Author's Personal Copy

Author's personal copy

Molecular Phylogenetics and Evolution 71 (2014) 261–273

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Molecular Phylogenetics and Evolution

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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 Gvozˇdík 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 b University of Basel, Department of Environmental Sciences (Biogeography), Klingelbergstr. 27, Basel 4056, Switzerland c 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 article info abstract

Article history: Torrent frogs of the genus Petropedetes Reichenow, 1874 as currently understood have a disjunct distri- Received 17 August 2013 bution with species endemic to West, Central or East Africa. We herein present a phylogenetic analysis Revised 26 October 2013 including all but one of the currently described 12 species of the genus. Maximum Likelihood and Bayes- Accepted 3 November 2013 ian analyses of combined nuclear (rag1, SIA, BDNF) and mitochondrial (16S, 12S, cytb) genes of more than Available online 15 November 2013 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 mono- Keywords: phyletic group with respect to closely related families Pyxicephalidae and Conrauidae and therefore Africa require taxonomic changes. We resurrect Arthroleptides Nieden, 1911 for the East African taxa. The Cen- Amphibians Taxonomy tral African taxa remain in the genus Petropedetes. The West African members are placed in the new Arthroleptides genus Odontobatrachus gen. nov. The taxonomic position of the new genus remains incertae sedis as it Petropedetes was not assigned to any of the four families included in our analyses. Potential new species have been Odontobatrachus gen. nov 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 documented in some taxa and several studies implicate ecological separation as a mechanism for diversiﬁcation among syntopic Cen- African torrent frogs of genus Petropedetes Reichenow, 1874 in- tral African taxa (Amiet, 1983, 1991; Barej et al., 2010a; Sanderson, habit the splash-water zone of clear running streams in predomi- 1936). nantly undisturbed forests of West, Central and East Africa. Prominent morphological characters of these medium to large Although torrent frogs are very similar in their general ecology, dif- sized frogs (up to 7 cm SVL) are T-shaped terminal phalanges with ferences in tadpole morphology and their habitat requirements heart-shaped digital discs and toe tips, presence of femoral glands, have been recognised; tadpoles being either semi-terrestrial on tympanic papillae, carpal spikes and tusks, but not all characters rocks in the spray zone or fully aquatic in zones of the strongest are present in all species (e.g. Barej et al., 2010a; Boulenger, currents (Barej et al., 2010a; Lamotte and Zuber-Vogeli, 1954; 1905; Klemens, 1998; Perret, 1966). Even some of the characters Lamotte et al., 1959; Lawson, 1993). Further information on the provided in Reichenow’s (1874) diagnosis of the genus, e.g. extent species’ biology is scarce; however, clutch guarding has been 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 syn- ⇑ Corresponding author. apomorphies support this grouping (Frost et al., 2006). E-mail addresses: [email protected] (M.F. Barej), [email protected] While femoral glands evolved independently in several non-re- (M.-O. Rödel), [email protected] (S.P. Loader), [email protected] (M. Menegon), [email protected] (N.L. Gonwouo), [email protected] lated families (e.g. Mantellidae, Phrynobatrachidae, Ranixalidae, (J. Penner), [email protected] (V. Gvozˇdík), [email protected] Nyctibatrachidae, and Pyxicephalidae) and probably play a role in (R. Günther), [email protected] (R.C. Bell), [email protected] (P. Nagel), reproduction (Vences et al., 2007), tympanic papillae are a unique [email protected] (A. Schmitz).

