Zoologica Scripta
Sky Islands of the Cameroon Volcanic Line: a diversification hot spot for puddle frogs (Phrynobatrachidae: Phrynobatrachus) BREDA M. ZIMKUS &VACLAV GVOZDIK
Submitted: 26 February 2013 Zimkus, B. M. & Gvo zd ık, V. (2013). Sky Islands of the Cameroon Volcanic Line: a Accepted: 28 June 2013 diversification hot spot for puddle frogs (Phrynobatrachidae: Phrynobatrachus). —Zoologica doi:10.1111/zsc.12029 Scripta, 42, 591–611. The continental highlands of the Cameroon Volcanic Line (CVL) represent biological ‘sky islands’ with high levels of species richness and endemism, providing the ideal opportunity to understand how orogenesis and historical climate change influenced species diversity and distribution in these isolated African highlands. Relationships of puddle frogs (Phrynobatra- chus) endemic to the CVL are reconstructed to examine the patterns and timing of puddle frog diversification. Historical distributions were reconstructed using both elevation and geography data. Puddle frogs diversified in the CVL via several dispersal and vicariance events, with most of the locally endemic species distributed across the northern part of the montane forest area in the Bamenda-Banso Highlands (Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and medium elevation areas connecting these mountains). Two new species, P. jimzimkusi sp. n. and P. njiomock sp. n., are also described based on molecular analyses and morphological examination. We find that these new species are most closely related to one another and P. steindachneri with the ranges of all three species overlapping at Mt. Oku. Phrynobatrachus jimzimkusi sp. n. is distributed in the southern portion of the continental CVL, P. njiomock sp. n. is endemic to Mt. Oku, and P. steindachneri is present in the northeastern part of the montane forest area. Both new species can be distinguished from all other puddle frogs by a combination of morphological characters, including their large size, ventral coloration and secondary sexual characteristics present in males. These results highlight the Bamenda-Banso Highlands, and specifically emphasize Mt. Oku, as a centre of diversification for puddle frogs, supporting the conservation importance of this region. Our results also provide new insights into the evolutionary processes shaping the CVL ‘sky islands’, demonstrating that lineage diversification in these montane amphibians is significantly older than expected with most species diverging from their closest relative in the Miocene. Whereas climatic changes during the Pliocene and Pleistocene shaped intra- specific diversification, most speciation events were significantly older and cannot be linked to Africa’s aridification in response to Pleistocene climate fluctuations. Corresponding author: Breda Zimkus, Museum of Comparative Zoology and Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. E-mail: [email protected] V aclav Gvo zd ık, Laboratory of Molecular Ecology, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Lib echov, Czech Republic; Department of Zool- ogy, National Museum, 193 00 Prague, Czech Republic; Department of Environmental Sciences, Biogeography, University of Basel, Klingelbergstrasse 27, 4056 Basel, Switzerland. E-mail: [email protected]
Introduction islands, the dispersal of sky island species is limited by low- Sky islands are high-elevation habitats that are geographi- elevation habitat, facilitating divergence of isolated popula- cally separated and isolated among different mountains tions. Like their oceanic counterparts, sky islands are gen- (Heald 1951). Similar to the waters separating oceanic erators of diversity over multiple spatial and temporal
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 591 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık scales and offer considerable potential to investigate how dif- number of puddle frogs of the genus Phrynobatrachus (Zim- ferent evolutionary processes lead to species formation. Ver- kus 2009; Blackburn 2010; Blackburn & Rodel€ 2011). tebrates within sky island complexes have experienced Puddle frogs are one of the most diverse sub-Saharan multiple climate-induced distributional shifts, most notably anuran lineages with 85 species currently recognized in association with the Pleistocene glacial cycles. It is sug- (Zimkus et al. 2010; Frost 2013). A clade of species was gested that many temperate vertebrate lineages have evolved found to be endemic to the highlands of Cameroon and as a result of the Pleistocene refugial isolation and subse- Nigeria using molecular analyses of mitochondrial DNA quent lineage formation, following the ‘species pump’ model (mtDNA), supporting the view that montane regions have of diversification (Avise & Walker 1998; Avise et al. 1998; acted as centre of speciation for Phrynobatrachus (e.g. Zim- Johnson & Cicero 2004). However, a pattern of pre-Pleisto- kus 2009; Zimkus et al. 2010). This clade currently com- cene origination has been found in numerous vertebrates, prises seven species from submontane (901–1800 m) and challenging this hypothesis (Zink & Slowinski 1995; Klicka montane (>1800 m) elevations, including P. chukuchuku, & Zink 1997; Roy et al. 2000; Bowie et al. 2004; Pyron & P. cricogaster, P. danko, P. manengoubensis, P. schioetzi, Burbrink 2009). Little is known regarding the patterns and P. steindachneri and P. werneri, and a single lowland timing of diversification of taxa within tropical sky islands, (<900 m) species, P. batesii (Blackburn 2010; Blackburn & which have been explored less than their lowland, temperate Rodel€ 2011). Zimkus (2009) found two lineages within counterparts. A small number of studies on birds and small P. steindachneri, differing from each other by an uncorrected mammals of the Afromontane ecoregion suggest that Africa’s genetic distance of approximately 3% in 12S-16S, which is aridification in response to Pleistocene glaciation in temper- considered a threshold value in the identification of unrec- ate zones has had an influence on montane speciation (Smith ognized amphibian species (Vieites et al. 2009). This was et al. 2000; Bowie et al. 2006; Missoup et al. 2012). It is, how- not a surprising result given that Amiet (1978) had identi- ever, unclear how climatic changes may have influenced fied a number of potential new species from Cameroon, other montane vertebrate species, including amphibians. including Phrynobatrachus sp. 11, collected only near Lake The mainland highlands of the Cameroon Volcanic Line Oku, and Phrynobatrachus sp. 2, which was believed to be (CVL) are Afrotropical sky islands that comprise the moun- distributed from Mt. Manengouba to Tchabal Mbabo (see tains of western Cameroon and eastern Nigeria (Burke also Amiet 1971 as ‘Phrynobatrachus sp.’, Amiet 1976; Gart- 2001). These highlands extend from Mt. Cameroon in the shore 1986). Unfortunately, these species were never for- southwest to the Bamenda-Banso Highlands and Adamawa mally described. These results prompted further Plateau (including Gotel Mountains and Tchabal Mbabo) investigation of this highland endemic clade with a focus on in the northeast of Cameroon and also include the Obudu P. steindachneri, distributed across mountains of the CVL. and Mambilla Plateaus in eastern Nigeria. The CVL is of Here, we examine the patterns and timing of puddle frog tectonic and volcanic origin but with unclear chronological diversification across the CVL to test three hypotheses. history (Marzoli et al. 2000; Montigny et al. 2004; D eruelle First, we use dispersal–vicariance analysis of elevational and et al. 2007; Koch et al. 2012; Milelli et al. 2012). The oldest geographical data to infer the biogeographical history of part in the extreme north near Lake Chad dates to the Late puddle frogs of this region to determine if: (i) puddle frogs Cretaceous (Mbowou et al. 2012). However, the vast major- colonized highland habitats multiple times independently ity of the CVL was uplifted during the Cenozoic (Bamen- and (ii) the ancestor to the clade that includes all puddle da-Banso Highlands likely from the Oligocene to Miocene) frogs of the CVL was distributed in the large, central high- with a trend of decreasing age of volcanic activity in the land region, the Bamenda-Banso Highlands, which includes southwestern direction (Marzoli et al. 2000). The youngest Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and med- volcanoes, Mt. Cameroon and Mt. Manengouba, are of Pli- ium elevation areas connecting these mountains. We also ocene to Pleistocene origin. High species richness and use dating methods to estimate divergence times to deter- endemism are thought to be a result of in situ speciation mine if: (iii) Pleistocene glaciation in temperate zones had over this region’s long and complex orogenic history (Lou- a major impact on speciation within puddle frogs of the ette 1981; Stuart 1986; Lawson 1993; Bowden & Andrews CVL. Lastly, we describe two new species of puddle frogs, 1994; Stattersfield et al. 1998; Myers et al. 2000; Lovett & which are diagnosed on the basis of morphological charac- Taplin 2004). Numerous amphibian species have been ters and supported by mtDNA sequence divergences. The recently discovered in this region as a result of both addi- description of these two species of puddle frogs and reanal- tional fieldwork and re-examination of museum specimens ysis of biogeography of this clade reaffirms the CVL as an (Amiet & Dowsett-Lemaire 2000; Boistel & Amiet 2001; important region for amphibian speciation and identifies Herrmann et al. 2004; Rodel€ et al. 2004; Plath et al. 2006; the Bamenda-Banso Highlands, and more specifically, Mt. Blackburn 2008; Blackburn et al. 2009, 2010), including a Oku, as a centre of speciation in puddle frog evolution.
