Diversity of Ptychadena in Rwanda and taxonomic status of P. chrysogaster Laurent, 1954 (Amphibia, Anura, Ptychadenidae)

Abstract We assess the diversity of Ptychadena species in Rwanda based on re-examination of voucher specimens in museum collections and our own data from recent assessment of the species composition of amphibian communities in Rwanda. We recognize five species which we allocate to the following available names: P. anchietae, P. chrysogaster, P. nilotica, P. porosissima, and P. uzungwensis. We did not find evidence for the presence of P. grandisonae and P. oxyrhynchus which have been listed for the country. The five species can be distinguished by quantitative morphometrics (discriminant analysis, success rate: 100 %) and a number of qualitative characters of external morphology. We provide an identification key to the Rwandan species and describe the morphology of each species in detail. The taxonomic status and the phylogenetic position of Ptychadena chrysogaster are further assessed based on the partial sequence of the mitochondrial 16S rRNA. The species differs genetically from available homologous sequences from congeners by an uncorrected p distance of at least 4.2 % and appears to be most closely related to specimens assigned to P. porosissima, P. mahnerti, “P. aff. uzungwensis” and “P. aff. bibroni”.


Introduction
Ridged Frogs of the genus Ptychadena Boulenger, 1917 are widespread in sub-Saharan Africa where approximately 50 species occur.Species of the genus share a similar general appearance and many are poorly delimited, having been described based on taxonomically doubtful characters.Several species names have been erroneously considered synonyms of others, thus confusing character diagnoses in subsequent accounts; and some taxa were described based on specimens later found to represent more than one species (e.g.Boulenger 1879;Loveridge 1932;Laurent 1954;Guibé and Lamotte 1957;Schmidt and Inger 1959;Lamotte 1967;Poynton 1970;Rödel 2000;Channing 2001; Channing and Howell 2006;Dehling and Sinsch in press).Therefore, even the local/regional diversity of these frogs is often difficult to assess.Herein, we address the diversity of Ridged Frogs in Rwanda.We have recently shown that three species (P.anchietae [Bocage, 1868], P. nilotica [Seetzen, 1855], and P. porosissima [Steindachner, 1867]) inhabit the wetlands along the upper Nile (Dehling and Sinsch in press).
Further taxa have been reported from Rwanda and it is currently unclear which species actually occur in the country.Nieden (1913) reported P. nilotica (referred to as Rana mascareniensis Duméril & Bibron, 1841) from several localities in Rwanda.Based on his own collections, Laurent (1954) reported P. uzungwensis (Loveridge, 1932) and described P. chrysogaster Laurent, 1954 andP. loveridgei Laurent, 1954 as new species, the latter now being considered a synonym of P. porosissima (Schmidt and Inger 1959).Poynton and Broadley (1985), Channing (2001), and Poynton and Channing (2004) stated that P. grandisonae Laurent, 1954 occurs in Rwanda.Fischer and Hinkel (1992) listed only "P.mascareniensis" [= P. nilotica].Poynton et al. (2004) stated that P. anchietae was likely to occur in Rwanda but confirmed records were missing.According to Spawls et al. (2006), P. chrysogaster, P. mascareniensis, and P. uzungwensis occur in Rwanda but not P. anchietae and P. porosissima.Branch (2005) included Rwanda in the geographic range of P. oxyrhynchus (Smith, 1849).Recently, we collected P. anchietae in Rwanda and resurrected the name P. nilotica for the populations which occur along the Nile and in Central Kenya and Tanzania and which had been formerly referred to as Rana mascareniensis or Ptychadena mascareniensis (Sinsch et al. 2012, Dehling and Sinsch in press).
In order to clarify how many and which species occur in Rwanda, we re-examined the specimens of Ptychadena in the herpetological collection of the Royal Museum for Central Africa in Tervuren, Belgium (RMCA), on which almost all previous Rwandan records are based.We herein report the results and compare the findings to our own data from recent assessment of the composition of amphibian communities at numerous locations in Rwanda.We further assess the taxonomic status and the phylogenetic position of Ptychadena chrysogaster based on examination of most of the available voucher material from Rwanda including the type series and on comparison of the partial sequence of the mitochondrial 16S rRNA gene with homologous sequences of its congeners.

