al.,
2016). Documentation and results from this test are
provided in the Supporting Information (File S1).
For the BI analyses, we used M R B AYES v.3.2.6
(Huelsenbeck & Ronquist, 2001; Ronquist &
Huelsenbeck, 2003) using two independent runs,
each with four chains, and running for 50 million
generations, with sampling every 1000 generations.
Convergence was assessed by examining the effective
sample sizes of all parameters in TRACER v.1.6
(Rambaut et al., 2014) after discarding the first 25%
of samples as burn-in. Both ML and BI analyses were
performed through the CIPRES Science Gateway v.3.3
(Miller et al., 2010) using default parameters.
Both ML and BI phylogenetic analyses were
a l s o p e r f o r m e d s e p a r a t e l y o n m i t o ch o n d r i a l
(12S + 16S+tRNA val + COI + ND2) and nuclear
loci (RAG + TYR) to assess potential mitonuclear
discordance (for trees generated from this analyses,
see Supporting Information, Fig. S1). However, the
concatenated dataset for all loci was used for optimal
tree reconstruction (Kluge, 1989, 2004). Also, to assess
the influence of heterozygote alleles, we phased the two
nuclear DNA (nuDNA) alignments (RAG and TYR)
separately using PHASE v.2.1 (Stephen et al., 2001).
SEQPHASE (Flot, 2010) was used to generate PHASE
input files from FASTA alignments and to regenerate
FASTA alignments from PHASE output files.
Phylogenetic trees (ML and BI) were then estimated
for each phased alignment (phased RAG + phased TYR)
in addition to a concatenated alignment comprising
mitochondrial DNA (mtDNA) and phased nuclear DNA
(12S + 16S+tRNAval + COI + ND2 + phased RAG + phased
TYR), following the same procedures mentioned above.
Trees generated from phased alignments are provided
in the Supporting Information (Fig. S2).
All ML and BI trees were visualized in FIGTREE v.1.4.3
(http://tree.bio.ed.ac.uk/software/figtree/). The trees
in Figure 2 and the Supporting Information (Figs S1,
S2, S3) were prepared using CORELDRAW X6. Node
support with bootstrap (BS) values ≥ 70 for ML (Hillis
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Figure 2. Maximum likelihood tree (concatenated dataset) depicting phylogenetic relationship of frogs previously
recognized as Huia (coloured branches, which also match with the island/locality of the samples; clades A–H) from Sumatra,
Java, Borneo and the mainland to other closely related species within the family Ranidae. Black circles represent highly
supported nodes [bootstrap support (BS) > 70] for unphased and phased dataset, whereas low support values are shown
with a slash (/) for unphased dataset (BS)/phased dataset 1 (BS1)/phased dataset 2 (BS2). Tadpole samples are represented
by ‘_Tad’. Topotypic samples are represented by numbers *1–*4 and the locality shown on the map. Pink triangles, orange
circles, blue stars and purple squares on the map represent the sampling sites.
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
taxon to Huia from mainland Asia (clade G; Fig. 2),
although with low support.
Compared with previous studies, which included
only one or two individuals of Huia, the significantly
larger number of Huia from Indonesia (Sumatra
and Java) included in this study (adults, N = 39;
tadpoles, N = 3) reveal, for the first time, high levels of
genetic diversity and phylogenetic structure. Based on
this study, the Indonesian clade (subclades A–F; Fig. 2)
is represented by at least six divergent lineages with
mitochondrial divergences ≤ 17.14% (Fig. 3; Supporting
Information, Table S3). The smallest genetic distance
is between clades C and D (3.43–4.76%), which is
similar to intrapopulation distances within clades
A and C (3.40–4.80%; Fig. 3). Genetic distances
between clades A and B are considerably higher
(6.90–7.40%), and the largest distances are observed
between H. cavitympanum and all other Huia species
(14.00–17.00%), between H. melasma (clade G) and
all Indonesian Huia (clades A–F; 12.40–16.80%), and
between H. cavitympanum and Meristogenys (14.10–
16.60%; Fig. 3).
Figure 3. Distribution of uncorrected genetic p-distance for 16S+tRNAval mitochondrial gene for Huia and its closely
related species.
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datasets and analyses (Fig. 2; Supporting Information,
Figs S1, S2). However, branching arrangements of
the abovementioned clades are weakly supported
and variable for each dataset (Fig. 2; Supporting
Information, Figs S1, S2).
The ML trees from concatenated datasets with and
without nuDNA phasing reveal the same topologies
(Fig. 2). This topology is also supported by the SH
test. Interestingly, the BI topology from the unphased
concatenated dataset is similar to the ML topology
using phased and unphased concatenated datasets
with regard to the arrangements of Huia, Meristogenys,
Sumaterana and Clinotarsus, but differs in the
relationships among the remaining clades (Amolops,
Odorrana, Hylarana, Pulchrana and Chalcorana). The
BI topology from the phased concatenated dataset infers
Sumaterana as the sister taxon to a clade comprising
Meristogenys and all Huia, while Clinotarsus is
sister to the Sumaterana + Huia + Meristogenys
clade (Supporting Information, Fig. S2). The clade
comprising Huia from Java and Sumatra (clades A–F;
Fig. 2) is highly supported and is inferred as the sister
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TADPOLE MORPHOLOGY
DISCUSSION
HIDDEN DIVERSITY OF HUIA FROGS
Our results are consistent with the conclusion by
Manthey & Denzer (2014) that there is hidden and
undescribed diversity within Huia in the Indonesian
islands of Sumatra and Java. Matsui et al. (2006)
indicated the occurrence of a distinct lineage of Huia
in Sumatra. They presented Huia sp. from Provinsi
Jambi as a distinct lineage of frogs previously known
as H. sumatrana from the same province with 18.0%
genetic divergence (see Matsui et al., 2006: fig. 2;
Kurabayashi et al., 2010: fig. 1). Matsui et al. (2006) also
mentioned that Huia sp. (N6468) and H. sumatrana
(N6474) are morphologically different but did not
present any characters in their study. However, in
a separate analysis that we conducted (Supporting
Information, Fig. S3), N6468 was recovered as
Sumaterana crassiovis (Boulenger, 1920) and N6474
was nested within clade A (Huia sp. 1).
The broad geographical sampling of Huia in this
study revealed three distinct lineages (clade A,
clade B and clade C+D; Fig. 2) within frogs currently
considered as H. sumatrana, with high genetic
divergence among them (> 5.00%; Fig. 3). The genetic
distance between clades C and D is small (3.43–4.76%;
Fig. 3), which is similar to intrapopulation distances
within clades A and C (3.40–4.80%; Fig. 3), indicating
that the divergence between clades C and D is likely
to reflect intra- as opposed to interspecies divergence.
We consider clade C to represent true H. sumatrana
based on the phylogenetic position of two topotypic
specimens (UTA A-64939 and UTA A-64940) within
this clade. Interestingly, this H. sumatrana clade is
more closely related to the Javan lineage (clade E)
than to other Sumatran lineages (clade A or clade B).
Clade A contained a sample (MZB. AMPH.23526;
Fig. 2) that was collected 170 km south of the
type locality of H. modigliani (Si Rambe, ~181 km
south-east of Bukit Lawang; the type locality of
H. sumatrana). Additional sampling, preferably at or
closer to the type locality, is required to determine
the validity of H. modigliani. This species is known
only from the type locality and has not been collected
since. Clade B from Lampung province is genetically
distinct from clade A (p-distance = 6.90–7.40%) and
could potentially represent a new undescribed lineage
(Huia sp. 2). However, clade B contained only one
sample; therefore, more data are needed to confirm the
distinction of this lineage.
Samples from Java are well represented by two
distinct lineages (clade E and clade F; Fig. 2), but only
one species (H. masonii) is currently recognized from
Java. Another taxon, H. javana, has been considered
a synonym of H. masonii (Iskandar, 1998; Frost
2019). However, a study by Manthey & Denzer (2014)
considered H. javana as a valid species distinct from
H. masonii based on morphology. Our sample (MZB.
Amph.22371; clade F) was collected from Cibodas,
the type locality of H. javana. Thus, we proffer that
clade F is H. javana and suggest that its synonymy
with H. masonii should be removed. Our data clearly
corroborated the presence of two Huia species on Java
(see detailed discussion below).