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Material and methods removed, the resulting consensus alignment was 2286 bp in DNA extraction, PCR, amplification and sequencing length, 110 base pairs less than the shortest alignment (gap The mitochondrial DNA fragment spanning approximately cost 20). 1.9 kb of the 12S rRNA, tRNA-valine and 16S rRNA Per cent pairwise uncorrected molecular distances genes (12S-16S hereafter) was chosen due to its previous between species were analysed using PAUP* 4b10 (Swof- use in the construction of a molecular phylogeny of this ford 2000). Estimates of phylogeny were obtained using group (Zimkus et al. 2010). DNA was extracted from mus- Bayesian inference (BI) and maximum likelihood (ML) cle or liver tissue fixed in 99% ethanol using Qiagen methods. Based on Zimkus et al. (2010), only those species DNeasy tissue kits (Qiagen Inc, Valencia, CA, USA). within the clade of interest were included; Phrynobatrachus Amplification followed the standard PCR conditions (Pa- africanus was used to root the phylogeny. Bayesian analyses lumbi 1996) with the following thermal cycle profile: were conducted with MRBAYES 3.2.1 (Ronquist et al. 2012). 2 min at 94 °C, followed by 35 cycles of 94 °C for 30 s, Four Markov chains were run for 20 million generations 46 °C for 30 s, and 72 °C for 60 s and a final extension that were sampled every 2000 generations using four phase at 72 °C for 7 min. All amplified PCR products were chains, at a temperature of 0.2. Three additional replicates verified using electrophoresis on a 1.0% agarose gel stained were performed to confirm that Markov chain Monte with SYBR Safe DNA gel stain (Invitrogen Corporation, Carlo (MCMC) searches were not trapped at local optima. Carlsbad, CA, USA). PCR products were purified using A model of molecular evolution was selected from the the Qiagen DNeasy DNA Purification System according to data using MRMODELTEST v. 2 (Nylander 2004) for BI and the manufacturer’s recommendations. DNA sequences were JMODELTEST v. 2.1.1 (Darriba et al. 2012) for ML. ML anal- obtained on an automated DNA sequencer (ABI PRISM ysis was implemented in PHYML v. 3.0 (Guindon et al. 2010) 3730xl Foster City, CA, USA). Editing and assembly of using the best approach, which combines nearest neighbour contigs were completed in SEQUENCHER 5.0 (Gene Codes interchanges with the subtree pruning and regrafting algo- Corp., Ann Arbor, MI, USA). Molecular data were com- rithm, and employing the transition model TIM2 + I + G piled with previously published sequence data, representing [A-C = A-T = 6.4174, A-G = 25.7378, A-T = 6.4174, a total of 12 species and including novel data generated C-T = 56.0317, C-G = G-T = 1.0000, p-inv = 0.6040, from 38 individuals (Table S1). All novel nucleotide gamma = 0.8510]. Both the hierarchical likelihood ratio sequence data were deposited in GenBank (KF020520– tests and the Akaike Information Criterion implemented in KF020557). MRMODELTEST selected a general time-reversible (GTR) + gamma (G) + proportion of invariable sites (I) model with Sequence alignment and phylogenetic analyses a gamma distribution of 0.7519 and a proportion of invari- Previous estimates of phylogeny of this clade have differed able sites of 0.2961 (estimated base frequencies: in several key respects (Zimkus 2009; Blackburn 2010; A = 0.3370, C = 0.2364, G = 0.1775, T = 0.2491; rate Zimkus et al. 2010; Blackburn & Rodel€ 2011). To explore matrix: A-C = 5.7004, A-G = 26.2128, A-T = 7.5313 the effect of alignment on phylogeny estimation of this C-G = 1.0437, C-T = 57.1910, G-T = 1.0000). For BI, clade, three different multiple alignments were generated plots of likelihood vs. generation number were used to with differing gap cost parameters (10, 15 and 20) in determine that stationarity was reached at <50 000 genera- CLUSTALX 2.1 (Larkin et al. 2007). Each alignment was tions; however, 10% were excluded from computation of checked manually using MACCLADE 4.08 to ensure that large posterior probabilities to ensure a sufficient burn-in value. stretches of data were not misaligned (Maddison & Maddison A total of 36 000 trees (9000 post-burn-in trees from four 2005). Subsequently, a consensus alignment was generated separate runs) were used to construct a 50% majority rule in which all nucleotide positions differing between the consensus tree. Topologies with posterior probabilities three alignments were excluded. This follows the exclusion >95% were considered well supported (Wilcox et al. 2002). alignment method advocated by Gatesy et al. (1993), Nodal support for ML topology was assessed through non- excluding characters for which the homology is uncertain. parametric bootstrap analysis; 1000 bootstrap replicates The results of phylogenetic analyses using the consensus were performed. Branches present in ≥70% of replicates alignment were then compared with those of the align- were considered well supported (Hillis & Bull 1993). ments generated using different gap costs. The final data Bayesian inference and ML analyses of the four align- matrices for the three alignments generated using differing ments (three produced using differing gap cost parameters gap costs varied slightly in length due to differences in the and a consensus alignment that removed the characters dif- numbers of insertions and deletions (gap cost 10: 2412 bp; fering between the previous three alignments) produced gap cost 15: 2398 bp; gap cost 20: 2396 bp). When the trees with largely congruent topologies. There were two characters that differed between the three alignments were major differences in the topologies among these analyses:
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 593 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık
(i) the relative placement of P. chukuchuku and (ii) the rela- Van Bocxlaer et al. (2006) using Sanderson’s penalized like- tionships among the three species of the P. steindachneri lihood method. Bayesian MCMC was performed for complex. In the phylogenies produced using alignments 20 million generations with a sample frequency of 1000 generated with differing gap costs, P. chukuchuku formed a generations, and a burn-in of 10%. Node ages were calcu- polytomy with the two major clades. In the consensus lated as mean node heights from the maximum clade credi- alignment, there were low measures of support for the sis- bility tree summarized from the posterior distribution of ter relationship between the two major clades, which sug- trees. gests that the placement of P. chukuchuku remains problematic. The resolution of the relationships among the Biogeographical analyses species of the P. steindachneri complex was also somewhat Historical distributions of puddle frogs from the CVL were variable. In the consensus alignment, the two new species reconstructed using both elevation and geography data. were most closely related to one another. Although there The first analysis was performed to determine whether spe- was high support for a monophyly of the P. steindachneri cific clades were partitioned by elevation, assigning individ- complex, there were low support values for the nodes uals to low (<900 m), medium (901–1800 m) or high within the complex. As statistical dispersal–vicariance analy- (>1800 m) elevation (Zimkus 2009). These elevational des- sis (S-DIVA) requires a fully resolved tree, we chose to use ignations correspond to key differences in vegetation the consensus alignment for final analyses. We henceforth among lowland, submontane and montane regions. The use only the consensus alignment and refer to the results elevational stratification that occurs across these mountains of these reconstructions in the Results Section. is quite distinct: sub-Saharan submontane forests exist We constructed haplotype networks based on a fragment between 900 and 1800 m, while above this exists a mixture of the 16S rRNA gene (approximately 500 bp) to investi- of montane elements, including distinct montane forests, as gate the evolutionary relationships among haplotypes well as patches of montane grasslands, bamboo forests and within the P. steindachneri complex using the algorithm subalpine communities (White 1983). Previous studies of given by Templeton et al. (1992) and implemented in the fauna across the CVL support delineation among lowland, TCS software package version 1.21 (Clement et al. 2000). submontane and montane zones, although the vegetation The statistical parsimony algorithm links haplotypes with of the CVL montane zone is sometimes considered transi- the smaller number of differences as defined by a 95% tional between submontane and montane in its characteris- confidence criteria and identifies the most probable ances- tics (Amiet 1971, 1976; Hofer et al. 1999; Gonwouo et al. tral haplotype according to coalescent theory (Castelloe & 2006). A second analysis defined eight biogeographical Templeton 1994). units in Cameroon and Nigeria: (i) Lowland (regions <900 m); (ii) Mt. Manengouba, Cameroon; (iii) Mt. Nlo- Dating estimates nako, Cameroon; (iv) Bamenda-Banso Highlands (including Diversification times were estimated through an uncorrelat- Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and med- ed relaxed-clock model for which the evolutionary rate in ium elevation areas connecting these mountains); (v) each branch is drawn independently from a lognormal dis- Obudu Plateau, Nigeria; (vi) Mambilla Plateau, Nigeria; tribution implemented in BEAST v1.7.1 (Drummond & (vii) Gotel Mountains, Cameroon and Nigeria; (viii) Tcha- Rambaut 2007). This relaxed-clock model performs well bal Mbabo, Cameroon. Both analyses relied on statistical with both clocklike and nonclocklike data (Drummond dispersal–vicariance analysis (S-DIVA; Yu et al. 2010) as et al. 2006). As the fossil record of sub-Saharan frogs is implemented in RASP 2.0 (Yu et al. 2011). This parsimony- deficient for Phrynobatrachidae and there is no unambigu- based approach reconstructs ancestral areas based on a ously dated paleogeographical vicariant event, we compared sample of trees, thereby generating credibility support val- divergence time estimates from previous large-scale studies ues for alternative phylogenetic hypotheses. Ancestral areas that contained Phrynobatrachus species (Bossuyt et al. 2006; were reconstructed using post-burn-in trees generated Van Bocxlaer et al. 2006; Roelants et al. 2007). We using BEAST v1.7.1 (see Dating Estimates for additional included sequence data of one additional outgroup, P. na- information). To explore the effects of area constraints, we talensis, to allow us to calibrate our tree using the diver- performed two S-DIVA runs; the first run allowed a maxi- gence between P. africanus and P. natalensis. As these three mum of two areas for a clade’s ancestral range, while the previous studies included the same sequences for P. afric- second did not constrain the maximum number of areas. anus and P. natalensis and resulted in similar divergence estimates, we constrained the age of the divergence Morphology between P. africanus and P. natalensis to 20.6 Æ 2.1 mya This work was based on study of specimens from museum with a uniform prior distribution, following the result of collections (Appendix S1). Assignment of particular spe-
594 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line cies to Phrynobatrachus follows Frost (2013). Museum tree was <50%. The second major clade was well supported abbreviations correspond to those of Sabaj P erez (2013). using both methods and contained P. cricogaster and the The following measurements were taken to the nearest P. steindachneri complex. The P. steindachneri complex con- 0.1 mm under a dissecting microscope using digital calipers: tained six geographically defined and well-supported lin- snout–vent length (body length), measured from snout tip eages (Fig. 2), two of them from the southwestern and to posterior margin of vent; head width, measured at great- central part of the range (Obudu Plateau, Mt. Manen- est head width in close proximity to the angles of the jaws; gouba, Bamboutos Mts., Mt. Lefo, Mt. Oku), which we head length, measured from to snout tip to angle of jaw; describe herein as two new species. The other four lineages tympanum diameter, measured at greatest tympanum width; that formed a clade were distributed in the northeastern eye diameter, measured at greatest anteroposterior diameter part of the range in the region of type locality of P. steind- of upper eyelid; interorbital distance, measured at the level achneri (‘Banjo’ [=Banyo, Adamawa Province, Cameroon]; of the interorbital bar; internarial distance, measured from Nieden 1910; Amiet 1971; Fig. 2B). Intraspecific sequence the centre of one external naris to the other; snout length, variation in P. steindachneri s.s. (N = 15) was high, ranging measured from anterior border of eye to snout tip; snout– up to 3.92%. As genetic data from specimens collected nostril length, measured from the centre of external naris from the type locality of P. steindachneri were unavailable, to snout tip, approximately in parallel to body axis; eyelid– we tentatively identified these four clades as P. steindachneri nostril length, measured from the corner of the eye to the sensu stricto (s.s.). The four lineages of P. steindachneri s.s. centre of external naris; humerus length, measured from were recorded from: (i) Bamenda-Banso Highlands (Mt. body wall to distal portion of elbow; radioulna length, mea- Mbam and Mt. Oku); (ii) Mambilla Plateau and Gotel sured from elbow to proximal edge of most proximal pal- Mountains; (iii) Gotel Mountains; (iv) Tchabal Mbabo. mar tubercle; femur length, measured from centre of vent Statistical parsimony analysis of the three major lineages to distal portion of knee; tibiofibula length, measured from within the P. steindachneri complex is shown in Fig. 3. knee to proximal edge of ankle; foot and tarsus length, Haplotypes of P. sp. 2 (sensu Amiet 1976, 1978; Gartshore measured from the proximal edge of the ankle to the tip of 1986) were from the Bamenda-Banso Highlands, Mt. Ma- the fourth toe; digital length, measured from proximal edge nengouba and the Obudu Plateau with the most probable of the most proximal subarticular tubercle to distal tip; inner ancestral haplotype from Mt. Oku within the Bamenda- and outer metatarsal tubercles lengths, measured parallel to Banso Highlands. Haplotypes of P. sp. 11 (sensu Amiet hind limb axis at the greatest lengths. Males were identified 1978) were restricted to Mt. Oku (Lake Oku and forest by the presence of testes or secondary sexual characteristics, reserve northeast of summit) within the Bamenda-Banso which includes dark-coloured throats, presence of vocal Highlands with the most probable ancestral haplotype from folds on the lateral margins of the throat, gular spines or Lake Oku. Haplotypes of P. steindachneri from the Bamen- nuptial excrescences on the medial and dorsal surface of the da-Banso Highlands (Mt. Mbam and Mt. Oku), including first finger (Stewart 1967; Zimkus & Blackburn 2008). the most probable ancestral haplotype from Mt. Oku, were Females were identified by either the presence of ova and/ linked with fewest connections to those from the Mambilla or large body size without exhibiting secondary sexual Plateau and Gotel Mts. A second group of P. steindachneri traits of mature males. The webbing formula used is haplotypes from the Gotel Mountains were linked to those based on Savage & Heyer (1997) without use of the + and from the Mambilla Plateau, rather than the Gotel Mts., À annotation (i.e. Myers & Duellman 1982). which was supported by results of molecular phylogenetic analysis (Figs 1 and 2). Haplotypes of P. steindachneri from Results Tchabal Mbabo were not found to be linked to others Molecular phylogenetics using a 95% confidence criteria. Relationships among Phrynobatrachus reconstructed utilizing the consensus alignment (see Materials and Methods: Dating estimates and biogeographical analyses Sequence alignment and Phylogenetic Analysis) resulted in the The monophyletic clade that includes all highland taxa same topology using BI and ML methods (Fig. 1). A clade restricted to the Cameroon Volcanic Line (CVL clade), as of highland endemics that included two lowland species, well as two lowland species, P. batesii and P. ruthbeateae, P. batesii and the recently described P. ruthbeateae, was well originated in the Early Miocene (Fig. 1). The majority of supported. Phrynobatrachus chukuchuku was sister to two speciation events within this clade took place within the major clades but with low support values. The first clade Middle to Late Miocene, while intraspecific diversifications contained the two lowland species, as well as P. danko, occurred in the very Late Miocene, continuing through the P. manengoubensis, P. schioetzi and P. werneri. This clade Pliocene and Pleistocene. S-DIVA analyses of elevation was well supported in BI, but the support value for the ML data revealed that the ancestor of the CVL clade was likely
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 595 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık
Fig. 1 Dispersal of puddle frogs into highlands of the Cameroon Volcanic Line as reconstructed using S-DIVA analyses and scaled using geological time based on BEAST analyses. For nodal support values see Fig. S1. Individuals of the P. steindachneri complex (P. jimzimkusi sp. n., P. njiomock sp. n. and P. steindachneri) are colour-coded by geographical region; all other species included are colour-coded by the ancestral reconstruction for the species with the number of individuals indicated in parentheses after the name. Species that could not be unambiguously reconstructed are shown in black. Ancestral ranges for nodes that could not be unambiguously resolved are shown with letters corresponding to the geographical regions with frequency of occurrence shown in parentheses; ancestral ranges reconstructed with <10% frequency of occurrence are not reported. The Bamenda-Banso Highlands includes Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and medium elevation areas connecting these mountains. Outgroups (P. africanus and P. natalensis) are not shown.