262 M.F. Barej et al. / Molecular Phylogenetics and Evolution 71 (2014) 261–273 feature in anuran acoustic communication (Narins et al., 2001). A as part of the Ranidae Rafinesque, 1814 (Bossuyt et al., 2006; Scott, carpal spike in males most probably serves a territorial and/or 2005; van der Meijden et al., 2005). aggressive behaviour in breeding males (Barej et al., 2010a; Later Frostetal.(2006)grouped these frogs with Conraua Nie- Sanderson, 1936). den, 1908 and Indirana Laurent, 1986 in the family Petropedeti- The genus type species is P. cameronensis Reichenow, 1874 from dae Noble, 1931, still using the name Arthroleptides for their Central Africa. Later, Boulenger (1888) and du Bocage (1895) de- East African taxon. However, Scott’s (2005) results have subse- scribed Cornufer johnstoni and Tympanoceros newtonii respectively, quently been adopted in Frost’s online database (Frost, 2013). In both of which are from Central Africa. Boulenger (1900) transferred a very recent phylogeny Pyron and Wiens (2011) likewise listed Cornufer johnstoni and Tympanoceros newtonii to the genus Petrope- East African taxa in the genus Petropedetes, but transferred the detes. One species, P. obscurus Ahl, 1924 was formerly described genus Conraua to its own family Conrauidae Dubois, 1992, while from East Africa but Perret (1984) placed it in synonymy with Indirana was placed in the Ranixalidae Dubois, 1987 (Blackburn the Central African P. cameronensis, because morphological differ- and Wake, 2011). The most recent changes have subsequently ences were insufficient and locality data most probably got con- left the genus Petropedetes the sole member of the family fused. Barej et al. (2010a) confirmed this point of view but Petropedetidae. placed P. newtonii in synonymy with P. johnstoni. All recent large-scale molecular phylogenies including Petr- Currently the highest species diversity in the genus is found in opedetinae / Petropedetidae only sampled Central and East Afri- western Central Africa around the Gulf of Guinea (= Biafra Bay). can representatives; the West African P. natator was absent. Eight species occur in the area from Nigeria to the Republic of Con- Solely Scott (2005) analysed P. natator; however, molecular data go (Petropedetes cameronensis Reichenow, 1874; P. euskircheni were unavailable to her and only morphological data were used. Barej et al., 2010a; P. johnstoni (Boulenger, 1888 "1887"), P. juli- According to Scott’s (2005) combined analysis of morphology awurstnerae Barej et al., 2010a; P. palmipes Boulenger, 1905; P. and genetics, African torrent frogs form a clade, being supported parkeri Amiet, 1983; P. perreti Amiet, 1973; P. vulpiae Barej et al., by a number of osteological apomorphies as well as general exter- 2010a and are either widely distributed in lowland forests or inha- nal morphology and secondary sexual characters. However, with- bit restricted mountainous areas (Amiet, 1986; Barej et al., 2010a; in this clade Scott (2005) recognised characters separating P. Perret, 1966, 1984). Various Central African (CA) species occur in natator from both Central African taxa (P. cameronensis, P. new- sympatry in the submontane zone (Amiet, 1975, 1983; Barej toni = P. vulpiae, and P. parkeri in her analysis) and a single East et al., 2010a; Herrmann et al., 2005; Parker, 1936) and Barej African representative (P. martiensseni). Furthermore, Scott et al. (2010a) already indicated the presence of additional unde- (2005) identified synapomorphies for the Central African and East scribed species in Central Africa. Three species, P. dutoiti (Love- African groupings. ridge, 1935), P. martiensseni (Nieden, 1911) and P. yakusini In order to resolve the systematic relationships between African (Channing, Moyer & Howell, 2002) are known from forests in iso- torrent frogs throughout their entire range, we herein provide the lated East African (EA) mountain peaks (e.g. Channing et al., first comprehensive molecular phylogeny, including 11 of 12 cur- 2002; Nieden, 1911; Loader et al., 2013) and only a single species, rently described 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 2. Material and methods to important centres of diversity and endemism (Burgess et al., 2007b and references therein). In western Central Africa this refers 2.1. Taxon sampling to parts of the Cameroon volcanic line, which shows e.g. the highest diversity in the frog genera Astylosternus and Leptodactylodon Our sampling includes all but one of the currently known spe- (Amiet, 1977, 1980), chameleons (Barej et al., 2010b) and mam- cies. Solely, the East African species Petropedetes dutoiti (Loveridge, mals (Missoup et al., 2012) in that region. In eastern Africa this re- 1935) from Kenya is missing. This species might be extinct since no fers to the East Arc Mountains, known for their exceptional individuals have been seen in the last five decades (Andreone et al., diversity amongst others in the frog genera Nectophrynoides and 2008; Groombridge, 1994; IUCN, 2012). However, rediscovery at- Arthroleptis (Blackburn, 2008; Menegon et al., 2004), but also in tempts are ongoing (B. Akoth in litt. 04.IV.2013). Where possible, other vertebrate groups (Burgess et al., 2007a). we included multiple specimens from across the known distribu- For P. natator Jean-Louis Amiet (footnote in Perret, 1984) sug- tion range of each taxon. Recently, Loader et al. (2013) pointed to gested this species be placed in its own genus based on adult and a higher diversity within East African mountains, indicating the larval morphology. Whereas, P. natator was originally placed in presence of new taxa. The latter taxa are included in our analysis Petropedetes, the three East African species were formerly placed as well. in the genus Arthroleptides Nieden, 1911. One of the crucial differ- Outgroup taxa include Conraua, which according to Frost et al. ences is the absence of vomerine teeth in Arthroleptides (Nieden, (2006) was regarded as a member of the family Petropedetidae 1911), a character often disclaimed on genus level differentiation (but see introduction). Moreover, we added members of the Afri- (e.g. Inger, 1954). However, Deckert (1938) recognised differ- canura sensu Frost et al. (2006) with representatives of the Phryno- ences in the pectoral girdle, which he regarded as sufficient to batrachidae Laurent, 1940 and its sister taxon Pyxicephaloidea keep both genera. Robert Drewes (in Largen, 1991)hadrecom- Bonaparte, 1850, respectively. mended expanding the definition of the genus Petropedetes in or- A full list of samples and respective GenBank numbers are given der to include Arthroleptides taxa. Finally, based on combined in Table 1. Museum abbreviations are as follow: The Natural His- morphological and molecular data Scott (2005) allocated Arthro- tory Museum, London, United Kingdom (BMNH); Institut Royal leptides to Petropedetes, given the latter genus was paraphyletic des Sciences Naturelles de Belgique, Bruxelles, Belgium (IRSNB- otherwise. KBIN); Muséum d’histoire naturelle, Genève, Switzerland (MHNG); On a higher systematic level, the genus Petropedetes has been Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN); placed in different taxonomic ranks (subfamilies and families). North Carolina Museum of Natural Sciences, Raleigh, USA (NCSM); Originally, Reichenow (1874) classified the genus as part of the National Museum, Museum of Natural History, Prague, Czech family Hylae Laurenti, 1768 (a synonym of Hylidae Rafinesque, Republic (NMP6V); Museo Tridentino di Scienze Naturali, Trento, 1815). Later Noble (1931) generated the subfamily Petropedetinae 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 Voucher ID Locality, country GenBank accession numbers 12S 16S cytb BDNF SIA rag1 Hyperolius ocellatus MHNG 2715.58 Big Massaka, CM KF693275 KF693379 KF693659 KF693477 KF693539 KF693599 Phrynobatrachus calcaratus ZFMK 89567 Big Massaka, CM KF693276 KF693380 KF693660 KF693478 KF693540 KF693600 Phrynobatrachus auritus MHNG 2715.89 Rumpi Hills: Mofako Balue, CM KF693277 KF693381 KF693661 KF693479 KF693541 KF693601 Phrynobatrachus africanus MHNG 2715.45 Mt Kupe: Nyasoso, CM KF693278 KF693382 KF693662 KF693480 KF693542 KF693602 Cacosternum boettgeri MHNG 2740.55 Kuruman, ZA KF693279 KF693383 KF693663 KF693481 KF693543 KF693603 Conraua goliath CAM1 CM KF693280 KF693384 KF693664 KF693482 KF693544 KF693604 Conraua robusta ZMB 78427 Mt. Manengouba: Manengouba Village, CM KF693281 KF693385 KF693665 KF693483 KF693545 KF693605 Conraua robusta MHNG 2731.52 Rumpi Hills: Mofako Balue, CM KF693282 KF693386 KF693666 KF693484 KF693546 KF693606 Conraua alleni ZMB 78428 East of Ghi Mtn., LR KF693283 KF693387 KF693667 KF693485 KF693547 KF693607 Conraua alleni ZMB 78429 Fouta Djallon: Konkouré, GN KF693284 KF693388 KF693668 KF693486 KF693548 KF693608

