DIVERSITY AND BIOGEOGRAPHY OF THE FROG GENUS

HYLARANA (RANIDAE) ACROSS SUB-SAHARAN AFRICA

A thesis submitted to the faculty of

San Francisco State University
In partial fulfillment of
The Requirements for
The Degree
Master of Science Biology:
Ecology, Evolution, and Conservation Biology

by

Gregory Forbes Mills Jongsma

San Francisco, California
August 2014

Copyright by
Gregory Forbes Mills Jongsma
2014

CERTIFICATION OF APPROVAL

I certify that I have read Diversity and Biogeography of the Frog Genus Hylarana
(Ranidae) across Sub-Saharan Africa by Gregory Forbes Mills Jongsma, and that in
my opinion this work meets the criteria for approving a thesis submitted in partial
fulfillment of the requirements for the degree Master of Science in Biology:
Ecology, Evolution, and Conservation Biology at San Francisco State University.

____________________________
David C. Blackburn, PhD

____________________________
Vance T. Vredenburg, PhD

____________________________
Eric J. Routman, PhD

DIVERSITY AND BIOGEOGRAPHY OF THE FROG GENUS HYLARANA

(RANIDAE) ACROSS SUB-SAHARAN AFRICA

Gregory Forbes Mills Jongsma

San Francisco, California
2014

The frog genus Hylarana (family Ranidae) is ideal for exploring the relationship between
diversification and geography across sub-Saharan Africa because of its wide distribution
that spans many potential biogeographic barriers. Using multiple loci from the
mitochondrial and nuclear genomes, we characterize the principal lineages and their
geographic extents as well as estimate phylogenetic relationships of Hylarana across
Africa. Our data supports that widespread Hylarana species likely represent several
cryptic undescribed species. Recognition of additional species of African Hylarana will
contribute to refining regions of endemism in Central Africa and identify barriers to gene
flow across the continent.

I certify that the Abstract is a correct representation of the content of this thesis.

____________________________
David C. Blackburn, PhD

_______________
Date

ACKNOWLEDGEMENTS

This project was only possible because of the collaborative spirit of the African
herpetological community. We received more than 300 samples from more than a dozen
institutions on three continents. A big thanks to C. Spencer at the MVZ, J. Vindum at
CAS, J. Rosado and B. Zimkus at the MCZ, W. Conradie at PEM, J. Feinstein at the
AMNH, E. Greenbaum at UTEP and M.-O. Rdel and M. Barej at the ZMB for
supplying tissue samples. I would like to thank S. Ghose and A. Sellas for their support
and patience with me in the genomics lab. A big thanks to D. Blackburn, D. Portik, R.
Tarvin, M. Talla-Kouete, D. Fotibu and B. Freiermuth for help and entertainment in the
field. I am grateful to E. Routman, V. Vredenburg, D. Portik, and E. Stanley for helpful
insight in the analyses and writing of this thesis. I deeply indebted to D. Blackburn for all
the opportunities he has afforded me. It has been so much fun. Thank you!

TABLE OF CONTENTS

Contents
LIST OF TABLES ............................................................................................................ xii
LIST OF FIGURES ......................................................................................................... xiii
LIST OF APPENDICES .................................................................................................... ix
1. Introduction ..................................................................................................................... 1
2. Materials and Methods .................................................................................................... 4
2.1 Taxon sampling ......................................................................................................... 4
2.2 Extraction, amplification, and DNA sequencing ...................................................... 5
2.3 Sequence alignment and phylogenetic analysis ........................................................ 7
2.4 Putative species delimitation ..................................................................................... 8
2.5 Biogeographic analysis ............................................................................................. 9
2.6 Estimating a times of divergences ........................................................................... 10
3. Results ........................................................................................................................... 10
3.1 Sequence alignment................................................................................................. 10
3.2 Phylogenetic analyses ............................................................................................. 11
3.3 Historical biogeography .......................................................................................... 12
4. Discussion ..................................................................................................................... 18
4.1 Diversity and phylogeny of African Hylarana ....................................................... 18
4.2 Biogeography and timing of divergences................................................................ 20
4.3 Timing of Arrival to Africa ..................................................................................... 22
References ......................................................................................................................... 23
Appendix 1: Sample summaries ....................................................................................... 28
Appendix 2: Putative species pairwise distances ............................................................ 36

vi

LIST OF TABLES

1. Loci summary..6
2. Best-fit models.9

vii

LIST OF FIGURES

1. Sampling map.5
2. mtDNA phylogeny..13
3. Combined dataset phylogeny..14
4. Time calibrated phylogeny..15
5. PTP and bPTP species delimitation phylogeny...16
6. Dispersal-vicariance analysis.......17