596 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line
AB
Fig. 2 Relationships among species of the P. steindachneri complex inferred from mitochondrial sequence data from the fragment comprising the 12S rRNA, valine t-RNA and 16S rRNA genes (A) and corresponding localities within the Cameroon Volcanic Line (B). Bayesian posterior probabilities are shown above branches, and maximum likelihood (ML) bootstrap values are shown below branches; dashes indicate Bayesian posterior probabilities that are <0.50 and ML values that are <50. distributed at high elevations with one subclade colonizing and the Bamenda-Banso Highlands (13/100). Mt. Oku, the lowland habitats and subsequently recolonizing highland most prominent and highest peak of the Bamenda-Banso regions (Fig. S1). The ancestor of the P. steindachneri com- Highlands, harbours the highest diversity of montane pud- plex was reconstructed as a high-elevation species. Biogeo- dle frogs (Doherty-Bone, Gvo zd ık and Ohler, in prep.) graphical analyses of geographical regions revealed that the with two new species being described herewith. Bamenda-Banso Highlands was an important region in the diversification of puddle frogs of the CVL (Fig. 1). The Descriptions of two new species provenance of the ancestor of the CVL clade could not be Genus Phrynobatrachus Gunther,€ 1862 reconstructed with certainty, but the Bamenda-Banso Phrynobatrachus jimzimkusi Zimkus, Gvo zd ık & Gonwouo sp. n. Highlands were inferred with highest frequency (87/100). Fig. 4, Table 1 In addition, the remaining reconstructions showed that the Jim Zimkus’ puddle frog ancestor of this clade was distributed in both the lowlands Phrynobatrachus sp. (partim) Amiet (1971, p. 92, Pl. I, Fig. 2)
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Blackburn, P. Huang, M. Talla (MCZ A-138075); one adult female, CAMEROON, West Province, Mt. Bamboutos summit, 5°39′41′′N10°7′37′′E, 2540 m, 8 August 2006, D.C. Blackburn, K.S. Blackburn, P. Huang, M. Talla (MCZ A-138076).
Referred specimens. See Appendix S1.
Etymology. The first author dedicates this species to James (Jim) Zimkus for his full and continuing support of her research.
Diagnosis. Phrynobatrachus jimzimkusi can be distin- guished from all other Phrynobatrachus by a combination of morphological characters, including its large size, ventral coloration and secondary sexual characteristics present in males. Dorsum exhibits chevron-shaped glands outlined in dark brown, appearing prominent on the medium brown dorsum, and a thick vertebral line may be present. The Fig. 3 Relationships among 16S rRNA haplotypes of the venter in males is mottled with a median zone that lacks P. steindachneri complex using statistical parsimony networks as pigment, extending from the chin to the vent. The venter implemented in TCS. Each circle in a given network corresponds in females is lighter than that of males, varying from light to one observed haplotype with the size of each circle proportional brown mottling on a cream background with little mottling to the number of individuals corresponding to the observed on the belly to dark mottling on a white background that haplotype (small circle, one individual; large circle, two or more individuals with number indicated). Circles are colour-coded by covers throat, pectoral region and abdominal region. Small, mountain region; small black dots represent hypothetical white spots or asperities extend from above the eye to the haplotypes needed to connect the network but not observed insertion of the hind limbs and may be present in either among the samples. The haplotype with the highest outgroup sex. Males also exhibit minute asperities on the dorsum, probability is displayed with a white outline. throat and undersides of feet and toes. The tympanum is either indistinct or only scarcely visible with a supratym- Phrynobatrachus sp. 2 (partim) Amiet (1976, p. 78, 82–83, panic fold present. Manual webbing is absent; pedal web- Tables 2 and 3; 1978, p. 6), Gartshore (1986, p. 218–219). bing is moderate to extensive. Manual and pedal digit tips Holotype. Adult male, CAMEROON, West Province, are expanded into small discs with circummarginal grooves. below Mt. Bamboutos summit, 5°39′41.0′′N, 10°07′37.9′′E, A mid-tarsal tubercle, elongate inner metatarsal tubercle 2050 m elevation, 24 September 2004, D.C. Blackburn, J. and outer metatarsal tubercle are present. Diffo and N. Gonwouo (MCZ A-136904). Differential diagnosis. A total of 18 valid puddle frog Paratypes. Same collection data as for holotype, three species are currently known from Cameroon with the adult females, one adult male (MCZ A-136903, 136905– ranges of many of these species extending into Nigeria 136908); two adult females, CAMEROON, West Province, (Fr etey & Blanc 2000; Fr etey 2008; Fr etey et al. 2012; Mt. Bamboutos summit, 2500–2600 m elevation, 25 August Rodel€ et al. 2012). A number of species from this region 2004, D.C. Blackburn, J. Diffo and N. Gonwouo (MCZ A- are considerably smaller in size when compared with 136891–136892); three adult females, CAMEROON, West P. jimzimkusi, including P. calcaratus (15.3–25.0 mm, Province, Mt. Bamboutos, 5°37′24′′N, 10°6′39′′E, 2020 m n = 3 mm), P. chukuchuku (15.7–18.9 mm, n = 5), P. cornu- elevation, 7 August 2006, D.C. Blackburn, K.S. Blackburn, P. tus (14.0–19.1 mm, n = 4), P. danko (19.6–21.2 mm, n = 5), Huang, M. Talla (MCZ A-138065–138066, 138071); one P. hylaios (14.8–22.0 mm, n = 3), P. latifrons (18.3– adult female, CAMEROON, West Province, Mt. Bamboutos, 20.5 mm, n = 4), P. manengoubensis (14.2–17.3 mm, n = 5), 5°39′41′′N, 10°7′37′′E, 2050 m elevation, 7 August 2006, P. ruthbeateae (13.8–19.5 mm, n = 9), P. sandersoni (21.2– D.C. Blackburn, K.S. Blackburn, P. Huang, M. Talla (MCZ 23.0, n = 3), P. schioetzi (20.6–27.7, n = 15; Blackburn & A-138072); one adult male, CAMEROON, West Province, Rodel€ 2011) and P. werneri (16.1–18.0, n = 2 mm). A num- below Mt. Bamboutos summit, 5°40′7′′N, 10°6′11′′E, ber of additional characteristics can also aid in distinguish- 2540 m elevation, 8 August 2006, D.C. Blackburn, K.S. ing the newly described species from these smaller-sized
598 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line
A B
C D
E F
G H
Fig. 4 Photographs of male holotype of P. jimzimkusi sp. n. in dorsal —A. and ventral —C. view; male holotype of P. njiomock sp. n. in dorsal —B. and ventral —D. view; female paratype of P. jimzimkusi sp. n. in dorsal —E. and ventral —G. view; female paratype of P. njiomock sp. n. in dorsal (—F. and ventral —H. view.
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 599 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık
Table 1 Descriptive morphometrics of Phrynobatrachus jimzimkusi sp. n
MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ MCZ A-136904 A-136903 A-136905 A-136907 A-136908 A-136891 A-136892 A-138065 A-138066 A-138071 A-138072 A-138075 A-138076 Male Female Female Male Female Female Female Female Female Female Female Male Female Character Holotype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype
Snout–vent length 32.3 31.4 31.1 34.0 32.4 31.3 30.2 32.4 29.1 29.2 31.5 31.8 29.2 Head width 11.6 9.9 10.1 11.5 11.5 11.1 10.8 11.4 10.1 11.0 10.2 11.0 9.7 Head length 11.5 10.0 10.8 11.6 10.8 11.4 10.4 10.5 9.5 9.0 10.0 9.7 10.0 Tympanum 2.0 1.9 1.8 2.5 2.5 1.8 1.9 2.5 2.2 2.0 2.5 2.0 1.9 diameter Eye diameter 3.8 3.4 3.0 3.9 3.6 3.2 3.6 4.1 3.5 3.6 4.1 3.8 3.5 Interorbital 3.3 2.7 2.9 3.1 3.2 2.1 2.8 3.2 2.7 2.8 2.8 3.3 2.4 distance Internarial 3.3 2.7 3.4 3.5 3.4 3.0 2.9 3.3 2.9 2.8 3.1 3.3 2.8 distance Snout length 5.2 4.6 4.5 4.8 4.8 4.5 5.0 4.7 4.2 4.8 4.9 5.0 4.3 Snout-nostril length 2.3 2.1 2.2 2.3 2.2 1.7 2.5 1.9 1.9 3.0 2.3 2.3 2.2 Eyelid-nostril 3.1 2.3 2.9 2.5 2.4 2.6 2.9 2.4 2.3 2.6 2.4 2.5 2.3 Humerus length 8.6 7.7 8.1 9.4 8.1 7.9 8.1 8.4 7.5 7.7 7.9 8.3 6.9 Radioulna length 8.2 7.1 7.9 8.6 7.8 7.2 7.0 8.0 7.3 7.1 7.6 8.2 6.5 Manus digit I 2.5 2.3 2.7 3.3 2.7 2.7 3.0 3.3 2.8 2.4 2.8 2.3 2.4 Manus digit II 3.8 3.2 3.5 3.9 3.4 3.0 3.4 3.7 3.2 3.3 3.5 3.5 3.1 Manus digit III 6.0 5.4 5.6 6.9 6.3 5.7 5.7 6.6 5.6 6.0 6.0 4.0 5.4 Manus digit IV 4.9 4.6 5.1 5.9 5.0 4.3 4.3 5.3 4.8 4.7 5.0 4.8 4.0 Femur length 18.0 16.9 17.1 20.0 17.5 16.1 16.4 18.2 16.0 15.8 16.4 18.4 15.4 Tibiofibula length 19.1 18.3 17.9 20.6 19.1 17.3 18.1 18.7 17.4 17.8 18.8 18.7 17.1 Foot and 29.6 27.4 27.1 31.9 28.5 25.9 27.9 27.4 26.2 26.9 26.8 28.5 25.5 tarsus length Pes digit I 3.5 3.2 3.2 4.3 3.1 3.1 3.3 3.7 4.0 3.3 3.2 3.5 2.9 Pes digit II 4.6 4.2 4.4 5.7 4.1 3.9 4.4 4.8 4.3 4.0 4.1 4.7 3.4 Pes digit III 8.2 7.2 7.1 8.6 8.1 7.0 7.5 7.9 7.1 7.0 7.1 7.3 6.5 Pes digit IV 12.0 11.1 11.3 13.3 11.6 11.2 11.0 12.3 10.9 10.7 10.9 11.6 10.0 Pes digit V 8.5 7.5 7.4 9.0 8.3 7.5 7.4 7.8 7.1 7.1 7.3 7.3 6.7 Inner metatarsal 1.2 1.3 1.2 1.2 1.3 1.4 1.6 1.6 1.2 1.1 1.1 1.5 1.4 tubercle length Outer metatarsal 0.6 0.6 0.7 0.6 0.8 0.9 0.9 0.9 0.6 0.8 0.7 0.6 0.7 tubercle length
All measurements are given in millimeters. species. Phrynobatrachus jimzimkusi can be differentiated 1966). Phrynobatrachus manengoubensis, a montane species, from P. calcaratus and P. cornutus, two lowland species, has only rudimentary webbing, and the posterior portion because it lacks an eyelid cornicle and has more extensive of the abdomen and undersides of hind limbs are uniformly webbing (Boulenger 1906; Perret 1966, 1988). Males of yellowish or with fine reddish-brown spots (Angel 1940; P. chukuchuku, a species endemic to Mt. Oku, exhibit a dis- Perret 1966; Blackburn 2010; Blackburn & Rodel€ 2011; tinctive ventral colour pattern with a black throat, dark Rodel€ et al. 2012). Phrynobatrachus ruthbeateae, a lowland brown to black pectoral and abdominal region, and a light species, exhibits a distinctive colour pattern with a black or white area on the proximal hind limbs (Zimkus 2009). lateral facemask, a white belly, and either a black throat in Males of P. danko, a species endemic to the Mambilla Pla- males or a white throat with uniform black lower mandi- teau, exhibit a dark throat and an orange or pale yellow bles in females. Phrynobatrachus sandersoni, also a lowland venter with small, grey spots (Blackburn 2010). Phrynoba- species, has well-developed T-shaped digital discs, and trachus hylaios, a lowland species, has only rudimentary breeding males exhibit nuptial pads on manual digit I, lat- webbing and exhibits dark spots on ventral surfaces, includ- eral vocal folds and odontoids (tooth-like projections) in ing the pectoral region, belly and lower sides of extremities the lower jaw (Perret 1966). Phrynobatrachus schioetzi, cur- (Perret 1959, 1966). Phrynobatrachus latifrons, also a lowland rently known only from the forests of the Obudu Plateau, species, exhibits extensive webbing like the newly described exhibits relatively little pedal webbing, a pale or white line species, but throats in males are yellow in colour (Perret along the supratympanic ridge, a pale band on the lateral
600 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line