Conraua alleni ZMB 78433 Nimba Mts.: Bangué, GN KF693285 KF693389 KF693669 KF693487 KF693549 KF693609 261–273 (2014) 71 Evolution and Phylogenetics Molecular / al. et Barej M.F. Odontobatrachus natator ZMB 78211 Diéké/Yonsonso, GN KF693286 KF693390 KF693670 KF693488 KF693550 KF693610 Odontobatrachus natator ZMB 78244 Grand Gedeh, LR KF693287 KF693391 KF693671 KF693489 KF693551 KF693611 Odontobatrachus natator ZMB 78206 Nimini FR, SL KF693288 KF693392 KF693672 KF693490 KF693552 KF693612 Odontobatrchus sp. nov. ZMB 78315 Fouta Djallon: Labé/Sala, GN KF693289 KF693393 KF693673 KF693491 KF693553 KF693613 Odontobatrchus sp. nov. ZMB 78317 Fouta Djallon: Dalabé, GN KF693290 KF693394 KF693674 KF693492 KF693554 KF693614 Odontobatrchus sp. nov. ZMB 78319 Fouta Djallon: Sala, GN KF693291 KF693395 KF693675 KF693493 KF693555 KF693615 Arthroleptides martiensseni ZFMK 77306 East Usambara Mts.: Amani, TZ KF693292 KF693396 KF693676 KF693494 KF693556 KF693616 Arthroleptides martiensseni ZFMK 77307 East Usambara Mts.: Amani, TZ KF693293 KF693397 KF693677 KF693495 KF693557 KF693617 Arthroleptides martiensseni BM 2002.576 East Usambara Mts.: Nilo TZ JX546951 JX546956 KF693678 KF693496 — — Arthroleptides martiensseni BM 2002.575 East Usambara Mts.: Nilo TZ JX546943 JX546958 JX546978 KF693497 — — Arthroleptides martiensseni BM 2000.826 East Usambara Mts.: Amani, TZ JX546950 JX546955 JX546970 KF693498 KF693558 KF693618 Arthroleptides yakusini BM 2005.014 Uluguru Mts., Kasanga, TZ JX546945 JX546961 JX546981 KF693499 KF693559 KF693619 Arthroleptides yakusini BM 2005.1382 Mahenge Mts., TZ JX546946 JX546963 JX546972 KF693500 KF693560 KF693620 Arthroleptides yakusini BM 2005.1383 Mahenge Mts., TZ JX546944 JX546966 JX546974 KF693501 KF693561 KF693621 Arthroleptides yakusini BM 2005.013 Uluguru Mts., Kasanga, TZ JX546942 JX546964 KF693679 — KF693562 — Arthroleptides yakusini BM 2005.567 Udzungwa, TZ JX546941 JX546965 KF693680 KF693502 KF693563 KF693622 Arthroleptides c MTSN 8358 Kanga, TZ JX546948 JX546960 KF693681 KF693503 KF693564 KF693623 Arthroleptides sp. nov. MTSN 8255 Nguru, TZ JX546947 JX546959 KF693682 KF693504 KF693565 KF693624 Petropedetes cameronensis ZFMK 81616 Mt. Nlonako, CM KF693294 KF693398 KF693683 KF693505 KF693566 KF693625 Petropedetes cameronensis ZFMK 81615 Mt. Nlonako, CM KF693295 GU256015 KF693684 KF693506 KF693567 KF693626 Petropedetes cameronensis ZFMK 81154 Mt. Nlonako, CM KF693296 GU256016 KF693685 KF693507 KF693568 KF693627 Petropedetes cameronensis N30Rhiho Rhoko Forest, NG KF693297 KF693399 Petropedetes cameronensis ZMB 73729 Rhoko Forest, NG KF693298 GU256017 Petropedetes cameronensis ZMB 73728 Mbe Mts., NG KF693299 GU256018 Petropedetes cameronensis ZFMK78018 Mt. Nlonako, CM — KF693400 Petropedetes cameronensis NMP6V 74646/2 Bakossi Mts.: Messaka, CM — KF693401 KF693686 KF693508 KF693569 KF693628 Petropedetes cameronensis NMP6V 74645/1 Bakingili, CM — KF693402 Petropedetes cameronensis NMP6V 74645/2 Bakingili, CM — KF693403 Petropedetes cameronensis NMP6V 73403/1 Bakingili, CM — KF693404 Petropedetes cameronensis NMP6V 73403/2 Bakingili, CM — KF693405 Petropedetes cameronensis NMP6V 73403/3 Bakingili, CM — KF693406 Petropedetes cameronensis ZMB 78204 Ebo Forest, CM KF693300 KF693407 KF693687 KF693509 KF693570 KF693629 Petropedetes sp. aff. euskircheni ZFMK 75539 Mt. Nlonako, CM KF693301 GU256020 KF693688 KF693510 KF693571 KF693630 Petropedetes sp. aff. euskircheni ZFMK 81103 Mt. Nlonako, CM KF693302 GU256022 — KF693511 KF693572 KF693631 Petropedetes sp.sp. aff. aff. euskirchenieuskircheni ZFMKZFMK 81168 78365 Mt. Mt. Nlonako, Nlonako, CM CM — — GU256023 GU256024 PetropedetesPetropedetes sp. aff. euskircheni ZMB 78385 Mt. Nlonako: Nguéngué, CM KF693303 KF693408 Petropedetes sp. aff. euskircheni ZMB 78386 Mt. Nlonako: Nguéngué, CM KF693304 KF693409 Petropedetes euskircheni ZFMK 75582 Mt. Kupe, CM KF693305 GU256019 Petropedetes euskircheni ZFMK 75586 Mt. Kupe, CM KF693306 GU256021

Petropedetes euskircheni MHNG 2713.10 Rumpi Hills: Mt. Rata, CM KF693307 KF693410 263 (continued on next page) Author's personal copy 264 Table 1 (continued)

Taxon Voucher ID Locality, country GenBank accession numbers 12S 16S cytb BDNF SIA rag1 Petropedetes euskircheni ZFMK 88865 Rumpi Hills: Mt. Rata, CM KF693308 KF693411 KF693689 KF693512 KF693573 KF693632 Petropedetes euskircheni MHNG 2713.8 Mt. Kupe, CM KF693309 GU256025 — KF693513 KF693574 KF693633 Petropedetes euskircheni MHNG 2713.5 Mt. Kupe, CM KF693310 KF693412 Petropedetes euskircheni ZFMK 88864 Mt. Kupe, CM KF693311 GU256026 Petropedetes euskircheni MHNG 2713.7 Mt. Kupe, CM KF693312 GU256027 KF693514 KF693575 KF693634 Petropedetes euskircheni ZMB 78387 Fotabong, CM KF693313 KF693413 Petropedetes johnstoni ZFMK 87710 Campo, CM KF693314 GU256028 KF693690 KF693515 KF693576 KF693635 Petropedetes johnstoni ZFMK 87709 Campo, CM KF693315 GU256029 KF693691 KF693516 KF693577 KF693636 Petropedetes johnstoni ZMB 78201 Miangasio Lendi, CM KF693316 KF693414 KF693692 Petropedetes juliawurstnerae ZFMK 75590 Mt. Kupe, CM KF693317 GU256030 Petropedetes juliawurstnerae ZFMK 68134 Mt. Kupe, CM KF693318 KF693415 Petropedetes juliawurstnerae ZFMK 68131 Mt. Kupe, CM KF693319 KF693416 ..Brje l oeua hlgntc n vlto 1(04 261–273 (2014) 71 Evolution and Phylogenetics Molecular / al. et Barej M.F. Petropedetes juliawurstnerae ZFMK 67360 Mt. Kupe, CM KF693320 GU256031 Petropedetes juliawurstnerae ZFMK 67987 Mt. Kupe, CM KF693321 KF693417 Petropedetes juliawurstnerae MHNG 2713.17 Rumpi Hills: Mofako Balue, CM KF693322 KF693418 Petropedetes juliawurstnerae MHNG 2713.19 Rumpi Hills: Mofako Balue, CM KF693323 KF693419 KF693693 KF693517 KF693578 KF693637 Petropedetes juliawurstnerae ZMB 73694 Mt. Kupe, CM KF693324 GU256032 Petropedetes juliawurstnerae NMP6V 74632/1 Bakossi Mts.: Messaka, CM KF693325 KF693420 KF693694 KF693518 KF693579 KF693638 Petropedetes juliawurstnerae NMP6V 74632/2 Bakossi Mts.: Messaka, CM KF693326 KF693421 Petropedetes juliawurstnerae ZFMK 89440 Rumpi Hills: Mofako Balue, CM KF693327 KF693422 Petropedetes juliawurstnerae MHNG 2713.12 Mt. Kupe: Nyasoso, CM KF693328 KF693423 Petropedetes palmipes NCSM 76813 Monts de Cristal NP, GA KF693329 KF693424 KF693695 KF693519 KF693580 KF693639 Petropedetes palmipes NCSM 76814 Monts de Cristal NP, GA KF693330 KF693425 — KF693520 KF693581 KF693640 Petropedetes palmipes NCSM 76815 Monts de Cristal NP, GA KF693331 KF693426 — KF693521 KF693582 KF693641 Petropedetes parkeri ZFMK 87702 Amebishu, CM KF693332 GU256033 — KF693522 KF693583 KF693642 Petropedetes parkeri ZMB 73739 Cross River NP, NG KF693333 GU256034 — KF693523 KF693584 KF693643 Petropedetes parkeri NMP6V 74680/1 Mt. Fungom, CM — KF693427 Petropedetes parkeri NMP6V 74680/2 Mt. Fungom, CM — KF693428 Petropedetes parkeri VP10-60 Mt. Fungom, CM — KF693429 Petropedetes parkeri NMP6V 74690 Mt. Fungom, CM — KF693430 Petropedetes parkeri NMP6V 74678/1 Mt. Fungom, CM — KF693431 Petropedetes parkeri NMP6V 74686/4 Mt. Fungom, CM — KF693432 Petropedetes parkeri NMP6V 74686/7 Mt. Fungom, CM — KF693433 Petropedetes parkeri NMP6V 74686/22 Mt. Fungom, CM — KF693434 Petropedetes parkeri NMP6V 74691 Munkep, CM — KF693435 Petropedetes perreti ZFMK 75524 Mt. Nlonako, CM KF693334 GU256035 Petropedetes perreti ZFMK 75519 Mt. Nlonako, CM KF693335 KF693436 Petropedetes perreti ZFMK 69227 Mt. Nlonako, CM KF693336 KF693437 Petropedetes perreti 0847N Manengouba Mts.: Esipa Village, CM KF693337 KF693438 — KF693524 KF693585 KF693644 Petropedetes perreti ZMB 73737 Manengouba Mts.: Esipa Village, CM KF693338 GU256036 Petropedetes perreti 0998N Manengouba Mts.: Ebonemin, CM KF693339 KF693439 Petropedetes perreti ZMB 73734 Manengouba Mts.: Ebonemin, CM KF693340 GU256037 — KF693525 KF693586 KF693645 Petropedetes perreti 0846N Manengouba Mts.: Esipa Village, CM KF693341 KF693440 Petropedetes perreti 0159LG Manengouba Mts., CM KF693342 KF693441 — KF693526 KF693587 KF693646 Petropedetes perreti 0175LG Manengouba Mts., CM KF693343 KF693442 Petropedetes perreti 0165LG Manengouba Mts., CM KF693344 KF693443 Petropedetes perreti ZMB 73731 Manengouba Mts., CM KF693345 GU256038 Petropedetes perreti 0158LG Manengouba Mts., CM KF693346 KF693444 Petropedetes perreti 0157LGZFMK 75588 Manengouba Mt. Kupe, CM Mts., CM KF693347 KF693348 KF693445 GU256039 KF693696 KF693527 KF693588 KF693647 PetropedetesPetropedetes vulpiae vulpiae ZFMK 81167 Mt. Nlonako, CM KF693349 GU256040 — KF693528 KF693589 KF693648 Petropedetes vulpiae ZFMK 81623 Mt. Nlonako, CM KF693350 GU256041 Petropedetes vulpiae ZMB 73726 Mbe Mts., NG KF693351 KF693446 KF693697 KF693529 KF693590 KF693649 Petropedetes vulpiae ZFMK 81554 Mt. Nlonako, CM KF693352 KF693447 — KF693530 — KF693650 Author's personal copy