viii

LIST OF APPENDICES

1. Voucher specimen summary..28

2. Pairwise distances of putative species...44

ix

1. Introduction

Due to political instability, lack of sound infrastructure and its large size, subSaharan Africa has seen few molecular phylogenetic or phylogeographic studies at the
continental scale. Over the past decade, however, increased sampling and strong global
collaborations have resulted in a significant increase in large-scale African phylogenetic
and biogeographic studies across an array of taxa, including amphibians (Blackburn 2008;
Zimkus et al. 2010), reptiles (Leach et al. 2009; Stanley et al. 2011; Shirley et al. 2014),
mammals (Lorenzen 2012), birds (Voelker et al. 2010; Fuchs et al. 2011) and invertebrates
(Dijstake 2007). These large-scale studies continue to shape our understanding of what
processes drive and maintain species diversity across sub-Saharan Africa and invariably
these studies reveal previously unknown cryptic diversity. Because extensive taxon
sampling is important to accurate phylogenetic estimation (Zwickl and Hillis 2002; Heath
et al. 2008), accounting for cryptic species is important for achieving a fuller understanding
of evolutionary processes and also key for designing effective, long-lasting conservation
efforts in Africa (Smith and Wayne 1996).
Two central hypothesis have been proposed since the 19th century to explain
heightened faunal diversity in the tropics: the Riverine Barrier Hypothesis (Wallace 1876)
and the Pleistocene Forest Refuge Hypothesis (Haffer 1969). Both models rely on
allopatric speciation to explain tropical diversity. Biogeographic barriers like rivers have
long been recognized as a major driver of speciation (Wallace 1876; Avise 2000). These
barriers are physical features of the landscape that impede gene flow between populations

2
(Avise 2000; Milner et al. 2012). Some barriers are absolute but most are permeable to
varying degrees. For example, amphibians were long thought to be largely intolerant of salt
water (Darwin 1859) but several recent molecular studies have demonstrated that overseas
dispersal is an important factor for explaining present day amphibian distributions around
the globe (Vences 2003; Blackburn et al. 2010; Pyron 2014). The permeability of a barrier
depends on dispersal capabilities, physiological restraints, and natural history traits of the
species involved. Recent studies using molecular approaches to investigate animal
diversity in Africa have found that cryptic species are often be separated from sister taxa
across biogeographic barriers such as rivers (Voelker et al. 2013).
Often when biogeographic barriers are not obvious boundaries, or when cryptic
species are found in sympatry, the Pleistocene Forest Refuge theory is invoked (Haffer
1969). This theory proposes that repeated forest contraction events since the Pleistocene
have been a major isolating force driving allopatric speciation across tropical regions,
including Central Africa, and that subsequent forest expansion events have brought closely
related species into sympatry (Haffer 1969; Anthony 2007). It is most likely that both
refugia and geographic barriers have together influenced species diversity and population
structure in the Afrotropics. Detailed study of widespread, tropical species with low
dispersal capabilities is especially attractive for understanding patterns of speciation in
Africa.
Amphibians are ideal candidates for studying biogeography because they are
species-rich (Pyron and Wiens 2011), occupy a high diversity of habitats (Duellman and
Trueb), and are poor dispersers (Wells 2007). Within Africa, some species of the frog genus
Hylarana have nearly pan-African distributions spanning numerous types of biogeographic

3
barriers. For this reason, we sought to investigate the intrageneric relationships of all
members of African Hylarana across their respective distributions.
The genus Hylarana Tschudi, 1838 is a member of the family Ranidae and part of
the larger radiation called the Natatanura (Chen et al. 2005; Bossuyt et al. 2006; Frost et
al. 2006; Blackburn and Wake 2011; Pyron and Wiens 2011). Ranidae is one of few
globally distributed frog families and comprises more than 350 species (Frost 2014) that
exhibit a wide range morphological and ecological diversity. This group has undergone
significant taxonomic revisions over the past three decades (Dubois 1987, 1992, 2005;
Inger 1996; Roelants et al. 2004; Chen et al. 2005; Bossuyt et al. 2006; Frost et al. 2006;
Pyron and Wiens 2011). The genus Hylarana is unique among the members of Ranidae
because its distribution spans two continents, covering much of sub-Saharan Africa and
southern Asia. This distribution is supported by numerous molecular studies including
Asian and African Hylarana species that recover the genus as monophyletic with respect
to other ranid genera (Kosuch et al. 2001; Bossuyt et al. 2006; Frost et al. 2006; Pyron and
Wiens 2011). This extensive distribution makes Hylarana ideal for investigating
biogeographic patterns both within and between Africa and Asia.
For this study, we concentrated on the poorly studied species of Hylarana restricted
to sub-Saharan Africa, which includes 11 currently recognized species. Our goals for this
project are (1) to identify undescribed cryptic diversity of African Hylarana and identify
potential drivers of this diveristy, (2) to identify barriers to gene flow across the continent,
and (3) to estimate divergence times among African species as well as between Africa and
Asia.

2. Materials and Methods

2.1 Taxon sampling

Tissues loans were acquired from numerous museums and institutions from around
the world (Appendix 1) as well as supplemented with field research in Cameroon (2013)
and Uganda (2014). In the field, specimens were collected, euthanized, and then liver tissue
samples were fixed in RNAlater storage buffer before the whole specimens were preserved
in 10% buffered formalin; newly collected specimens and tissues were deposited at the
California Academy of Sciences. Outgroup taxa include representatives of each of subgenera of Asian Hylarana (Dubois 1992; Chen et al. 2005) and the type species for this
genus, H. erythraea (Schlegal 1837). Taxonomy follows Frost (2014) unless otherwise
stated.
The ingroup sampling for this study included 313 individuals representing 9 of the
11 known species of Hylarana in Africa. The two species not included here are H.
parkeriana (Mertens, 1938), which is known only from the type series collected in Angola,
and H. lemairei (De Witte, 1921) which is found in southern Democratic Republic of
Congo, Zambia and eastern Angola. Despite the larger range of H. lemairei in comparison
to H. parkeriana, we were unable to obtain tissues samples of this species. Our sampling
includes specimens from 24 African countries, spanning nearly the entirety of the
distribution of Hylarana in Africa (Fig. 1). We chose four Asian Hylarana species, and
one species each of Amolops (Ranidae) and Nanorana (Dicroglossidae) as outgroup taxa
based on previous studies (Chen et al. 2005; Frost et al. 2006; Pyron and Wiens 2011).