Table 2 Mean per cent pairwise genetic distances (uncorrected) of ous prominent white spots and lateral vocal folds (Rodel€ Phrynobatrachus jimzimkusi sp. n. and P. njiomock sp. n. compared 2000). Phrynobatrachus plicatus (22.6–38.9, n = 6) exhibits with other Cameroonian Phrynobatrachus species using the mito- elongate and distinct dorsal ridges, and males have black chondrial 12S rRNA, valine-tRNA, and 16S rRNA genes throats with deep, lateral vocal folds (Gunther€ 1858). The newly described species is most similar to P. steindachneri Molecular Molecular distance (%) distance (%) s.s. and the other newly described species, P. njiomock sp. n. to to These three species share a similar size, extent of webbing P. jimzimkusi Standard P. njiomock Standard and variable dorsal coloration. Ventral coloration is simi- Species N sp. n. deviation sp. n. deviation larly mottled in the pectoral and abdominal regions (grey, P. africanus 7 12.98 0.00 12.68 0.45 but commonly also with yellow in between grey patches) in P. batesii 3 6.89 0.00 6.59 0.06 P. jimzimkusi and P. steindachneri s.s., while in P. njiomock P. chukuchuku 3 5.58 0.00 5.09 0.13 sp. n., it appears lighter, usually predominantly creamy- P. cricogaster 3 6.41 0.00 6.03 0.00 white. In all three species, the venter is mottled with the P. danko 3 7.42 0.00 6.81 0.00 P. jimzimkusi sp. n. 25 ––2.91 0.12 throat appearing darker when compared to the pectoral P. manengoubensis 5 7.06 0.00 6.66 0.00 and abdominal regions, but the mottling of P. njiomock sp. P. njiomock sp. n. 12 2.91 0.12 –– n. is comparatively much less prominent with the venter P. ruthbeateae 2 6.30 0.00 5.94 0.00 considerably lighter in some specimens. In addition, the P. schioetzi 4 8.68 0.89 8.11 0.01 clear median zone in the majority of specimens of P. jim- P. steindachneri 15 2.78 0.35 3.40 0.00 P. werneri 2 7.08 0.27 6.67 0.01 zimkusi and P. steindachneri s.s. extends from the chin to the vent, while it may be only barely visible in the pectoral See Table S1 for locality data and GenBank accession numbers of specific tissue and abdominal regions of P. njiomock sp. n. Females of vouchers. P. jimzimkusi and P. steindachneri s.s. exhibit darker ventral coloration when compared with P. njiomock sp. n.; the for- surface of the body and males have a uniformly dusky mer have darker mottling that is present on the throat and throat (Blackburn & Rodel€ 2011). In males of P. werneri, abdomen, while the latter generally exhibits light mottling which is distributed in submontane and montane forest and only on the throat. The lectotype of P. steindachneri (ZMB savannah within the CVL, the throat and pectoral region is 20429) and a female paralectotype (MCZ A-19577) from blackish with the remaining ventral surfaces yellowish ‘Banjo’ [=Banyo, Adamawa Province, Cameroon; Nieden (Nieden 1910; Perret 1966; Blackburn & Rodel€ 2011; 1910; Amiet 1971] appear to have mottling on the under- Rodel€ et al. 2012). side, but this pigmentation is faint due to the age of the The newly described species is morphologically most specimens. [ZMB 20429 was labeled as lectotype, although similar to other medium to large Cameroonian and Nige- it is unclear who made this designation, if the selection was rian species, including P. africanus, P. auritus, P. batesii, justified and whether this information was formally pub- P. cricogaster, P. natalensis, P. njiomock sp. n., P. plicatus and lished; therefore, we formally propose that this specimen, P. steindachneri. For those species that are similar in size to likely male with SVL 30.4 mm, be designated the lectotype the new species, a number of characteristics can be used to of Phrynobatrachus steindachneri Nieden, 1910.] Dorsal skin distinguish them. Males of P. africanus (17.8–30.4, n = 7) of P. njiomock sp. n. is generally smoother with less warts have odontoids in the lower jaw and prominent nuptial than in P. jimzimkusi and P. steindachneri s.s., which are pads on the dorsal surface of manual digit I (Perret 1966; more verrucose. Males of P. jimzimkusi and P. steindachneri Zimkus & Blackburn 2008). Phrynobatrachus auritus (30.9– s.s. exhibit minute asperities on the dorsum; males of both 35.6, n = 5) and P. batesii (22.6–31.0, n = 6) both have a newly described species and P. steindachneri s.s. also exhibit black lateral facemask, as well as elongate and distinct dor- minute asperities on undersides of feet and toes. In males sal ridges; the former also exhibits a blue band along the of P. jimzimkusi and P. steindachneri s.s., minute asperities interior half of the ventral shanks (Lamotte & Xavier 1966; are prominent on the throat (P. jimzimkusi: Figs 4C, 5B, Perret 1966; Rodel€ et al. 2012). Phrynobatrachus cricogaster D; P. steindachneri: Fig. 5J), whereas they are less conspicu- (19.8–23.8, n = 3; 20–32 mm, n = 54; Perret 1966), a sub- ous or variably present in males of P. njiomock sp. n. montane species, can be identified by its distinct bulls-eye (Figs 4D and 5F). ventral pattern, as well as four to five additional tarsal tubercles, one of which forms a small spur with the heel Genetic differentiation and variation. Phrynobatrachus jim- (Perret 1957, 1966). Like the newly described species, zimkusi differed from other Cameroonian and eastern Nige- P. natalensis (20.9–33.4, n = 14) exhibits extensive webbing, rian puddle frog species by mitochondrial (12S-16S) but males of this species exhibit a black throat with numer-
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 601 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık
Table 3 Descriptive morphometrics of Phrynobatrachus njiomock sp. n
NMP6V NMP6V NMP6V NMP6V NMP6V NMP6V MCZ MCZ MCZ MCZ MHNG MHNG 74523/2 74523/1 74523/3 74523/4 74523/5 74523/6 A-136875 A-136876 A-136877 A-136878 2452.017 2452.018 MCZ MCZ Male Female Female Female Female Female Female Female Female Female Male Male A-138118 A-138119 Character Holotype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Paratype Male Female
Snout–vent length 28.0 28.7 27.4 28.9 30.0 30.8 28.1 25.2 27.2 28.5 33.3 35.0 30.7 28.0 Head width 9.7 9.1 8.8 9.3 9.3 9.7 9.4 8.3 9.2 9.3 12.0 11.1 10.9 9.9 Head length 8.7 8.4 8.5 9.1 9.2 8.2 8.8 8.4 8.4 8.9 11.1 11.5 10.9 9.7 Tympanum diameter 1.6 1.8 1.6 1.7 1.7 1.9 1.5 1.6 1.5 2.0 1.7 1.9 1.3 1.4 Eye diameter 3.4 3.5 3.5 3.1 3.1 3.8 3.4 2.7 3.0 2.9 4.1 4.4 3.3 3.4 Interorbital distance 2.8 2.5 2.8 2.3 2.5 2.8 2.5 2.7 2.6 3.3 3.3 3.1 2.2 3.0 Internarial distance 2.1 2.8 2.7 2.7 2.6 2.9 2.8 2.7 2.8 2.8 3.6 3.2 3.4 2.7 Snout length 4.0 3.9 3.7 4.0 4.0 4.5 4.1 3.9 3.8 4.2 4.7 5.4 4.6 4.1 Snout-nostril length 1.7 1.6 1.9 1.9 1.9 2.0 2.0 1.6 1.8 1.8 2.3 2.6 1.8 1.9 Eyelid-nostril 2.1 1.7 1.9 1.9 1.9 2.3 2.1 1.8 1.8 2.2 2.4 2.7 2.9 2.2 Humerus length 6.8 6.3 6.8 6.3 7.0 6.8 6.7 6.6 5.7 7.2 8.7 9.6 6.9 6.8 Radioulna length 6.5 6.1 6.0 6.7 6.7 6.7 6.5 6.2 5.9 6.5 7.1 8.2 6.7 6.4 Manus digit I 2.1 2.2 2.0 2.2 2.5 2.3 2.2 2.2 2.3 2.4 2.8 3.2 2.6 2.2 Manus digit II 2.7 2.5 2.4 2.5 2.7 2.9 2.8 2.7 2.4 2.9 3.4 3.8 3.3 2.7 Manus digit III 5.1 4.7 4.5 4.7 5.3 4.7 5.1 5.0 4.7 5.1 6.0 6.4 5.9 4.7 Manus digit IV 4.2 3.7 3.6 3.7 4.4 4.0 3.7 3.7 4.1 4.1 5.2 5.0 4.2 3.9 Femur length 15.1 14.1 13.0 14.1 14.1 15.1 14.4 14.7 14.8 15.1 18.6 18.9 16.7 15.1 Tibiofibula length 17.6 14.8 15.0 15.0 16.4 16.3 16.7 15.6 16.0 16.6 20.4 20.2 18.2 16.8 Foot and tarsus 26.9 22.3 21.2 23.4 24.8 26.1 25.2 25.2 24.9 25.8 28.8 29.7 27.8 24.4 length Pes digit I 3.1 2.4 2.6 2.7 2.7 2.9 2.9 2.7 2.6 2.7 3.9 3.5 3.4 2.7 Pes digit II 3.8 3.4 3.3 3.3 3.4 3.8 3.7 4.0 3.8 3.8 4.7 5.2 4.5 3.8 Pes digit III 6.7 5.3 5.5 5.9 6.5 6.4 6.6 6.7 6.4 6.8 7.3 7.6 7.6 6.0 Pes digit IV 10.4 8.9 8.5 9.2 10.2 9.7 10.1 10.0 9.9 10.4 11.7 12.2 11.1 9.6 Pes digit V 7.0 5.7 6.0 6.3 7.1 7.2 6.7 6.8 6.6 7.3 7.6 7.9 7.3 6.2 Inner metatarsal 1.1 1.0 0.8 0.8 1.1 0.9 1.0 1.1 0.9 0.9 1.3 1.2 1.2 1.0 tubercle length Outer metatarsal 0.6 0.6 0.5 0.4 0.4 0.5 0.5 0.4 0.4 0.6 0.8 0.7 0.5 0.6 tubercle length
All measurements are given in millimeters. sequence divergence of 2.78–12.98% (Table 2). Intraspecific but distinct discs with circummarginal grooves; tibiofibula sequence variation ranged up to 0.94% for P. jimzimkusi. slightly longer than femur; relative length of toes IV > V > III > II > I; tarsal tubercle present in close prox- Description of the holotype. Adult male. SVL 32.3 mm imity to conspicuous, elongate inner metatarsal tubercle; (Fig. 4; Table 1). Large with compact body shape and outer metatarsal tubercle present but significantly smaller robust hind limbs; head as wide as long; canthus rostralis than inner metatarsal tubercle; toes with single, round sharp; loreal region vertical and slightly concave; distance subarticular tubercles; pes webbing moderate to extensive from naris to rostral tip less than that from naris to with the following webbing formula I[1]-[0]II[1]-[1]III[2]- anterior eye; eye diameter approximately 1.7 times snout– [2]IV[2]-[2]V; toe tips expanded into small discs with nostril length; internarial distance equal to interorbital dis- circummarginal grooves; dorsal skin smooth with tiny tance; tympanum barely visible; eye diameter 1.9 times asperities; venter (pectoral and abdominal regions) smooth; tympanum diameter; tympanic annulus not evident exter- small, light-coloured asperities on the upper lip, below the nally; supratympanic fold distinct, extending from posterior tympanum and supratympanic fold and extending down the corner of eye to base of the forelimb; premaxillary and flanks; gular skin finely granular but without lateral folds; maxillary teeth present; conical papilla present in centre of undersides of feet and toes covered with small asperities; heart-shaped tongue. Relative length of fingers: pair of chevron-shaped glands present behind the eyes, III > IV ≥ II > I; hand with large and oval palmar and the- converging in the scapular region. nar tubercles and smaller tubercles surrounding; fingers with single, round subarticular tubercles; no webbing pres- Coloration of holotype (in ethanol). Medium to dark ent between manus digits; finger tips expanded into small brown with some darker and lighter brown spots and a
602 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line
A B C D
E F G H
I J K O
L M N
Fig. 5 Photographs of frogs and habitats of the P. steindachneri complex. Phrynobatrachus jimzimkusi sp. n. from the Bamenda-Banso Highlands (A–D): —A. An adult male from dorsolateral view; —B. The same male from ventral view; —C. A specimen in typical sleeping position on a leaf above a stream; —D. Ventral side of a specimen showing variation in colour pattern. Phrynobatrachus njiomock sp. n. from Lake Oku (E–H): —E. An adult male; —F. The same male from ventral view (Photo E, F: T. M. Doherty-Bone); —G. A female displaying reddish patches, which are a common for the species; —H. Ventral side of another female (Photo H: A. Kod adkov a). Phrynobatrachus steindachneri from the Gotel Mountains (I–K): —I. An adult male; —J. The same adult from ventral view; —K. Another specimen showing colour pattern variation. Lectotype of P. steindachneri (ZMB 20429) from the Banyo District, Adamawa, Cameroon (L). Habitats of the P. steindachneri complex (M–O): —M. General habitat of P. jimzimkusi sp. n. in northwestern Bamenda-Banso Highlands (above Big Babanki village); —N. Lake Oku on Mt. Oku, the centre of distribution of P. njiomock sp. n. where the species is abundant; —O. A stream habitat of P. steindachneri in the Gotel Mountains. thick, light brown, mid-dorsal stripe. Chevron-shaped the venter, extending from the chin to the vent. Palmar, scapular glands are light brown and outlined in darker thenar and surrounding tubercles on hand are greyish- brown. Two elongate spots found behind the scapular white in colour. glands are similarly light-coloured with a darker outline. Light-coloured asperities are found on the upper lip and Variation. Descriptive morphometrics of Phrynobatra- extend from below the eye and supratympanic fold to chus jimzimkusi are provided in Table 1. Morphological anterior of the hind limb. Dorsal sides of hind limbs proportions in paratypes are generally consistent with those barred. Venter mottled with the pectoral and abdominal in the holotype. All specimens examined have a snout–vent region appearing lighter in colour when compared with length between 29 and 34 mm; males (31.8–34.0 mm, the throat. Throat exhibits small asperities that appear mean 32.7, n = 3) are generally slightly larger than females whitish in colour. A clear median zone is apparent on (29.1–32.4 mm, mean 30.8, n = 10; t-test: P = 0.04). Dor-
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 603 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık sum in some individuals lacks the light brown mid-dorsal ecologically restricted habitat, we recommend that this stripe and varies from medium to dark brown, appearing species be categorized as Vulnerable, following the Interna- uniform in colour or exhibiting lighter and darker spots. tional Union for Conservation of Nature criteria (IUCN Dorsal head coloration and presence of a light-coloured Standards & Petitions Subcommittee 2011). interorbital line varies among paratypes and referred speci- mens examined; interorbital line appears more prominent Genus Phrynobatrachus Gunther,€ 1862 in those individuals that lack a mid-dorsal stripe. Dorsum Phrynobatrachus njiomock Zimkus & Gvo zd ık sp. n. in females is smooth with some small, white asperities on Fig. 4, Table 3 the sides; undersides of feet and toes lack small asperities. Lake Oku puddle frog Ventral coloration of females varies from light brown mot- Phrynobatrachus sp. 11 Amiet (1978, p. 6). tling on a cream background on the throat with scant Holotype. Adult male, CAMEROON, Northwest Prov- mottling on the belly to dark mottling on a white back- ince, Mt. Oku, Lake Oku, 06°12′07.6″N10°27′33.3″E, ground that covers the throat, pectoral region and abdomi- 2219 m elevation, 24 December 2009, A. Kod adkov a, R. nal region. Tropek (NMP6V 74523/2).