Petropedetes vulpiae ZFMK 78364 Mt. Nlonako, CM KF693353 KF693448 Petropedetes vulpiae ZFMK 88863 Big Massaka, CM KF693354 GU256042 Petropedetes vulpiae ZMB 73692 Big Massaka, CM KF693355 GU256043 Petropedetes vulpiae ZFMK 88862 Mt. Kupe, CM KF693356 KF693449 Petropedetes vulpiae ZFMK 88859 Mt. Kupe, CM KF693357 GU256044 Petropedetes vulpiae MHNG 2713.4 Mt. Kupe, CM KF693358 KF693450 Petropedetes vulpiae MHNG 2713.9 Mt. Kupe, CM KF693359 KF693451 Petropedetes vulpiae NMP6V 74641 Bakossi Mts.: Edib/Messaka, CM KF693360 KF693452 Petropedetes vulpiae NMP6V 74646/1 Bakossi Mts.: Messaka, CM KF693361 KF693453 Petropedetes vulpiae NMP6V 73439/1 Bakingili, CM KF693362 KF693454 — KF693531 KF693591 KF693651 Petropedetes vulpiae NMP6V 73439/2 Bakingili, CM KF693363 KF693455 Petropedetes vulpiae ZMB 78425 Ebo Forest, CM KF693364 KF693456 KF693698 KF693532 KF693592 KF693652 Petropedetes vulpiae MNCN 50403 Bioko Island: Rio Osa, GQ — KF693457 Petropedetes vulpiae MNCN 50405 Bioko Island: Rio Osa, GQ KF693365 KF693458 Petropedetes vulpiae MNCN 50406 Bioko Island: Rio Osa, GQ — KF693459 Petropedetes vulpiae MNCN 50407 Bioko Island: Rio Osa, GQ — KF693460 Petropedetes vulpiae MNCN 50411 Bioko Island: Rio Ole, GQ KF693366 KF693461

Petropedetes vulpiae MNCN 50417 Caldera de Luba; Bioko, GQ — KF693462 261–273 (2014) 71 Evolution and Phylogenetics Molecular / al. et Barej M.F. Petropedetes vulpiae MNCN 50465 Bioko Island: Rio Sibitá, Bococo, GQ KF693367 KF693463 KF693699 KF693533 KF693593 KF693653 Petropedetes vulpiae MNCN 50466 Bioko, GQ — KF693464 Petropedetes vulpiae ZMB 78421 Foot of Mt. Etinde: Etome, CM KF693368 KF693465 Petropedetessp.nov. 1 NMP6V 74577/1 Mt. Tchabal Gangdaba, CM KF693369 KF693466 KF693700 KF693534 KF693594 KF693654 Petropedetes sp.nov. 1 NMP6V 74577/2 Mt. Tchabal Gangdaba, CM KF693370 KF693467 Petropedetes sp.nov. 1 NMP6V 74577/3 Mt. Tchabal Gangdaba, CM — KF693468 Petropedetes sp.nov. 1 NMP6V 74592/1 Banyo, CM KF693371 KF693469 KF693701 KF693535 KF693595 KF693655 Petropedetes sp.nov. 1 NMP6V 74592/2 Banyo, CM KF693372 KF693470 Petropedetes sp.nov. 1 NMP6V 73389/1 Big Babanki, Bamenda Highlands, CM KF693373 KF693471 Petropedetes sp.nov. 1 NMP6V 73389/2 Big Babanki, Bamenda Highlands, CM KF693374 KF693472 Petropedetes sp.nov. 1 NMP6V 73391/1 Mejung, Bamenda Highlands, CM KF693375 KF693473 KF693702 KF693536 KF693596 KF693656 Petropedetes sp.nov. 1 NMP6V 73391/2 Mejung, Bamenda Highlands, CM KF693376 KF693474 Petropedetes sp. nov. 2 NCSM 76811 Monts de Cristal NP, GA KF693377 KF693475 — KF693537 KF693597 KF693657 Petropedetes sp. nov. 3 NCSM 76812 Monts de Cristal NP, GA KF693378 KF693476 KF693703 KF693538 KF693598 KF693658 265 Author's personal copy