Fig 1: Sampling map of African Hylarana sequenced for this study. Symbols represent
recognized and/or candidate species used in this study.

2.2 Extraction, amplification, and DNA sequencing

All molecular work (extractions, PCR, and sequencing) was conducted at the Center for
Comparative Genomics (CCG) at the California Academy of Sciences (CAS). Genomic
DNA was extracted from tissues (liver, muscle or toe clips) using Qiagen Spin-Column
Kits and following their protocol for animals. A 815 bp fragment of mitochondrial DNA
that encodes part of the mitochondrial ribosomal 16S gene was amplified by polymerase
chain reaction (PCR; 94C 30 s, 52C 30 s, 72C 1 min) for 35 cycles using the primers
pairs 16Sc and 16Sd (Moriarty and Cannatella 2004) (Table 1). Four nuclear genes were

6
amplified using a nested PCR protocol as outlined in Shen et al. (2013) (for additional
details, see Table 1). Amplified PCR products were purified using ExoSAP-IT
(Affymetrixs) and then sequenced in both directions by direct double-strand cyclesequencing using BigDye v3.1 chemistry. The amplifying primers for 16S were used in the
cycle sequencing reactions for the mitochondrial gene. Universal forward and reverse
sequencing

primers

(5-AGGGTTTTCCCAGTCACGAC-3

and

5-

AGATAACAATTTCACACAGG-3) were used for all four nuclear genes following Shen
et al. (2013). Cycle sequencing products were precipitated with ethanol, 125 mM EDTA,
pH 8.0 and sequenced on a Applied Biosystems 3130 Genetic Analyzer at the CCG.
Sequences will be deposited in GenBank at the time of publication (Appendix 1).

Table 1
Summary of the mitochondrial DNA (16S) and 4 nuclear protein coding loci, including their
primer sequences, and the number of base pairs analyzed per loci in this study.
Locus

Primer pair and sequence

Reference

# of bp

16S

16Sc: GTRGGCCTAAAAGCAGCCAC
16Sd: CTCCGGTCTGAACTCAGATCACGTAG

Moriarty and
Cannatella 2004

815

DISP2

DISPf1: CCKAGYTGGTCYYTNGGNAAYTA
DISPr1: AARAARCAGCANAGNGAYTGRAARAA
DISPf2: AGGGTTTTCCCAGYCGRTAYATHTGG
DISPf2: AGATAACAATTTCCNGTRAAGTCNAC

Chen et al. 2013

876

FICD

FICDf1: CCKCTNGTNGARGARATHGAYCA
FICDr1: TYTCNGTRCAYTTNGCDATRAA
FICDf2: AGGGTTTTCCCAGAYATHTAYCAYAC
FICDr2: AGATAACAATTTCCNCTRAACCARAA

Chen et al. 2013

515

KIAA2013

KIAAf1: CTCAANTAYGCNGAYCAYTGYTT
KIAAr1: CCNGGNCCRCARTAYTCRTTRTA
KIAAf2:AGGGTTTTCCCAGNGAGAAYYTGTGG
KIAAr2: AGATAACAATTTCCNCTRAACCARAA

Chen et al. 2013

528

REV3L

REV3Lf1: CGYTGYTGGTCNCCNACNGAYCC REV3Lr1:

TTNCTRCARAANGGYTCYTGRTA
REV3Lf2: AGGTTTTCCCAGTYGARAGNTGYTA
REV3Lr2: AGATAACAATTTCACATNAGNGGYTT

Chen et al. 2013

594

2.3 Sequence alignment and phylogenetic analysis

Contigs for sequences for each gene region were assembled in Geneious v7.2
(Biomatters; available from http://www.geneious.com/). Next the consensus sequences
were aligned for each gene using MUSCLE v3.5 (Edgar, 2004) and checked manually for
gross errors. The resulting block of aligned sequences was trimmed on both ends to ensure
all individuals had complete data. The 16S mitochondrial locus and all four nuclear loci
were concatenated in Geneious 7.2.
We used the Akaike Information Criterion (AIC) as implemented in jModelTest
v2.0 (Posada and Crandall, 1998) to select the best-fit model of nucleotide substitution for
each gene partition (Table 2). Each respective model was used in all model-based
phylogenetic inferences discussed below.
Phylogenies were reconstructed using maximum likelihood and Bayesian
methods for mtDNA, and of partitioned mtDNA and nDNA for a sub-set of samples. ML
analyses were conducted using RAxML v7.2.8 (Randomized Accelerated Maximum
Likelihood; Stamatakis, 2006) and Garli v2.0 (Genetic Algorithm for Rapid Likelihood
Inference; Bazinet et al. 2014). To obtain a preliminary estimate of lineage diversity, we
first constructed a mtDNA phylogeny based on 815 bp of 16S for 313 ingroup samples
and eight outgroup samples. The nucleotide substitution model, GTR+I+ was
implemented in RAxML. The AutoMRE option in RAxML was first used to estimate the
necessary bootstrap replicates to achieve convergence, and we then doubled this number
for each ML estimate. Using our 321 taxon mtDNA ML phylogeny as a road map, we
chose two or more individuals from each distinct and well supported (>70 bootstrap
support) clade or lineage for sequencing of the four nuclear loci. This resulted in a subset