Distribution and natural history. Phrynobatrachus jim- Paratypes. Same collection data as holotype, five adult zimkusi is known from submontane and montane regions females (NMP6V 74523/1, NMP6V 74523/3–6); eight (1300–2900 m a.s.l.) within the southern portion of the females, CAMEROON, Northwest Province, Lake Oku, CVL, including Mt. Manengouba, Bamboutos Mts., Mt. 06°12.144′N, 10°27.638′E, 2220 m elevation, 22 September Lefo and Mt. Oku in Cameroon and the Obudu Plateau in 2004, D.C. Blackburn, J. Diffo and N. Gonwouo (MCZ A- Nigeria (Fig. 2B). Amiet (1971, 1976) listed this species (as 136875–136882); five females, CAMEROON, Northwest ‘P. sp.’ and ‘P. sp. 2’, respectively) also from Tchabal Province, Lake Oku, forest at edge of lake, 06°12.144′N, Mbabo, but we conclude that this mountain is inhabited by 10°27.638′E, 2220 m elevation, 18 August 2006, D.C. P. steindachneri. Phrynobatrachus jimzimkusi predominantly Blackburn, K.S. Blackburn, P. Huang, and M. Talla (MCZ occupies calm, pool-like passages of rapid streams in mon- A-138135–138137, 138139–138140); two males, CAMER- tane forests, including typically stream-fringing forests and OON, Northwest Province, Lake Oku, J.-L. Amiet bushes in montane savanna, but it sometimes (especially in (MHNG 2452.017–2452.018). high elevations) can be found along streams in open grass- lands. It may be locally abundant. The species seems to Referred specimens. See Appendix S1. have diurnal activity; at night, it usually rests on mossy stones or leaves and stems up to 1 m high in the vegetation Etymology. The specific epithet is derived from Oku, a around streams. They have never been heard to call (cf. Bantu language of Cameroon, and means eleventh in refer- Gartshore 1986). Females lay clutches of eggs in small ence to Phrynobatrachus sp. 11 of Amiet (1978), which was groups, attaching them to root hairs or vegetation floating collected near Lake Oku. We propose to use it as a noun in the water column (observed in the early dry season of in apposition. late November in the Bamenda-Banso Highlands). Tad- poles are of typical ranoid-like appearance and inhabit Diagnosis. Phrynobatrachus njiomock is readily distin- pools in streams. guished from all other Phrynobatrachus by a combination of morphological characters, including its relatively large size, Conservation status. The habitat of P. jimzimkusi is dorsal markings and secondary sexual characteristics pres- highly threatened by agricultural pressure, including use of ent in males. Dorsum exhibits chevron-shaped glands out- herbicides and pesticides, cattle grazing, burning of mon- lined in dark brown, appearing prominent on the medium tane grasslands, harvesting for firewood and expanding brown-grey, and reddish patches are common. In males, human settlements. In particular, the type locality of light brown mottling is present on the throat, appearing P. jimzimkusi in the Bamboutos Mts. supports a high slightly darker when compared with the pectoral and human population density that solely relies on the moun- abdominal regions, which are generally creamy-white with tain, and deforestation through cutting and burning for some areas of indistinct mottling; clear median zone may farming has modified the forest and grassland habitats. In be distinct or barely visible on throat. Undersides of addition, the use of herbicides and pesticides is having a females are generally lighter than males; light brown mot- negative impact on amphibian communities, which rely on tling is present on the throat; belly is most often immacu- these water sources. Although P. jimzimkusi is locally abun- late but may have some areas of indistinct mottling. Small, dant, considering the pressure on its geographically and light-coloured asperities may be present in either sex and
604 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line are present below the tympanum and supratympanic fold, num and supratympanic fold and extending down the extending down the flanks. Males also exhibit minute flanks; lateral vocal folds absent; undersides of feet and asperities on undersides of feet and toes. Tympanum is toes covered with small asperities; pair of chevron-shaped either indistinct or only scarcely visible with a supratym- glands are present behind the eyes, converging in the scap- panic fold present. Manual webbing is absent; pedal web- ular region. bing is moderate to extensive. Manual and pedal digit tips are expanded into small discs with circummarginal grooves. Coloration of holotype (in ethanol). Light brown to grey A mid-tarsal tubercle, elongate inner metatarsal tubercle with some darker and lighter brown spots. Chevron-shaped and outer metatarsal tubercle are present. scapular glands are light brown and outlined in darker brown. Light-coloured asperities are found on the upper Differential diagnosis. Differentiation of P. njiomock from lip and extend from below the eye and supratympanic fold other Cameroonian and eastern Nigerian species was simi- to anterior of the hind limb. Dorsal sides of hind limbs lar to that of P. jimzimkusi. See differential diagnosis of slightly barred. Venter slightly mottled in the anterior por- P. jimzimkusi. tion with the throat appearing darker when compared with the pectoral region; abdomen mostly white. An indistinct Genetic differentiation and variation. Phrynobatrachus clear median zone is barely visible on the throat. njiomock differed from other Cameroonian and eastern Nigerian species of Phrynobatrachus by mtDNA (12S-16S) Variation. Descriptive morphometrics of Phrynobatra- sequence divergence of 2.91–12.68% (Table 2). Intraspe- chus njiomock are provided in Table 3. Morphological cific sequence variation ranged up to 0.54% for Phrynoba- proportions in selected paratypes and additional material trachus njiomock. examined for morphology (Appendix S1) are generally con- sistent with those of the holotype. All specimens have a Description of holotype. Adult male, SVL 28.0 mm snout–vent length between 25.2 and 35.0 mm; males (28.0– (Fig. 4; Table 3). Large with compact body shape and 35.0, mean 31.8, n = 4) are generally slightly larger than strong hind limbs; head as wide as long; canthus rostralis females (25.2–30.8, mean 28.3, n = 10; t-test, P = 0.01). sharp; loreal region vertical and slightly concave; distance Dorsum in most individuals lacks the light brown mid-dor- from naris to rostral tip slightly less than that from naris sal stripe but may be present (MCZ A-138118; MHNG to anterior of eye; eye diameter two times snout–nostril 2452.017–2452.018); colour of dorsum varies from greyish- length; interorbital distance only slightly larger than in- brown to dark brown, appearing uniform in colour or ternarial distance; tympanum barely visible; eye diameter exhibiting lighter and darker spots. In life, many adults typ- more than two times tympanum diameter; tympanic annu- ically possessed reddish patches on the dorsal side, includ- lus not evident externally; supratympanic fold present but ing the limbs and head. Presence of a light-coloured not prominent, extending from posterior corner of eye to interorbital line and dorsal head coloration varies among base of forelimb; premaxillary and maxillary teeth present; paratypes and additional material examined; interorbital conical papilla present in centre of heart-shaped tongue. line appears more prominent in those individuals that lack Relative length of fingers: III > IV > II > I; hand with a mid-dorsal stripe. Ventral coloration of females generally large and oval palmar and thenar tubercles and smaller very light; light brown mottling present on the throat, tubercles surrounding; fingers with single, round subarticu- appearing one shade darker than abdomen; belly most lar tubercles; no webbing present between manus digits; often immaculate but may have some marginal areas with finger tips expanded into small but distinct discs with cir- mottling. cummarginal grooves; tibiofibula longer than femur; rela- tive length of toes IV > III > V > II > I; tarsal tubercle Distribution and natural history. Phrynobatrachus njiomock present in close proximity to conspicuous, elongate inner is known only from the Kilum-Ijim Forest on Mt. Oku metatarsal tubercle; outer metatarsal tubercle present but (2219–2400 m a.s.l.), including shores of Lake Oku, a significantly smaller than inner metatarsal tubercle; toes freshwater crater lake found 2219 m above sea level, where with single, round subarticular tubercles; pes webbing it is the most abundant frog (excluding the aquatic Xenopus extensive with the following webbing formula: I[1]-[1]II longipes). There is little information about its distribution [1]-[2]III[2]-[2]IV[2]-[2]V; toe tips expanded into small and niche preference in the surrounding forests where all discs with circummarginal grooves. Dorsal skin with tiny three species of the P. steindachneri complex might be asperities and warts laterally but relatively smooth; venter found. In addition to its abundance in the vicinity of Lake rather smooth but with minute asperities present on Oku, this species occurs in or close to streams, which is throat; small, light-coloured asperities below the tympa- similar to P. jimzimkusi, the closely related species from the
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 605 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık complex. Unlike P. jimzimkusi, P. njiomock seems to have sequence data from P. ruthbeateae, a second species distrib- predominantly crepuscular and nocturnal activity, similar to uted at lower elevations. Phrynobatrachus ruthbeateae was P. steindachneri (as observed in the Gotel Mts.). Tadpoles found to be sister to P. batesii, the other low-elevation commonly develop directly in the shallow waters of Lake species, and the clade that contains smaller-bodied, higher Oku. More ecological studies on this species and the entire elevation species (P. danko, P. manengoubensis, P. schioetzi P. steindachneri complex in the Mt. Oku area are needed to and P. werneri). better understand their mutual ecological relationships and Closer examination of the clade lineage currently recog- niche differentiation. nized as P. steindachneri s.s. is needed to determine whether additional species should be described. Uncorrected pair- Conservation status. Although a major part of the known wise genetic divergences of 12S-16S within P. steindachneri distribution range of P. njiomock is protected as the Kilum- exceeded 3% in some cases, suggesting that there may be Ijim Plantlife Sanctuary, a strong anthropogenic pressure unconfirmed candidate species; however, at this time, we still persists in the area due to livestock grazing and agri- categorize these lineages as deep conspecific lineages due cultural expansion. Considering the very restricted range of to lack of other, independent sources of evidence (e.g. mor- this species with an area of occupancy likely <10 km2, the phological or acoustic differences; sensu Vieites et al. fact that most of individuals are concentrated around a sin- 2009). Although morphological differences may exist, the gle location (Lake Oku), an observed decline in the lectotype and paralectotypes of P. steindachneri (originally number of individuals (T. M. Doherty-Bone, pers. comm.) 28 ZMB syntypes) are in poor condition, making it difficult and a high risk of a fish introduction into Lake Oku, we to assess the morphology in detail. Hence, the collection of propose P. njiomock to be listed as Critically Endangered specimens from the vicinity of the type locality (Banyo, according to the