Author's personal copy
Molecular Phylogenetics and Evolution 71 (2014) 261–273
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Light shines through the spindrift – Phylogeny of African torrent
frogs (Amphibia, Anura, Petropedetidae)
Michael F. Barej a,⇑, Mark-Oliver Rödel a, Simon P. Loader b, Michele Menegon c, Nono L. Gonwouo d,
Johannes Penner a, Václav Gvoždík b,e,f, Rainer Günther a, Rayna C. Bell g, Peter Nagel b, Andreas Schmitz h
a
Museum für Naturkunde, Leibniz Institute for Research on Evolution and Biodiversity, Invalidenstrasse 43, D-10115 Berlin, Germany
University of Basel, Department of Environmental Sciences (Biogeography), Klingelbergstr. 27, Basel 4056, Switzerland
Museo Tridentino di Scienze Naturali, Via Calepina 14, 38100 Trento, Italy
d
Université of Yaoundé I, Faculty of Science, Laboratory of Zoology, P.O. Box 812, Yaoundé, Cameroon
e
Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Kvetna 8, 603 65 Brno, Czech Republic
f
National Museum, Department of Zoology, Cirkusová 1740, CZ-19300 Prague, Czech Republic
g
Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA
h
Natural History Museum of Geneva, Department of Herpetology and Ichthyology, C.P. 6434, 1211 Geneva 6, Switzerland
b
c
a r t i c l e
i n f o
Article history:
Received 17 August 2013
Revised 26 October 2013
Accepted 3 November 2013
Available online 15 November 2013
Keywords:
Africa
Amphibians
Taxonomy
Arthroleptides
Petropedetes
Odontobatrachus gen. nov
a b s t r a c t
Torrent frogs of the genus Petropedetes Reichenow, 1874 as currently understood have a disjunct distribution with species endemic to West, Central or East Africa. We herein present a phylogenetic analysis
including all but one of the currently described 12 species of the genus. Maximum Likelihood and Bayesian analyses of combined nuclear (rag1, SIA, BDNF) and mitochondrial (16S, 12S, cytb) genes of more than
3500 base pairs, revealed clades corresponding to the three sub-Saharan regions. Molecular results are
confirmed by morphological differences. Surprisingly, the three geographic clades do not form a monophyletic group with respect to closely related families Pyxicephalidae and Conrauidae and therefore
require taxonomic changes. We resurrect Arthroleptides Nieden, 1911 for the East African taxa. The Central African taxa remain in the genus Petropedetes. The West African members are placed in the new
genus Odontobatrachus gen. nov. The taxonomic position of the new genus remains incertae sedis as it
was not assigned to any of the four families included in our analyses. Potential new species have been
detected within all three major clades, pointing to a still not fully clarified diversity within African torrent
frogs.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
African torrent frogs of genus Petropedetes Reichenow, 1874 inhabit the splash-water zone of clear running streams in predominantly undisturbed forests of West, Central and East Africa.
Although torrent frogs are very similar in their general ecology, differences in tadpole morphology and their habitat requirements
have been recognised; tadpoles being either semi-terrestrial on
rocks in the spray zone or fully aquatic in zones of the strongest
currents (Barej et al., 2010a; Lamotte and Zuber-Vogeli, 1954;
Lamotte et al., 1959; Lawson, 1993). Further information on the
species’ biology is scarce; however, clutch guarding has been
⇑ Corresponding author.
E-mail addresses: [email protected] (M.F. Barej), [email protected]
(M.-O. Rödel), [email protected] (S.P. Loader), [email protected] (M.
Menegon), [email protected] (N.L. Gonwouo), [email protected]
(J. Penner), [email protected] (V. Gvoždík), [email protected]
(R. Günther), [email protected] (R.C. Bell), [email protected] (P. Nagel),
[email protected] (A. Schmitz).
1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2013.11.001
documented in some taxa and several studies implicate ecological
separation as a mechanism for diversification among syntopic Central African taxa (Amiet, 1983, 1991; Barej et al., 2010a; Sanderson,
1936).
Prominent morphological characters of these medium to large
sized frogs (up to 7 cm SVL) are T-shaped terminal phalanges with
heart-shaped digital discs and toe tips, presence of femoral glands,
tympanic papillae, carpal spikes and tusks, but not all characters
are present in all species (e.g. Barej et al., 2010a; Boulenger,
1905; Klemens, 1998; Perret, 1966). Even some of the characters
provided in Reichenow’s (1874) diagnosis of the genus, e.g. extent
of webbing, distinct tympana, presence of vomerine teeth, are absent in many of the species. Overall, the genus is morphologically
very heterogeneous, and apart from a forked omosternum, no synapomorphies support this grouping (Frost et al., 2006).
While femoral glands evolved independently in several non-related families (e.g. Mantellidae, Phrynobatrachidae, Ranixalidae,
Nyctibatrachidae, and Pyxicephalidae) and probably play a role in
reproduction (Vences et al., 2007), tympanic papillae are a unique
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M.F. Barej et al. / Molecular Phylogenetics and Evolution 71 (2014) 261–273
feature in anuran acoustic communication (Narins et al., 2001). A
carpal spike in males most probably serves a territorial and/or
aggressive behaviour in breeding males (Barej et al., 2010a;
Sanderson, 1936).
The genus type species is P. cameronensis Reichenow, 1874 from
Central Africa. Later, Boulenger (1888) and du Bocage (1895) described Cornufer johnstoni and Tympanoceros newtonii respectively,
both of which are from Central Africa. Boulenger (1900) transferred
Cornufer johnstoni and Tympanoceros newtonii to the genus Petropedetes. One species, P. obscurus Ahl, 1924 was formerly described
from East Africa but Perret (1984) placed it in synonymy with
the Central African P. cameronensis, because morphological differences were insufficient and locality data most probably got confused. Barej et al. (2010a) confirmed this point of view but
placed P. newtonii in synonymy with P. johnstoni.
Currently the highest species diversity in the genus is found in
western Central Africa around the Gulf of Guinea (= Biafra Bay).
Eight species occur in the area from Nigeria to the Republic of Congo (Petropedetes cameronensis Reichenow, 1874; P. euskircheni
Barej et al., 2010a; P. johnstoni (Boulenger, 1888 "1887"), P. juliawurstnerae Barej et al., 2010a; P. palmipes Boulenger, 1905; P.
parkeri Amiet, 1983; P. perreti Amiet, 1973; P. vulpiae Barej et al.,
2010a and are either widely distributed in lowland forests or inhabit restricted mountainous areas (Amiet, 1986; Barej et al., 2010a;
Perret, 1966, 1984). Various Central African (CA) species occur in
sympatry in the submontane zone (Amiet, 1975, 1983; Barej
et al., 2010a; Herrmann et al., 2005; Parker, 1936) and Barej
et al. (2010a) already indicated the presence of additional undescribed species in Central Africa. Three species, P. dutoiti (Loveridge, 1935), P. martiensseni (Nieden, 1911) and P. yakusini
(Channing, Moyer & Howell, 2002) are known from forests in isolated East African (EA) mountain peaks (e.g. Channing et al.,
2002; Nieden, 1911; Loader et al., 2013) and only a single species,
P. natator Boulenger, 1905, is known from the Upper Guinea forests
of West Africa (WA; Boulenger, 1905; Rödel et al., 2004).
Areas occupied by Central and East African torrent-frogs belong
to important centres of diversity and endemism (Burgess et al.,
2007b and references therein). In western Central Africa this refers
to parts of the Cameroon volcanic line, which shows e.g. the highest diversity in the frog genera Astylosternus and Leptodactylodon
(Amiet, 1977, 1980), chameleons (Barej et al., 2010b) and mammals (Missoup et al., 2012) in that region. In eastern Africa this refers to the East Arc Mountains, known for their exceptional
diversity amongst others in the frog genera Nectophrynoides and
Arthroleptis (Blackburn, 2008; Menegon et al., 2004), but also in
other vertebrate groups (Burgess et al., 2007a).