8
of 70 specimens for which we sampled mitochondrial and nuclear loci. Our final
concatenated alignment had little missing data (96% complete) and no samples were
missing more than one locus. Partitioned ML analyses were conducted in Garli v2.0 on
the concatenated alignment, and using the best-fit model for each partition.
Partitioned Bayesian phylogenetic estimates were obtained using MrBayes v3.2.2
(Huelsenbeck and Ronquist 2001) for our complete 5-locus concatenated alignment for 70
taxa, using the same partitioning strategy and corresponding substitution models as our ML
analyses. Sampling every 1000 generations, we ran four independent Markov Chain Monte
Carlo (MCMC) for 20 million generations. The first 25% of the sampled trees were
discarded as burn-in. The convergence of runs was assessed by the effective sample size
(ESS) values of parameters, likelihood scores through time plots using TRACER v1.6
(Rambaut and Drummond 2009).

2.4 Putative species delimitation

To further evaluate species boundaries, we took several approaches using our
partitioned alignment. We conducted analyses using both PTP (Poisson Tree Process) and
bPTP (Bayesian Poisson Tree Process) as implemented in the web-server for these analyses
(http://species.h-its.org; Zhang et al. 2013). As a prior for these analyses, we used the ML
tree from Garli analyses for the partitioned alignment and treated clades with posterior
probabilities and bootstrap support above 0.95 and 70%, respectively, as putative species.
The PTP approach assumes that each substitution has a fixed probability as being the root
of a speciation event (Zhang et al. 2013) and outperforms the more widely used GMYC
method (Fujisawa and Barraclough 2013) for species delimitation (Zhang et al. 2013). Four

9
independent MCMC analyses were run for 100,000 generations, sampling every 100
generations. We discarded the first 25% of samples as burn-in, and convergence was
confirmed using Tracer v1.6.

Table 2
Summary of best and next best-fit models of nucleotide substitution
for each gene as implemented in jModelTest v2.0
Locus

Best-fit
model

Next best- Delta

fit model
AIC

16S mtDNA

GTR+I+

TIM2+I+ 0.9163

DISP

GTR+

GTR+I+

1.1777

FICD

HKY+I+

K80+I+

2.9481

KIAA

K80+I+

SYM+I+

0.9881

REVL13

HKY+

HKY+I+

2.1461

2.5 Biogeographic analysis

We performed biogeographic analyses using S-DIVA (Statistical DispersalVicariance Analyses) as implemented in RASP (Yu et al. 2010) on the ML phylogeny
constructed for 70 specimens using the complete of alignment of mitochondrial and
nuclear data. Coarse and large-scale geographic units follow Blackburn (2008) and
include West Africa (G), Central Africa (C), East Africa (E), southern Africa (B), and
Asia (A). Here, for West Africa this includes all samples west of Nigeria, for Central
Africa this includes the lower Guinean forest and the Congo Basin, for East Africa this
includes all samples east of the Albertine Rift, for Southern Africa this includes samples
south of -6 latitude, and all outgroups were coded as Asian. The analyses were run
without constraining the number of unit areas allowed in an ancestral state reconstruction.

10

2.6 Estimating a times of divergences

We estimated divergence times using BEAST v1.7.5 (Drummond et al. 2012).
Based on recent empirical studies, amphibian mitochondrial sequence divergence rates are
between 0.8% and 1.9% total divergence per million years (Sanguila et al. 2011). We used
the mean of this range (1.4%) to estimate divergences among African Hylarana. Using our
partitioned alignment for 70 specimens with mitochondrial and nuclear data, we used the
same models of evolution as in the above ML and Bayesian analyes. We applied the relaxed
clock approach, as implemented in BEAST, which allows branch lengths to vary according
to an uncorrelated lognormal distribution, and a Yule process with a random starting tree.
All other prior settings were left as default in BEAST. Four independent MCMC analyses
were run for 200 million generations, sampling every 200 generations. We discarded the
first 25% of samples as burn-in. Convergence was confirmed using Tracer v1.6.

3. Results

3.1 Sequence alignment

The complete 16S mtDNA aligned matrices spans 886 bp for 321 samples. The
complete nDNA alignment spans 2516 bp across four unlinked gene regions for 70
samples. The final concatenated mtDNA and nDNA alignment spanned 3288 bp for 70
samples.