THE IMPORTANCE OF TADPOLE MORPHOLOGY IN HUIA
Tadpole morphology can be a powerful tool for
differentiating species, particularly for ranids with
gastromyzophorous tadpoles (Yang, 1991). Shimada
et al. (2015) demonstrated that larval morphology was
particularly useful for diagnosing species of Meristogenys,
many of which are notoriously difficult to distinguish in
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Based on the 16S+tRNA val sequence, the three
unidentified Huia tadpoles are matched to clade F
(UA20150061; Fig. 2) and clade A (ZMH A12659 and
ZMH A12660; Fig. 2). The UA20150061 tadpole is
matched with adult specimens from the same locality
and with the topotypic material of H. javana (MZB.
AMPH.22371). In contrast, the other two tadpoles
from Sumatra (ZMH A12659 and ZMH A12660) are
not matched with the clade comprising topotypic
H. sumatrana (UTA A-64939 and UTA A-64940; clade
C; Fig. 2). Variation in the LTRF of Huia tadpoles from
Sumatra, Java and Borneo from this and previous
studies (total N = 200; including the two sequenced
tadpoles, ZMH A12659 and ZMH A12660) are
provided in Table 2. In general, Huia tadpoles from
Borneo (N = 47) have at least ten keratodont rows
on the upper lip and five or six keratodont rows on
the lower lip; Huia tadpoles from Java (N = 64) have
seven to nine keratodont rows on the upper lip and
six to ten keratodont rows on the lower lip; and Huia
tadpoles from Sumatra (N = 89) have six to eight
and six to nine keratodont rows on the upper and
lower lip, respectively (see Table 2). Additionally, the
upper jaw sheath of Bornean tadpoles is consistently
an undivided Λ-shape, as opposed to an undivided
M-shape for tadpoles from Java and Sumatra. Also,
we observed no glands on the tail fin of any Bornean
tadpoles, whereas tadpoles from Java and Sumatra
possess scattered glands on their tail fin. We also
observed the presence of an oblique gland cluster
immediately below the origin of the spiracular tube
and running upward and backward in the Bornean
tadpoles, but this gland is absent in the Sumatran
and Javan tadpoles.
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
681
Table 2. Variations on labial tooth row formula for all tadpoles belonging to frogs previously recognized as Huia species
Island
Locality
Borneo
NA
Borneo
Sources
1
10(3–10)/6(2)
NA
7
11(4–11)/6(1)
Borneo
NA
1
12(4–12)/5
Borneo
NA
2
12(4–12)/6(1)
Borneo
Borneo
Borneo
Malaysia, Borneo, Kinabalu Park, Poring, Sungai Langanan 1
Malaysia, Borneo, Sabah, Ranau, Poring
4
Malaysia, Borneo, Inman river between Kalawat and
5
Bonggol
Malaysia, Borneo, Sabah, Ranau, Sungai Bundu Tuhan 13
Indonesia, Borneo, Provinsi Kalimantan Timur, Kayan
2
Basin, Bahau, En’nggeng B’io, drains into Bahau river
Indonesia, Java, Provinsi Jawa Barat, Situ Gunung,
10
Sukabumi
12(4–12)/6(1)
12(4–12)/6(1)
11(4–11)/6(1)
Inger (1966); Manthey
& Denzer (2014)
Inger (1966); Manthey
& Denzer (2014)
Inger (1966); Manthey
& Denzer (2014)
Inger (1966); Manthey
& Denzer (2014)
This study
This study
This study
11(4–11)/6(1)
11(4–11)/6(1)
This study
This study
7(4–7)/9(1)
7(4–7)/8(1)
8(4–8)/8(1)
8(4–8)/9(1)
8(5–8)/8(1)
8(5–8)/9(1)
9(5–9)/8(1)
7(4–7)/6(1)
This study
This study
9(4–9)/10(1)
This study
Java
Java
Indonesia, Java, Provinsi Jawa Barat, Situ Gunung
waterfall, Sukabumi
Java
Java
Indonesia, Java, Provinsi Jawa Barat, Gunung Halimun- 1
Salak National Park, tributary of Cimadus River
Indonesia, Java, Provinsi Jawa Barat, Gunung
1
Halimun-Salak National Park, Cikaniki River
Indonesia, Java, Provinsi Jawa Barat, Pasir Datar
10
Java
Indonesia, Java, Provinsi Jawa Barat, Cibodas
4
8(5–8)/9(1)
Java
Indonesia, Java, Provinsi Jawa Barat, Cibodas
24
7(4–7)/7(1)
7(4–7)/8(1)
Java
Indonesia, Java, Provinsi Jawa Barat, Posata Bantam
3
8(5–8)/7(2)
Sumatra
Indonesia, Sumatra, Provinsi Aceh, Sungai Alas
Sumatra
Indonesia, Sumatra, Provinsi Aceh, Kabupaten Gayo
Lues, Sungai Moelawak, jeeder stream to Alas river,
along road Ketambe-Blangkajeren
6
3
1
1
39
Sumatra
Sumatra
Indonesia, Sumatra, Provinsi Aceh, Mane
Indonesia, Sumatra, Provinsi Sumatera Utara, Alah
Basin, Lae Renum, Kampung Tigalingga,
Indonesia, Sumatra, Provinsi Sumatera Utara, Deli
7(4–7)/8(1)
7(4–7)/9(1)
6(3–6)/8(1)
7(4–7)/6(1)
7(4–7)/8(1)
7(4–7)/9(1)
8(4–8)/8(1)
8(4–8)/9(1)
7(4–7)/7(1)
8(4–8)/7(1)
8(4–8)/8(1)
7(4–7)/8(1)
Java
Sumatra
11
5
1
1
4
7(4–7)/8(1)
8(5–8)/9(1)
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
This study
Van Kampen (1910);
Manthey & Denzer
(2014)
Van Kampen (1907);
Manthey & Denzer
(2014)
Van Kampen (1910);
Manthey & Denzer
(2014)
Boulenger (1882);
Smith (1930);
Manthey & Denzer
(2014)
Manthey & Denzer
(2014)
This study
This study
This study
Yang (1991); Manthey
& Denzer (2014)
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Denticle formula
Borneo
Borneo
N
682
U. ARIFIN ET AL.
Table 2. Continued
Locality
Sumatra
Indonesia, Sumatra, Provinsi Sumatera Utara,
1
Kabupaten Karo, Mata air (Long Pagam), hill stream
along road Lau Kawar-Berastagi, jeeder stream to
sungai Lau Kawar
Indonesia, Sumatra, Provinsi Sumatera Barat, Batang
1
Si Joontour
Indonesia, Sumatra, Provinsi Sumatera Barat, Kota Pa- 11
dang, Pantai Purus area, from hill-side stream
Sumatra
Sumatra
Sumatra
Sumatra
Sumatra
Sumatra
Sumatra
N
Indonesia, Sumatra, Provinsi Sumatera Barat, Taman
Nasional Kerinci Seblat, Muara Labuh
Indonesia, Sumatra, Provinsi Sumatera Barat, Sumpur
Kudus, Batang Karangan
Indonesia, Sumatra, Provinsi Sumatera Barat, Sumpur
Kudus, Batang Karangan
Indonesia, Sumatra, Provinsi Jambi, Danau Lingkat,
Kerinci
Indonesia, Sumatra, Provinsi Bengkulu, Taman
Nasional Kerinci-Seblat, Gunung Baru
Denticle formula
Sources
7(4–7)/8(1)
This study
8(4–8)/7(1)
This study
This study
1
7(4–7)/8(1)
7(4–7)/9(1)
8(4–8)/8(1)
7(4–7)/8(1)
This study, sequenced
1
7(4–7)/7(1)
This study, sequenced
4
7(4–7)/7(1)
This study
8
7(4–7)/8(1)
This study
1
7(4–7)/6(1)
This study
Abbreviation: NA, not available.
their adult forms. Likewise, our study demonstrated that
tadpole morphology was more effective at differentiating
between species of Huia, which are also phenotypically
indistinguishable as adults.
Huia tadpoles from Borneo, Java and Sumatra
examined in this study are highly variable (Table 2).
Interestingly, tadpoles from Java are morphologically
more similar to tadpoles from Sumatra than to the
Bornean H. cavitympanum tadpoles (see Results
section), which is consistent with results from our
molecular phylogenetic analyses (Fig. 2). Huia from Java
and Sumatra can be distinguished from Bornean Huia
by the following: (1) shape of the upper jaw sheath; (2)
number in the LTRF (upper and lower lip); (3) presence/
absence of glands on the tail fin; and (4) presence/
absence of the oblique gland cluster below the origin
of the spiracular tube, running upward and backward.
Sumatran and Javan tadpoles have an undivided,
M-shaped upper jaw sheath, whereas the upper jaw
sheath of Bornean tadpoles is undivided but Λ-shaped.