For P. natator Jean-Louis Amiet (footnote in Perret, 1984) suggested this species be placed in its own genus based on adult and
larval morphology. Whereas, P. natator was originally placed in
Petropedetes, the three East African species were formerly placed
in the genus Arthroleptides Nieden, 1911. One of the crucial differences is the absence of vomerine teeth in Arthroleptides (Nieden,
1911), a character often disclaimed on genus level differentiation
(e.g. Inger, 1954). However, Deckert (1938) recognised differences in the pectoral girdle, which he regarded as sufficient to
keep both genera. Robert Drewes (in Largen, 1991) had recommended expanding the definition of the genus Petropedetes in order to include Arthroleptides taxa. Finally, based on combined
morphological and molecular data Scott (2005) allocated Arthroleptides to Petropedetes, given the latter genus was paraphyletic
otherwise.
On a higher systematic level, the genus Petropedetes has been
placed in different taxonomic ranks (subfamilies and families).
Originally, Reichenow (1874) classified the genus as part of the
family Hylae Laurenti, 1768 (a synonym of Hylidae Rafinesque,
1815). Later Noble (1931) generated the subfamily Petropedetinae
as part of the Ranidae Rafinesque, 1814 (Bossuyt et al., 2006; Scott,
2005; van der Meijden et al., 2005).
Later Frost et al. (2006) grouped these frogs with Conraua Nieden, 1908 and Indirana Laurent, 1986 in the family Petropedetidae Noble, 1931, still using the name Arthroleptides for their
East African taxon. However, Scott’s (2005) results have subsequently been adopted in Frost’s online database (Frost, 2013). In
a very recent phylogeny Pyron and Wiens (2011) likewise listed
East African taxa in the genus Petropedetes, but transferred the
genus Conraua to its own family Conrauidae Dubois, 1992, while
Indirana was placed in the Ranixalidae Dubois, 1987 (Blackburn
and Wake, 2011). The most recent changes have subsequently
left the genus Petropedetes the sole member of the family
Petropedetidae.
All recent large-scale molecular phylogenies including Petropedetinae / Petropedetidae only sampled Central and East African representatives; the West African P. natator was absent.
Solely Scott (2005) analysed P. natator; however, molecular data
were unavailable to her and only morphological data were used.
According to Scott’s (2005) combined analysis of morphology
and genetics, African torrent frogs form a clade, being supported
by a number of osteological apomorphies as well as general external morphology and secondary sexual characters. However, within this clade Scott (2005) recognised characters separating P.
natator from both Central African taxa (P. cameronensis, P. newtoni = P. vulpiae, and P. parkeri in her analysis) and a single East
African representative (P. martiensseni). Furthermore, Scott
(2005) identified synapomorphies for the Central African and East
African groupings.
In order to resolve the systematic relationships between African
torrent frogs throughout their entire range, we herein provide the
first comprehensive molecular phylogeny, including 11 of 12 currently described species.
2. Material and methods
2.1. Taxon sampling
Our sampling includes all but one of the currently known species. Solely, the East African species Petropedetes dutoiti (Loveridge,
1935) from Kenya is missing. This species might be extinct since no
individuals have been seen in the last five decades (Andreone et al.,
2008; Groombridge, 1994; IUCN, 2012). However, rediscovery attempts are ongoing (B. Akoth in litt. 04.IV.2013). Where possible,
we included multiple specimens from across the known distribution range of each taxon. Recently, Loader et al. (2013) pointed to
a higher diversity within East African mountains, indicating the
presence of new taxa. The latter taxa are included in our analysis
as well.
Outgroup taxa include Conraua, which according to Frost et al.
(2006) was regarded as a member of the family Petropedetidae
(but see introduction). Moreover, we added members of the Africanura sensu Frost et al. (2006) with representatives of the Phrynobatrachidae Laurent, 1940 and its sister taxon Pyxicephaloidea
Bonaparte, 1850, respectively.
A full list of samples and respective GenBank numbers are given
in Table 1. Museum abbreviations are as follow: The Natural History Museum, London, United Kingdom (BMNH); Institut Royal
des Sciences Naturelles de Belgique, Bruxelles, Belgium (IRSNBKBIN); Muséum d’histoire naturelle, Genève, Switzerland (MHNG);
Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN);
North Carolina Museum of Natural Sciences, Raleigh, USA (NCSM);
National Museum, Museum of Natural History, Prague, Czech
Republic (NMP6V); Museo Tridentino di Scienze Naturali, Trento,
Italy (MTSN); Zoologisches Forschungsmuseum Alexander Koenig,
Author's personal copy
Table 1
List of taxa included in this study including voucher ID, locality data (country codes: CM = Cameroon, GA = Gabon, GN = Guinea, GQ = Equatorial Guinea, LR = Liberia, NG = Nigeria, SL = Sierra Leone, TZ = Tanzania, ZA = South Africa) and
GenBank accession numbers (Genbank# GUxxxxxx after Barej et al., 2010a; JXxxxxxx after Loader et al., 2013; new GenBank# KF693275–KF693703).
Taxon
MHNG 2715.58
ZFMK 89567
MHNG 2715.89
MHNG 2715.45
MHNG 2740.55
CAM1
ZMB 78427
MHNG 2731.52
ZMB 78428
ZMB 78429
ZMB 78433
ZMB 78211
ZMB 78244
ZMB 78206
ZMB 78315
ZMB 78317
ZMB 78319
ZFMK 77306
ZFMK 77307
BM 2002.576
BM 2002.575
BM 2000.826
BM 2005.014
BM 2005.1382
BM 2005.1383
BM 2005.013
BM 2005.567
MTSN 8358
MTSN 8255
ZFMK 81616
ZFMK 81615
ZFMK 81154
N30Rhiho
ZMB 73729
ZMB 73728
ZFMK78018
NMP6V 74646/2
NMP6V 74645/1
NMP6V 74645/2
NMP6V 73403/1
NMP6V 73403/2
NMP6V 73403/3
ZMB 78204
ZFMK 75539
ZFMK 81103
ZFMK 81168
ZFMK 78365
ZMB 78385
ZMB 78386
ZFMK 75582
ZFMK 75586
MHNG 2713.10
Locality, country
Big Massaka, CM
Big Massaka, CM
Rumpi Hills: Mofako Balue, CM
Mt Kupe: Nyasoso, CM
Kuruman, ZA
CM
Mt. Manengouba: Manengouba Village, CM
Rumpi Hills: Mofako Balue, CM
East of Ghi Mtn., LR
Fouta Djallon: Konkouré, GN
Nimba Mts.: Bangué, GN
Diéké/Yonsonso, GN
Grand Gedeh, LR
Nimini FR, SL
Fouta Djallon: Labé/Sala, GN
Fouta Djallon: Dalabé, GN
Fouta Djallon: Sala, GN
East Usambara Mts.: Amani, TZ
East Usambara Mts.: Amani, TZ
East Usambara Mts.: Nilo TZ
East Usambara Mts.: Nilo TZ
East Usambara Mts.: Amani, TZ
Uluguru Mts., Kasanga, TZ
Mahenge Mts., TZ
Mahenge Mts., TZ
Uluguru Mts., Kasanga, TZ
Udzungwa, TZ
Kanga, TZ
Nguru, TZ
Mt. Nlonako, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Rhoko Forest, NG
Rhoko Forest, NG
Mbe Mts., NG
Mt. Nlonako, CM
Bakossi Mts.: Messaka, CM
Bakingili, CM
Bakingili, CM
Bakingili, CM
Bakingili, CM
Bakingili, CM
Ebo Forest, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Mt. Nlonako: Nguéngué, CM
Mt. Nlonako: Nguéngué, CM
Mt. Kupe, CM
Mt. Kupe, CM
Rumpi Hills: Mt. Rata, CM
GenBank accession numbers
12S
16S
cytb
BDNF
SIA
rag1
KF693275
KF693276
KF693277
KF693278
KF693279
KF693280
KF693281
KF693282