11

3.2 Phylogenetic analyses

Our mtDNA phylogeny with full sampling demonstrated high levels of lineage
diversity throughout African Hylarana (Fig. 2). As is expected with mtDNA, Support
values for deeper nodes in the tree are less well-supported than those towards the tips.
Adding four nuclear loci to sub-samples of the major lineages observed in the larger
mtDNA-only phylogeny added support to deeper node (Fig. 3). Likelihood and Bayesian
analyses of the five-gene concatenated alignment produced virtually identical tree
topologies with high bootstrap support and posterior probabilities throughout the trees (Fig.
3). African species of Hylarana are supported as monophyletic with respect to the Asian
Hylarana included here, including the subgenera proposed by Dubois (1987, 1992) (Figs.
2,3).
Hylarana albolabris is revealed as polyphyletic (Figs. 2, 3). Nominotypical
samples of H. albolabris from the Lower Guinean Forest in Gabon are more closely related
to other Hylarana species in Central Africa (H. amnicola and H. asperrima) than to
populations recognized as H. albolabris in the Upper Guinea forests of West Africa. This
distinct West African lineage is sister to the sympatric species H. fonensis. A topotypic
specimen of Hylarana longipes is nested within the Central African H. albolabris clade
(Fig. 2) and was not supported as distinct by out PTP or bPTP species delimitation analyses.
However, the species delimitation analyses did support the existence of several
undescribed cryptic species throughout the phylogeny. These include two cryptic H.
galamensis species in East Africa, a cryptic species of H. amnicola and H. lepus in southern
Republic of Congo, and a cryptic H. albolabris which is sister to H. fonensis in West Africa
(Fig. 3).

12

3.3 Historical biogeography

Dispersal-vicariance analysis did not support Hylarana as having an Asian origin, contrary
to other studies (Kosuch et al. 2001). The analysis also predicts some extinction events
have occurred between H. occidentalis, the earliest diverging African lineage and H.
leptoglossa, the most closely related Asian Hylarana in out phylogeny. Our divergence
time analyses estimate that the divergence between African and Asian Hylarana occurred
approx. 14 mya (95% credible interval: 1711 mya). While our analyses did not produce
clear patterns for the geographic origins of Hylarana diversity within Africa, the late
Miocene is revealed as an important time of lineage diversification among the forestdwelling Hylarana, including the most recent common ancestor of H. lepus and H.
asperrima. The large subclade comprising H. darlingi, H. albolabris, H. asperrima and H.
amnicola originated in Central and/or southern Africa in the late Miocene (approx. 6
million years ago). Notably there is a marked increase in lineage diversification, especially
in Central African taxa, during the Pleistocene (2.5 million 10,000 years ago).

13

Figure 2: ML phylogeny estimated using RAxML based on partial 16S mitochondrial

sequence for all samples. Black dots represent nodes with both bootstrap support above
70% and posterior probabilities above 0.90.

14

Figure 3: Species-level phylogeny of African Hylarana, based on concatenated mDNA and

nDNA datasets. Black dots represent nodes with bootstrap values >70% and posterior
probability >0.95. Green dots represent nodes with high support from only ML analysis
(bootstrap support >70%) and red dots are nodes with only high support from Bayesiana
analysis (posterior probability >0.95).

15

Figure 4: Time calibrated phylogeny based on a molecular clock for mitochondrial gene
(0.014% / million years; Sanguila et al. 2011). The node bars (blue) represent 95%

16
credibity intervals. The size of the the green dots represent posterior probabilities of >
0.95.

Fig 5: PTP species delimitation tree. Red clades represent putative species. bPTP
analyses resulted in an identical topology and putative species.

17

Fig 6: Dispersal-Vicariance phylogeny as produced by S-DIVA. West Africa (G), Central

Africa (C), East Africa (E), southern Africa (B), and Asia (A).

18

4. Discussion

4.1 Diversity and phylogeny of African Hylarana

We provide the first phylogenetic study of the African species of the genus
Hylarana, which provides insights into amphibian diversification across the continent. This
phylogeny includes nine of the 11 recognized species in Africa, including samples that
span much of the geographic distribution for even the most widespread species. All of our
analyses support African Hylarana as monophyletic, suggesting there was a single
dispersal event into Africa. However, more Asian sampling could alter this finding.
Hylarana occidentalis from the Upper Guinean forests of West Africa is the earliest
diverging extant lineage of African Hylarana. Both H. occidentalis and the H. galamensis
species-group are highly divergent from other African Hylarana (pairwise distance
between 13% and 19% based on the concatenated alignment). Interestingly, the highest
pairwise divergence between the remaining diversity of African Hylarana (excluding H.
occipitalis and galamensis) with any Asian Hylarana is approximately 17% (Appendix 2).
Some African Hylarana are as or more divergent from one another as they are from Asian
lineages.
Our phylogenetic and species delimitation analyses strongly suggest that diversity
has been underestimated for Hylarana across sub-Saharan Africa. There are five
undescribed cryptic Hylarana species, two that are separated from their sister taxa by a
biogeographic barrier and three that are sympatric. Hylarana albolabris is polyphyletic:

19
one species occurs in West Africa and is sister to H. fonensis, while the other nominotypical
H. albolabris is found in Central Africa and is sister to H. asperrima (Fig. 3). Our species
delimitation analyses suggests that a number of lineages referred to H. albolabris in Central
Africa should be considered a single species. Based on morphological characters
distinguishing it from H. albolabris, Perret (1960) described H. longipes as a new species
of Hylarana from high elevation sites (>1000 m asl) on the Bamilk and Adamawa
Plateaus in Cameroon. Our phylogenetic analyses include a topotypic sample of H.
longipes that agrees with Perrets (1960) description, including larger size and
proportionately longer feet relative to H. albolabris. However, this sample is nested within
the diversity of lineages comprising the Central African species H. albolabris and we
therefore recommend that H. longipes be considered a junior synonym of H. albolabris.
Our analyses suggest that the widespread species H. galamensis is composed of
three distinct species. The West African lineage is distributed from Senegal east across the
Dahomey Gap to eastern Nigeria; the Central/East African lineage occurs across eastern
DRC and Uganda; and the third lineage is restricted to the Kenyan and Tanzanian coast.
Despite the strong support for each of these lineages as distinct species, we note that there
is a major sampling gap (~2200 km) between our West African and eastern DRC samples
where H. galamensis is known to occur. Therefore, it is difficult to say whether the support
for this lineage derives principally from isolation-by-distance or because of this sampling
gap. The East African coastal lineage, however, that is supported as a distinct species here,
was originally described as Limnodytes bravanus Peters 1882 and later recognized as a
subspecies of H. galamensis by Loveridge (1936). Based on the morphological and

20
genealogical distinctiveness of this lineage, we recommend that Hylarana galamensis
bravana be elevated to H. bravana.
Interestingly, a number of cryptic lineages exist in sympatry with their sister taxa,
including H. cf. albolabris with H. fonensis in West Africa and cryptic and nominotypical
lineages of both H. amnicola and H. lepus in Central Africa (Figs. 2,3). The Pleistocene
Forest Refuge Hypothesis (Haffer 1969) has been proposed as an explanation of elevated
species diversification in tropics during the past 2.5 million years that is driven by cyclical
expansion and retraction of forests. As forests contract, populations of plants and animals
are isolated leading to allopatric speciation. Subsequently, as forests again expand
divergent populations and species that share a recent common ancestor are brought into
secondary contact. In Central and West Africa, this pattern has been detected in birds (Mayr
and O'Hara 1987; Voelker et al. 2010), gorillas (Anthony et al. 2007), and rodents (Nicolas
et al. 2011) and may also explain the pattern observed here of sympatric close relatives of
Hylarana.

4.2 Biogeography and timing of divergences

Our results demonstrate that different biogeographic barriers (rivers, mountains and
arid divides) have played an important role in shaping Hylarana diversity in Africa. The
effectiveness of a barrier varies between species as is highlighted within Hylarana when
considering the arid Dahomey Gap as a barrier between and West and Central Africa. The
forest dwelling species Hylarana cf. albolabris and H. fonensis are genetically distinct (8
10% total pairwise distance) from their Central African counterparts (H. albolabris, H.

21
asperrima and H. amnicola), whereas H. galamensis, a savannah specialist, shows little
differentiation across the Dahomey Gap. The Dahomey Gap is an important barrier for
many forest-dwelling amphibian species and appears to be a major driver of the Upper
Guinean forests biological distinctiveness from the Guineo-Congolian forest (Schitz
1967; Poynton 1999; Penner et al. 2011).
Central Africas Guineo-Congolian forests host the greatest Hylarana species
diversity within Africa and most of this diversity is concentrated within the albolabris
subclade. Several species exhibit substantial population structure, which is largely driven
by barriers such as the Congo and Ogoou Rivers and the salt-water strait between Bioko
Island and mainland Cameroon (H. albolabris, H. amnicola and H. lepus). All of the intraand interspecific diversity observed in this clade arose during the Pleistocene (2.5 million
10,000 years ago). This pattern is similar to that found in other species in the region
including primates (Anthony et al. 2007; Telfer et al. 2003), birds (Voelker et al. 2013),
and rodents (Kennis et al. 2011), and serves to highlight the important role that rivers play
as a driver of allopatric speciation in Central Africa. Hylarana albolabris on Bioko Island
is genetically most similar to mainland Cameroon, which supports similar findings for
other species (ex. Arthroleptis: Blackburn 2010) that Bioko is an extension of the mainland
Cameroon fauna.
Central Africa has long been considered an evolutionary museum suggesting that
the regions fauna is dominated by old lineages (Mayr and OHara 1986; Kingdon 1990).
However, our results support other recent studies (e.g., Anthony et al. 2007; Nicolas et al.
2011; Telfer et al. 2003; Voelker et al. 2010, 2013) that demonstrate the Guineo-Congolian
forests as an active evolutionary region where lineages have diversified at both the

22
population and species-level since the Plio-Pleistocene. The combination of stable forest
refugia maintaining old lineages (Mayr and O'Hara 1986) with both oscillating forest cover
and riverine barriers driving evolution of new lineages leads the Guinea-Congolian to be
an important center of diversification and endemicity within Africa.