Furthermore, there are also distinct differences in the
number of keratodont rows on the upper and lower
lips. Javan and Sumatran tadpoles have seven to nine
and six to eight rows on the upper lip respectively, as
opposed to Bornean tadpoles, which have at least ten
rows. On the lower lip, tadpoles from Java and Sumatra
have six to ten and six to nine rows, respectively, in
contrast to five or six rows in Bornean tadpoles. Apart
from characters in the mouth, all Huia tadpoles from
Borneo lacked glands on the tail fin, whereas a large
number of glands are present on the tail fin of tadpoles
from Sumatra and Java. An oblique row of glands below
the origin of the spiracular tube and running upward
and backward (Inger, 1985) was also observed in all
Bornean tadpoles of Huia, but not in any tadpole from
Java or Sumatra. This finding confirmed previous
observations by Inger (1985) and Stuart (2008). These
distinct differences clearly indicate that Bornean Huia
are different from those in Java and Sumatra.
Another noteworthy finding is that although
molecular data for Bornean H. cavitympanum were
limited, we observed high levels of variation in
tadpole morphology, suggesting that this taxon could
harbour more species diversity. Apart from variation
in their LTRF (Table 2), tadpoles from Inman River,
Sabah, Malaysia (Fig. 4C) has no skin projections
on the posterior part of the body compared with the
specimens from Kinabalu Park, Sabah, Malaysia (Fig.
4B) and Kayan Basin, Kalimantan, Indonesia (Fig. 4A).
The size of the abdominal sucker relative to the body
length also differed in those specimens (Fig. 4A–C).
Unfortunately, tissue samples for those tadpoles
are unavailable. Thus, it remains unclear whether
those differences represent intraspecific variation
or unrecognized species-level divergences. Future
molecular and morphological studies incorporating
more samples (adults and larvae) from a wider
geographical range are necessary to verify the identity
of these tadpoles and to assess the true diversity of
H. cavitympanum.
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Island
Parameter
Amolops (Yang, 1991)
Huia (this study)
Wijayarana
(this study)
Meristogenys (Yang, 1991;
‘Wijayarana’
melasma (Stuart Shimada & Matsui, 2019)
& Chan-ard, 2005)
Sumaterana
(Arifin et al., 2018)
Geographical ranges Nepal, northern India,
western and southern
China to Peninsular
Malaysia
Tadpoles
Gastromyzophorous
Upper beak
Divided or undivided
Borneo
Sumatra, Java
Thailand, Laos
Borneo
Sumatra
Gastromyzophorous
Λ-shaped; undivided
Gastromyzophorous
M-shaped; undivided
Unknown
Unknown
Gastromyzophorous
With M-shape at the base;
divided
Lower beak
V-shaped; undivided
V-shaped; undivided
V-shaped; undivided
Unknown
LTRF on upper lip
LTRF on lower lip
≥ 4 rows
10–12 rows
3 rows (except for Amolops 5–6 rows
larutensis 5 rows; Yang,
1991; and Amolops
cremnobatus 6 rows;
Inger & Kottelat, 1998)
Postorbital; prespiracular
Postorbital; posterior
(some individuals);
mid-lateral present in
oblique cluster below
A. cremnobatus (Inger &
spiracle, running
Kottelat, 1998)
upward and backward
Present
Absent
6–9 rows
6–10 rows
Unknown
Unknown
V-shaped; divided or undivided
≥ 5 rows
≥ 6 rows
Gastromyzophorous
Broadly arched, with
thinner medial
section; undivided
V-shaped; undivided
Infraorbital; postorbital; Unknown
prespiracular; posterior mid-lateral
Infraorbital; postorbital;
prespiracular; posterior
mid-lateral
Infraorbital;
postorbital
Present/absent
Unknown
Present/absent
Absent
Absent
Absent
Present
Unknown
Present/absent
Absent
Visible
On surface; not
transparent; not
encased by dark
Π-shaped marking
Visible
Deep in cavity; clear/
transparent; not
encased by Π-shaped
marking
Visible
On surface; not transparent; not encased by
dark Π-shaped marking
Visible
On surface; not
transparent; not
encased by dark
Π-shaped marking
Post-tympanic fold
Ratio of (length)
Finger I to
Finger II
Present
Finger I ≤ Finger II
Present
Finger I = Finger II
(Yang, 1991)
Visible
Not visible
On surface; not
On surface; clear/
transparent;
transparent; encased
not encased by
by dark Π-shaped
dark Π-shaped
marking
marking
Present
Present
Finger I ≥ Finger II
Finger I = Finger
II
Present
Finger I > Finger II
Absent
Finger I ≤ Finger II
Ratio of disc
width of Finger
III to toe IV
Wider
Subequal (Yang, 1991)
Subequal
Slightly larger or equal,
except for Meristogenys
kinabaluensis small
Wider or almost
equal
Glands on
ventral body
Glands on fin
Adults
Pineal body
Tympanum
Subequal
683
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Glands on head
and body
8–9 rows
8–9 rows
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
Table 3. Morphological comparison of adults and larvae
684
Table 3. Continued
Huia (this study)
Wijayarana
(this study)
Ratio of length
of metacarpal
of Finger I and
Finger II
Metacarpal of Finger I ≥
60% of metacarpal of
Finger II
Metacarpal of Finger
I 54% of metacarpal of
Finger II (Yang, 1991)
Metacarpal of Finger I Unknown
subequal to metacarpal of Finger II
Ratio of crossbar
width and length
of terminal
phalanx of
Finger III
Crossbar width > 60%
length of terminal
phalanx
Crossbar width 50%
length of terminal
phalanx (Yang, 1991)
Crossbar width < 50%
length of terminal
phalanx
Unknown
Crossbar width < 60%
length of terminal
phalanx
Tibia 73.00–75.00% of
SVL (Yang, 1991)
Present or absent (absent according to Yang,
1991)
Tibia > 70% of SVL
Tibia > 70% of
SVL
Present
Tibia > 70% of SVL
Ratio of tibia length Tibia < 70% of SVL
to SVL
Outer metatarsal
Absent
tubercle
Present/absent
Metacarpal of Finger I
< 60% of metacarpal of
Finger II
Present (except for
M. kinabaluensis;
Yang, 1991)
Abbreviations: LTRF, labial tooth row formula; SVL, snout–vent length.
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Amolops (Yang, 1991)
Sumaterana
(Arifin et al., 2018)
Metacarpal of
Finger I subequal
to metacarpal of
Finger II (this
study)
Crossbar width
subequal to
length of terminal
phalanx, except
for Sumaterana
montana crossbar
width 33% length
(this study)
Tibia 58.08–78.39%
of SVL
Absent
U. ARIFIN ET AL.
Meristogenys (Yang, 1991;
‘Wijayarana’
melasma (Stuart Shimada & Matsui, 2019)
& Chan-ard, 2005)
Parameter
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
685
Our study also corroborates the conclusion
of Manthey & Denzer (2014) and Arifin et al.
(2018) that the shape of beaks on tadpoles can be
effective at distinguishing the four ranid genera
with gastromyzophorous tadpoles (Amolops, Huia,
Meristogenys and Sumaterana; Fig. 5), including Huia
frogs from Sumatra and Java and from Borneo. This
also indicates that characters pertaining to the beaks
of tadpoles are synapomorphic at the generic level and
indicate that Huia from Sumatra and Java should
be considered a different genus. We recommend that
morphological characters of tadpoles should be used
for future systematic and species delimitation studies
involving these genera.
TAXONOMIC AMMENDMENTS
Our study assembled the most comprehensive genetic
dataset of Huia to date, which includes the genus
Sumaterana (that also possess gastromyzophorous
tadpoles), in addition to dense and expanded
population-level sampling across Sumatra and Java.
Our results corroborate findings of previous studies in
showing that Huia is paraphyletic. In conjunction with
new morphological data from tadpoles, we provide the
following justification and taxonomic amendments to
resolve this long-standing paraphyly.
With the aim of maintaining the natural groupings,
our phylogenetic analyses reveal three possible options
to resolve Huia taxonomy: (1) subsume all current
members of Huia and Meristogenys into one genus,
i.e. Huia (both genera were described at the same
time, but Huia was mentioned first in the original
publication); (2) merge Bornean H. cavitympanum
(genus name bearer) and Meristogenys into a single
genus (Huia) and erect a new genus to accommodate
the remaining species of Huia (clades A–G); or (3)
restrict Huia to comprise only H. cavitympanum
(monotypic genus), retain Meristogenys and erect a
new genus to accommodate the remaining species of
Huia (clades A–G).
