A new species of puddle frog from an unexplored mountain in southwestern Ethiopia (Anura, Phrynobatrachidae, Phrynobatrachus)

Abstract A new species of Phrynobatrachus is described from the unexplored and isolated Bibita Mountain, southwestern Ethiopia, based on morphological characters and sequences of the mitochondrial rRNA16s. The new species can be distinguished from all its congeners by a small size (SVL = 16.8 ± 0.1 mm for males, 20.3 ± 0.9 mm for females), a slender body with long legs and elongated fingers and toes, a golden coloration, a completely hidden tympanum, and a marked canthus rostralis. The phylogenetic hypothesis based on 16s sequences places the new species as sister to the species group that includes P.natalensis, although it is morphologically more similar to other dwarf Phrynobatrachus species, such as the Ethiopian P.minutus.


Introduction
The highlands of Ethiopia are known for their high degree of diversity and endemism (Williams et al. 2004). Approximately half of all species of anurans (frogs and toads) of Ethiopia are endemic, including five endemic genera (Largen andSpawls 2010, Gower et al. 2013). Despite a recent increase in studies on the diversity of the Ethiopian fauna, the southwestern part of the country remains poorly studied, in part due to the difficulty in accessing the region. A small number of collections of amphibians have been obtained in this area, most of them from the vicinity of the towns of Bonga and Mizan Teferi ( Figure 1A). Recent sampling campaigns conducted in the forests of the southwest revealed the existence of multiple undescribed taxa (Reyes-Velasco et al. 2018a, b). Additionally, the area harbors some of the last remaining forests in Ethiopia, giving it great potential for undiscovered diversity and making it of particular interest for taxonomists and conservationists.
While conducting fieldwork in the southwestern part of the country, we came across an undescribed species member of the genus Phrynobatrachus. This genus is one of the most species rich genera of African anurans, with 91 described and multiple undescribed taxa (Zimkus andSchick 2010, Frost 2018). This genus is widespread across sub-Saharan Africa. At least five species of Phrynobatrachus are found in Ethiopia: P. bullans Crutsinger, Pickersgill, Channing, andMoyer, 2004, P. inexpectatus Largen, 2001, P. minutus (Boulenger, 1895), P. natalensis (Smith, 1849), as well as P. sp. n. "Oromia" (Zimkus 2008, Largen and Spawls 2010, Frost 2018). The new species described here has multiple morphological characters that differentiate it from all other members of the genus, which is also supported by molecular evidence. Here, we describe the species as new to science.

Taxonomic sampling
The mountain of Bibita in southwestern Ethiopia (6.8034N, 35.0602E), an isolated plateau located between the Gambela and the Southern Nations, Nationalities, and Peoples' Regions, in the Bench Maji Zone, was explored ( Figure 1). This mountain is approximately 50 km southwest of Mizan Teferi and ~18 km east from the border with South Sudan. Bibita Mountain is separated from the rest of the Ethiopian highlands by the lowlands formed by the tributaries of the Akobo River, including the Gilo River. This mountain is also sometimes referred to as Gurra Farda (Hundera and Deboch 2008), which translates to "donkey's ears" in Oromo. On 16 and 17 June 2018, ten specimens of an undescribed species of Phrynobatrachus (two males, eight females) were collected, as well as multiple egg clutches from a pond in Bibita Mountain (6. 82293N, 35.0938E; 1972 m a. s. l.). We photographed the specimens in situ, and took live photographs in captivity. We then euthanized them using a ventral application of 20% benzocaine gel (Chen and Comb 1999). After euthanasia, we took additional photographs of dorsal and ventral views of each specimen. We collected liver tissue samples from all specimens and egg clutches and stored them in RNAlater (ThermoFisher Scientific), and then fixed all specimens in 10% formalin for 48 hours and stored them in 70% Ethanol. All specimens are deposited at the Herpetology Collection of the Addis Ababa University, Addis Ababa, Ethiopia.