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Bonn, Germany (ZFMK); Museum für Naturkunde, Berlin, Germany 2.4. Morphological analysis (ZMB). Remaining material refers to the national collection of Cam- eroon (CAM-, LG- & N-numbers). 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 para- 2.2. DNA extraction and sequencing type of P. juliawurstnerae ZMB 73694) and East Africa (female holotype of P. martiensseni, ZMB 21793, male P. yakusini ZMB 48472) DNA was extracted from ethanol-preserved liver, thigh or ton- were subjected to a micro-tomographic analysis at the Museum gue muscle. These tissues were taken either from fresh specimens für Naturkunde Berlin using a Phoenix nanotom X-ray|s tube at collected in the field or preserved museum specimens. DNA was 70–90 kV and 100lA, generating 1000 projections per scan. The extracted using Qi-Amp tissue extraction kits (Qiagen) and the different kV- and projection-settings depended on the respective peqGold Tissue DNA Mini Kit (PEQLAB Biotechnologie GmbH) or specimen size. Effective voxel size ranged between 8.3 to High Pure PCR Template Preparation kits (Roche) following the 31.2 lm and the exposure time was 750 ms. The cone beam recon- manufacturers protocols. Three nuclear [Seven-in-Absentia struction was performed using the datos|x-reconstruction software (SIA), Recombination Activation gene 1 (rag1) and Brain-derived (GE Sensing & Inspection Technologies GMBH phoenix|X-ray) and neurotrophic factor gene (BDNF)] and three mitochondrial genes the data were visualised and modified in VG Studio Max 2.0. [(12S rRNA, 16S rRNA, cytochrome b gene (cytb)] were amplified. Primers are given in Appendix Table A1. Amplification of 25 llor 3. Results and discussion 50 ll PCR reactions follow protocols given in Appendix Table A2. PCR products were purified using Qiaquick purification kits 3.1. Generated sequences (Qiagen). For quality assurance we sequenced both directions of the All six target loci, three mitochondrial and three nuclear, were amplified PCR product (using an external vendor Macrogen). analysed for a total of 52 ingroup and 11 outgroup terminals. In All sequences have been deposited in GenBank addition, a second dataset, containing a considerably larger num- (KF693275–KF693378 for 12S; KF693379–KF693476 for 16S; ber of samples with only 12S and 16S data were analysed contain- KF693477–KF693538 for BDNF; KF693539–KF693598 for SIA; ing a total of 103 ingroup and 11 outgroup terminals. KF693599–KF693658 for rag1; KF693659–KF693703 for cytb). 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,588bpofcytb, 396 bp of SIA,675bpofBDNF,930bpof 2.3. Molecular analyses rag1. Our second dataset including only 12S and 16S data consisted of 924 aligned base pairs (354 bp of 12S, 570 bp of 16S). Sequences were aligned using ClustalX (Thompson et al., 1997; default parameters) and manually checked using the original chro- matograph data in the program BioEdit (Hall, 1999). Ambiguities 3.2. Phylogenetic reconstructions were identified by eye and excluded from the analyses. Protein coding partitions (cytb, SIA, rag1, BDNF) were translated with the Herein, we present the first comprehensive analysis of African program TranslatorX (Abascal et al., 2010) to amino acids to deter- torrent frogs, based on several nuclear and mitochondrial gene par- mine codon positions and to check for absence of stop codons. titions. At present twelve Petropedetes species are recognised In order to test congruence in the topology of the present data- throughout their range in East (EA), Central (CA) and West Africa set we tested: (1) reduced dataset of mitochondrial sequences only (WA). However, the present phylogenetic analysis was inconsistent (12S & 16S as in Scott, 2005) and (2) combined nuclear and mito- with this arrangement, and the main clades corresponding to geo- chondrial genes (12S, 16S, cytb, BDNF, SIA, rag1). Two well estab- graphic regions do not form a monophyletic group (Figs. 1 and 2). lished techniques for phylogenetic estimation were applied: The results support some previous insights but are discussed anew. Bayesian Inference (BI; MrBayes, version 3.21 Â 64; Huelsenbeck Individual gene trees are not shown but, respective clades are and Ronquist, 2001; Ronquist et al., 2012) and Maximum Likeli- marked as bars and level of support indicated in Fig. 2. hood (ML; RAxML version 7.0.4; Stamatakis, 2006 using the rapid hill climbing algorithm following Stamatakis et al., 2007 and the 3.2.1. Analyses of molecular data GTR + G model). Our molecular analyses show that the monophyly of Petropede- The best-fit model of sequence evolution for each gene partition tes is not supported. Tree topologies with respective node support or respectively codon position was selected using jModeltest 2.1.2 values are shown in Fig. 1 for the 12S + 16S-tree and in Fig. 2 for the (Darriba et al., 2012, Appendix Table A3) using the Bayesian infor- nuclear and mitochondrial dataset. In both datasets the two ap- mation criterion (BIC). BIC was implemented in the partitioned BI plied phylogenetic approaches strongly agree in the overall topol- and additional single-gene analyses. Single-gene analyses of BI ogy, supporting the same terminal clades and taxa (including and ML (following aforementioned procedure) were performed to described and undescribed lineages). Three deeply divergent analyse clade stability of species lineages and major groupings in clades have been uncovered in African torrent frogs, each consis- different genes. tent with geography and reflecting the sub-Saharan distribution Bootstrap analyses with 1000 pseudoreplicates in the ML anal- (three separate clades EA, CA and WA). All three major clades were ysis were employed to evaluate the relative branch support in the supported by ML and Bayesian analyses (Figs. 1 and 2). Mean phylogenetic analysis. Bayesian analyses were run under parti- uncorrected genetic distances in the partial 16S rRNA gene among tioned schemes for 5 million generations using four chains sam- these major clades range between 14.12% and 20.83% (Table 2); in pling every 100 generations, with a burn-in of 1000 trees. Clades comparison, mean uncorrected genetic distances within these ma- with posterior probabilities (PP) P 95% were considered strongly jor clades were considerably lower (4.22–8.51%). Clades CA and EA supported. Stationarity has been checked with Tracer V1.5 (Ram- were more similar to each other 11.78–16.59%) than to WA (WA- baut and Drummond, 2007). Uncorrected p-distances between ma- CA: 17.35–22.91%; WA-EA 19.75–21.45%). jor clades and mean values of maxima within each region were Our analyses identified under-estimated species diversity in all calculated with PAUP* 4.0b10 for the partial 16S rRNA gene. regions. Clade WA includes Petropedetes natator and a second Author's personal copy

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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. Author's personal copy

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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.) Author's personal copy