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Figure 4. Morphological comparison between tadpoles of Huia cavitympanum from Kayan Basin River, East Kalimantan,
Indonesia (ZRC.1.4347; A), Sungai Langanan, Sabah, Malaysia (ZRC.1.11536; B) and Inman River, North Borneo, Malaysia
(ZRC.1.488; C). Photographs by Ruzaini Bin Ghazali (A, C) and by U.A. (B).
686
U. ARIFIN ET AL.
Option 1 is the most inclusive but, in our view, the
least desirable because it combines numerous welldefined, antiquated, highly divergent, morphologically
disparate and allopatrically distributed lineages
across a large geographical area. Option 2
synonymizes Meristogenys and H. cavitympanum,
thereby forming a clade that is endemic to Borneo.
Although this is cogent from a biogeographical and
evolutionary perspective, it is inconsistent with the
deep genetic divergence between H. cavitympanum
and the rest of Meristogenys (14.1–16.6%, in contrast
to interspecies distances within Meristogenys, which
were < 8%; Fig. 3; Supporting Information, Table S3).
Moreover, the fact that H. cavitympanum and all
other Meristogenys co-occur in Borneo, yet maintain
a clear and large genetic divergence between them
indicates strong genetic isolation that is derived from
an antiquated diversification event that pre-dates
the more recent species-level diversification events
within Meristogenys by a considerable amount of
time (Fig. 2). Furthermore, the large genetic distances
between H. cavitympanum and Meristogenys are
consistent with intergeneric distances between
other closely related genera (e.g. Clinotarsus and
Sumaterana; Fig. 3; Supporting Information, Table
S3), and there are discrete differences in tadpole
morphology that distinguish H. cavitympanum from
all other Meristogenys. Therefore, there are clearly
more lines of evidence to support the recognition of
H. cavitympanum and Meristogenys as distinct genera
than to combine them (see also Stuart, 2008). Our
data clearly support option 3; erecting a new genus to
accommodate the Huia frogs from Java, Sumatra and
Thailand/Laos would not only resolve the paraphyly
of Huia, but would also accurately and consistently
reflect the evolutionary history of each newly
demarcated genus, while simultaneously preserving
distinct, biologically meaningful and diagnosable
differences among them. The genus Meristogenys will
be retained, and the genus Huia will be restricted
to include only H. cavitympanum, which was also
previously considered by Manthey & Denzer (2014).
Although this proposal renders Huia as a monotypic
genus, this study provides preliminary data that
demonstrates the potential presence of additional and
undescribed lineages within H. cavitympanum (see
tadpole discussion above).
Our study not only corroborates the findings of
Manthey & Denzer (2014), but also provides the
following additional evidence to support both their and
our proposed amendments: (1) the paraphyly of Huia
s.s. as demonstrated using the most comprehensive
phylogeny to date, which includes dense and expanded
population-level sampling across Sumatra and Java
(Fig. 2); (2) high levels of genetic divergence between
H. cavitympanum and Meristogenys (14.1–15.6%) and
all other Huia s.s. (14–17%) that are consistent with
intergeneric distances between other closely related
genera (Fig. 3; Supporting Information, Table S3);
and (3) discrete differences in tadpole morphology
(LTRF, shape of jaw sheath and presence/absence
of the glands on the tail fin; see Table 3). These
independent yet complementary lines of evidence from
morphological and genetic data support the distinction
of H. cavitympanum, Meristogenys and all other
Huia s.s. (clade A–G; Fig. 2) as distinct, supraspecific
evolutionary units.
Therefore, under the framework of integrative
taxonomy, we formally propose the following
amendments to the taxonomic classification of all
currently recognized Huia frogs: (1) Huia is restricted
to comprise the single taxon H. cavitympanum,
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Figure 5. Morphological comparison of the shape of beaks among Huia tadpoles (minus Huia modigliani and Huia
melasma) and with other genera that have gastromyzophorous tadpoles. A, Amolops panhai. B, Meristogenys penrissenensis.
C, Huia cavitympanum. D, Huia sumatrana. E, Sumaterana crassiovis. Photographs by Matsui & Nabhitabhata (2006; A),
A.H. (B, C) and U.A. (D, E).
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
which is reciprocally monophyletic with the genus
Meristogenys; (2) a new genus (proposed herein and
described below) is erected to accommodate all other
Huia, including populations from Indonesia (Sumatra
and Java) and provisionally Indochina (see new genus
description below); and (3) H. javana is resurrected
from the synonymy of H. masonii (see Remarks on the
new genus below for more details).
FAMILY RANIDAE BOIE, 1828
(FIG. 6A)
Type species: Rana cavitympanum Boulenger, 1893,
Proceedings of the Zoological Society of London: 525.
Holotype: BMNH 1893.5.30.26 (BMNH 1947.2.4.16);
adult male).
Type locality: Kina Baloo, North Borneo (Kinabalu,
Sabah, Malaysia). Collector: A. Everett.
Common name: Sabah Huia frog (English) following
Frank & Ramus (1995) and Frost (2019); Kongkang
Jeram Sabah (Bahasa Indonesia).
male with nuptial pad and paired lateral vocal sacs;
humeral gland absent. In life, dorsum generally dark
brown; flanks light brown with markings; head light
brown with scattered dark brown markings; lips with
dark markings.
Geographical range: Huia is endemic to Borneo.
Remarks: Genetic distances among H. cavitympanum
from Kalimantan (RMBR2283), Sabah (FMNH
237299) and Sarawak (NMBE 1061482) are relatively
high (4.00–4.38%; Supporting Information Table S3).
Noteworthy differences are also found in tadpole
morphology among these populations (see discussion
above). Unfortunately, tissue samples for those
tadpoles are unavailable. Thus, it remains unclear
whether those differences represent intrapopulation
variation or unrecognized species-level divergences.
Future molecular and morphological studies
incorporating more samples (adults and larvae) from
a wider geographical range are necessary to verify
the identity of these tadpoles and to assess the true
diversity of Huia.
GENUS WIJAYARANA GEN. NOV.
Phylogenetic definition and content: Huia is a
distinct and independently evolving genus, which
shares a most recent common ancestor with the genus
Meristogenys (Fig. 2). It is currently understood to
be monotypic, containing H. cavitympanum, which is
endemic to the island of Borneo.
Diagnosis: (Tadpole characters presented here are
based on observation by U.A., whereas morphological
characters of adults are taken from published studies
(e.g. Inger, 1966; Yang, 1991; Manthey & Denzer, 2014).
Larvae gastromyzophorous; upper beak Λ-shaped,
lower beak V-shaped, both undivided; LTRF 10–12((3–
4)–(10–12))/5–6(0–2); postorbital glands present;
an oblique cluster of glands immediately below the
spiracular tube, running upward and backward
present; prespiracular glands present in some
individuals (Inger, 1985); glands on the tail fin absent.
Adults medium in size, body stocky; snout rounded;
tympanum clear, deep in cavity; supra- and posttympanic folds present, dorsolateral folds present;
ratio of tibia length to snout–vent length > 70%;
outer metatarsal tubercle present or absent (absent
according to Yang, 1991), inner metatarsal tubercle
present, small; length of Finger I = Finger II; length
of metacarpal of Finger I 54% of Finger II; width of
crossbar terminal phalanx of Finger III 50% length;
(FIG. 6B–F)
Zoobank registration:
lsid:zoobank.org:act:
4A7EC577-93DC-4A0D-851D-82B277E060E3
Type species: Huia sumatrana Yang, 1991, Fieldiana
Zoology 63: 31. Holotype: FMNH 209222, an adult
male, by original designation.
Type locality: Bukit Lawang Forest Reserve, Bohorok,
Sumatra (3°31′N, 98°8′E), Indonesia. Collected by
Harold K. Voris, 22 July 1976.
Etymology: Wijayarana is a compound of the words
‘wijaya’ (Vijaya, in Sanskrit, meaning victory) and
‘rana’ (Latin for frog, feminine). In this context,
Wijaya alludes to the Sriwijaya (or Sri Vijaya) empire
based in Palembang (Sumatra) between the 7 th and
14th centuries CE, with primarily maritime realms
(i.e. a thalassocracy). Sriwijaya became one of the
most powerful and expansive kingdoms of Indonesia,
spanning across most of Sumatra, Java and the Malay
Peninsula. With the exception of the Malay Peninsula,
these landmasses also represent the geographical
range of the genus.
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GENUS HUIA YANG, 1991
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Figure 6. A, Huia cavitympanum male, Taman Nasional Bukit Baka Bukit Raya, Provinsi Kalimantan Barat, Indonesia. B,
Wijayarana (clade A); UA20140663, male, Padang Aro, Taman Nasional Kerinci-Seblat, Provinsi Sumatera Barat, Indonesia.