Recordings of advertisement calls
A single calling male was recorded during the night of 17 June 2018 using a Sony Handycam camcorder. The soundtrack of the video was extracted using Adobe Premiere Pro and analyzed using Praat (Boersma and Weenink 2013). We analyzed two call bouts and fourteen notes produced by a single individual for dominant frequency, note duration, and interval inter-notes. We compared these calls to the advertisement calls of Phrynobatrachus minutus (recorded between Bonga and Jimma; 7.5350N, 36.5606E, 2216 m a. s. l.; SB233) and P. natalensis (east of Kibre Mengist; 5.8679N, 39.0490E, 1665 m a. s. l.; specimen not collected) which we recorded using a Marantz PMD661 MKII solid-state recorder and Sennheiser ME66 microphone. Unfortunately, calls for the other three Phrynobatrachus that occur in Ethiopia (P. bullans, P. inexpectatus, and P. sp. n. "Oromia" of Zimkus and Schick 2010) were not available for comparison.

Morphological measurements
For each specimen of the type series, we used an SPI dial caliper (model #31-415-3, precision: 0.1 mm) to take the following measurements as defined in Watters et al. (2016)

DNA extraction, PCR amplification, and phylogenetic analyses
We extracted DNA from the liver tissue samples stored in RNAlater with the use of standard potassium acetate protocol, or with the use of a DNeasy Blood & Tissue Kit (Qiagen). We also extracted DNA from eggs collected on leaves where females and the amplected pair of the new species were found, to ensure that these belonged to the new species. We then measured DNA concentration for each one of the samples using a broad range kit in a Qubit fluorometer (Life Technologies). We amplified a fraction of the 16s rRNA mitochondrial gene with the use of the primers LX12SN1a (forward) and LX16S1Ra (reverse) of Zhang et al. (2013). We performed Polymerase Chain Reaction (PCR) in total volumes of 48 μl with the use of regular Taq (Invitrogen), with the following conditions: initial denaturation at 94 °C (two minutes), followed by 35 cycles consisting of a denaturation step at 94 °C (30 seconds), annealing step at 48 °C (30 seconds), and extension step at 72 °C (one minute). The final extension step consisted of one minute at 72 °C. We shipped the unpurified PCR products for sequencing at BGI Tech Solutions (Hong Kong). We used the program Geneious v 9.1.6 (Biomatters Ltd., Auckland, NZ) to manually trim and edit the raw chromatograms. We included additional sequences of Phrynobatrachus from GenBank in order to infer the phylogenetic positions of the new material. We deposited all new sequences in GenBank (Suppl. material 1: Table S1). We aligned all sequences in MAFFT (Katoh et al. 2017) version 7, with the Q-INS-I option, which resulted in a final alignment of 589 bp. We then selected the best-fit model of nucleotide substitution in PartitionFinder v.1.1.1 (Lanfear et al. 2012). The model selected was the K2P + I + gamma. We performed Bayesian inference of phylogeny (BI) in MrBayes v 3.2.2 (Ronquist et al. 2012) in the CIPRES science gateway server (Miller et al. 2010). The Bayesian analysis consisted of four runs of 10 7 generations each, with four chains (one cold and three heated), sampling every 1,000 generations. We used Tracer v1.6 (Drummond and Rambaut 2007) to confirm that independent runs had converged, based on the overlap in likelihood and parameter estimates among runs, as well as effective sample size (ESS) and Potential Scale Reduction Factor value estimates (PSRF). PSRF indicated that individual runs had converged by 10 5 generations, so we discarded the first 25% of the runs as burn-in. We then annotated posterior probability values on the resulting topology in the program TreeAnnotator v 1.8.3 (Rambaut 2014). Diagnosis. Small species (SVL = 16.8 ± 0.1 mm for males, 20.3 ± 0.9 mm for females) attributed to the genus Phrynobatrachus by the presence of tarsal and outer metatarsal tubercles (Suppl. material 2: Figure S1A). Body slender, with long legs (tibia length/SVL = 0.6 in both sexes) rather long snout for the genus and very elongated fingers (hand length/SVL = 0.3 in both sexes) and toes (foot length/SVL = 0.6 in both sexes) in comparison to its congeners. Webbing absent between fingers and minimal between toes. Tympanum not visible. Canthus rostralis marked and concave from nostril to eye. Snout pointed. Nostrils not visible from above. Eyelid spine absent. Throat of adult males white with light grey freckles on the anterior third, without any spinulae. Femoral glands hardly distinguishable but present in adult males. Two ridges in the scapular region and two short, oblique ridges behind the eyes. These four ridges may be all