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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 CA EA Mean intra lineage p-distance WA 17.35–22.91 19.75–21.45 4.22 ± 0.24 CA 19.39 ± 0.90 11.78–16.59 4.89 ± 1.04 EA 20.83 ± 0.40 14.12 ± 1.08 8.51 ± 2.04

undescribed taxon from West Africa. Clade CA includes P. cameron- Arthroleptides. Further research on the historical biogeography of ensis, P. euskircheni, P. johnstoni, P. juliawurstnerae, P. palmipes, P. these frogs is necessary and likely to be insightful on deep-time parkeri, P. perreti, P. vulpiae and four additional lineages from Cen- biogeography of African forests. tral 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 3.4. Systematic readjustment of the genus Petropedetes Reichenow, between terminal taxa still remained weakly supported and were 1874 therefore partially unresolved. Support values in the 12S + 16S- dataset were low in both analyses, yielding in a polytomy in the Our molecular and morphological analyses support the division 50% majority-rule consensus tree of the Bayesian analysis. In con- of Petropedetes into clades corresponding to geographically distinct trast, PP-values in the complete dataset supported a few of the in- and disjunct distributions of species in eastern, central, and wes- ner nodes while ML support values remained mostly weak tern Africa. The original reasoning for synonymizing EA Arthrolep- (support values below 70% not shown). Lineages from EA include tides into Petropedetes was it’s grouping with CA Petropedetes,to P. martiensseni, P. yakusini and one additional taxon (Nguru popu- the exclusion of the WA P. natator (Scott, 2005). However, this tax- lation) from East Africa (as outlined in Loader et al., 2013). onomic change rested on the assumptions that 1. EA Petropedetes While terminal taxa were in almost all cases clearly separated were relatively similar to CA Petropedetes, and 2. WA P. natator by the mitochondrial genes, nuclear markers did possess concor- was the nearest grouping to EA and CA Petropedetes. Our results dances in haplotypes of different taxa forming different clusters challenge these assumptions and suggest taxonomic changes. Lin- (not shown; rag1: P. euskircheni – P. sp. aff. euskircheni; BDNF: P. eages from CA and EA formed a well-supported clade; the type spe- euskircheni – P. sp. aff. euskircheni, P. juliawurstnerae – P. perreti, cies Petropedetes cameronensis belongs to clade CA (Figs. 1 and 2). P. sp. nov.2 – P. vulpiae; SIA: P. euskircheni – P. sp. aff. euskircheni, Both of these clades are assigned to the family Petropedetidae. P. parkeri – P. sp. nov.1). However, two of the nuclear markers However, given their substantial differences, we favour generic (rag1 and BDNF) supported the three major groupings and more- recognition, as was originally formulated, resurrecting Arthrolep- over, all three nuclear markers signiﬁcantly supported a closer tides for EA species. In contrast, positioning of clade WA remains relationship of the taxa grouped in EA and CA, than to those of unclear in relation to the included outgroup taxa and cannot be as- WA (bars in Fig. 2). signed 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 analy- 3.3. Biogeography of African torrent frogs sis including representatives of all Ranoidea families was beyond the scope of this work. As formerly conceived the family Petropedetidae showed distinct disjunct distributions across West, Central and East African biogeographic regions. From a historical biogeographical perspec- 3.4.1. Clade WA tive, interpreting the relationships among these taxa would have The substantial divergence of WA species from the remaining revealed the historical relationships among these areas. Such African torrent frogs necessitates the description of a new genus. groups are of particular importance, given that torrent frogs are of- The only named taxon P. natator becomes the genus type species. ten restricted to forest, and therefore their phylogenetic patterns Odontobatrachus gen. nov. Barej, Rödel, Loader, Schmitz might reﬂect historical changes in forest habitats. With the distant Type species: Petropedetes natator Boulenger, 1905 nov. comb. relationship of the West African clade, and its uncertain phyloge- Diagnosis: Osteology: large nasals, rectangular, in median con- netic relationships relative to the family Petropedetidae, our re- tact; nasals overlapping sphenethmoid; anterior end of frontopa- sults can only provide biogeographic information on the close rietals running rather rectangular to axis; vomer with a strongly relationship found between East African Arthroleptides and Central expressed posteromedial ramus (dentigerous process) that covers African Petropedetes. Given the considerable genetic distance the proximal end of the neopalatine and bears well developed between the two genera Arthroleptides and Petropedetes, one can teeth; large and posteriorly curved teeth on premaxillaries and assume the split of those two clades is likely an ancient event - anterior maxillaries as well as a tusk-like odontoid on the mandi- potentially linked to the early separation of East and West African ble; anterior ramus of pterygoid does not reach neopalatine; forests (Kingdon, 1990; Maley, 1996). The application of molecular zygomatic ramus of the squamosal longer than the otic ramus dating will be necessary to examine this in further detail. It would and with regular outlines; base of omosternum convex; medial be intriguing to understand the close phylogenetic relationship edges of coracoids not overlapping (Fig. 3). External morphology: that the West African clade has, and what this might suggest about tympanum indistinct; males with external vocal sacs; nuptial biogeography of the West African faunal region (compare Penner excrescences in breeding males velvety; femoral glands present et al., 2011). Furthermore, the Ethiopian monotypic genus Ericaba- in males only. trachus Largen, 1991, potentially closely related to Petropedetidae Genetics: All taxa included in this genus form a clade and can be (sensu Largen, 1991), might provide further information on the clearly differentiated from the remaining torrent-frog genera. biogeographic relationships across African biogeographic regions Within our coding nuclear dataset, the genus possesses 77 unique in Petropedetidae. It might be predicted if Ericabatrachus is a petro- character states in both taxa (rag1: 45, BDNF: 17, SIA: 15). A total of pedetid, that it would cluster with the geographically close genus 93 unique character states in nuclear genes distinguish Odontoba- Author's personal copy