C, Wijayarana (clade B); UA20150202, male, Wiyono Waterfall, Provinsi Lampung, Indonesia. D, Wijayarana (clade C);
UA20150464–65, male and female in amplexus, Batang Karangan, Provinsi Sumatera Barat, Indonesia. E, Wijayarana
(clade E); UA20150032, male, Banyumas, Provinsi Jawa Tengah, Indonesia. F, Wijayarana (clade F); UA20150049;
Palutungan, Provinsi Jawa Barat, Indonesia. Size not to scale. Photographs by U.A.
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
689
Common name: Wijaya cascade frogs (English);
Kongkang Jeram Wijaya (Bahasa Indonesia).
depending on the status of W. melasma (see remarks
under that species).
Included species: Wijayarana javana (Yang, 1991)
comb. nov., Wijayarana masonii (Boulenger, 1884)
comb. nov., Wijayarana melasma (Stuart & Chanard, 2005) comb. nov., Wijayarana modigliani
(Doria, Salvidio, & Tavano, 1999) comb. nov. and
Wijayarana sumatrana (Yang, 1991), comb. nov.
Remarks: Yang (1991) suggested that the length of the
metacarpal of Finger I for W. javana is 61% of Finger II
and for W. sumatrana the length of the metacarpal of
Finger I is 69% of Finger II. However, according to our
findings, the length of the metacarpal of of Finger I for
Wijayarana is subequal to Finger II (see Supporting
Information, Fig. S4).
a crown clade consisting of lineages from Indonesia
(Sumatra and Java) and, provisionally, Thailand and
Laos (see Remarks). Species previously assigned to
H. masonii, H. sumatrana and H. javana (resurrected
herein) are transferred to the new genus Wijayarana.
The phylogenetic position of frogs previously
recognized as H. melasma is still uncertain, but based
on the results from this study we assign it provisionally
to the genus Wijayarana. We also tentatively assign
frogs previously known as H. modigliani to the genus
Wijayarana. However, owing to ambiguous diagnostic
characters and the absence of molecular samples from
the type locality, the validity of this species is still in
question (for more details, see Remarks).
Diagnosis: Larvae gastromyzophorous; upper beak
M-shaped, lower beak V-shaped, both undivided;
LTRF 6–9((3–5)–(6–9))/6–10(1); an oblique cluster
of glands immediately below the spiracular tube,
running upward and backward absent; infraorbital,
postorbital, prespiracular and posterior mid-lateral
glands present; abdominal glands absent, except
for few individuals; glands on the tail fin present.
Adults medium in size, body slender; snout pointed;
tympanum not depressed, clear, framed by dark
Π-shaped marking; supra-, post-tympanic and
dorsolateral folds present; ratio of tibia length to body
length > 70%; outer metatarsal tubercle present or
absent; inner metatarsal tubercle present; Finger I ≥
Finger II; length of metacarpal of Finger I subequal to
Finger II (see Supporting Information, Fig. S4); width
of crossbar terminal phalanx of Finger III < 50%
length (Supporting Information, Fig. S4); male with
nuptial pad and paired lateral vocal sacs; humeral
gland absent. In life, head and dorsal body generally
brown, with or without scattered white markings; lips
with dark markings.
Geographical range: Wijayarana has been recorded
from Java and Sumatra, Indonesia. This genus might
also (provisionally) occur in Thailand and Laos,
Yang (1991) described W. javana (as Huia javana
Yang, 1991) without mentioning W. masonii (Rana
masonii Boulenger, 1884) in his description. Iskandar
(1998) synonymized W. javana with W. masonii based
on morphological similarities between both species.
Subsequently, Manthey & Denzer (2014) pointed out
that W. javana differs from W. masonii by the presence
of a small outer metatarsal tubercle (vs. absent) and
relative length of Finger I = Finger II (vs. Finger I >
Finger II). Thus, Manthey & Denzer (2014) proposed
that both W. masonii and W. javana should be
considered preliminarily as valid species. This study
supports the hypothesis of Manthey & Denzer (2014)
by providing the following evidence: (1) sample MZB.
Amph.22371 was collected from Cibodas, the type
locality of W. javana (clade F) and was inferred as a
distinct and divergent lineage; and (2) morphological
examination of adult specimens of clade F (N = 7)
matched the original description of W. javana. Thus,
we support the proposal by Manthey & Denzer (2014)
to consider W. javana as a valid species. Furthermore,
we are confident that clade E does not represent
W. masonii, because an outer metatarsal tubercle
was observed in the specimens of this clade (N = 10).
It is also important to note that clade E is more
closely related to the true W. sumatrana (clade C–D)
than to W. javana (clade F). Thus, clade E might
constitute a new, undescribed species (Huia sp. 3) that
is conspecific with neither W. javana nor W. masonii.
These results also indicate that the true W. masonii
has yet to be sampled, and its identity remains dubious.
Additionally, given that we resurrected W. javana in
this study, several localities that were mentioned in
previous studies (e.g. Iskandar, 1998; Kurniati, 2003;
Kusrini, 2013) as part of the geographical distribution
of W. masonii should be re-evaluated.
We assign W. sumatrana as the type species of
Wijayarana because: (1) the identity of W. masonii
is still unclear, although it is the oldest available
name within species recognized under Wijayarana;
(2) W. sumatrana is confirmed based on the
abovementioned evidence; (3) W. sumatrana was
described at the same time as W. javana, but given
that the presence of another species in Java remains
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Phylogenetic definition and content: Wijayarana is
690
U. ARIFIN ET AL.
IMPLICATIONS OF THE AMENDMENTS
We are fully aware that our taxonomic proposal to
resolve paraphyly of frogs previously assigned as Huia
might be deemed controversial, especially considering
the low support of nodes for unstable branches
representing the sister relationship among the newly
erected genus Wijayarana and W. melasma, Huia,
Meristogenys, Sumaterana and Clinotarsus.
A l l av a i l a b l e st u d i e s, i n cl u d i n g o u r stud y,
despite some differences in sampling, converge to
similar conclusions, i.e. there is a problem with
Huia/Clinotarsus. There is some ambiguity (low
nodal support) in the region where species of these
taxa connect. Our sequence lengths were much longer
than those used by Stuart (2008), Wiens et al. (2009),
Chan & Brown (2017) and Arifin et al. (2018) and
contained fewer gaps than those of Pyron & Wiens
(2011) but still could not resolve the nodes in question.
Moreover, even after phasing the alignments of the
two nuclear loci in this study for the abovementioned
problematic nodes, the nodes still had low support
(see Fig. 2; Supporting Information Figs S1, S2).
Also, according to the SH test, the ML tree topology
presented in this study is accepted to represent the
phylogenetic relationships among Wijayarana in
Java and Sumatra, W. melasma, Huia, Sumaterana,
Clinotarsus and Meristogenys. Thus, we believe
that these poorly supported nodes are probably
attributable to rapid radiation at the time of origin
rather than data deficiency (Whitfield & Lockhart,
2007; Whitfield & Kjer, 2008). If we are correct, the
effort to solve this problem probably requires highthroughput molecular data, which is a venture that is
beyond the scope of the present project.
CONCLUSION
This study resolved the long-standing systematic
problems of Huia using new genetic sequences and
morphological data from tadpoles. We demonstrated
the efficacy of wide geographical sampling (especially
at type localities) of adults and larvae and the
integration of multiple sources of data to inform
taxonomic classification. This study also confirmed
that ranids with gastromyzophorous tadpoles can be
differentiated by the shape of the jaw sheaths.
For Huia s.s., larval morphology was particularly
informative and can be an important source of data
for species delimitation. This study resolved certain
taxonomic issues, but also raised a number of important
questions regarding the validity of W. modigliani,
the true identity of W. masonii, the phylogenetic and
taxonomic placement of W. melasma and the hidden
diversity within Wijayarana and Huia, especially
in the region of Indonesia, including Kalimantan in
Borneo. Another missing piece of evidence is that the
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unclear, we prefer not to select any recognized species
from this island as the type species for the genus.
We tentatively place the species modigliani in
Wijayarana solely based on its distribution in
Sumatra. Unfortunately, no tissue samples are
available to corroborate this. Manthey & Denzer
(2014) pointed out several ambiguous diagnostic
characters for W. modigliani that were originally
proposed by Doria et al. (1999), which lead to confusion
in species identification (for example, by Kurniati,
2009). However, given that Huia is now restricted
to Borneo and that all confirmed Wijayarana
(W. sumatrana and W. javana) and W. masonii (to
be confirmed) are restricted to Java and Sumatra,
we find it reasonable and most parsimonious to
place W. modigliani tentatively within Wijayarana
pending new evidence.