KF693283
KF693284
KF693285
KF693286
KF693287
KF693288
KF693289
KF693290
KF693291
KF693292
KF693293
JX546951
JX546943
JX546950
JX546945
JX546946
JX546944
JX546942
JX546941
JX546948
JX546947
KF693294
KF693295
KF693296
KF693297
KF693298
KF693299
—
—
—
—
—
—
—
KF693300
KF693301
KF693302
—
—
KF693303
KF693304
KF693305
KF693306
KF693307
KF693379
KF693380
KF693381
KF693382
KF693383
KF693384
KF693385
KF693386
KF693387
KF693388
KF693389
KF693390
KF693391
KF693392
KF693393
KF693394
KF693395
KF693396
KF693397
JX546956
JX546958
JX546955
JX546961
JX546963
JX546966
JX546964
JX546965
JX546960
JX546959
KF693398
GU256015
GU256016
KF693399
GU256017
GU256018
KF693400
KF693401
KF693402
KF693403
KF693404
KF693405
KF693406
KF693407
GU256020
GU256022
GU256023
GU256024
KF693408
KF693409
GU256019
GU256021
KF693410
KF693659
KF693660
KF693661
KF693662
KF693663
KF693664
KF693665
KF693666
KF693667
KF693668
KF693669
KF693670
KF693671
KF693672
KF693673
KF693674
KF693675
KF693676
KF693677
KF693678
JX546978
JX546970
JX546981
JX546972
JX546974
KF693679
KF693680
KF693681
KF693682
KF693683
KF693684
KF693685
KF693477
KF693478
KF693479
KF693480
KF693481
KF693482
KF693483
KF693484
KF693485
KF693486
KF693487
KF693488
KF693489
KF693490
KF693491
KF693492
KF693493
KF693494
KF693495
KF693496
KF693497
KF693498
KF693499
KF693500
KF693501
—
KF693502
KF693503
KF693504
KF693505
KF693506
KF693507
KF693539
KF693540
KF693541
KF693542
KF693543
KF693544
KF693545
KF693546
KF693547
KF693548
KF693549
KF693550
KF693551
KF693552
KF693553
KF693554
KF693555
KF693556
KF693557
—
—
KF693558
KF693559
KF693560
KF693561
KF693562
KF693563
KF693564
KF693565
KF693566
KF693567
KF693568
KF693599
KF693600
KF693601
KF693602
KF693603
KF693604
KF693605
KF693606
KF693607
KF693608
KF693609
KF693610
KF693611
KF693612
KF693613
KF693614
KF693615
KF693616
KF693617
—
—
KF693618
KF693619
KF693620
KF693621
—
KF693622
KF693623
KF693624
KF693625
KF693626
KF693627
KF693686
KF693508
KF693569
KF693628
KF693687
KF693688
—
KF693509
KF693510
KF693511
KF693570
KF693571
KF693572
KF693629
KF693630
KF693631
263
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Hyperolius ocellatus
Phrynobatrachus calcaratus
Phrynobatrachus auritus
Phrynobatrachus africanus
Cacosternum boettgeri
Conraua goliath
Conraua robusta
Conraua robusta
Conraua alleni
Conraua alleni
Conraua alleni
Odontobatrachus natator
Odontobatrachus natator
Odontobatrachus natator
Odontobatrchus sp. nov.
Odontobatrchus sp. nov.
Odontobatrchus sp. nov.
Arthroleptides martiensseni
Arthroleptides martiensseni
Arthroleptides martiensseni
Arthroleptides martiensseni
Arthroleptides martiensseni
Arthroleptides yakusini
Arthroleptides yakusini
Arthroleptides yakusini
Arthroleptides yakusini
Arthroleptides yakusini
Arthroleptides c
Arthroleptides sp. nov.
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes cameronensis
Petropedetes sp. aff. euskircheni
Petropedetes sp. aff. euskircheni
Petropedetes sp. aff. euskircheni
Petropedetes sp. aff. euskircheni
Petropedetes sp. aff. euskircheni
Petropedetes sp. aff. euskircheni
Petropedetes euskircheni
Petropedetes euskircheni
Petropedetes euskircheni
Voucher ID
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Table 1 (continued)
Taxon
euskircheni
euskircheni
euskircheni
euskircheni
euskircheni
euskircheni
johnstoni
johnstoni
johnstoni
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
juliawurstnerae
palmipes
palmipes
palmipes
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
parkeri
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
perreti
vulpiae
vulpiae
vulpiae
vulpiae
vulpiae
ZFMK 88865
MHNG 2713.8
MHNG 2713.5
ZFMK 88864
MHNG 2713.7
ZMB 78387
ZFMK 87710
ZFMK 87709
ZMB 78201
ZFMK 75590
ZFMK 68134
ZFMK 68131
ZFMK 67360
ZFMK 67987
MHNG 2713.17
MHNG 2713.19
ZMB 73694
NMP6V 74632/1
NMP6V 74632/2
ZFMK 89440
MHNG 2713.12
NCSM 76813
NCSM 76814
NCSM 76815
ZFMK 87702
ZMB 73739
NMP6V 74680/1
NMP6V 74680/2
VP10-60
NMP6V 74690
NMP6V 74678/1
NMP6V 74686/4
NMP6V 74686/7
NMP6V 74686/22
NMP6V 74691
ZFMK 75524
ZFMK 75519
ZFMK 69227
0847N
ZMB 73737
0998N
ZMB 73734
0846N
0159LG
0175LG
0165LG
ZMB 73731
0158LG
0157LG
ZFMK 75588
ZFMK 81167
ZFMK 81623
ZMB 73726
ZFMK 81554
Locality, country
Rumpi Hills: Mt. Rata, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Fotabong, CM
Campo, CM
Campo, CM
Miangasio Lendi, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Rumpi Hills: Mofako Balue, CM
Rumpi Hills: Mofako Balue, CM
Mt. Kupe, CM
Bakossi Mts.: Messaka, CM
Bakossi Mts.: Messaka, CM
Rumpi Hills: Mofako Balue, CM
Mt. Kupe: Nyasoso, CM
Monts de Cristal NP, GA
Monts de Cristal NP, GA
Monts de Cristal NP, GA
Amebishu, CM
Cross River NP, NG
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Mt. Fungom, CM
Munkep, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Manengouba Mts.: Esipa Village, CM
Manengouba Mts.: Esipa Village, CM
Manengouba Mts.: Ebonemin, CM
Manengouba Mts.: Ebonemin, CM
Manengouba Mts.: Esipa Village, CM
Manengouba Mts., CM
Manengouba Mts., CM
Manengouba Mts., CM
Manengouba Mts., CM
Manengouba Mts., CM
Manengouba Mts., CM
Mt. Kupe, CM
Mt. Nlonako, CM
Mt. Nlonako, CM
Mbe Mts., NG
Mt. Nlonako, CM
GenBank accession numbers
12S
16S
cytb
BDNF
SIA
rag1
KF693308
KF693309
KF693310
KF693311
KF693312
KF693313
KF693314
KF693315
KF693316
KF693317
KF693318
KF693319
KF693320
KF693321
KF693322
KF693323
KF693324
KF693325
KF693326
KF693327
KF693328
KF693329
KF693330
KF693331
KF693332
KF693333
—
—
—
—
—
—
—
—
—
KF693334
KF693335
KF693336
KF693337
KF693338
KF693339
KF693340
KF693341
KF693342
KF693343
KF693344
KF693345
KF693346
KF693347
KF693348
KF693349
KF693350
KF693351
KF693352
KF693411
GU256025
KF693412
GU256026
GU256027
KF693413
GU256028
GU256029
KF693414
GU256030
KF693415
KF693416
GU256031
KF693417
KF693418
KF693419
GU256032
KF693420
KF693421
KF693422
KF693423
KF693424
KF693425
KF693426
GU256033
GU256034
KF693427
KF693428
KF693429
KF693430
KF693431
KF693432
KF693433
KF693434
KF693435
GU256035
KF693436
KF693437
KF693438
GU256036
KF693439
GU256037
KF693440
KF693441
KF693442
KF693443
GU256038
KF693444
KF693445
GU256039
GU256040
GU256041
KF693446
KF693447
KF693689
—
KF693512
KF693513
KF693573
KF693574
KF693632
KF693633
KF693514
KF693575
KF693634
KF693690
KF693691
KF693692
KF693515
KF693516
KF693576
KF693577
KF693635
KF693636
KF693693
KF693517
KF693578
KF693637
KF693694
KF693518
KF693579
KF693638
KF693695
—
—
—
—
KF693519
KF693520
KF693521
KF693522
KF693523
KF693580
KF693581
KF693582
KF693583
KF693584
KF693639
KF693640
KF693641
KF693642
KF693643
—
KF693524
KF693585
KF693644
—
KF693525
KF693586
KF693645
—
KF693526
KF693587
KF693646
KF693696
—
KF693527
KF693528
KF693588
KF693589
KF693647
KF693648
KF693697
—
KF693529
KF693530
KF693590
—
KF693649
KF693650
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vulpiae
vulpiae
vulpiae
vulpiae
vulpiae
vulpiae
vulpiae
vulpiae
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sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp.nov. 1
sp. nov. 2
sp. nov. 3
ZFMK 78364
ZFMK 88863
ZMB 73692
ZFMK 88862
ZFMK 88859