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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 ap- Fig. 3. CT-scans of Odontobatrachus natator (ZMB 78203) scanned with 80 kV, plied nuclear genes distinguish all Arthroleptides spp. from Odonto- 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 batrachus spp. and 26 from Petropedetes spp. (Appendix Table A4). upper jaw and tusks on lower jaw, lower jaw virtually moved to open the mouth, Tadpole morphology and behaviour: parrot-beak-like jaw scale bar: 1.5 mm (d) pectoral girdle in ventral view – remaining skeleton in all sheaths; labial tooth rows present; oral disc with papillae; dorsal figures virtually removed, scale bar: 1.5 mm. fin reduced; ventral fin absent; development of limbs in early stages (Channing et al., 2002, 2012 and references therein). Tad- trachus spp. from Arthroleptides spp. and 88 from Petropedetes spp. poles persist on wet rock surfaces and live within a water film. (Appendix Table A4). Distribution: East Africa (Tanzania, Kenya; Appendix Fig. A1). Tadpole morphology and behaviour: head broad sucker-like mouth Included species: A. dutoiti Loveridge, 1935, A. martiensseni Nie- with enlarged lateral labial discs; anterior margin of mouth flap-like; den, 1911, A. yakusini Channing, Moyer & Howell, 2002. body dorso-ventrally compressed; lateral skin folds along body; Comment: Available data indicate presence of additional un- flattened belly extended as flap; tail muscular with narrow fin named taxa in East Africa. Menegon et al. (2008, 2011) pointed (Channing et al., 2012; Guibé and Lamotte, 1958; Lamotte and to the presence of morphologically divergent torrent frogs in Zuber-Vogeli, 1954). Tadpoles cling with their sucker-like mouth Nguru and Mahenge Mountains. This was supported by recent parts to rocks in the strongest currents in water falls and rapids. analyses in Loader et al. (2013) who discussed the phylogenetic Distribution:West Africa (Guinea, Sierra Leone, Liberia, western relationships and biogeography of this group. Côte d’Ivoire; Appendix Fig. A1). Included species: Odontobatrachus natator (Boulenger, 1905) nov. comb. 3.4.3. Clade CA Comment: Available data indicate the presence of more than one Based on the type species of the genus Petropedetes (P. cameron- species in West Africa. A detailed analysis of West African taxa is ensis) all species belonging to the Central African clade remain in beyond the scope of this work and will be treated separately. the genus Petropedetes.AsCornufer johnstoni (consequently also Differences between populations of the West African taxon have al- its synonym Tympanoceros newtonii, Barej et al., 2010a but see ready been recognised by Rödel and Bangoura (2004). A thorough below) is embedded within the genus Petropedetes, both names revision throughout the distribution range is under preparation. remain in the synonymy of the latter one. Etymology: The genus name refers to the Greek words odot1 Petropedetes Reichenow, 1874 (odous = tooth, genitive: odóntos) and basqavor (batrachos = Type species: Petropedetes cameronensis Reichenow, 1874. frog) and points to exceptionally long maxillary teeth and large Reference: Reichenow, 1874 Archiv für Naturgeschichte 1874, tusks on lower jaws. The ending is Latinised hence, following the 287–298 + 3 plates. ICZN the generic name Odontobatrachus is of male gender. Synonyms: Cornufer Boulenger, 1888 – in part; Tympanoceros du Common name: We advise to use the term ‘‘West African torrent Bocage, 1895. frogs’’ in English and ‘‘grenouilles des torrents d’Afrique de l’Ouest’’ Diagnosis: Osteology: small dorsolateral oriented nasals that do in French. not meet medially; anterior end of frontoparietals running acutely Remark: In accordance with article 8.5 of the International code angled to axis; vomer consisting of two parts and without poster- of Zoological Nomenclature (International Commission on Zoolog- omedial ramus; vomerine teeth present; small teeth on maxillaries ical Nomenclature 2012) the present publication (LSID: and no tusks on mandibles; pterygoid and neopalatine in contact; urn:lsid:zoobank.org:pub:67865554-55C4-443F-B56D-F8AA652EAA zygomatic ramus shorter than the otic ramus and with irregular 77) and nomenclatural act (LSID: urn:lsid:zoobank.org:act: outlines; metacarpals forming a spike in males (not all taxa); base 2E208480-FD27-4E01-AF8C-BDDD59874A4C) have been registered of omosternum concave to bifurcate; medial edges of coracoids not in ZooBank. overlapping. External morphology: tympanum indistinct or distinct; external vocal sacs absent; nuptial excrescences in breeding 3.4.2. Clade EA males spiny; minuscule spines may be present on chin, chest, dor- Assignment of all East African taxa to a single supported clade sum and flanks in breeding males; femoral glands present in both being clearly distinguished from clades CA and WA requires resur- sexes (larger in males); reversed sexual size dimorphism with rection of the genus Arthroleptides Nieden, 1911. Although not males growing larger than females in a few taxa. Author's personal copy

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Genetics: All taxa included in this genus are most similar to each more data are needed to clarify the resolution of populations. other, forming a clade and can be clearly differentiated from the Based on external morphological features, taxa P. sp. 2 and sp. 3 remaining torrent-frog genera. Within our coding nuclear dataset, (both from the Cristal Mts., Gabon) clearly resemble P. vulpiae the genus possesses 10 unique character states throughout all taxa and would therefore fit with a biogeographical split for P. vulpiae (rag1:7,BDNF:2,SIA: 1). A total of 88 unique character states in as already assumed by Barej et al. (2010a). Petropedetes samples applied nuclear genes distinguish all Petropedetes spp. from Odonto- collected on Fernando Po (= Bioko, Equatorial Guinea) genetically batrachus spp. and 26 from Arthroleptides spp. (Appendix Table A4). match samples of P. vulpiae from the mainland – including the type Tadpole morphology and behaviour: parrot-beak-like jaw locality. Bioko samples cluster with nearby coastal P. vulpiae pop- sheaths; labial tooth rows present; oral disc with papillae; tail very ulations on the foot of Mt. Cameroon, proving the existence of this muscular, ventral fin absent; development of limbs in early stages species distribution on the island of Bioko. Type material of Tympa- (Barej et al., 2010a; Channing et al., 2012 and references therein). noceros newtonii is lost, and du Bocage’s (1895) species description Tadpoles persist on wet rock surfaces and live within a water film. and figures are insufficient to distinguish the taxon from Petropede- Lawson (1993) reported on semi-terrestrial tadpoles that are tes johnstoni. Barej et al. (2010a) had placed P. newtonii in synon- attached to leaves. ymy of P. johnstoni, as topotypic vouchers collected by L. Fea on Distribution: Central Africa (Nigeria, Cameroon, Gabon, Equato- Fernando Po (= Bioko, Equatorial Guinea) from the collection of rial Guinea, Republic of Congo; Appendix Fig. A1). Museo Civico di Storia Naturale di ‘‘Giacomo Doria’’ (MSNG) mor- Included species: Petropedetes cameronensis Reichenow, 1874, P. phologically correspond to the latter species. Consequently, a euskircheni Barej, Rödel, Gonwouo, Pauwels, Böhme & Schmitz, new name was given to a clearly defined taxon on the mainland 2010, P. johnstoni (Boulenger, 1888 ‘‘1887’’), P. juliawurstnerae (Barej et al., 2010a). Hence, the validity of P. newtonii still remains Barej, Rödel, Gonwouo, Pauwels, Böhme & Schmitz, 2010, P. palm- uncertain. ipes Boulenger, 1905, P. parkeri Amiet, 1983, P. perreti Amiet, 1973, Drewes and Vindum (1994) reported Petropedetes tadpoles on P. vulpiae Barej et al., 2010a. the eastern side of the Congo Basin from Uganda. The identification Comments and taxonomic remarks: Unexpectedly and despite was later revised and assigned to Amietia (Frost, 2013). However, the use of several molecular markers including mitochondrial recently Behangana et al. (2009) report the finding of Petropedetes and nuclear genes, the relationships within the most species-rich sp. in one site of the Albertine Rift without giving any additional clade CA could not be solved unambiguously. The present phyloge- data on their finding or collection numbers of respective vouchers. netic analysis supports the occurrence of additional taxa in Central Hence, occurrence of the genus east of the Congo Basin still needs Africa (see Barej et al., 2010a). Thereafter, four more taxa have to be verified and remains intriguing. been uncovered, two occurring in Cameroon and two in Gabon. A detailed morphological analysis of species diagnosis is beyond 4. Conclusion the scope of this work and will be treated separately. Petropedetes sp. nov. 1 occurs in the Bamenda Highlands and Based on molecular analyses and morphology, African torrent western part of the Adamawa Plateau in Cameroon. A second un- frogs represent three geographic clades occupying three distinct named taxon (P. sp. aff. euskircheni) from Mt. Nlonako formerly sub-Saharan regions of West, Central and East Africa. Based on tentatively assigned to P. euskircheni by Barej et al. (2010a), has clear molecular distinctiveness and morphological and osteological proven to be distinct in our analyses. Despite repeated surveys of characters (adults, and/or tadpoles), these clades are recognised as the locality, only females have been collected; however, only sec- three distinct genera: Arthroleptides, Odontobatrachus gen. nov. and ondary sexual characters of breeding males allow for an unambig- Petropedetes. While East (Arthroleptides) and Central (Petropedetes) uous determination in terms of morphological traits (Amiet, 1973, African taxa belong to the family Petropedetidae, the new West 1983; Barej et al., 2010a). Mitochondrial DNA data clearly separate African genus could not be assigned to any of the herein included a taxon restricted to Mt. Nlonako, compared to the more widely families and is kept as incertae sedis until more data are available. distributed P. euskircheni, but this taxon lacks differences in the nu- All three clades contain additional undescribed taxa. clear 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 Author contributions (P. sp. nov. 2 and P. sp. nov. 3) are represented by a single sequence each. Both specimens were collected in the Cristal Mountains Ga- MFB, AS, MOR, SPL designed the study. MM, NLG, JP, VG, RB and bon, supporting the presence of new taxa south of Cameroon (Barej PN provided important samples and data. Molecular analyses were et al., 2010a). performed by MFB, AS, SPL. Morphological analyses were per- New samples of P. juliawurstnerae not only confirm the assumed formed by MFB and RG. Data interpretation and writing was done distribution (Bakossi Mts., Cameroon, see Barej et al., 2010a) but by MFB. All authors read, commented on and approved the final also extend the currently known range to the West (Rumpi Hills, manuscript. Cameroon). In the case of P. euskircheni, the distribution has been likewise extended to the west (Fotabong and Rumpi Hills, Camer- Acknowledgments oon), while the nearby Mt. Nlonako is inhabited by a distinct taxon (see above). Research and export permits were provided by the Ministries The highest genetic diversity has been found within P. vulpiae, from Guinea, Sierra Leone, Liberia, Nigeria, Cameroon, Gabon, including three distinct subclades. Although the final analysis of Equatorial Guinea and Tanzania. We thank two anonymous the concatenated data depicts a well-supported terminal clade reviewers and the editor for their comments on our manuscript for the species, the situation is more complex. Within our single- and their valuable suggestions, how to improve the present work. gene analyses three terminal taxa (P. johnstoni, sp. 2 and sp. 3) shift We thank the Tanzania Commission for Science and Technology their position between P. vulpiae subclades, preventing a clear (COSTECH research permit RCA 2001-272; RCA 2007-153), TAWIRI understanding of their relationships, despite a high pairwise genet- and Wildlife Division for granting permission to conduct research ic similarity between populations of P. vulpiae. Distributions of in Tanzania and export these specimens. For research conducted recognised subclades indicate potential biogeographical splits in Gabon, RCB thanks the Wildlife Conservation Society for logisti- along the lowlands of the Biafra Bay within this taxon; however, cal support, the Centre National de la Recherche Scientifique et Author's personal copy