We also tentatively place the species melasma in
the genus Wijayarana. This species was previously
known only from western and northern Thailand
(Stuart & Chan-Ard, 2005; Frost, 2019), but has
been confirmed by our study also to occur in Laos
(FMNH 271377 was collected from Luang Namtha
Prov, Vieng Phou Kha District). In both our ML
and BI trees (including ML and BI trees after
phasing the nuclear DNA alignments; see Fig. 2;
Supporting Information Fig. S2), W. melasma is
sister to Wijayarana clades from Java and Sumatra.
The position of melasma has never been stable in
previous studies (Fig. 1), including ours (e.g. Fig. 2),
despite obtaining the longest concatenated dataset
from both mitochondrial and nuclear markers, with a
low percentage of missing data. However, its genetic
divergence from all other closely related lineages
was high (> 10.00%) and consistent with divergence
observed between other genera. Wijayarana melasma
is morphologically more similar to other Wijayarana
species than to H. cavitympanum; for example, its
body is slender (vs. body stout in Huia), the snout
is pointed (vs. rounded in Huia) and the tympanum
is not depressed (vs. deep cavity in Huia). On the
contrary, we also notice several morphological
characters of adult W. melasma (Stuart & Chan-ard,
2005) that differ from Wijayarana, e.g. the pineal
body is not visible (vs. visible in Wijayarana), and the
tympanum not transparent and not encased by a dark
Π-shaped marking (vs. clear tympanum and encased
by Π-shaped marking in Wijayarana); see Table 3.
More importantly, tadpoles of W. melasma have never
been collected; hence, it remains to be determined
whether this species even has gastromyzophorous
tadpoles. As such, the taxonomic and phylogenetic
placement of W. melasma remains an open question
that will be likely to require additional molecular
and/or tadpole data from expanded geographical
sampling in Thailand/Laos to resolve.
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
tadpoles of W. melasma and W. modigliani remain
unknown (Manthey & Denzer, 2014). More fieldwork in
the region is needed to resolve the persisting problems
completely and elucidate the full extent of diversity
within this unique group of frogs.
ACKNOWLEDGEMENTS
FUNDING STATEMENT
This study was funded by Deutsche
Forschungsgemeinschaft (DGF; German Research
Foundation; 246870126 to A.H.) and through a stipend
to U.A. provided by the Deutscher Akademischer
Austauschdienst-Indonesian German Scholarship
Program (DAAD-IGSP; 91548731) and the Merit
Scholarship for International Students at Universität
Hamburg, Germany. Part of the study was funded by the
National Science Foundation (NSF; DEB-1146324 to
E.N.S. and M.B. Harvey), Volkswagen Foundation (I/79
405 to A.H.), Idea Wild (to U.A.) and LKCNHM Student
Research Grant (to U.A.). The European Commission
through the synthesis of systematic resources
(SYNTHESYS) access programme (NL-TAF-4882 and
GB-TAF-4412) supported U.A. during examination of
type specimens and other materials. The funders had
no role in study design, data collection and analysis,
decision to publish or preparation of the manuscript.
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Talavera G, Castresana J. 2007. Improvement of phylogenies
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher's web-site:
File S1. Documentation and results of Shimodaira–Hasegawa test.
Table S1. Primer information used in this study.
Table S2. Best model selected by PARTITIONFINDER.
Table S3. Genetic p-distance between Huia clades calculated in MEGA7 using 16S-tRNAval.
Figure S1. Trees generated from original dataset. A, maximum likelihood (ML) tree for mitochondrial
DNA only (12S + 16S+tRNAval + COI + ND2). B, Bayesian inference (BI) tree for mitochondrial DNA only
(12S + 16S+tRNAval + COI + ND2). C, ML tree for nuclear DNA only (RAG + TYR). D, BI tree for nuclear DNA
only (RAG + TYR). E, BI tree for concatenated DNA (12S + 16S+tRNAval + COI + ND2 + RAG + TYR).
Figure S2. Trees generated from phased alignments. A, maximum likelihood (ML) tree for phased nuclear DNA
only (RAG + TYR) for individual a (1) and individual b (2). B, Bayesian inference (BI) tree for phased nuclear
DNA only (RAG + TYR) for individual a (1) and individual b (2). C, BI tree for phased concatenated sequences
(12S + 16S+tRNAval + COI + ND2 + RAG + TYR).
Figure S3. Maximum likelihood tree [concatenated dataset, which included two Huia samples from Mastui et al.
(2006): N6468 and N6474].
Figure S4. X-Ray palmar image of Wijayarana (clade C; UA20140188).
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
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phylogenetic analysis and post-analysis of large phylogenies.
Bioinformatics 30: 1312–1313.
693
Voucher no.
GenBank accession no. (12S;
16S+tRNAval; COI; ND2; RAG; TYR)
Locality
Study
Amolops afghanus
CAS 221313
Amolops indoburmanensis
CAS 221675
Amolops marmoratus
CAS 234720
Amolops panhai
CAS 229816
MG909643; MG909570; MW322843;
MW292240; MG909608; MG909683
MG909644; MG909571; MW322844;
MW292241; MG909610; MG909686
MG909645; MG909572; MW322845;
MW292242; MG909611; MG909684
MG909646; MG909606; MW322846;
MW292243; MG909609; MG909685
Arifin et al. (2018); this
study
Arifin et al. (2018); this
study
Arifin et al. (2018); this
study
Arifin et al. (2018); this
study
Chalcorana chalconota
Tadpole Java 1
MZB.AMPH.30399 MW221997; NA; MW322847;
MW292244; MW292308; MW292356
FMNH 263424
MG909673; MG909604; MW322848;
MW292245; MG909641; MG909717
FMNH 268338
MG909672; MG9096603; MW322849;
MW292246; MG909640; MG909716
FMNH 237299
NA; NA; NA; NA; EF088246
EU076769
NMBE 1061482
MW221998; MW219540; MW322850;
NA; MW292309; NA
RMBR2283
MG909681; MG909602; MW322851;
MW292247; MG909632; MG909710
MZB.AMPH.22371 MW221999; NA; NA; MW292248;
MW292310; MW292357
MZB.AMPH.29424 MG909676; MG909599; MW322852;
MW292249; MG909634; MG909712
UA20150051
MW222000; MW219541; NA;
MW292250; MW292311; MW292358
UA20150033
MW222001; MW219542; MW322853;
MW292251; MW292312; MW292359
UA20150061
NA; MW219543; NA; NA; NA; NA
Myanmar, Kachin State, Putao District,
Machanbaw Township, Ahtonga Village
Myanmar, Chin State, Saw Stream,
Kanpetlet Township, Mindat Division
Myanmar, Shan State, Kalaw Township,
What Phu Ye Camp
Myanmar, Tanintharyi Division, Da Wei
District, Thayet Chaung Township, east of
Mal Ke Village, border of Nwa La Bo Reserve Forest along Ngwe Taung Stream
Indonesia, Java, Provinsi Jawa Barat,
Kabupaten Banyumas, Curug Cipendok
Thailand, Prachuap Kirikhan, Hua Hin
Specimen Thailand 1
FMNH 270711
Clinotarsus penelope
Specimen 1 Borneo
Specimen 2 Borneo
Specimen 3 Borneo
Specimen 1 Java
Specimen 2 Java
Specimen 3 Java
Specimen 4 Java
MG909674; NA; MW322854; NA; NA;
MG909714
Thailand, Ranong Province
Malaysia, Borneo, Sabah, Tenom District
Malaysia, Borneo, Pa Rabata, Gunung
Murud
Indonesia, Borneo, Kalimantan, Bukit Baka
Bukit Raya National Park
Indonesia, Java, Provinsi Jawa Barat,
Cibodas
Indonesia, Java, Provinsi Jawa Barat,
Kabupaten Sumedang
Indonesia, Java, Provinsi Jawa Barat,
Palutungan
Indonesia, Java, Provinsi Jawa Barat,
Kabupaten Banyumas
Indonesia, Java, Provinsi Jawa Barat,
Palutungan
Thailand, Nan, Pua District, Tambon Uan
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Clinotarsus alticola
This study
Arifin et al. (2018); this
study
Arifin et al. (2018); this
study
Stuart (2008)
This study
Arifin et al. (2018); this
study
This study
This study
This study
This study
This study
Arifin et al. (2018);
this study
U. ARIFIN ET AL.
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
Species
694
Appendix 1. Information for GenBank accession numbers, localities and related information for each voucher specimen used for molecular analyses
in this study
Species
Voucher no.