MHNG 2713.4
MHNG 2713.9
NMP6V 74641
NMP6V 74646/1
NMP6V 73439/1
NMP6V 73439/2
ZMB 78425
MNCN 50403
MNCN 50405
MNCN 50406
MNCN 50407
MNCN 50411
MNCN 50417
MNCN 50465
MNCN 50466
ZMB 78421
NMP6V 74577/1
NMP6V 74577/2
NMP6V 74577/3
NMP6V 74592/1
NMP6V 74592/2
NMP6V 73389/1
NMP6V 73389/2
NMP6V 73391/1
NMP6V 73391/2
NCSM 76811
NCSM 76812
Mt. Nlonako, CM
Big Massaka, CM
Big Massaka, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Mt. Kupe, CM
Bakossi Mts.: Edib/Messaka, CM
Bakossi Mts.: Messaka, CM
Bakingili, CM
Bakingili, CM
Ebo Forest, CM
Bioko Island: Rio Osa, GQ
Bioko Island: Rio Osa, GQ
Bioko Island: Rio Osa, GQ
Bioko Island: Rio Osa, GQ
Bioko Island: Rio Ole, GQ
Caldera de Luba; Bioko, GQ
Bioko Island: Rio Sibitá, Bococo, GQ
Bioko, GQ
Foot of Mt. Etinde: Etome, CM
Mt. Tchabal Gangdaba, CM
Mt. Tchabal Gangdaba, CM
Mt. Tchabal Gangdaba, CM
Banyo, CM
Banyo, CM
Big Babanki, Bamenda Highlands, CM
Big Babanki, Bamenda Highlands, CM
Mejung, Bamenda Highlands, CM
Mejung, Bamenda Highlands, CM
Monts de Cristal NP, GA
Monts de Cristal NP, GA
KF693353
KF693354
KF693355
KF693356
KF693357
KF693358
KF693359
KF693360
KF693361
KF693362
KF693363
KF693364
—
KF693365
—
—
KF693366
—
KF693367
—
KF693368
KF693369
KF693370
—
KF693371
KF693372
KF693373
KF693374
KF693375
KF693376
KF693377
KF693378
KF693448
GU256042
GU256043
KF693449
GU256044
KF693450
KF693451
KF693452
KF693453
KF693454
KF693455
KF693456
KF693457
KF693458
KF693459
KF693460
KF693461
KF693462
KF693463
KF693464
KF693465
KF693466
KF693467
KF693468
KF693469
KF693470
KF693471
KF693472
KF693473
KF693474
KF693475
KF693476
—
KF693531
KF693591
KF693651
KF693698
KF693532
KF693592
KF693652
KF693699
KF693533
KF693593
KF693653
KF693700
KF693534
KF693594
KF693654
KF693701
KF693535
KF693595
KF693655
KF693702
KF693536
KF693596
KF693656
—
KF693703
KF693537
KF693538
KF693597
KF693598
KF693657
KF693658
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Bonn, Germany (ZFMK); Museum für Naturkunde, Berlin, Germany
(ZMB). Remaining material refers to the national collection of Cameroon (CAM-, LG- & N-numbers).
2.2. DNA extraction and sequencing
DNA was extracted from ethanol-preserved liver, thigh or tongue muscle. These tissues were taken either from fresh specimens
collected in the field or preserved museum specimens. DNA was
extracted using Qi-Amp tissue extraction kits (Qiagen) and the
peqGold Tissue DNA Mini Kit (PEQLAB Biotechnologie GmbH) or
High Pure PCR Template Preparation kits (Roche) following the
manufacturers protocols. Three nuclear [Seven-in-Absentia
(SIA), Recombination Activation gene 1 (rag1) and Brain-derived
neurotrophic factor gene (BDNF)] and three mitochondrial genes
[(12S rRNA, 16S rRNA, cytochrome b gene (cytb)] were amplified.
Primers are given in Appendix Table A1. Amplification of 25 ll or
50 ll PCR reactions follow protocols given in Appendix Table A2.
PCR products were purified using Qiaquick purification kits
(Qiagen).
For quality assurance we sequenced both directions of the
amplified PCR product (using an external vendor Macrogen).
All
sequences
have
been
deposited
in
GenBank
(KF693275–KF693378 for 12S; KF693379–KF693476 for 16S;
KF693477–KF693538 for BDNF; KF693539–KF693598 for SIA;
KF693599–KF693658 for rag1; KF693659–KF693703 for cytb).
2.3. Molecular analyses
Sequences were aligned using ClustalX (Thompson et al., 1997;
default parameters) and manually checked using the original chromatograph data in the program BioEdit (Hall, 1999). Ambiguities
were identified by eye and excluded from the analyses. Protein
coding partitions (cytb, SIA, rag1, BDNF) were translated with the
program TranslatorX (Abascal et al., 2010) to amino acids to determine codon positions and to check for absence of stop codons.
In order to test congruence in the topology of the present dataset we tested: (1) reduced dataset of mitochondrial sequences only
(12S & 16S as in Scott, 2005) and (2) combined nuclear and mitochondrial genes (12S, 16S, cytb, BDNF, SIA, rag1). Two well established techniques for phylogenetic estimation were applied:
Bayesian Inference (BI; MrBayes, version 3.21 64; Huelsenbeck
and Ronquist, 2001; Ronquist et al., 2012) and Maximum Likelihood (ML; RAxML version 7.0.4; Stamatakis, 2006 using the rapid
hill climbing algorithm following Stamatakis et al., 2007 and the
GTR + G model).
The best-fit model of sequence evolution for each gene partition
or respectively codon position was selected using jModeltest 2.1.2
(Darriba et al., 2012, Appendix Table A3) using the Bayesian information criterion (BIC). BIC was implemented in the partitioned BI
and additional single-gene analyses. Single-gene analyses of BI
and ML (following aforementioned procedure) were performed to
analyse clade stability of species lineages and major groupings in
different genes.
Bootstrap analyses with 1000 pseudoreplicates in the ML analysis were employed to evaluate the relative branch support in the
phylogenetic analysis. Bayesian analyses were run under partitioned schemes for 5 million generations using four chains sampling every 100 generations, with a burn-in of 1000 trees. Clades
with posterior probabilities (PP) P 95% were considered strongly
supported. Stationarity has been checked with Tracer V1.5 (Rambaut and Drummond, 2007). Uncorrected p-distances between major clades and mean values of maxima within each region were
calculated with PAUP* 4.0b10 for the partial 16S rRNA gene.
2.4. Morphological analysis
Taxa from West (P. natator males ZMB 78203, ZMB 78243, female: ZMB 78216), Central (female holotype of P. cameronensis
ZMB 8222, male paratype of P. euskircheni ZMB 73693, male paratype of P. juliawurstnerae ZMB 73694) and East Africa (female holotype of P. martiensseni, ZMB 21793, male P. yakusini ZMB 48472)
were subjected to a micro-tomographic analysis at the Museum
für Naturkunde Berlin using a Phoenix nanotom X-ray|s tube at
70–90 kV and 100lA, generating 1000 projections per scan. The
different kV- and projection-settings depended on the respective
specimen size. Effective voxel size ranged between 8.3 to
31.2 lm and the exposure time was 750 ms. The cone beam reconstruction was performed using the datos|x-reconstruction software
(GE Sensing & Inspection Technologies GMBH phoenix|X-ray) and
the data were visualised and modified in VG Studio Max 2.0.
3. Results and discussion
3.1. Generated sequences
All six target loci, three mitochondrial and three nuclear, were
analysed for a total of 52 ingroup and 11 outgroup terminals. In
addition, a second dataset, containing a considerably larger number of samples with only 12S and 16S data were analysed containing a total of 103 ingroup and 11 outgroup terminals.
The alignment including nuclear and mitochondrial loci consisted
of 3513 base pairs. Sequences lengths were as follows: 354 bp of 12S,
570 bp of 16S, 588 bp of cytb, 396 bp of SIA, 675 bp of BDNF, 930 bp of
rag1. Our second dataset including only 12S and 16S data consisted of
924 aligned base pairs (354 bp of 12S, 570 bp of 16S).