disjointed, the two scapular ridges may be jointed to form a chevron shape, or the ridges may be jointed laterally in an hourglass shape. Comparisons. This species can easily be distinguished from other Ethiopian Phrynobatrachus: the body is slenderer, the hind limbs, fingers and toes are longer than all four other described Ethiopian Phrynobatrachus, P. bullans, P. natalensis, P. minutus, and P. inexpectatus. Additionally, the tips of fingers and toes are more enlarged than in any of these species, particularly in females. It is similar in size as P. minutus, slightly larger than P. inexpectatus and much smaller than P. bullans and P. natalensis. It can be further distinguished from P. natalensis by a more marked canthus rostralis and a completely hidden tympanum. Adult males of P. bibita sp. n. can be distinguished from P. bullans, P. natalensis, P. minutus, and P. inexpectatus by the white coloration of their throat (Largen 2001, Crutsinger et al. 2004. The new species is also distinct from the three junior synonyms of P. natalensis described from Ethiopia: Arthroleptis bottegi Boulenger, 1895, described from Auta, Bale province, possesses a distinct tympanum, a rounded snout, and is much larger than P. bibita (SVL of female holotype = 31 mm vs. 20.3 ± 0.9 mm in female P. bibita). Phrynobatrachus sciangallarum Scortecci, 1943, described from a presumably adult male from Murle, Gemu-Gofa province, differs from P. bibita by the presence of a considerably darkened throat and the lack of femoral glands. Additionally, it has more extensive foot webbing than P. bibita (Largen 2001). Arthroleptis-Phrynobatrachus zavattarii Scortecci, 1943, described from Caschei, Gemu-Gofa province, only differs from P. sciangallarum by the extent of the foot webbing, and all measurements of the type specimens fall within the range of P. natalensis (Largen 2001). Blackburn (2014) provided color photographs of the holotypes of these last two junior synonyms of P. natalensis. Based on those photographs, it appears that P. bibita has a slenderer head and more elongated fingers than these individuals. Finally, the type localities for these three junior synonyms of P. natalensis are in the southern part of the country, an area that is dry and at low elevation, in contrast with the habitat occupied by P. bibita sp. n.
Coloration of holotype in life. The body is golden ( Figure 2A); a dark chevron is present in the scapular region underlining two short back ridges. A faint dark triangle is present between the eyes and a faint dark bar is present between the eye and the shoulder. The upper eyelids are darker than the rest of the head. Loreal region faintly darker than the rest of the head. Upper and lower jaws lack any blotching. The flanks and the sides of the head present numerous small white spots, extending to the upper lip below the eye. Iris dark gold with a golden ring around the iris interrupted by a black dot at the bottom of the iris. The thighs and legs are faintly and irregularly banded. The back of the thighs is yellowish with irregular coverage of melanophores. A dark triangle is present in the vent region. Ventral region is white molted with light grey. The throat is white, with the anterior third light grey with white freckles. Ventral side of the thighs and legs shows irregular melanophores and white spots.
Coloration of holotype in preservative. After euthanasia, the individual's coloration darkened notably ( Figure 2B). In preservative, the dorsum presents a grey coloration.
Coloration of the body varies from golden to light brown, with few large light green blotches in some individuals. Most specimens present a dark chevron in the scapular region underlining two short back ridges that are either jointed or disjointed with two oblique ridges between the back of the eye and the shoulders. A more or less pronounced dark stripe is present between the nostril and the eye, and between the eye and the arm. A more or less distinctive dark bar is present between the eyes and some individuals present a lighter or green snout. The flanks and the sides of the head present numerous small white spots, extending to the upper lip in some individuals. The thighs and legs of some individuals are very faintly and irregularly banded. The back of the thighs is cream with small light grey spots. Vocal sac in adult males is white with the frontal third of the throat light grey with white freckles, while in females the throat and the ventral skin is light grey or yellowish molted with white. Some individuals have a thin light line on the backside of the thigh from the vent to the tarsal tubercle. A dark triangle is generally present in the back of the thigh, around the vent.