International Union for Conservation of Cameroon; Fig. 2B) and other localities within the Adama- Nature criteria (IUCN Standards & Petitions Subcommit- wa Plateau (Gotel Mts. and Tchabal Mbabo) is needed to tee 2011). determine whether one or more species are currently iden- tified as P. steindachneri. Discussion Phylogenetic patterns Biogeographical patterns and dating estimates Results of this study further clarify the diversification of Puddle frogs of the Sky Islands of the CVL provide a puddle frogs across the highlands of Cameroon and Nige- unique opportunity to understand the effect of historical ria. The pattern of phylogenetic relationships is similar to climate change on a lineage that includes both populations that found by Zimkus (2009), Blackburn (2010), Zimkus that are endemic to isolated mountains, as well as those et al. (2010) and Blackburn & Rodel€ (2011). The major that are more widespread across highland regions. This difference between this study and previous studies is the study also reveals how cyclical climatic changes from the placement of P. chukuchuku, but this is not surprising given Miocene through the Pleistocene influenced interspecific that low nodal support for P. chukuchuku found in these and intraspecific diversification in a clade endemic to the previous studies. Blackburn (2010) found P. chukuchuku to CVL. Dispersal–vicariance analysis using geographical data be sister to the clade that includes P. cricogaster and suggests that the main body of mountains of the CVL, P. steindachneri, while Zimkus (2009), Zimkus et al. (2010) the Bamenda-Banso Highlands, which includes the Bam- and Blackburn & Rodel€ (2011) found this species to be sis- boutos Mts., Mt. Lefo, Mt. Mbam and Mt. Oku, and the ter to the clade that included P. batesii and the small-to medium elevation areas connecting these mountains are an medium-bodied species of the highlands (P. danko, P. schi- important region for diversification in puddle frogs (Fig. oetzi, P. werneri and P. manengoubensis). Unlike these previ- S1). More than half of the highland species currently ous studies, we found that P. chukuchuku was sister to two known are distributed in this central highland region, and major clades of puddle frogs of the CVL using a consensus the ancestor of the CVL clade was likely distributed in alignment that excluded characters that differed among this region. Dispersal–vicariance analysis using elevational three alignments generated using varying gap costs. In the data suggests that the ancestor of the CVL clade was phylogenies produced with alignments generated using dif- likely endemic to the highest elevations (Fig. S1). During fering gap costs, P. chukuchuku formed a polytomy with subsequent radiation, the ancestor of all recent species, these two major clades. Results of this study and all excluding P. chukuchuku, descended into lower elevations previous studies support the sister relationship between from where one subclade further radiated into lowland P. cricogaster and the P. steindachneri clade, which now habitats while another subclade recolonized highland areas. includes P. jimzimkusi and P. njiomock. These analyses dif- After this diversification in lowlands, one more radiation fered from previous studies as they are the first to include into higher elevations took place. This scenario indicates
606 ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 B. M. Zimkus & V. Gvo zd ık Puddle frogs of the Cameroon Volcanic Line that there were at least two independent recolonizations W. chirioi are listed as Critically Endangered on the IUCN of the highlands within this clade. Both reconstructions of Red List of Threatened Species (Amiet 2004b; Tinsley & ancestral distributions in regards to elevation and geogra- Measey 2004). In addition, Mt. Oku is the habitat of other phy reject the hypothesis that Phrynobatrachus invaded near-endemic endangered species, including Astylosternus highland zones a single time. Although the placement of ranoides, Cardioglossa oreas and Werneria bambutensis (Amiet P. chukuchuku continues to be problematic, it is unlikely 2004a; Amiet & Schiøtz 2004). that the discovery of new species will support a monophy- Few parts of the CVL are formally protected within letic clade that contains only highland species. We can Cameroon at the national level (Bergl et al. 2007), even therefore confidently support the hypothesis that puddle though this region comprises a significant portion of the frogs colonized highland habitats of the CVL multiple West African biodiversity hot spot (Myers et al. 2000). The times independently. It is possible that the discovery of International Council for Bird Preservation has recom- new species may reveal that the patterns of diversification mended the protection of habitats within the CVL with into the highlands are even more complex than currently the establishment of parks on Mt. Cameroon, Mt. Kupe reconstructed with more than two colonizations of high- and Mt. Oku (Stuart 1986). Mt. Oku was further high- land regions. lighted as one of the most important areas for avian con- Dating estimates suggest that the CVL clade originated servation in Cameroon with the protection of the in the Bamenda-Banso Highlands in the Early Miocene, Bamenda-Banso Highlands suggested as a way to maximize ca. 22–21 mya (Fig. 1), during the time of a rich volcanic the preservation of intraspecific avian diversity (Smith et al. activity in the region (Mt. Oku, 31–22 mya; Bamboutos 2000). Zimkus (2009) suggested that the mid-highlands of Mts., 21–14 mya; Marzoli et al. 2000). The majority of the CVL, including Mt. Manengouba and the Bamenda- intraspecific diversifications occurred during the Pliocene Banso Highlands, should be a focus of conservation efforts and Pleistocene (<5.3 mya), suggesting that this period based on the number of unique amphibian taxa present in played an important role in shaping intraspecific genetic these habitats. The montane forest on Mt. Oku, Kilum- structure and distribution patterns. Pliocene changes, lead- Ijim Forest, is now the only extensive area of forest left ing to a cooler and drier climate, and Pleistocene fluctua- anywhere in the Bamenda-Banso Highlands. Although the tions drove periodic montane forest expansions and montane forest surrounding Lake Oku is considered a contractions, which allowed species to disperse into adja- ‘Plantlife Sanctuary’, degradation of the forest, incursions cent mountains and subsequently isolated them. Dating of livestock, increased development and traffic are major estimates should be considered preliminary as only a single concerns (Doherty-Bone 2011; V. Gvo zd ık, unpubli. data). constraint was used in dating estimates and no fossil cali- It is also important to note that the grasslands found at the bration points were available. A follow-up study, possibly summit of Mt. Oku (summit 3011 m) are, besides Mt. using nuclear DNA data, could be completed to verify Cameroon, at the highest elevation within the CVL diversification times estimated in this study. (Mt. Kupe, summit 2064 m; Mt. Nlonako, summit 1825 m; Mt. Manengouba, summit 2411 m; Tchabal Mbabo, Conservation of habitats and endemic species highest elevation approximately 2380 m; Obudu Plateau, Cameroon exhibits incredible species diversity with over highest elevation approximately 1700 m). These high-ele- 200 described frog species, ranking highest in the percent- vation grasslands thereby represent a unique habitat that age of endemic amphibians found on mainland Africa (Am- should be protected in addition to montane forest habitats. iet 1989; Jenkins & Hamilton 1992; Poynton 1999; Herrmann et al. 2005). The CVL, and more specifically Acknowledgements the Bamenda-Banso Highlands, appears to be especially Many thanks to R. Drewes (CAS, San Francisco), A. important for the diversification of puddle frogs. The pres- Kod adkov a and R. Tropek (Biology Centre of the Acad- ence of the two new puddle frog species, P. jimzimkusi and emy of Sciences of the Czech Republic, Cesk e Bud ejovice), P. njiomock, on Mt. Oku reinforces the importance of this M.-O. Rodel€ (ZMB, Berlin), J. Rosado (MCZ, Harvard), mountain as critical habitat for the conservation for puddle A. Schmitz (MHNG, Geneva), M. Vences (Technical Uni- frog diversity. In addition to the two puddle frog species versity of Braunschweig) and D. Wake (MVZ, San Fran- endemic to Mt. Oku (P. chukuchuku and P. njiomock), two cisco) who made specimens under their care available and/ other anurans are known to be endemic to habitats on Mt or donated tissues for this study. Thank you to C. S. Oku. Xenopus longipes is found only within Lake Oku (Lou- Walker of the Harvard Map Collection for assistance with mont & Kobel 1991; Kobel et al.1998), while Wolterstorffi- the map used in Fig. 2. We thank D. C. Blackburn (CAS, na chirioi is distributed in the grasslands on the summit of San Francisco) and A. Schmitz for use of unpublished Mt. Oku (Boistel & Amiet 2001). Both X. longipes and molecular data and ecological data, J.-L. Amiet for discus-
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 607 Puddle frogs of the Cameroon Volcanic Line B. M. Zimkus & V. Gvo zd ık sion regarding species of the P. steindachneri complex, Republic of Cameroon, with an analysis of morphological diversity T. M. Doherty-Bone for additional data and photographs, in the genus. Zoological Journal of the Linnean Society, 154, 611–630. and Y. Wu for assistance with dating methods. The specific Blackburn, D. C. (2010). A new puddle frog (Phrynobatrachidae: Phrynobatrachus) from the Mambilla Plateau in eastern Nigeria. epithet for P. njiomock was suggested by the Fon of Elak African Journal of Herpetology, 59,33–52. Oku (pers. comm. to D. C. Blackburn). Field research was Blackburn, D. C. & Rodel,€ M.-O. (2011). A new puddle frog made possible through the permission from the Cameroo- (Phrynobatrachidae: Phrynobatrachus) from the Obudu Plateau in nian Ministry of Scientific Research and Innovation Eastern Nigeria. Herpetologica, 67, 271–287. and the Ministry of Forestry and Wildlife (Nos. 00132/ Blackburn, D. C., Gonwouo, L. N., Ernst, R. & Rodel,€ M.-O. MINRESI/B00/C00/C10/C13, 1010/PRBS/MINFOF/SG/ (2009). A new squeaker frog (Arthroleptidae: Arthroleptis) from DFAP/SDVEF/SC). V.G. would like to extend his thanks the Cameroon Volcanic Line with redescriptions of Arthroleptis adolfifriederici Nieden, 1911 “1910” and A. variabilis Matschie, to E. Vunan (SATEC, Big Babanki), M. LeBreton and N. – 1893. Breviora, 515,1 22. L. Gonwouo (Yaounde) for their hospitality, assistance with Blackburn, D. C., Gvo zd ık, V. & Leach e, A. D. (2010). A new logistics and valuable advice. Funding was provided by the squeaker frog (Arthroleptidae: Arthroleptis) from the mountains Ministry of Culture of the Czech Republic (NAKI of Cameroon and Nigeria. Herpetologica, 66, 335–348. #DF12P01OVV021). Boistel, R. & Amiet, J.-L. (2001). 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(Anura: Phrynobatrachidae) endemic to Mount Oku, Cameroon. Supporting Information – Zoological Journal of the Linnean Society, 157, 795 813. Additional Supporting Information may be found in the Zimkus, B. M. & Blackburn, D. C. (2008). Distinguishing features of online version of this article: the sub-Saharan frog genera Arthroleptis and Phrynobatrachus: a short Appendix S1. Specimens examined morphologically. guide for field and museum-based researchers. Breviora, 513,1–12. Zimkus, B. M., Rodel,€ M.-O. & Hillers, A. (2010). Complex Fig. S1. Dispersal of puddle frogs within highlands of patterns of continental speciation: molecular phylogenetics and the Cameroon Volcanic Line as reconstructed using S- biogeography of sub-Saharan puddle frogs (Phrynobatrachus). DIVA analyses and scaled using geological time based on Molecular Phylogenetics and Evolution, 55, 883–900. BEAST analyses. Zink, R. M. & Slowinski, J. B. (1995). Evidence from molecular Table S1. Phrynobatrachus specimens included in molec- fi systematics for decreased avian diversi cation in the Pleistocene ular analyses. epoch. Proceedings of the National Academy of Sciences of the United States of America, 92, 5832–5835.