GenBank accession no. (12S;
16S+tRNAval; COI; ND2; RAG; TYR)
Locality
Study
Specimen Laos 1
FMNH 271377
Specimen 1 Sumatra
UA20140009
UA20140018
Specimen 3 Sumatra
UA20140022
Specimen 4 Sumatra
UA20140120
Specimen 5 Sumatra
UA20140158
Specimen 6 Sumatra
UA20140188
Specimen 7 Sumatra
UA20140422
Specimen 8 Sumatra
UA20141019
Specimen 9 Sumatra
UA20140981
MW222010; MW219552; MW322860;
MW292261; MW292320; MW292368
Specimen 10 Sumatra
UA20141095
Specimen 11 Sumatra
UA20141061
Specimen 12 Sumatra
UA20141109
Specimen 13 Sumatra
MVZ 271197
Specimen 14 Sumatra
MVZ 271199
MW222011; MW219553; MW322861;
MW292262; MW292321; MW292369
MW222012; NA; MW322862;
MW292263; MW292322; MW292370
MW222013; MW219554; MW322863;
MW292264; MW292323; MW292371
MW222014; MW219555; MW322864;
MW292265; MW292324; MW292372
MW222015; MW219556; MW322865;
MW292266; MW292325; MW292373
Laos, Luang Namtha Prov, Vieng Phou Kha
District
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuseur
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuseur, Jambur Gele
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuseur, Jambur Gele
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuseur, Ketambe
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuseur, Ketambe
Indonesia, Sumatra, Provinsi Aceh, Taman
Buru Linge-Isaq
Indonesia, Sumatra, Provinsi Aceh, Kab.
Ulu Masen, Mane
Indonesia, Sumatra, Provinsi Bengkulu,
Taman Nasional Keinci-Seblat, Gunung
Baru
Indonesia, Sumatra, Provinsi Bengkulu,
Taman Nasional Keinci-Seblat, Gunung
Baru
Indonesia, Sumatra, Provinsi Bengkulu,
Taman Nasional Keinci-Seblat
Indonesia, Sumatra, Provinsi Bengkulu,
Taman Nasional Keinci-Seblat
Indonesia, Sumatra, Provinsi Bengkulu,
Taman Nasional Keinci-Seblat
Indonesia, Sumatra, Provinsi Bengkulu,
Kec. Agra Makmur, Air Terjun Kemumu
Indonesia, Sumatra, Provinsi Bengkulu,
Kec. Agra Makmur, Air Terjun Kemumu
Arifin et al. (2018);
this study
This study
Specimen 2 Sumatra
MG909675; MG909601; MW322855;
MW292252; MG909637; MG909715
MW222002; MW219544; NA;
MW292253; MW292313; MW292360
MW222003; MW219545; MW315884;
MW292254; MW292314; MW292361
MW222004; MW219546; NA;
MW292255; MW292315; MW292362
MW222005; MW219547; NA;
MW292256; NA; MW292363
MW222006; MW219548; MW322856;
MW292257; MW292316; MW292364
MW222007; MW219549; MW322857;
MW292258; MW292317; MW292365
MW222008; MW219550; MW322858;
MW292259; MW292318
MW222009; MW219551; MW322859;
MW292260; MW292319; MW292367
Specimen 15 Sumatra
UA20140828
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Indonesia, Sumatra, Provinsi Jambi, Taman This study
Nasional Kerinci-Seblat, Bukit Tapan
Indonesia, Sumatra, Provinsi Sumatera
This study
Utara, Taman Nasional Batang Gadis
695
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Specimen 16 Sumatra
MW222016; MW219557; NA;
MW292267; MW292326; MW292374
MZB.AMPH.23526 MW222017; MW219558; MW315885;
MW292268; MW292327; MW292375
This study
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
Appendix 1. Continued
696
Appendix 1. Continued
Voucher no.
GenBank accession no. (12S;
16S+tRNAval; COI; ND2; RAG; TYR)
Locality
Study
Specimen 17 Sumatra
Specimen 20 Sumatra
UA20150116
MW222018; MW219559; MW322866,
MW292269; MW292328; MW292376
MG909677; MG909600; MW322867;
MW292270; MG909635; MG909713
NA; MW219560; MW322868;
MW292271; MW292329; NA
MW222019; MW219561; MW315886;
MW292272; MW292330; MW292377
Specimen 21 Sumatra
UA20150202
Indonesia, Sumatra, Provinsi Sumatera
Utara, road to Bukit Lawang
Indonesia, Sumatra, Provinsi Sumatera
Utara, outskirts of Bukit Lawang
Indonesia, Sumatra, Provinsi
lampung, Way Sindalapai, south of Liwa
Indonesia, Sumatra, Provinsi
Lampung, Kabupaten Lampung Barat,
Kec. Gedong Surian
Indonesia, Sumatra, Provinsi
Lampung, Air Terjun Wiyono
Indonesia, Sumatra, Provinsi
Lampung, Air Terjun Talang Ogan
Indonesia, Sumatra, Provinsi Sumatera
Selatan, Kabupaten. Ogan Komering Ulu
Selatan
Indonesia, Sumatra, Provinsi Sumatera
Barat, trail to Pingara up to Talakmau
Indonesia, Sumatra, Provinsi Sumatera
Barat, trail to Pingara up to Talakmau
Indonesia, Sumatra, Provinsi Sumatera
Barat, Kab. Tanah Datar, Lembah Anai
Indonesia, Sumatra, Provinsi Sumatera
Barat, Cagar Alam Rimbo Panti
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Gunung Bontak
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Muara Labuh
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Padang Aro
This study
Specimen 19 Sumatra
UTA A-64940
(topotypic)
UTA A-64939
(topotypic)
UTA A-62547
Indonesia, Sumatra, Provinsi Sumatera
Barat, Kabupaten Pauh,
Universitas Andalas
Indonesia, Sumatra, Provinsi Sumatera
Barat, Cagar Alam Malampah Alahan
Panjang
This study
Specimen 18 Sumatra
Specimen 23 Sumatra
Specimen 24 Sumatra
Specimen 25 Sumatra
Specimen 26 Sumatra
Specimen 27 Sumatra
Specimen 28 Sumatra
UTA A-62437
MW222023; MW219565; NA;
MW292276; MW292333; MW292381
MZB.AMPH.22350 MW222024; MW219566; MW322871;
MW292277; MW292334; MW292382
MVZ 271217
MW222025; MW219567; MW322872;
MW292278; MW292335; MW292383
MVZ 271219
MW222026; MW219568; MW315887;
MW292279; MW292336; MW292384
UA20140712
MW222027; MW219569; MW322873;
MW292280; MW292337; MW292385
Specimen 29 Sumatra
UA20140729
MW222028; MW219570; MW322874;
MW292281; MW292338; MW292386
Specimen 30 Sumatra
UA20140663
MW222029; MW219571; MW322875;
MW292282; MW292339; MW292387
Specimen 31 Sumatra
UA20140455
MW222030; MW219572; MW322876;
MW292283; MW292340; MW292388
Specimen 32 Sumatra
UA20140460
MW222031; MW219573; MW322877;
MW292284; MW292341; MW292389
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© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
Specimen 22 Sumatra
MW222020; MW219562; NA;
MW292273; MW292331; MW292378
UA20150195
MW222021; MW219563; MW322869;
MW292274; MW292332; MW292379
MZB.AMPH.23246 MW222022; MW219564; MW322870;
MW292275; NA; MW292380
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
U. ARIFIN ET AL.
Species
Species
Voucher no.