Etymology. The specific name refers to Bibita Mountain, the type and only known locality for the species. It is an invariable noun used in apposition.
Habitat, distribution, and natural history. All individuals were collected in a single large overgrown forest pond ( Figure 3A), at night. The surrounding forest consisted of large trees with overhanging epiphytes and dense undergrowth. All females and the amplected pair were found on vegetation ca. 30 cm above water ( Figure 3B). A single male was found in the water, presumably while calling. All collected females were gravid, and bicolor eggs were visible through the skin. Females seemed to aggregate in specific areas of the pond, were numerous egg clutches were found on leaves overhanging the water ( Figure 3B). Laying eggs on vegetation overhanging the water is unusual in Phrynobatrachus, most species laying their eggs directly in the water (Zimkus et al. 2012). We thus confirmed that these eggs belonged to Phrynobatrachus bibita sp. n. by sequencing their mitochondrial rRNA 16s. Various forms of terrestrial egg deposition have been described in the genus Phrynobatrachus (Zimkus et al. 2012): most similarly to P. bibita, P. sandersoni (Parker, 1935) lays its eggs on vegetation up to 2 m above small puddles, small streams or water-saturated soil (Amiet 1981) and P. krefftii lays its eggs above the water, on rocks or vegetation (Harper and Vonesh 2010). Phrynobatrachus guineensis Guibé & Lamotte, 1961 lays its eggs on the bark of trees above water-filled tree holes (Rödel 1998) and P. dendrobates lays its eggs in tree holes or above streams (Zimkus et al. 2012). Finally, P. phyllophilus Rödel & Ernst, 2002, P. tokba (Chabanaud, 1921, and P. villiersi Guibé, 1969 lay their eggs on the leaf litter or the forest floor (Rödel andErnst 2002a, 2002b;Zimkus et al. 2012). Phrynobatrachus bibita sp. n. thus adds to the diversity of reproductive modes in the genus.
Five egg clutches were photographed and contained approximately 30 eggs each (range 22 to 33). All observed clutches were found between 20 -40 cm above water on vegetation, and up to two clutches were found on a single leaf. When laid, the eggs are bicolor, heavily pigmented, and encased in a thin gelatinous layer; as the egg develops, the pigmentation is more evenly distributed at its surface, with dark brown freckle, and the gelatinous layer becomes much thicker. It is possible that female P. bibita sp. n. are guarding their egg clutches in a similar manner as P. sandersoni females, which attend their eggs at night by standing over them or staying in the close proximity (Amiet 1981). Additional behavioral observations are necessary to determine whether females P. bibita sp. n. truly display such parental care. Other frog species were found in the same pond or nearby, including Afrixalus enseticola Largen, 1974, Leptopelis vannutellii (Boulenger, 1898, Paracassina obscura (Boulenger, 1895), and Ptychadena cf. erlangeri (Ahl, 1924).
The forest in Bibita Mountain appears to be well preserved, as there are no settlements inside the forest, and a footpath is the only way to cross the forest. People from the surrounded villages harvest wild honey from the forest, but this is the only   noticeable human activity there. Based on satellite imagery, no human disturbance is apparent at the higher elevations of the mountain; however, most areas below 1900 m have been transformed into agricultural land.
Call. The advertisement call is composed of a series of pulsed notes with a slight upward frequency modulation within each note ( Figure 4A). It is similar to that of Phrynobatrachus minutus ( Figure 4B) in spectral structure, note length, and dominant frequency. The vocalization of P. bibita sp. n. can be distinguished from that of P. natalensis ( Figure 4C) by its higher dominant frequency, indicative of its smaller body size. Dominant frequency: 3318 ± 94 Hz. Note duration = 630 ± 80 ms. Interval inter-notes = 1085 ± 630 ms. The mediocre quality of our recording prevents us from analyzing the advertisement call of P. bibita sp. n. in further details, and a more complete description of their vocalization will be needed in the future, in particular to distinguish it from other species of dwarf Phrynobatrachus.