ª 2013 The Norwegian Academy of Science and Letters, 42, 6, November 2013, pp 591–611 611 APPENDIX S1 SPECIMENS EXAMINED MORPHOLOGICALLY Type status, sex and specific locality are provided. P. africanus- CAS 199303 (male), Cameroon, East Province, Dja Reserve, Boumir Camp; CAS 207779 (male), Equatorial Guinea, Bioko Id., ca 3.6 km N (by rd) of Luba; MCZ A-136731 (male), Cameroon, E of Samakeke, Mangombe Forest Reserve, Littoral Province; MCZ A-136756 (male), MCZ A-136757 (male), Cameroon, Efolofo, N slope of Mt. Cameroon, Southwest Province; MCZ A-136800 (female), Cameroon, E of Ntale Village, Banyang Mbo Forest Reserve, Southwest Province; MCZ A-136839 (female), Cameroon, Ekoneman Awa, Ejagham Forest Reserve, Southwest Province; MCZ A- 136944 (male), MCZ A-136945 (female), Cameroon, East of Campo, Campo-Ma'an National Park, South Province; MCZ A-138103 (male), Cameroon, Obang, Northwest Province. P. auritus- BMNH 1947.2.30.2 (syntype; male), BMNH 1947.2.30.3 (syntype; male?), BMNH 1947.2.30.4 (syntype; male?), Equatorial Guinea, Benito River; CAS 199344 (male), Cameroon, East Province, Dja Reserve, Boumir Camp; CAS 207937 (male), Equatorial Guinea, Bioko Island, vic Moka Malabo; MCZ A-136736 (male?), Cameroon, E of Samakeke, Mangombe Forest Reserve, Littoral Province; MCZ A-136792 (female), Cameroon, E of Ntale Village, Banyang Mbo Forest Reserve, Southwest Province; MCZ A-138094 (female), MCZ A-138095 (male), MCZ A-138096 (female), MCZ A-138097 (female), MCZ A-138098 (female), MCZ A-138099 (female), Cameroon, Obang, Northwest Province. P. batesii- MCZ A-136783 (female), MCZ A-136784 (female), MCZ A-136793 (female), Cameroon, E of Ntale Village, Banyang Mbo Forest Reserve. P. calcaratus- MCZ A-12837 (female), Cameroon, Buea, Southwest Province; MVZ 245139 (female?), MVZ 245140 (female), Ghana, Togo Hills, Kyabobo National Park, Volta Region. P. chukuchuku- MCZ A-138127 (holotype; male), MCZ A-138124 (paratype; male), MCZ A-138125 (paratype; male), MCZ A-138126 (paratype; female), MCZ A-138128 (paratype; female), MCZ A-138129 (paratype; male), MCZ A-138130 (paratype; female), MCZ A-138131 (paratype; male), MCZ A-138132 (paratype; male), MCZ A-138134 (paratype; male), P. cornutus- CAS 199268 (female), Cameroon, East Province, Dja Reserve, Boumir Camp; CAS 207799 (female), Equatorial Guinea, Bioko Island, ca 3.6 km N of Luba; CAS 207877 (male), Equatorial Guinea, Bioko Island, vic Moka Malabo, Rio Lomo; MCZ A-136837 (female), Cameroon, Ekoneman Awa, Ejagham Forest Reserve, Southwest Province. P. cricogaster- MCZ A-31572 (male), MCZ A-31573 (male), Cameroon, Nsoung Nkongsamba, Littoral Province; MCZ A-138030 (male), Cameroon, Nsoung, Southwest Province. P. danko- MCZ A-139529 (holotype; male), MCZ A-139530 (paratype; female), MCZ A-139531 (paratype; male), MCZ A-139532 (paratype; male), MCZ A-139533 (paratype; male), Nigeria, Kurmin Danko Forest Reserve, Taraba. P. hylaios- MHNG 964.100 (holotype; male), Cameroon, Foulassi, Sangmelima; AMNH 8976 (male?), Belgian Congo (Democratic Republic of the Congo), Matadi; AMNH 8978 (male?), Begian Congo (Democratic Republic of the Congo), Zambi. P. jimzimkusi- MCZ A-136927 (female), MCZ A-136928 (female), MCZ A-136929 (female), Cameroon, Southwest Province, Mt. Manengouba; MCZ A-138052 (female), MCZ A-138053 (female), Cameroon, Southwest Province, Mt. Manengouba. P. latifrons- MCZ A-17536 (syntype; male), Cameroon, Dodo; MVZ 245150 (female), Ghana, ca. 10 mi N of Accra, Greater Accra Region; MVZ 245154 (male?), Ghana, Togo Hills, Nkwanta, stream ca. 8 km NE of Kyabobo, Volta Region; ZMB 28344 (male?), Ghana, Accra, Greater Accra Region. P. manengoubensis- MCZ A-136923 (female), MCZ A-136924 (female), MCZ A- 138047 (female), Cameroon, Mt. Manengouba, Southwest Province; MCZ A-138036 (female), MCZ A-138043 (male), MCZ A-138044 (male), MCZ A-138050 (male), MCZ A-138054 (female), Cameroon, Manengouba, Southwest Province. P. njiomock- MCZ A-138118 (male), MCZ A-138119 (female), Cameroon, Northwest Province, Mt. Oku, Kilum-Ijim Forest northeast of summit. P. plicatus- BMNH 1947.2.29.97 (holotype; male), Guinea, coast of Guinea; MVZ 245164 (male), MVZ 245165 (female), Ghana, Ankasa National Park, Western Region. P. ruthbeateae- MCZ A-147865 (female), MCZ A-147866 (female), MCZ A-147867 (female), MCZ A-147870 (male), MCZ A-147883 (male), MCZ A-147888 (female), MCZ A-147889 (male), MCZ A-147890 (female), MCZ A-147891 (male), Gabon, Batéké Plateau National Park, Haut-Ogooué. P. schioetzi- MVZ 253334 (juvenile), Nigeria, Cross River National Park, Obudu Cattle Ranch, Cross River State. P. steindachneri- ZMB 20429 (lectotype; male?), Cameroon, “Banjo-Bezirk” [= Banyo District]; MCZ A-19577 (paralectotype; female), Cameroon, Banyo, Adamaoua (or Adamawa) Region; MCZ A-138056 (female), MCZ A-138057 (female), MCZ A-138058 (female), MCZ A-138059 (female), MCZ A-138060 (female), Cameroon, village at Mt. Mbam; MCZ A-138104 (female), MCZ A-138105 (female), MCZ A-138106 (female), MCZ A-138107 (male?), MCZ A-138108 (female), MCZ A-138109 (female), MCZ A- 138110 (female), MCZ A-138112 (female), MCZ A-138113 (female), Cameroon, Elak Oku Village, Northwest Province; NMP6V 74519/2 (male), Cameroon – Nigeria border, Gotel Mts., Tchabal Wadi (Gangirwal), locality 1; NMP6V 74520/1 (female), Cameroon – Nigeria border, Gotel Mts., Tchabal Wadi (Gangirwal), locality 2; NMP6V 74522/1 (female), Cameroon – Nigeria border, Gotel Mts., Tchabal Wadi (Gangirwal), locality 4. P. werneri- ZMB 20434 (lectotype; male), Cameroon, Banjobezirk [= Banyo District], Adamawa Province; ZMB 20789 (paralectotype; female), ZMB 74532 (paralectotype; female), ZMB 74533 (paralectotype; female), Cameroon, Bamenda, Northwest Province; MVZ 234835 (female), Cameroon, Ndop, Northwest Province.