GenBank accession no. (12S;
16S+tRNAval; COI; ND2; RAG; TYR)
Locality
Study
Specimen 33 Sumatra
UA20140504
MW222032; MW219574; MW315888;
MW292285; MW292342; MW292390
This study
Specimen 34 Sumatra
UA20150431
Specimen 35 Sumatra
UA20150464
Tadpole Sumatra 1
ZMH A12660
MW222033; MW219575; MW322878;
MW292286; MW292343; MW292391
MW222034; MW219576; MW322879;
MW292287; MW292344; MW292392
NA; MW219577 ; NA; NA; NA; NA
Tadpole Sumatra 2
ZMH A12659
NA; MW219578 ; NA; NA; NA; NA
Indonesia, Sumatra, Provinsi Sumatera
Barat, Cagar Alam Malampah Alahan
Panjang
Indonesia, Sumatra, Provinsi Sumatera
Barat, Tanjung Bungo
Indonesia, Sumatra, Provinsi Sumatera
Barat, Batang Karangan, Sumpur Kudus
Indonesia, Sumatra, Provinsi Sumatera
Barat, Batang Karangan, Sumpur Kudus
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Muara Labuh
Indonesia, Provinsi Aceh, Taman Nasional
Gunung Leuser
Malaysia, Borneo, Sabah, Gunung Mulu
National Park
Malaysia, Borneo, Sarawak, Usun Apau
National Park
Malaysia, Borneo, Sabah, Kinabalu
National Park
Malaysia, Borneo, Sarawak, Matang
Malaysia, Borneo, Sarawak, Paya Maga
Malaysia, Borneo, Sarawak, Paya Maga
This study
Hylarana erythraea
MZB.AMPH.29423 MG909680; MG909597; MW322880;
NA; MG909639; MG909689
Meristogenys amoropalamus NMBE 1056400
MW222035; MW219579; MW322881;
MW292288; MW292345; MW292393
Meristogenys amoropalamus NMBE 1057304
MW222036; MW219580; MW322882;
MW292289; MW292346; MW292394
Meristogenys amoropalamus NMBE 1061885
MW222037; NA; MW322883;
MW292290; MW292347; MW292395
Meristogenys jerboa
KUHE 12055
NA; NA; NA; NA; AB360202; NA
Meristogenys kinabaluensis NMBE 1066029
MW222038; MW219581; MW322884;
MW292291; MW292348; MW292396
Meristogenys kinabaluensis NMBE 1065985
MW222039; MW219582; MW322885;
MW292292; MW292349; MW292397
Meristogenys kinabaluensis NMBE 1064112
MG909678; MG909598; MW322886;
MW292293; MG909633; MG909711
This study
This study
This study
Arifn et al. (2018); this
study
This study
This study
This study
Shimada et al. (2008)
This study
Meristogenys kinabaluensis
NMBE 1066113
MW222040; MW219583; MW322887;
MW292294; MW292350; MW292398
Malaysia, Borneo, Sabah, Gunung Kinabalu Arifin et al. (2018); this
National Park, Sayap Substation, Sungai
study
Kemantis trail
Malaysia, Borneo, Sarawak, Pulong Tau
This study
National Park
Meristogenys orphocnemis
NMBE 1064105
Meristogenys poecilus
NMBE 1064092
Meristogenys sp.
NMBE 1057344
MW222041; MW219584; MW322888;
MW292295; MW292351; MW292399
MW222042; MW219585; MW322889;
MW292296; MW292352; MW292400
MW222043; NA; MW322890;
MW292297; MW292353; MW292401
Malaysia, Borneo, Sabah, Bundu Tuhan,
This study
Sungai Winokok
Malaysia, Borneo, Sabah, Tawau Hills Park, This study
Sungai Tawau
Malaysia, Borneo, Sarawak, Usun Apau
This study
National Park
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
697
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Appendix 1. Continued
698
Appendix 1. Continued
Voucher no.
GenBank accession no. (12S;
16S+tRNAval; COI; ND2; RAG; TYR)
Locality
Study
Meristogenys sp.
NMBE 1065861
NMBE 1061612
Pulchrana picturata
UA20140782
Sumaterana crassiovis
ZMH A14197
Sumaterana crassiovis
ZMH A14185
Malaysia, Borneo, Sarawak, Merarap
Hotspring
Malaysia, Borneo, Sarawak, Palungan,
Gunung Murud
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Muara Labuh
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Padang Aro
Indonesia, Sumatra, Provinsi Lampung, Air
Terjun Talang Ogan
Indonesia, Sumatra, Provinsi Aceh, Mane
This study
Odorrana hosii
MW222044; MW219586; MW322891;
MW292298; MW292354; MW292402
MG909647; MG909605; MW322892;
MW292299; MG909612; MG909687
MW222045; MW219586; MW322893;
MW292300; MW292355; MW292403
Indonesia, Sumatra, Provinsi Aceh, Taman
Buru Linge-Isaq
Indonesia, Sumatra, Provinsi Sumatera
Barat, Taman Nasional Kerinci-Seblat,
Gunung Baru
Indonesia, Sumatra, Provinsi Aceh, Taman
Nasional Gunung Leuser, Marpunge
Malaysia, Sarawak, Gunung Mulu National
Park
This study
Sumaterana dabulescens
Sumaterana dabulescens
Sumaterana montana
MG909655; MG909581; MW322894;
MW292301; MG909617; MG909695
MG909649; MG909576; MW322895;
MW292302; MG909624; MG909700
MZB.AMPH.29378 MG909666; MG909595; MW322896;
MW292303; MG909631; MG909708
MZB.AMPH.29396 MG909665; MG909594; MW322897;
MW292304; MG909630; MG909707
MZB.AMPH.29377 MG909671; MG909593; MW322898;
MW292305; MG909629; MG909698
Sumaterana montana
ZMH A14164
Staurois guttatus
NMBE1056532
MG909670; MG909592; MW322899;
MW292306; MG909628; MG909697
MG909682; MG909607; MW322900;
MW292307; MG909642; MG909718
Abbreviation: NA, Not Available.
This study
This study
This study
This study
This study
This study
This study
Arifin et al. (2018); this
study
U. ARIFIN ET AL.
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© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
Species
TAXONOMY AND SYSTEMATICS OF HUIA FROGS
APPENDIX 2
Java (N = 23)
Indonesia, Java, West Java, Sukabumi, Situ Gunung
[ZRC.1.6682–91 (N = 10)]; Situ Gunung waterfall
[ZRC.1.3851–61 (N = 11)]; Taman Nasional Gunung
Halimun-Salak, Sungai Cikaniki [ZMH A12665
(N = 1)]; Taman Nasional Gunung Halimun-Salak,
tributary of Sungai Cimadus [ZMH A12666 (N = 1)].
Sumatra (N = 74)
I n d o n e s i a , S u m a t r a , A c e h , K a b u p a t e n G ay o
Lues, Sungai Moelawak, jeeder stream to Alas
river, along road Ketambe-Blangkajeren [total
N = 39; ZRC.1.13198 (N = 5); ZRC.1.13199 (N = 2);
ZRC.1.13200 (N = 4); ZRC.1.13201 (N = 3); ZRC.1.13202
(N = 2); ZRC.1.13203 (N = 3); ZRC.1.13204 (N = 3);
Z R C. 1 . 1 3 2 0 5 ( N = 4 ) ; Z R C. 1 . 1 3 2 0 6 ( N = 5 ) ;
ZRC.1.13207 (N = 4); ZRC.1.13208 (N = 4)]; Mane
[total N = 5; ZMH A12657 (N = 2); ZMH A12658
(N = 3)]; Kabupaten Karo, Mata air (Long Pagam),
hill stream along road Lau Kawar-Berastagi,
jeeder stream to sungai Lau Kawar [ZRC.1.13209
(N = 1)]; Kabupaten Dairi, Alah Basin, Lae Renum,
Kampung Tigalingga [ZRC.1.5627–28 (N = 2)];
Sumatera Barat, Batang Si Joontour [ZRC.1.5404
(N = 1)]; Kota Padang, Pantai Purus area, from hillside stream [total N = 11; ZRC.1.10845–46 (N = 2);
ZRC.1.10842–44 (N = 3); ZRC.1.10847–52 (N = 6)];
Taman Nasional Kerinci-Seblat, Muara Labuh
[ZMH A12660* (N = 1)]; Batang Karangan [total
N = 5; ZMH A12660* (N = 1), ZMH A12661 (N = 1);
ZMH A12662 (N = 3)]; Jambi, Kabupaten Kerinci,
Danau Lingkat [ZRC.1.4033–40 (N = 8)]; Bengkulu,
Taman Nasional Kerinci-Seblat, Gunung Baru
[ZMH A12663 (N = 1)].
*Sequenced for 16S+tRNAval loci.
© 2021 The Linnean Society of London, Zoological Journal of the Linnean Society, 2021, 193, 673–699
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TADPOLE SPECIMENS EXAMINED IN THIS STUDY
Borneo (N = 25)
Malaysia, Sabah, Poring, Sungai Kapungit II ([ZMH
A11927 (N = 4)]; Malaysia, Borneo, Sabah, Kinabalu
Park, Poring, Sungai Langanan [ZRC.1.11536 (N = 1)];
Malaysia, Sabah, Ranau, Sungai Bundu Tuhan [ZMH
A11930 (N = 13)]; Indonesia, Borneo, Kalimantan
Timur, Kayan Basin, Bahau, En’nggeng B’io, drains
into Bahau river [ZRC.1.4347–48 (N = 2)]; Malaysia,
Borneo, north Borneo, Inman river between Kalawat
and Bonggol [total N = 5; ZRC.1.488 (N = 1); ZRC.1.490
(N = 1); ZRC.1.492–94 (N = 3)].
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Eric Smith