The first endemic West African vertebrate family – a new anuran family highlighting the uniqueness of the Upper Guinean biodiversity hotspot

Background Higher-level systematics in amphibians is relatively stable. However, recent phylogenetic studies of African torrent-frogs have uncovered high divergence in these phenotypically and ecologically similar frogs, in particular between West African torrent-frogs versus Central (Petropedetes) and East African (Arthroleptides and Ericabatrachus) lineages. Because of the considerable molecular divergence, and external morphology of the single West African torrent-frog species a new genus was erected (Odontobatrachus). In this study we aim to clarify the systematic position of West African torrent-frogs (Odontobatrachus). We determine the relationships of torrent-frogs using a multi-locus, nuclear and mitochondrial, dataset and include genera of all African and Asian ranoid families. Using micro-tomographic scanning we examine osteology and external morphological features of West African torrent-frogs to compare them with other ranoids. Results Our analyses reveal Petropedetidae (Arthroleptides, Ericabatrachus, Petropedetes) as the sister taxon of the Pyxicephalidae. The phylogenetic position of Odontobatrachus is clearly outside Petropedetidae, and not closely related to any other ranoid family. According to our time-tree estimation Odontobatrachus has been separated from other frog lineages since the Cretaceous (90.1 Ma; confidence interval: 84.2-97.1 Ma). Along with this molecular evidence, osteological and external diagnostic characters recognize West African torrent-frogs as distinct from other ranoids and provide strong support for the necessity of the recognition of a new family of frogs. This is the only endemic vertebrate family occurring in the Upper Guinea biodiversity hotspot. Conclusion Based on molecular and morphological distinctiveness, the West African torrent-frog Odontobatrachus natator is allocated to a newly described anuran family. The discovery of an endemic vertebrate family in West Africa highlights the Upper Guinean forests as an outstanding, but highly endangered biodiversity hotspot.


Molecular analyses 1.1 GenBank accession numbers
The origin of the family Odontobatrachidae dates back to major splits within the higher-level taxon Ranoidae [4]. Interestingly, molecular timescales could provide a means to estimate taxonomic rank for amphibians [5]. Avise and Liu' [5] estimation for amphibians fits well within the taxonomic rank of family for Odontobatrachidae. Given are mean estimate values (above the 95% congruence interval bar, CI), posterior probabilities (below CI) and node numbers behind the respective node. Divergence times are provided in Table A2. The tree is rooted with hierarchical outgroups Sarcopterygii (Latimeria), Caudata (Cryptobranchidae, Hynobiidae); outgroups not shown (for GenBank numbers of outgroups see Table A3).

Topology tests
Likelihood values of seven alternative topologies were tested against the unconstraint optimal tree ( Fig. 1). Results of the AU-test and the SH-test are provided in Table A4. The genus Odontobatrachus was placed either basal to the Petropedetidae (Table A4.1), sister to any genus within the Petropedetidae (Table A4.

2-A4.4) or sister to a clade comprising Central and East
African torrent-frog genera (Petropedetes + Arthroleptides; Table A4.5). All placements related to the family Petropedetidae were clearly rejected. However, due to a weak resolution in basal nodes (lacking resolved relationships within the Natatanura), topologies placing Odontobatrachidae fam. nov. sister to other families cannot be excluded completely -as shown in the potential grouping with Conrauidae, which cannot be statistically significantly rejected.
However, alternative topologies show lower probability values and were not shown to have any strong support in any phylogenetic analyses (compare Table A4.1, A4.6 and A4.7).   Pectoral girdle: pectoral girdle firmisternal ( Figure A2e); coracoids robust, perpendicular to each other and fused with the scapulae, their proximal ends not overlapping, proximal and distal epiphyses of the same size; omosternum completely ossified, base of omosternum convex and not divided as in many other African ranids [7,8]; episternum cartilaginous with shape of transversal section of a mushroom whose "head´s" centre is slightly concave and whose stipe extends anteriorly; clavicle with broad base that is united with the pars acromialis of the scapula,

Osteological characters distinguishing Petropedetidae and Odontobatrachidae fam. nov.
Using micro-tomographic analysis we differentiated Odontobatrachus from formerly assumed African torrent frogs Barej et al. [9] synonymised P. newtoni with P. johnstoni and described three new taxa. Vouchers examined by Scott [8] were not available to us; hence, species assignments cannot be reviewed to ensure comparison with our material. Recognized differences between Odontobatrachidae fam. nov and Petropedetidae are summarized on genus level in Table A5 and illustrated in Figures A3-A15, respectively.          Figure   A4.   Species assignments as in Figure A4.  Figure A4; Petropedetes cameronensis (ZMB 27159). P. cameronensis lacks the enlarged crista as brachial hypertrophy is not present in the species [9]; comparable to the on-related genus Leptodactylodon [10], in which males of species with brachial hypertrophy likewise possess enlarged cristae on the humerus.  Figure A4; Petropedetes cameronensis (ZMB 27159). P. cameronensis lacks a spike [9].

Amphibian diversity in African realms
In order to get an overview of overall diversity and number of endemic taxa, numbers of families and genera occurring in the Upper Guinea forests, we compared endemicity on different systematic levels in the four African biogeographic regions of western, central, eastern and southern Africa as recognized in published literature [11][12][13][14][15][16]. We refer to rough geographic sub-divisions, as present knowledge is incomplete, differing between vertebrate groups and applied data [16,17]. Sub-division is as follow: western Africa: Senegal to west of Cross River,  Table   A6 provides data on shared higher taxa between at least two biogeographic regions, endemics and total numbers at generic and family level are given (Table A6). Data extracted from Frost [18].