4.3 Timing of Arrival to Africa

In contrast to previous studies (e.g., Kosuch et al. 2001), the dispersal-vicariance
analysis does not support an Asian origin for Hylarana. However, this is likely a sampling
artifact driven by our emphasis on African taxa. Sampling of additional Asian species,
including the vast majority of species of Hylarana and related genera (Pyron and Wiens,
2011; Frost, 2014) may significantly alter this biogeographic scenario for the origin of
Hylarana. Our time-calibrated phylogeny estimates that the divergence between African
and Asian Hylarana occurred during the Miocene between 17 and 10 mya. The only
previous time calibrated phylogeny that included both African and Asian Hylarana
estimated the divergence to be substantially older at ~30 mya during the Oligocene
(Bossuyt et al. 2006). During the Oligocene, Africa was a continental island with no land
connections to Asia or Europe. Geological evidence supports that the first permanent landbridge, likely since the Cretaceous, connecting Eurasia with north-eastern Africa formed
during the middle Miocene around 15 million years ago (Bosworth et al. 2005). This makes
a Miocene Era dispersal a more plausible senerio then one during the Oligocene, which
would have required over sea dispersal events. Given the lack of relevant fossil, adding
sampling of Asian Hylarana to a time-calibrated molecular phylogeny will be the best
approach for adding confidence to our proposed biogeographic scenario.

23
Given our proposed entry point of Hylarana into Africa under this scenario, it is
surprising that the earliest diverging lineage within African Hylarana (H. occidentalis) is
restricted to the Upper Guinean forests of West Africa, nearly 5000 km west of the Arabian
Peninsula. This suggests that extinction events of populations in the intervening region
followed subsequent to the arrival of Hylarana in Africa and is supported by our S-DIVA
analyses. Interestingly, West Africas Upper Guinean forest is well appreciated as an
important region of endemism including for old lineages of amphibian taxa including
Pseudhymenochirus (Evans et al. 2004), Nimbaphrynoides (Sandberger et al., 2010),
Morerella (Rdel et al. 2009) and Odontobatrachidae (Barej et al. 2014) as well as for an
ancient mammal lineage, the tenrecoid Micropotamogale (Poux et al. 2008). The overall
pattern of having only old and young Hylarana lineages in West Africa is similar to other
sub-Saharan frogs including pipids (Evans et al. 2004) and arthroleptid frogs (Blackburn
2008).

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Appendix 1: Sample summaries

Institution
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS

Voucher ID
199321
199323
199326
199327
202203
202204
202205
207621
207650
207651
207652
207653
229991
229992
214840
202399
249985
249986
249987
250824

Genus
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

Species
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
galamensis
sp.
sp.
sp.
sp.
galamensis

Country
Cameroon
Cameroon
Cameroon
Cameroon
Uganda
Uganda
Uganda
Equatorial Guinea
Equatorial Guinea
Equatorial Guinea
Equatorial Guinea
Equatorial Guinea
Sierra Leone
Sierra Leone
Kenya
Uganda
Cameroon
Cameroon
Cameroon
Burundi

29
Drex
Drex
Drex
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
LSU
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ

PJM405
PJM406
PJM408
20072
20074
20111
20114
20115
20310
20324
20071
20073
20075
20112
20113
20260
20311
117597
117601
117606
117618
117621
117643
117646
117652
117733
A-138200
A-138198
A-138055
A-139632
A-139637
A-139641
A-139643
A-139712
A-139717
A-139718
A-139746
A-139747
A-139748
A-139749
A-139750
A-139751
A-139752

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
albolabris
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
sp.
albolabris
amnicola
sp.
amnicola
lepus
albolabris
albolabris
lepus
lemairii
lemairii
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.

Equatorial Guinea
Equatorial Guinea
Equatorial Guinea
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
DRC
DRC
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon

30
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
MCZ
ROM
ROM
PEM
PEM
PEM
PEM
PEM
PEM
PEM
PEM
SAIAB
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ

A-139753
A-139773
A-139774
A-139815
A-140074
A-140179
A-140180
A-140255
A-140258
A-140259
A-140260
A-140281
A-140282
A-140283
A-140293
A-140294
A-140301
A-140296
A-147856
A-147869
A-147879
19861
19863
10005
10006
10016
10017
10603
ANG-0064
ANG-0069
ANG-0070
185858
145467
234091
234147
234148
234166
234167
234593
234830
234831
234832
234833

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

sp.
sp.
sp.
sp.
sp.
galamensis
galamensis
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
sp.
albolabris
albolabris
albolabris
albolabris
albolabris
lepus
lepus
lepus
darlingi
galamensis
TADPOLE
TADPOLE
TADPOLE
darlingi
albolabris
galamensis
albolabris
sp.
galamensis
galamensis
albolabris
albolabris
albolabris
albolabris
albolabris

Gabon
Gabon
Gabon
Gabon
DRC
DRC
DRC
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Gabon
Liberia
Liberia
Angola
Angola
Angola
Angola
Angola
Angola
Angola
Angola
Malawi
Ghana
Tanzania
Kenya
Tanzania
Kenya
Kenya
Kenya
Cameroon
Cameroon
Cameroon
Cameroon

31
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ

234834
234850
234851
245197
245198
245199
245200
245201
245202
245203
245204
245205
245206
245207
245208
245209
245210
245211
245212
245213
245214
245215
245216
245217
245218
245219
245220
245221
245222
245223
245224
245225
245427
249561
249562
249563
249564
249565
249566
249567
249568
249569
249570

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

sp.
sp.
sp.
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris

Cameroon
Cameroon
Cameroon
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana

32
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
MVZ
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB

249571
249572
249573
249574
249575
249576
249577
249578
249579
249580
249581
249582
249583
253119
253120
253122
253123
253124
253125
253126
253127
253128
MBUR 02910
MBUR 03011
MBUR 03026
MBUR 03151
MBUR 03202
MBUR 03317
MBUR 03318
MBUR 03358
MBUR 03384
MBUR 03414
P.LI 12 004
P.LI 12 005
P.LI 12 061
P.LI 12 076
P.LI 12 077
MH 0169
MH 0225
MH 0416
MH 0417
MH 0439
MH 0440

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
albolabris
albolabris
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
galamensis
albolabris
albolabris
albolabris
albolabris
lepus
amnicola
amnicola
amnicola
amnicola
lepus
albolabris
albolabris
occidentalis
occidentalis
albolabris
albolabris
amnicola
amnicola
amnicola
albolabris
albolabris

Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Nigeria
Nigeria
Nigeria
Nigeria
Nigeria
Nigeria
Nigeria
Nigeria
Nigeria
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Republic of Congo
Liberia
Liberia
Liberia
Liberia
Liberia
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon

33
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB

MD 010
MD 039
MD 0136
MD 0139
MD 0195
MD 0226
MH 0145
CB2010 046
(40?)
CB2010 047
CB2010 120
CB2010 169
CB2010 186
CB2010 188
L09 200
L09 224
L09 600
L09 601
L09 609
L09 626
BE 026
BE 067
BE 083
BE 112
ANK 015
ANK 008
KAK 039
LE 011
LE 022
OWS 028
Gu 115
Gu 138
Gu 149
Gu 150
Gu 188
DS 060
DS 097
DS 104
G 078
GO 012
GO 057
GOL 060
GRE 010

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
sp.
albolabris
albolabris
sp.
fonensis

Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Guinea

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
galamensis
fonensis
occidentalis
fonensis
albolabris
occidentalis
occidentalis
fonensis
fonensis
albolabris
galamensis
albolabris
galamensis
galamensis
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
occidentalis
occidentalis
occidentalis
occidentalis
albolabris
albolabris
albolabris
albolabris
galamensis
occidentalis
occidentalis
albolabris
albolabris

Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Guinea
Benin
Benin
Benin
Benin
Ghana
Ghana
Ghana
Ghana
Ghana
Ghana
Guinea
Guinea
Guinea
Guinea
Guinea
CAR
CAR
CAR
Ghana
Liberia
Liberia
Sierra Leone
Liberia

34
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
ZMB
EBG
EBG

JP 036
JP 039
JP 040
JP 056
JP 148
LOM 114
MTN 242
SI 032
SI 033
SI 050
TI 001
TI 002
TI 004
OWS 028
TI 020
TI 029
TI 060
HD 023
NIM 079
NIM 097
NIM 099
NIM 101
TIW 008
TIW 023
(28?)
TIW 027
TIW 052
TOG 013
TOG 044
TOG 056
SA 008
SA 009
BTR 203
74296
RIO 005
RIO 006
DS 060
DS 061
DS 104
DS 105
LG 776
1575
1576

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

amnicola
amnicola
amnicola
galamensis
fonensis
albolabris
sp.
albolabris
fonensis
galamensis
sp.
sp.
sp.
sp.
albolabris
sp.
albolabris
albolabris
sp.
albolabris
sp.
sp.
albolabris

DRC
DRC
DRC
DRC
DRC
Sierra Leone
Guinea
Guinea
Guinea
Guinea
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone
Cote D'Ivoire
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone
Sierra Leone

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
galamensis
galamensis
occidentalis
galamensis
sp.
sp.
albolabris
albolabris
albolabris
albolabris
lepus
galamensis
galamensis

Sierra Leone
Sierra Leone
Sierra Leone
Togo
Togo
Togo
Senegal
Senegal
Senegal

CAR
CAR
CAR
CAR
Cameroon
DRC
DRC

35
EBG
ELI
ELI
ELI
EBG
EBG
EBG
EBG
EBG
EBG
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
ZFMK
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DCB

1984
251
265
1362
1810
2459
2500
2501
2478
2660
67983
67984
67289
67290
90468
51850
92976
68736
68739
87900
87829
87830
87901
87833
87823
87824
87825
266
273
297
298
304
340
405
406
407
408
409
516
517
520
521
528

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

galamensis
galamensis
galamensis
galamensis
albolabris
albolabris
albolabris
albolabris
albolabris
albolabris
asperrima
asperrima
asperrima
asperrima
galamenensis
galamenensis
albolabris
albolabris
albolabris
albolabris
amnicola
amnicola
albolabris
amnicola
amnicola
amnicola
amnicola
albolabris
albolabris
albolabris
albolabris
albolabris
lepus
albolabris
albolabris
albolabris
albolabris
albolabris
lepus
lepus
lepus
lepus
albolabris

DRC
DRC
DRC
DRC
DRC
DRC
DRC
DRC
DRC
DRC
Cameroon
Cameroon
Cameroon
Cameroon
Mali
Gambia
Ivory Coast
Ghana
Ghana
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon

36
DCB
DCB
DCB
DCB
DCB
DCB
DCB
DMP

529
530
566
567
568
569
586
1160

Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana
Hylarana

albolabris
albolabris
albolabris
sp.
sp.
lepus
amnicola
longipes

Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon
Cameroon

Appendix 2: Putative species pairwise distances