3.2. Phylogenetic reconstructions
Herein, we present the first comprehensive analysis of African
torrent frogs, based on several nuclear and mitochondrial gene partitions. At present twelve Petropedetes species are recognised
throughout their range in East (EA), Central (CA) and West Africa
(WA). However, the present phylogenetic analysis was inconsistent
with this arrangement, and the main clades corresponding to geographic regions do not form a monophyletic group (Figs. 1 and 2).
The results support some previous insights but are discussed anew.
Individual gene trees are not shown but, respective clades are
marked as bars and level of support indicated in Fig. 2.
3.2.1. Analyses of molecular data
Our molecular analyses show that the monophyly of Petropedetes is not supported. Tree topologies with respective node support
values are shown in Fig. 1 for the 12S + 16S-tree and in Fig. 2 for the
nuclear and mitochondrial dataset. In both datasets the two applied phylogenetic approaches strongly agree in the overall topology, supporting the same terminal clades and taxa (including
described and undescribed lineages). Three deeply divergent
clades have been uncovered in African torrent frogs, each consistent with geography and reflecting the sub-Saharan distribution
(three separate clades EA, CA and WA). All three major clades were
supported by ML and Bayesian analyses (Figs. 1 and 2). Mean
uncorrected genetic distances in the partial 16S rRNA gene among
these major clades range between 14.12% and 20.83% (Table 2); in
comparison, mean uncorrected genetic distances within these major clades were considerably lower (4.22–8.51%). Clades CA and EA
were more similar to each other 11.78–16.59%) than to WA (WACA: 17.35–22.91%; WA-EA 19.75–21.45%).
Our analyses identified under-estimated species diversity in all
regions. Clade WA includes Petropedetes natator and a second
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267
Fig. 1. Phylogeny of African torrent frogs based on mitochondrial data (genes encoding 12S + 16S; 114 sequences, 924 bp). Numbers along branches indicate Bayesian
posterior probabilities and thorough bootstrap values as obtained using RAxML 7.0.4. Asterisks point to maximum support under both methods (ML: 100/PP: 1.00), remaining
significance values (ML: >70/PP: >0.95) are provided.
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Fig. 2. Phylogeny of African torrent frogs based on concatenated mitochondrial and nuclear genes (12S + 16S + cytb, BDNF + SIA + rag1; 63 sequences, 3513 bp). Numbers
along branches indicate Bayesian posterior probabilities and thorough bootstrap values as obtained using RAxML 7.0.4. Asterisks point to maximum support under both
methods (ML: 100/PP: 1.00), remaining significance values (ML: >70/PP: >0.95) are provided. Major geographic clades are marked (CA = Central Africa, EA = East Africa,
WA = West Africa). Bars along terminal taxa indicate results and support within morphological and single-gene analyses inferred from Bayesian and Maximum Likelihood
analyses. Colouration indicates support for clades in ML or BI and additional diagonal lines refer to support in both approaches (red = EA, green = CA, blue = WA, black = no
geographical clade supported). A sign of [+] shows a supported close relationship between EA and CA. Coding of terminal taxon bars as following: filled bar = supported (PP:
P0.95 or ML: P70), empty bar = not supported (PP: <0.95 or ML: <70; even if a clade has been recognised), dash = data missing (sequence not available or morphology data
not present). The first column (morphology) refers to lineages diagnosable by morphology alone (either scientifically described or morphological characters analysed). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 2
Values of uncorrected p-distances for the partial 16S gene. Given are percentages of range (min–max), mean value and standard deviation (mean ± stdv) between major clades.
Additionally, mean values of maximum p-distances between terminal taxa have been calculated for an intra-lineage comparison (mean ± stdv).
WA
WA
CA
EA
19.39 ± 0.90
20.83 ± 0.40
CA
EA
Mean intra lineage p-distance
17.35–22.91
19.75–21.45
11.78–16.59
4.22 ± 0.24
4.89 ± 1.04
8.51 ± 2.04
14.12 ± 1.08
undescribed taxon from West Africa. Clade CA includes P. cameronensis, P. euskircheni, P. johnstoni, P. juliawurstnerae, P. palmipes, P.
parkeri, P. perreti, P. vulpiae and four additional lineages from Central Africa; two of them being represented by a single sample only.
The most basal node within CA in all analyses separated P. palmipes
from all other species. Unexpectedly, within clade CA relationships
between terminal taxa still remained weakly supported and were
therefore partially unresolved. Support values in the 12S + 16Sdataset were low in both analyses, yielding in a polytomy in the
50% majority-rule consensus tree of the Bayesian analysis. In contrast, PP-values in the complete dataset supported a few of the inner nodes while ML support values remained mostly weak
(support values below 70% not shown). Lineages from EA include
P. martiensseni, P. yakusini and one additional taxon (Nguru population) from East Africa (as outlined in Loader et al., 2013).
While terminal taxa were in almost all cases clearly separated
by the mitochondrial genes, nuclear markers did possess concordances in haplotypes of different taxa forming different clusters
(not shown; rag1: P. euskircheni – P. sp. aff. euskircheni; BDNF: P.
euskircheni – P. sp. aff. euskircheni, P. juliawurstnerae – P. perreti,
P. sp. nov.2 – P. vulpiae; SIA: P. euskircheni – P. sp. aff. euskircheni,
P. parkeri – P. sp. nov.1). However, two of the nuclear markers
(rag1 and BDNF) supported the three major groupings and moreover, all three nuclear markers significantly supported a closer
relationship of the taxa grouped in EA and CA, than to those of
WA (bars in Fig. 2).
3.3. Biogeography of African torrent frogs
As formerly conceived the family Petropedetidae showed distinct disjunct distributions across West, Central and East African
biogeographic regions. From a historical biogeographical perspective, interpreting the relationships among these taxa would have
revealed the historical relationships among these areas. Such
groups are of particular importance, given that torrent frogs are often restricted to forest, and therefore their phylogenetic patterns
might reflect historical changes in forest habitats. With the distant
relationship of the West African clade, and its uncertain phylogenetic relationships relative to the family Petropedetidae, our results can only provide biogeographic information on the close
relationship found between East African Arthroleptides and Central
African Petropedetes. Given the considerable genetic distance
between the two genera Arthroleptides and Petropedetes, one can
assume the split of those two clades is likely an ancient event potentially linked to the early separation of East and West African
forests (Kingdon, 1990; Maley, 1996). The application of molecular
dating will be necessary to examine this in further detail. It would
be intriguing to understand the close phylogenetic relationship
that the West African clade has, and what this might suggest about
biogeography of the West African faunal region (compare Penner
et al., 2011). Furthermore, the Ethiopian monotypic genus Ericabatrachus Largen, 1991, potentially closely related to Petropedetidae
(sensu Largen, 1991), might provide further information on the
biogeographic relationships across African biogeographic regions
in Petropedetidae. It might be predicted if Ericabatrachus is a petropedetid, that it would cluster with the geographically close genus
Arthroleptides. Further research on the historical biogeography of
these frogs is necessary and likely to be insightful on deep-time
biogeography of African forests.
3.4. Systematic readjustment of the genus Petropedetes Reichenow,
1874
Our molecular and morphological analyses support the division
of Petropedetes into clades corresponding to geographically distinct
and disjunct distributions of species in eastern, central, and western Africa. The original reasoning for synonymizing EA Arthroleptides into Petropedetes was it’s grouping with CA Petropedetes, to
the exclusion of the WA P. natator (Scott, 2005). However, this taxonomic change rested on the assumptions that 1. EA Petropedetes
were relatively similar to CA Petropedetes, and 2. WA P. natator
was the nearest grouping to EA and CA Petropedetes. Our results
challenge these assumptions and suggest taxonomic changes. Lineages from CA and EA formed a well-supported clade; the type species Petropedetes cameronensis belongs to clade CA (Figs. 1 and 2).
Both of these clades are assigned to the family Petropedetidae.
However, given their substantial differences, we favour generic
recognition, as was originally formulated, resurrecting Arthroleptides for EA species. In contrast, positioning of clade WA remains
unclear in relation to the included outgroup taxa and cannot be assigned to a family and is placed as incertae sedis pending further
analyses. The aim of our study herein was to clarify the phylogenetic placement of all known torrent frogs; the higher-level analysis including representatives of all Ranoidea families was beyond
the scope of this work.