Estimates of evolutionary relationships. The goal of our phylogenetic analysis was to test the phylogenetic position of the new species, not to obtain a phylogeny of the genus, which has been previously done elsewhere ). Our phylogenetic estimates recovered three main lineages in the genus Phrynobatrachus ( Figure 5); however, several of the nodes have low support. We briefly describe the resulting topology below.
We recovered a basal group of Phrynobatrachus, which included the species P. acutirostris Nieden, 1912, P. krefftii, P. dendrobates, P. petropedetoides Ahl, 1924, and sandersoni. This clade is consistent with the "Clade A" of , however this clade received low support (posterior probability (pp) < 0.5). All remaining species in the genus were grouped into two major clades, which received strong support (pp = 1). The first one of these two groups is similar to the "Clade B" of , which includes the Ethiopian species P. minutus, P. inexpectatus, and P. sp. n. "Oromia" of Zimkus and Schick (2010), while the second group is consistent with the "Clade C" of , and includes the Ethiopian species P. natalensis. Phrynobatrachus bibita sp. n. was not closely related to any of the other Ethiopian species, and instead basal to the last group ("Clade C" of ), however with high support (pp = 1). Our analysis grouped the egg clutch collected (SB422) with the adults P. bibita sp. n. (holotype SB440 and paratopotypes SB419, SB420, SB425), ensuring that it belonged to the new species. We did not include samples of three junior synonyms of P. natalensis from Ethiopia (see Comparisons), however, samples referable to these junior synonyms have previously been included on a phylogeny of members of the P. natalensis group (Lara 2016), and they are well nested within P. natalensis, we can thus conclude that they do not represent P. bibita.

Discussion
Most of the natural vegetation of the Ethiopian highlands has been transformed for agriculture or into grazing fields for cattle (Williams et al. 2004). However, in the southwestern end of the country, a few areas still conserve their original vegetation, and several of these areas remain largely unexplored. Bibita Mountain is one of such areas, and to our knowledge, no vertebrate collections has ever been obtained there. The only published account on the flora or fauna of Bibita Mountain comes from a study on plant composition and structure (Hundera and Deboch 2008). The discovery of a distinctly new species of Phrynobatrachus after only two days of survey calls out for further exploration of the biological diversity in this area. Our previous research showed that the amphibian fauna of Ethiopia is underestimated by the current taxonomy, and we believe that further research on Bibita Mountain, as well as in other nearby areas might greatly increase the number of species of amphibians in Ethiopia.
The phylogenetic relationships of many members of the genus Phrynobatrachus remains unresolved.  recovered three main clades of Phrynobatrachus in their analysis, which they designated as groups 'A', 'B' and 'C'. Our results show that P. bibita sp. n. is not related to other currently described species of Ethiopian Phrynobatrachus, and instead, it appears to be sister to all the members of the P. natalensis group ( Figure 5). Interestingly, the morphology of P. bibita sp. n., including long limbs, elongated fingers and toes, enlarged toe tips and pointed snout, is most similar to the more distantly related species P. sandersoni, P. dendrobates, and P. krefftii. In addition, P. bibita sp. n. shares with these species a rare behavior for the genus consisting of laying eggs on vegetation above the water (Amiet 1981, Harper and Vonesh 2010, Zimkus et al. 2012). Finally, P. bibita sp. n. might display parental care, as observed in P. sandersoni and P. dendrobates (Amiet 1981, Zimkus et al. 2012 and unknown from any other species of the genus. Whether P. bibita sp. n. retained ancestral morphological and behavioral characters shared with the P. krefftii species group, or whether it is a case of convergence remains to be investigated. Additional work is necessary to better understand the evolutionary history of this diverse genus.