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Technologique (CENAREST) and Agence Nationale des Parcs Boulenger, G.A., 1905. Descriptions of new West-African frogs of the genera Nationaux (ANPN) for research permits and the Direction de la Petropedetes and Bulua. Ann. Mag. Nat. Hist. 15, 281–283. Burgess, N.D., Butynski, T.M., Cordeiro, N.J., Doggart, N., Fjeldså, J., Howell, K., Faune et de la Chasse for export permits. This work was funded Kilahama, F., Loader, S.P., Lovett, J.C., Mbilinyi, B., Menegon, M., Moyer, D., by various organisations including the Swiss National Science Nashanda, E., Perkin, A., Stanley, W., Stuart, S., 2007a. The biological importance Foundation (31003A-133067) to SPL. MFB got a SYNTHESYS grant of the Eastern Arc Mountains of Tanzania and Kenya. Biol. Conserv. 134, 209– 231. (GB-TAF-1474) to examine the type material of O. natator; this stay Burgess, N.D., Balmford, A., Cordeiro, N.J., Fjeldså, J., Küper, W., Rahbek, C., was supported by D. Gower (BMNH). Thanks to K. 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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 primer sequences study/source 16sar-L 5' - CGC CTG TTT ATC AAA AAC AT - 3' Palumbi et al.,1991 16S 16sbr-H 5' - CCG GTC TGA ACT CAG ATC ACG T - 3' Palumbi et al.,1991 12S L1091 5' - AAA CTG GGA TTA GAT ACC CCA CTA T - 3' Kocher et al., 1989 12S 12S H1478 5' - GAG GGT GAC GGG CGG TGT GT - 3' Kocher et al., 1989 Bossuyt and Milinkovitch, CBJ10933 5' - TAT GTT CTA CCA TGA GGA CAA ATA TC - 3' 2000 cytb Bossuyt and Milinkovitch, cytb C 5' - CTA CTG GTT GTC CTC CGA TTC ATG T - 3' 2000 CB2F 5' - TGA GGA CAA ATA TCT TTT TGA GGG - 3' Hillis et al., 1996 cytb CB3R 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, BDNF-F ATA CTT - 3' 2006 BDNF 5' - CTA TCT TCC CCT TTT AAT GGT CAG TGT ACA Noonan and Chippindale, BDNF-R AAC - 3' 2006 SIA1 (T3) 5 ' -TCGAGTGCCCCGTGTGYTTYGAYTA - 3' Bonacum et al., 2001 SIA SIA2 (T7) 5' - GAAGTGGAAGCCGAAGCAGSWYTGCATCAT - 3' Bonacum et al., 2001 Mart-FL1 5' - AGC TGC AGY CAR TAY CAY AAR ATG TA - 3' Pramuk et al., 2008 rag1 AMP-R1 5' - AAC TCA GCT GCA TTK CCA ATR TCA - 3' Pramuk et al., 2008

Supplementary data 3

Applied PCR protocols.

12S 16S cytb BDNF SIA rag1 initial denaturation 94°C à 90s 94°C à 90s 94°C à 120s 94°C à 300s 94°C à 120s denaturation 94°C à 45s 94°C à 45s 94°C à 30s 94°C à 20s 94°C à 60s 94°C à 60s 62°C à 60s annealing 35x 50°C à 60s 30x 54°C à 30s 40x 54°C à 45s 39x 57°C à 57s 17x [modify: - 35x 50°C à 60s 1° each cycle] extension 74°C à 120s 72°C à 60s 72°C à 60s 72°C 120s 72°C à 60s 72°C à 60s denaturation 94°C à 60s annealing 20x 48°C à 60s extension 72°C à 60s final extension 72°C à 420s 72°C à 420s 72°C à 600s 72°C à 600s 72°C à 420s 72°C à 420s

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 aligned locus sequence BIC sequences taxon coverage lengths 12S 354 bp TIM2ef+G 91 22 16S 570 bp TIM2+I+G 109 22 Pos.1 TPM2+G cytb 588 bp Pos.2 TrN+G 39 19 Pos.3 TrN+G Pos.1 K80+G rag1 930 bp Pos.2 K80+G 52 22 Pos.3 K80+I Pos.1 K80+G BDNF 675 bp Pos.2 JC 54 22 Pos.3 JC Pos.1 K80+G SIA 396 bp Pos.2 K80+I 52 22 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 8 9 10 11 12 13 14 15 16 17 18 19 20 base postion 9 12 30 32 39 58 63 105 111 129 147 169 195 199 227 240 246 267 283 284 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 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 T T G T G A C T G T T T Arthroleptides A T A A G T C A T G A C A G T C A C/T C C Petropedetes A T G A A/G C C A C A/G G C A G T C A C T T

rag1 61 62 63 64 65 66 67

base postion 849 886 894 900 907 924 924

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 8 9 10 11 12 13 14 15 16 17 18 19 20 base postion 43 45 141 144 146 162 174 186 309 339 375 408 411 432 447 459 465 504 528 543 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

SIA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

bp 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