3.4.1. Clade WA
The substantial divergence of WA species from the remaining
African torrent frogs necessitates the description of a new genus.
The only named taxon P. natator becomes the genus type species.
Odontobatrachus gen. nov. Barej, Rödel, Loader, Schmitz
Type species: Petropedetes natator Boulenger, 1905 nov. comb.
Diagnosis: Osteology: large nasals, rectangular, in median contact; nasals overlapping sphenethmoid; anterior end of frontoparietals running rather rectangular to axis; vomer with a strongly
expressed posteromedial ramus (dentigerous process) that covers
the proximal end of the neopalatine and bears well developed
teeth; large and posteriorly curved teeth on premaxillaries and
anterior maxillaries as well as a tusk-like odontoid on the mandible; anterior ramus of pterygoid does not reach neopalatine;
zygomatic ramus of the squamosal longer than the otic ramus
and with regular outlines; base of omosternum convex; medial
edges of coracoids not overlapping (Fig. 3). External morphology:
tympanum indistinct; males with external vocal sacs; nuptial
excrescences in breeding males velvety; femoral glands present
in males only.
Genetics: All taxa included in this genus form a clade and can be
clearly differentiated from the remaining torrent-frog genera.
Within our coding nuclear dataset, the genus possesses 77 unique
character states in both taxa (rag1: 45, BDNF: 17, SIA: 15). A total of
93 unique character states in nuclear genes distinguish Odontoba-
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Fig. 3. CT-scans of Odontobatrachus natator (ZMB 78203) scanned with 80 kV,
100 mA, Voxelsize. Scans of (a) skull in dorsal view, scale bar: 2 mm (b) skull in
ventral view, lower jaw, scale bar: 2 mm (c) skull in lateral view showing teeth on
upper jaw and tusks on lower jaw, lower jaw virtually moved to open the mouth,
scale bar: 1.5 mm (d) pectoral girdle in ventral view – remaining skeleton in all
figures virtually removed, scale bar: 1.5 mm.
trachus spp. from Arthroleptides spp. and 88 from Petropedetes spp.
(Appendix Table A4).
Tadpole morphology and behaviour: head broad sucker-like mouth
with enlarged lateral labial discs; anterior margin of mouth flap-like;
body dorso-ventrally compressed; lateral skin folds along body;
flattened belly extended as flap; tail muscular with narrow fin
(Channing et al., 2012; Guibé and Lamotte, 1958; Lamotte and
Zuber-Vogeli, 1954). Tadpoles cling with their sucker-like mouth
parts to rocks in the strongest currents in water falls and rapids.
Distribution:West Africa (Guinea, Sierra Leone, Liberia, western
Côte d’Ivoire; Appendix Fig. A1).
Included species: Odontobatrachus natator (Boulenger, 1905) nov.
comb.
Comment: Available data indicate the presence of more than one
species in West Africa. A detailed analysis of West African taxa is
beyond the scope of this work and will be treated separately.
Differences between populations of the West African taxon have already been recognised by Rödel and Bangoura (2004). A thorough
revision throughout the distribution range is under preparation.
Etymology: The genus name refers to the Greek words odot1
(odous = tooth, genitive: odóntos) and basqavor (batrachos =
frog) and points to exceptionally long maxillary teeth and large
tusks on lower jaws. The ending is Latinised hence, following the
ICZN the generic name Odontobatrachus is of male gender.
Common name: We advise to use the term ‘‘West African torrent
frogs’’ in English and ‘‘grenouilles des torrents d’Afrique de l’Ouest’’
in French.
Remark: In accordance with article 8.5 of the International code
of Zoological Nomenclature (International Commission on Zoological Nomenclature 2012) the present publication (LSID:
urn:lsid:zoobank.org:pub:67865554-55C4-443F-B56D-F8AA652EAA
77) and nomenclatural act (LSID: urn:lsid:zoobank.org:act:
2E208480-FD27-4E01-AF8C-BDDD59874A4C) have been registered
in ZooBank.
3.4.2. Clade EA
Assignment of all East African taxa to a single supported clade
being clearly distinguished from clades CA and WA requires resurrection of the genus Arthroleptides Nieden, 1911. Although not
included in our analysis due to missing data, we herein list A. dutoiti as a part of this eastern African torrent frog radiation.
Arthroleptides Nieden, 1911
Type species: Arthroleptides martiensseni Nieden, 1911
Reference: Nieden, 1911 Sitzungsberichte der Gesellschaft
Naturforschender Freunde zu Berlin 1910, 441–452.
Diagnosis: Osteology: small dorsolateral oriented nasals that do
not meet medially; anterior end of frontoparietals running slightly
angular to axis; vomer without a posteromedial ramus; vomerine
teeth absent; small teeth on maxillaries and no tusks on mandibles;
pterygoid and neopalatine in contact; metacarpals forming a spike in
males (not all taxa); zygomatic ramus shorter than the otic ramus
and with irregular outlines; base of omosternum bifurcate; medial
edges of coracoids overlapping. External morphology: tympanum
distinct; external vocal sacs absent; nuptial excrescences in breeding males spiny; femoral glands present in males only.
Genetics: All taxa included in this genus are most similar to each
other, forming a clade and can be clearly differentiated from the
remaining torrent frog genera. Within our coding nuclear dataset,
the genus possesses 16 unique character states throughout all taxa
(rag1: 14, BDNF: 1, SIA: 1). A total of 93 unique character states in applied nuclear genes distinguish all Arthroleptides spp. from Odontobatrachus spp. and 26 from Petropedetes spp. (Appendix Table A4).
Tadpole morphology and behaviour: parrot-beak-like jaw
sheaths; labial tooth rows present; oral disc with papillae; dorsal
fin reduced; ventral fin absent; development of limbs in early
stages (Channing et al., 2002, 2012 and references therein). Tadpoles persist on wet rock surfaces and live within a water film.
Distribution: East Africa (Tanzania, Kenya; Appendix Fig. A1).
Included species: A. dutoiti Loveridge, 1935, A. martiensseni Nieden, 1911, A. yakusini Channing, Moyer & Howell, 2002.
Comment: Available data indicate presence of additional unnamed taxa in East Africa. Menegon et al. (2008, 2011) pointed
to the presence of morphologically divergent torrent frogs in
Nguru and Mahenge Mountains. This was supported by recent
analyses in Loader et al. (2013) who discussed the phylogenetic
relationships and biogeography of this group.
3.4.3. Clade CA
Based on the type species of the genus Petropedetes (P. cameronensis) all species belonging to the Central African clade remain in
the genus Petropedetes. As Cornufer johnstoni (consequently also
its synonym Tympanoceros newtonii, Barej et al., 2010a but see
below) is embedded within the genus Petropedetes, both names
remain in the synonymy of the latter one.
Petropedetes Reichenow, 1874
Type species: Petropedetes cameronensis Reichenow, 1874.
Reference: Reichenow, 1874 Archiv für Naturgeschichte 1874,
287–298 + 3 plates.
Synonyms: Cornufer Boulenger, 1888 – in part; Tympanoceros du
Bocage, 1895.
Diagnosis: Osteology: small dorsolateral oriented nasals that do
not meet medially; anterior end of frontoparietals running acutely
angled to axis; vomer consisting of two parts and without posteromedial ramus; vomerine teeth present; small teeth on maxillaries
and no tusks on mandibles; pterygoid and neopalatine in contact;
zygomatic ramus shorter than the otic ramus and with irregular
outlines; metacarpals forming a spike in males (not all taxa); base
of omosternum concave to bifurcate; medial edges of coracoids not
overlapping. External morphology: tympanum indistinct or distinct; external vocal sacs absent; nuptial excrescences in breeding
males spiny; minuscule spines may be present on chin, chest, dorsum and flanks in breeding males; femoral glands present in both
sexes (larger in males); reversed sexual size dimorphism with
males growing larger than females in a few taxa.
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M.F. Barej et al. / Molecular Phylogenetics and Evolution 71 (2014) 261–273
Genetics: All taxa included in this genus are most similar to each
other, forming a clade and can be clearly differentiated from the
remaining torrent-frog genera. Within our coding nuclear dataset,
the genus possesses 10 unique character states throughout all taxa
(rag1: 7, BDNF: 2, SIA: 1). A total of 88 unique character states in
applied nuclear genes distinguish all Petropedetes spp. from Odontobatrachus spp. and 26 from Arthroleptides spp. (Appendix Table A4).