Table S1. Phrynobatrachus specimens included in molecular analyses. Museum abbreviations follow Sabaj Pérez (2013). See Fig. 2B for map of montane localities where specimens of the P. steindachneri complex were collected. Species Collection No. GenBank No. Locality Elevation Reference P. africanus CAS 207779 FJ769116 Equatorial Guinea: Bioko Id.: ca 0-20 Zimkus (2009) 3.6 km N (by rd) of Luba P. africanus MCZ A-136731 FJ769117 Cameroon: Littoral Province: E 0-200 Zimkus (2009) of Samakeke, Mangombe Forest Reserve P. africanus MCZ A-136756 FJ769118 Cameroon: Southwest Province: 500 Zimkus (2009) Efolofo, N slope of Mt. Cameroon P. africanus MCZ A-136800 FJ769119 Cameroon: Southwest Province: 600-800 Zimkus (2009) E of Ntale Village, Banyang Mbo Forest Reserve, N/NW of Ntale Plateau P. africanus MCZ A-136839 FJ769120 Cameroon: Southwest Province: 150-200 Zimkus (2009) Ekoneman Awa, Ejagham Forest Reserve P. africanus MCZ A-136944 FJ769121 Cameroon: South Province: East 150-200 Zimkus (2009) of Campo, Campo-Ma'an National Park P. africanus MCZ A-136945 FJ769122 Cameroon: South Province: East 150-200 Zimkus (2009) of Campo, Campo-Ma'an National Park P. batesii MCZ A-136783 FJ769113 Cameroon: Southwest Province: 600-800 Zimkus (2009) East of Ntale Village, Banyang Mbo Forest Reserve, N/NW of Ntale Plateau P. batesii MCZ A-136784 FJ769114 Cameroon: Southwest Province: 600-800 Zimkus (2009) East of Ntale Village, Banyang Mbo Forest Reserve, N/NW of Ntale Plateau P. batesii MCZ A-136793 FJ769115 Cameroon: Southwest Province: 600-800 Zimkus (2009) E of Ntale Village, Banyang Mbo Forest Reserve, N/NW of Ntale Plateau P. chukuchuku MCZ A-138124 FJ769103 Cameroon: Northwest Province: 2800 Zimkus (2009) Mt. Oku, near summit P. chukuchuku MCZ A-138125 FJ769104 Cameroon: Northwest Province: 2800 Zimkus (2009) Mt. Oku, near summit P. chukuchuku MCZ A-138126 FJ769105 Cameroon: Northwest Province: 2800 Zimkus (2009) Mt. Oku, near summit P. cricogaster MCZ A-138030 FJ769101 Cameroon: Southwest Province: 1380 Zimkus (2009) Nsoung (Mt. Manengouba) P. cricogaster ZMB 71458 FJ769100 Nigeria: Cross River State: 1500-1600 Zimkus (2009) Cross River National Park, Okwangwo (Obudu Plateau) P. cricogaster MV 9.8 FJ769102 Cameroon: Littoral Province: 1100-1200 Zimkus (2009) Mt. Nlonako P. danko MCZ A-139529 GU732281 Nigeria: Taraba: Kurmi Danko 1500 Blackburn (2010) (Mambilla Pleateau) P. danko MCZ A-139530 GU732282 Nigeria: Taraba: Kurmi Danko 1500 Blackburn (2010) (Mambilla Pleateau) P. danko MCZ A-139531 GU732283 Nigeria: Taraba: Kurmi Danko 1500 Blackburn (2010) (Mambilla Pleateau) P. jimzimkusi sp. n. MCZ A-136891 FJ769090 Cameroon: West Province: Mt. 2500-2600 Zimkus (2009) Bamboutos summit P. jimzimkusi sp. n. MCZ A-136892 FJ769091 Cameroon: West Province: Mt. 2500-2600 Zimkus (2009) Bamboutos summit P. jimzimkusi sp. n. MCZ A-136903 FJ769085 Cameroon: West Province: 2050 Zimkus (2009) Below Mt. Bamboutos P. jimzimkusi sp. n. MCZ A-136904 FJ769086 Cameroon: West Province: 2050 Zimkus (2009) Below Mt. Bamboutos P. jimzimkusi sp. n. MCZ A-136909 FJ769084 Cameroon: West Province: 2050 Zimkus (2009) Below Mt. Bamboutos P. jimzimkusi sp. n. MCZ A-136928 FJ769093 Cameroon: Southwest Province: 2000-2100 Zimkus (2009) Mt. Manengouba P. jimzimkusi sp. n. MCZ A-136927 FJ769092 Cameroon: Southwest Province: 2000-2100 Zimkus (2009) Mt. Manengouba P. jimzimkusi sp. n. MCZ A-138079 HM441259 Cameroon: West Province: Lake 2040 Blackburn Awing (Mt. Lefo) (unpublished) P. jimzimkusi sp. n. MCZ A-138080 HM441260 Cameroon: West Province: Lake 2040 Blackburn Awing (Mt. Lefo) (unpublished) P. jimzimkusi sp. n. MCZ A-138081 HM441261 Cameroon: West Province: Lake 2040 Blackburn Awing (Mt. Lefo) (unpublished) P. jimzimkusi sp. n. MCZ A-138064 FJ769083 Cameroon: West Province: Mt. 2020 Zimkus (2009) Bamboutos P. jimzimkusi sp. n. MCZ A-139119 FJ769089 Cameroon: Northwest Province: 2800 Zimkus (2009) Mt. Oku, vicinity summit P. jimzimkusi sp. n. MVZ 253341 FJ769088 Nigeria: Cross River State: 1500-1600 Zimkus (2009) Cross River National Park, Obudu Cattle Ranch (Obudu Plateau) P. jimzimkusi sp. n. MVZ 253342 FJ769133 Nigeria: Cross River State: 1500-1600 Zimkus (2009) Cross River National Park, Obudu Cattle Ranch (Obudu Plateau) P. jimzimkusi sp. n. NMP6V 73385/1 KF020520 Cameroon: Northwest Province: 2100 This study Kedjom Keku (= Big Babanki) summit (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 73385/2 KF020521 Cameroon: Northwest Province: 2100 This study Kedjom Keku (= Big Babanki) summit (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 73385/8 KF020522 Cameroon: Northwest Province: 2100 This study Kedjom Keku (= Big Babanki) summit (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 73390/1 KF020523 Cameroon: Northwest Province: 1310 This study Kedjom Keku (= Big Babanki) slope (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 73390/2 KF020524 Cameroon: Northwest Province: 1310 This study Kedjom Keku (= Big Babanki) slope (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74525/1 KF020525 Cameroon: Northwest Province: 1770 This study Babungo (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74525/2 KF020526 Cameroon: Northwest Province: 1770 This study Babungo (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74525/7 KF020527 Cameroon: Northwest Province: 1770 This study Babungo (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74526/1 KF020528 Cameroon: Northwest Province: 2070 This study Bat (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74526/2 KF020529 Cameroon: Northwest Province: 2070 This study Bat (Bamena-Banso Highlands) P. jimzimkusi sp. n. NMP6V 74526/3 KF020530 Cameroon: Northwest Province: 2070 This study Bat (Bamena-Banso Highlands) P. manengoubensis MCZ A-136923 FJ769109 Cameroon: Southwest Province: 1800-1900 Zimkus (2009) Mt. Manengouba P. manengoubensis MCZ A-136924 FJ76910 Cameroon: Southwest Province: 1800-1900 Zimkus (2009) Mt. Manengouba P. manengoubensis MCZ A-138043 FJ769106 Cameroon: Southwest Province: 1850 Zimkus (2009) Mt. Manengouba P. manengoubensis MCZ A-138044 FJ769107 Cameroon: Southwest Province: 1850 Zimkus (2009) Mt. Manengouba P. manengoubensis MCZ A-138047 FJ769108 Cameroon: Southwest Province: 2100 Zimkus (2009) Mt. Manengouba P. njiomock sp. n. MCZ A-136875 FJ769095 Cameroon: Northwest Province: 2220 Zimkus (2009) Lake Oku P. njiomock sp. n. MCZ A-136876 FJ769096 Cameroon: Northwest Province: 2220 Zimkus (2009) Lake Oku P. njiomock sp. n. MCZ A-136877 FJ769097 Cameroon: Northwest Province: 2220 Zimkus (2009) Lake Oku P. njiomock sp. n. MCZ A-136879 FJ769098 Cameroon: Northwest Province: 2220 Zimkus (2009) Lake Oku P. njiomock sp. n. MCZ A-138118 FJ769094 Cameroon: Northwest Province: 2370 Zimkus (2009) Mt. Oku, forest reserve northeast of summit P. njiomock sp. n. MCZ A-138135 FJ769099 Cameroon: Northwest Province: 2220 Zimkus (2009) Lake Oku, forest at edge of lake P. njiomock sp. n. MCZ A-138136 KF020531 Cameroon: Northwest Province: 2220 This study Lake Oku, forest at edge of lake P. njiomock sp. n. NMP6V 73382/1 KF020532 Cameroon: Northwest Province: 2219 This study Lake Oku P. njiomock sp. n. NMP6V 73382/2 KF020533 Cameroon: Northwest Province: 2219 This study Lake Oku P. njiomock sp. n. NMP6V 74523/1 KF020534 Cameroon: Northwest Province: 2219 This study Lake Oku P. njiomock sp. n. NMP6V 74523/2 KF020535 Cameroon: Northwest Province: 2219 This study Lake Oku P. njiomock sp. n. NMP6V 74524 KF020536 Cameroon: Northwest Province: 2400 This study stream 2km NW of Lake Oku P. ruthbeateae MCZ A-147865 KF020537 Gabon: Haut-Ogooué Province: 414 This study Batéké Plateau National Park P. ruthbeateae MCZ A-147891 KF020538 Gabon: Haut-Ogooué Province: 395 This study Batéké Plateau National Park P. schioetzi MVZ 253334 FJ769112 Nigeria: Cross River State: 1500-1600 Zimkus (2009) as Cross River National Park, P. sp (werneri B); Obudu Cattle Ranch (Obudu reidentified by Plateau) Blackburn & Rödel (2011) P. schioetzi ZMB 74523 JF828577 Nigera: Cross River State, 1500 Blackburn & Rödel Obudu Plateau, tributary of (2011) Afundi River P. schioetzi ZMB 74524 JF828578 Nigera: Cross River State, 1500 Blackburn & Rödel Obudu Plateau, tributary of (2011) Afundi River P. schioetzi ZMB 74527 JF828579 Nigera: Cross River State, 1500 Blackburn & Rödel Obudu Plateau, tributary of (2011) Afundi River P. steindachneri MCZ A-138057 HM441256 Cameroon: West Province: - Blackburn village at Mt. Mbam (unpublished) P. steindachneri MCZ A-138058 HM441257 Cameroon: West Province: - Blackburn village at Mt. Mbam (unpublished) P. steindachneri MCZ A-138059 HM441258 Cameroon: West Province: - Blackburn village at Mt. Mbam (unpublished) P. steindachneri MCZ A-138104 FJ769087 Cameroon: Northwest Province: 1900 Zimkus (2009) Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138105 KF020539 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138108 KF020540 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138109 KF020541 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138112 KF020542 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138116 KF020543 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-138117 KF020544 Cameroon: Northwest Province: 1900 This study Elak Oku Village (Mt. Oku)_ P. steindachneri MCZ A-139608 KF020545 Nigeria: Taraba: Ngel Nyaki 1460 This study (Mambilla Plateau) P. steindachneri MCZ A-139609 KF020546 Nigeria: Taraba: Ngel Nyaki 1460 This study (Mambilla Plateau) P. steindachneri NMP6V 74519/1 KF020547 Cameroon – Nigeria border: 1940 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 1 P. steindachneri NMP6V 74519/2 KF020548 Cameroon – Nigeria border: 1940 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 1 P. steindachneri NMP6V 74519/3 KF020549 Cameroon – Nigeria border: 1940 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 1 P. steindachneri NMP6V 74519/5 KF020550 Cameroon – Nigeria border: 1940 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 1 P. steindachneri NMP6V 74520/1 KF020551 Cameroon – Nigeria border: 2250 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 2 P. steindachneri NMP6V 74520/5 KF020552 Cameroon – Nigeria border: 2250 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 2 P. steindachneri NMP6V 74520/6 KF020553 Cameroon – Nigeria border: 2250 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 2 P. steindachneri NMP6V 74521/1 KF020554 Cameroon – Nigeria border: 2010 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 3 P. steindachneri NMP6V 74522/1 KF020555 Cameroon – Nigeria border: 2330 This study Gotel Mts.: Tchabal Wadi (Gangirwal), locality 4 P. steindachneri ZFMK 75705 KF020556 Cameroon: Tchabal Mbabo, 5 2060 This study km NEE of Fungoi, creek in gallery forest on southern slope P. steindachneri ZFMK 75706 KF020557 Cameroon: Tchabal Mbabo, 5 2060 This study km NEE of Fungoi, creek in gallery forest on southern slope P. werneri MVZ 234835 FJ769111 Cameroon: Northwest Province: 1100 Zimkus (2009) Ndop (Bamenda-Banso Highlands) P. werneri MCZ A-138074 GU732284 Cameroon: West Province: Mt. 2050 Blackburn (2010) Bamboutos