Tadpole morphology and behaviour: parrot-beak-like jaw
sheaths; labial tooth rows present; oral disc with papillae; tail very
muscular, ventral fin absent; development of limbs in early stages
(Barej et al., 2010a; Channing et al., 2012 and references therein).
Tadpoles persist on wet rock surfaces and live within a water film.
Lawson (1993) reported on semi-terrestrial tadpoles that are
attached to leaves.
Distribution: Central Africa (Nigeria, Cameroon, Gabon, Equatorial Guinea, Republic of Congo; Appendix Fig. A1).
Included species: Petropedetes cameronensis Reichenow, 1874, P.
euskircheni Barej, Rödel, Gonwouo, Pauwels, Böhme & Schmitz,
2010, P. johnstoni (Boulenger, 1888 ‘‘1887’’), P. juliawurstnerae
Barej, Rödel, Gonwouo, Pauwels, Böhme & Schmitz, 2010, P. palmipes Boulenger, 1905, P. parkeri Amiet, 1983, P. perreti Amiet, 1973,
P. vulpiae Barej et al., 2010a.
Comments and taxonomic remarks: Unexpectedly and despite
the use of several molecular markers including mitochondrial
and nuclear genes, the relationships within the most species-rich
clade CA could not be solved unambiguously. The present phylogenetic analysis supports the occurrence of additional taxa in Central
Africa (see Barej et al., 2010a). Thereafter, four more taxa have
been uncovered, two occurring in Cameroon and two in Gabon. A
detailed morphological analysis of species diagnosis is beyond
the scope of this work and will be treated separately.
Petropedetes sp. nov. 1 occurs in the Bamenda Highlands and
western part of the Adamawa Plateau in Cameroon. A second unnamed taxon (P. sp. aff. euskircheni) from Mt. Nlonako formerly
tentatively assigned to P. euskircheni by Barej et al. (2010a), has
proven to be distinct in our analyses. Despite repeated surveys of
the locality, only females have been collected; however, only secondary sexual characters of breeding males allow for an unambiguous determination in terms of morphological traits (Amiet, 1973,
1983; Barej et al., 2010a). Mitochondrial DNA data clearly separate
a taxon restricted to Mt. Nlonako, compared to the more widely
distributed P. euskircheni, but this taxon lacks differences in the nuclear markers sampled. Based on mitochondrial data, a substantial
divergence of all specimens originating from Mt. Nlonako suggests
their possible distinction as a new species. Two putative new taxa
(P. sp. nov. 2 and P. sp. nov. 3) are represented by a single sequence
each. Both specimens were collected in the Cristal Mountains Gabon, supporting the presence of new taxa south of Cameroon (Barej
et al., 2010a).
New samples of P. juliawurstnerae not only confirm the assumed
distribution (Bakossi Mts., Cameroon, see Barej et al., 2010a) but
also extend the currently known range to the West (Rumpi Hills,
Cameroon). In the case of P. euskircheni, the distribution has been
likewise extended to the west (Fotabong and Rumpi Hills, Cameroon), while the nearby Mt. Nlonako is inhabited by a distinct taxon
(see above).
The highest genetic diversity has been found within P. vulpiae,
including three distinct subclades. Although the final analysis of
the concatenated data depicts a well-supported terminal clade
for the species, the situation is more complex. Within our singlegene analyses three terminal taxa (P. johnstoni, sp. 2 and sp. 3) shift
their position between P. vulpiae subclades, preventing a clear
understanding of their relationships, despite a high pairwise genetic similarity between populations of P. vulpiae. Distributions of
recognised subclades indicate potential biogeographical splits
along the lowlands of the Biafra Bay within this taxon; however,
271
more data are needed to clarify the resolution of populations.
Based on external morphological features, taxa P. sp. 2 and sp. 3
(both from the Cristal Mts., Gabon) clearly resemble P. vulpiae
and would therefore fit with a biogeographical split for P. vulpiae
as already assumed by Barej et al. (2010a). Petropedetes samples
collected on Fernando Po (= Bioko, Equatorial Guinea) genetically
match samples of P. vulpiae from the mainland – including the type
locality. Bioko samples cluster with nearby coastal P. vulpiae populations on the foot of Mt. Cameroon, proving the existence of this
species distribution on the island of Bioko. Type material of Tympanoceros newtonii is lost, and du Bocage’s (1895) species description
and figures are insufficient to distinguish the taxon from Petropedetes johnstoni. Barej et al. (2010a) had placed P. newtonii in synonymy of P. johnstoni, as topotypic vouchers collected by L. Fea on
Fernando Po (= Bioko, Equatorial Guinea) from the collection of
Museo Civico di Storia Naturale di ‘‘Giacomo Doria’’ (MSNG) morphologically correspond to the latter species. Consequently, a
new name was given to a clearly defined taxon on the mainland
(Barej et al., 2010a). Hence, the validity of P. newtonii still remains
uncertain.
Drewes and Vindum (1994) reported Petropedetes tadpoles on
the eastern side of the Congo Basin from Uganda. The identification
was later revised and assigned to Amietia (Frost, 2013). However,
recently Behangana et al. (2009) report the finding of Petropedetes
sp. in one site of the Albertine Rift without giving any additional
data on their finding or collection numbers of respective vouchers.
Hence, occurrence of the genus east of the Congo Basin still needs
to be verified and remains intriguing.
4. Conclusion
Based on molecular analyses and morphology, African torrent
frogs represent three geographic clades occupying three distinct
sub-Saharan regions of West, Central and East Africa. Based on
clear molecular distinctiveness and morphological and osteological
characters (adults, and/or tadpoles), these clades are recognised as
three distinct genera: Arthroleptides, Odontobatrachus gen. nov. and
Petropedetes. While East (Arthroleptides) and Central (Petropedetes)
African taxa belong to the family Petropedetidae, the new West
African genus could not be assigned to any of the herein included
families and is kept as incertae sedis until more data are available.
All three clades contain additional undescribed taxa.
Author contributions
MFB, AS, MOR, SPL designed the study. MM, NLG, JP, VG, RB and
PN provided important samples and data. Molecular analyses were
performed by MFB, AS, SPL. Morphological analyses were performed by MFB and RG. Data interpretation and writing was done
by MFB. All authors read, commented on and approved the final
manuscript.
Acknowledgments
Research and export permits were provided by the Ministries
from Guinea, Sierra Leone, Liberia, Nigeria, Cameroon, Gabon,
Equatorial Guinea and Tanzania. We thank two anonymous
reviewers and the editor for their comments on our manuscript
and their valuable suggestions, how to improve the present work.
We thank the Tanzania Commission for Science and Technology
(COSTECH research permit RCA 2001-272; RCA 2007-153), TAWIRI
and Wildlife Division for granting permission to conduct research
in Tanzania and export these specimens. For research conducted
in Gabon, RCB thanks the Wildlife Conservation Society for logistical support, the Centre National de la Recherche Scientifique et
Author's personal copy
272
M.F. Barej et al. / Molecular Phylogenetics and Evolution 71 (2014) 261–273
Technologique (CENAREST) and Agence Nationale des Parcs
Nationaux (ANPN) for research permits and the Direction de la
Faune et de la Chasse for export permits. This work was funded
by various organisations including the Swiss National Science
Foundation (31003A-133067) to SPL. MFB got a SYNTHESYS grant
(GB-TAF-1474) to examine the type material of O. natator; this stay
was supported by D. Gower (BMNH). Thanks to K. Mahlow (ZMB)
for generating CT-scans and F.S. Ceccarelli (UB) for her assistance.
Thanks to W. Böhme (ZFMK) and T. Poiss (HU Berlin) for assistance
in etymological concerns. Thanks to J. Barej, M. Hirschfeld, O. Kopecký, M.T. Kouete, H.C. Liedtke and B.L. Stuart for support in the
field and to all local chiefs for permission to carry out field work
within their area of supervision. S. Castroviejo Fisher and B. Alvarez
Dorda (MNCN) as well as B.L. Stuart (NCSM) made available additional tissue samples. W. Böhme (ZFMK) kindly sent material on
loan. Photos of living torrent frogs have been kindly provided by
D.C. Blackburn (CAS), A. Channing (University of Western Cape)
and M. Dehling (University of Koblenz-Landau).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2013.11.
w001.
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GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – Phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Appendix A. Supplementary material
A1. Graphical abstract
A2. Distribution of African „Torrent Frogs“
BAREJ, M.F., M.-O. RÖDEL, S.P. LOADER, M. MENEGON, N.L. GONWOUO, J. PENNER, V.
GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – Phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Supplementary data 1
References
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BAREJ, M.F., M.-O. RÖDEL, S.P. LOADER, M. MENEGON, N.L. GONWOUO, J. PENNER, V.
GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – Phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Supplementary data 2
List of applied primers and respective sources.
gene
16S
12S
cytb
cytb
BDNF
SIA
rag1
16sar-L
16sbr-H
12S L1091
12S H1478
CBJ10933
cytb C
CB2F
CB3R
BDNF-F
BDNF-R
SIA1 (T3)
SIA2 (T7)
Mart-FL1
AMP-R1
primer sequences
5' - CGC CTG TTT ATC AAA AAC AT - 3'
5' - CCG GTC TGA ACT CAG ATC ACG T - 3'
5' - AAA CTG GGA TTA GAT ACC CCA CTA T - 3'
5' - GAG GGT GAC GGG CGG TGT GT - 3'
study/source
Palumbi et al.,1991
Palumbi et al.,1991
Kocher et al., 1989
Kocher et al., 1989
Bossuyt and Milinkovitch,
5' - TAT GTT CTA CCA TGA GGA CAA ATA TC - 3'
2000
Bossuyt and Milinkovitch,
5' - CTA CTG GTT GTC CTC CGA TTC ATG T - 3'
2000
5' - TGA GGA CAA ATA TCT TTT TGA GGG - 3'
Hillis et al., 1996
5' - GGC GAA TAG GAA RTA TCA TTC - 3'
Hillis et al., 1996
5' - GAC CATCCT TTT CCT KAC TAT GGT TAT TTC Noonan and Chippindale,
ATA CTT - 3'
2006
5' - CTA TCT TCC CCT TTT AAT GGT CAG TGT ACA Noonan and Chippindale,
AAC - 3'
2006
5 ' -TCGAGTGCCCCGTGTGYTTYGAYTA - 3'
Bonacum et al., 2001
5' - GAAGTGGAAGCCGAAGCAGSWYTGCATCAT - 3' Bonacum et al., 2001
5' - AGC TGC AGY CAR TAY CAY AAR ATG TA - 3'
Pramuk et al., 2008
5' - AAC TCA GCT GCA TTK CCA ATR TCA - 3'
Pramuk et al., 2008
BAREJ, M.F., M.-O. RÖDEL, S.P. LOADER, M. MENEGON, N.L. GONWOUO, J. PENNER, V.
GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – Phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Supplementary data 3
Applied PCR protocols.
12S
initial denaturation
denaturation
annealing
extension
94°C à 45s
35x 50°C à 60s
74°C à 120s
16S
cytb
BDNF
rag1
94°C à 90s
94°C à 120s
94°C à 300s
94°C à 120s
94°C à 45s
94°C à 30s
94°C à 20s
94°C à 60s
94°C à 60s
30x 54°C à 30s
40x 54°C à 45s
72°C à 60s
72°C à 60s
39x 57°C à 57s
72°C 120s
denaturation
62°C à 60s
17x [modify:
1° each cycle]
72°C à 60s
35x 50°C à 60s
72°C à 60s
94°C à 60s
annealing
20x 48°C à 60s
extension
final extension
SIA
94°C à 90s
72°C à 60s
72°C à 420s
72°C à 420s
72°C à 600s
72°C à 600s
72°C à 420s
72°C à 420s
BAREJ, M.F., M.-O. RÖDEL, S.P. LOADER, M. MENEGON, N.L. GONWOUO, J. PENNER, V.
GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – Phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Supplementary data 4
Partitioning schemes for applied mitochondrial and nuclear genes. Given are loci, sequence
length, best fitting model (position dependent in coding genes), number of sequences and
taxon coverage (including outgroups).
molecular data set
locus
aligned
sequence
lengths
12S
354 bp
16S
570 bp
cytb
rag1
BDNF
SIA
588 bp
930 bp
675 bp
396 bp
BIC
sequences
taxon coverage
TIM2ef+G
91
22
TIM2+I+G
109
22
39
19
52
22
54
22
52
22
Pos.1
TPM2+G
Pos.2
TrN+G
Pos.3
TrN+G
Pos.1
K80+G
Pos.2
K80+G
Pos.3
K80+I
Pos.1
K80+G
JC
Pos.2
Pos.3
JC
Pos.1
K80+G
Pos.2
K80+I
Pos.3
K80+I
BAREJ, M.F., M.-O. RÖDEL, S.P. LOADER, M. MENEGON, N.L. GONWOUO, J. PENNER, V.
GVOŽDÍK, R. GÜNTHER, R.C. BELL, P. NAGEL & A. SCHMITZ (2014): Light shines through the
spindrift – the phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae). –
Molecular Phylogenetics and Evolution, 71: 261-273. doi.org/10.1016/j.ympev.2013.11.001.
Supplementary data 5
Unique base states in nuclear coding genes (rag1, BDNF, SIA) of African torrent frogs
genera Odontobatrachus gen. nov., Arthroleptides and Petropedetes. Given are base positions
according to our alignment, colour coding as in Fig. 1 (CA: green, EA: red, WA: blue).
rag1
1
2
3
4
5
6
7
base postion
9
12
30
32
39
58
63
8
9
10
11
12
13
14
15
16
17
18
19
20
Odontobatrachus gen. nov.
A
A
A
T
A
C
A
G
C
C
A
C
A
G
T
A
A
C
C
T
Arthroleptides
G
A
G
C
G
A
T
C
T
G
C
G
G
A
C
A
G
T
T
C
Petropedetes
G
G
G
C
G
A
T
C
T
C
C
A
A
A
C
C
G
C
T
C
rag1
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
base postion
294 306 327 369 385 423 441 449 450 454 475 507 516 525 530 564 631 635 645 648
105 111 129 147 169 195 199 227 240 246 267 283 284
Odontobatrachus gen. nov.
G
C
G
G
C
C
C
A
G
G
T
T
A
T
T
T
C
A
T
C
Arthroleptides
G
A
A
A
G
T
C
G
T
G
G
C
C
C
C
C
A
G
C
C
Petropedetes
A
A
G
A
G
T
T
G
T
A
G
C
C
A
A
C
A
G
C
A
rag1
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
base postion
657 660 672 675 681 711 714 715 726 744 747 748 753 762 765 774 801 804 838 843
Odontobatrachus gen. nov.
T
A/G
G
G
C
C
T
G
Arthroleptides
A
Petropedetes
A
T
A
A
T
G
A
rag1
61
base postion
849 886 894 900 907 924 924
62
63
64
65
T
T
G
T
G
A
C
T
G
T
T
T
G
T
C
A
T
A/G
C
C
A
C
G
A
C
A
G
T
C
A
C/T
C
C
A/G
G
C
A
G
T
C
A
C
T
T
66
67
8
9
10
11
12
13
14
15
16
17
18
19
20
Odontobatrachus gen. nov.
T
G
G
T
C
C
C
Arthroleptides
C
T
A
G
A
T
T
Petropedetes
T
G
A
G
C
C
C
BDNF
1
2
3
4
5
6
7
base postion
43
45
Odontobatrachus gen. nov.
G
T
C
A
C
T
A
G
C
A
G
G
T
C
G
C
C
T
T
G
Arthroleptides
G
A
T
G
T
G
G
A
T
G
A
A
T
A
T
T
T
C
A
A
Petropedetes
A
A
T
G
C
G/A
G
A
T
G
A
A
C
A
T
T
T
C
A
A
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
SIA
bp
141 144 146 162 174 186 309 339 375 408 411 432 447 459 465 504 528 543
123 150 162 165 168 198 204 219 246 255 276 279 280 312 321 324 378
Odontobatrachus gen. nov.
G
C
T
T
G
G
A
C
T
G
A
T
T
G
T
T
G
Arthroleptides
A
T
A
C
T
A
G
T
C
G
T
C
C
A
C
C
A
Petropedetes
A
T
A
C
T
A
G
T
C
A
T
C
C
G
C
C
A