Morphological examination
We examined voucher specimens deposited at RMCA.Additional specimens including our recently collected material are deposited in the collection of the Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany (ZFMK).See Appendix 1 for a complete list of examined specimens.
For the morphological analysis, we took the following 18 measurements to the nearest 0.1 mm using digital calipers, following Dehling and Sinsch (in press): (1) Snout-vent length (SVL); (2) tibiofibula length (TFL, measured with both knee and tibio-tarsal articulation flexed); (3) thigh length (THL, from vent to knee with thigh being held vertically to median body plane and knee flexed); (4) total hindlimb length (LEG, from vent to tip of fourth toe with leg fully extended and being held vertically to median body plane); (5) tarsus + foot length (TarL, from tibio-tarsal articulation to tip of fourth toe); (6) foot length (FOT, from proximal end of inner metatarsal tubercle to tip of fourth toe); (7) forearm + hand length (ARM, distance from elbow to tip of third finger); (8) hand length (HND, distance from proximal end of inner palmar tubercle to tip of third finger); (9) head width (HW, measured at the level of the jaw joint); (10) head length (HL, distance from posterior end of mandible to tip of snout); (11) interorbital distance (IO, shortest distance between upper eyelids); (12) upper eyelid width (EW); (13) horizontal eye diameter (ED); ( 14) horizontal tympanum diameter (TD); (15) eye to nostril distance (EN, distance between anterior margin of eye and centre of nostril); ( 16) nostril to snout distance (NS, distance between centre of nostril and tip of snout); (17) snout length (SL, distance between anterior margin of eye to tip of snout); (18) internarial distance (NN, distance between centres of nostrils).To avoid an interobserver bias, all measurements were taken by JMD.Additionally, we recorded the following qualitative characters: (1) position of external vocal sac aperture in males; (2) number of longitudinal dorsal dermal ridges; (3) texture of ventral skin; (4) extent of nuptial pads in males; (5) number of supernumerary metacarpal tubercles; (6) size and shape of thenar and palmar tubercles; (7) extent of toe webbing; (8) presence of outer metatarsal tubercle; (9) relative size of inner metatarsal tubercle; (10) ventral colouration; (11) presence of light line on dorsal face of tibia; (12) presence of light band on dorsum; (13) presence of dark brown stripe on preaxial side of tarsus; (14) colour of external dorsal fold; (15) colour pattern on postaxial side of femur.Sex of males was determined by presence of secondary sexual characteristics (vocal slits, nuptial pads), that of females by either examination of gonads through dissection or size (female if larger than smallest 10 percent of adult males).The webbing formulae are given as proposed by Myers and Duellman (1982).Terminology for dermal dorsal ridges and orientation of external vocal sac aperture follows Perret (1979).

Statistical Analyses
Descriptive statistics depended on the outcome of the test for normality.Normally distributed data were described by the arithmetic mean and corresponding standard error and/or range, those deviating significantly by median and range.Principal component analyses were run on the morphometric data set including 18 variables and 89 observations each (P.anchietae: 15 males, 3 females; P. chrysogaster: 13 males, 10 females; P. nilotica: 13 males, 10 females; P. porosissima: 11 males, 7 females; P. uzungwensis: 6 males, 1 female).We compared the scores obtained for the principal components 2 and 3 describing shape to distinguish taxa without a priori assignment to taxa.The morphometric distances were adjusted for SVL by calculating a linear regression of each variable against SVL and storing the residuals as representatives of size-independent shape variables.This transformed data set was used for discriminant analyses with taxa as predefined groups to optimize distinction.To account for sexual dimorphism, discriminant analyses were run separately for males (n=58) and females (n=31).Significance level was set at alpha = 0.05.All calculations were based on the procedures of the program package STATGRAPHICS centurion for Windows, version XV.

DNA barcoding and phylogenetic analyses
We isolated DNA from a liver tissue sample from a specimen of Ptychadena chrysogaster (ZFMK 58797), collected in southern Rwanda by H. Hinkel in 1993.DNA was used to sequence a fragment of the 16S mitochondrial rRNA gene, a universal marker to barcode amphibian species (Vences et al. 2005).Protocols of DNA extraction, PCR, purification, and sequencing follow Dehling and Sinsch (in press).The obtained sequence was compared with those in GenBank using a standard nucleotide-nucleotide BLAST search and with our own sequences from Rwandan specimens and was incorporated into an existing alignment (see Dehling and Sinsch in press for a list of sequences and GenBank Accession numbers).Editing and alignment were completed in MEGA5 (Tamura et al. 2011).Sequences were trimmed to the same length.The final alignment consisted of 548 base pairs.Calculations of pairwise distances and phylogenetic analysis (Maximum Likelihood) were carried out in MEGA5.Maximum Likelihood analysis was run using the GTR + G + I model and the Nearest-Neighbor-Interchange with 1000 bootstrap replicates.

Results
Examination of specimens suggested that five morphologically distinct species were present in Rwanda to which we assign the following names: Ptychadena anchietae, P. chrysogaster, P. nilotica, P. porosissima, and P. uzungwensis (Figures 1 and 2).For allocation of specimens to P. anchietae, P. nilotica, and P. porosissima and discussion thereof see Dehling and Sinsch (in press).The examined material included type specimens of both of P. chrysogaster and P. uzungwensis (Appendix 1).Allocation of other specimens to the latter two species is based on direct comparison with the type material.We reassigned several specimens that had been deposited in the museum collections under wrong names.Noteworthy are two of the paratypes of P. chrysogaster (RMCA 41989, 41994) which belong in fact to P. porosissima.

Morphological differentiation
The morphometric features of the five species are summarized in Table 2. Principal component analysis yielded three PCs accounting for 89.6% of total variation (Table 3A).PC1 represented variation in size, whereas the shape-related PC2 and PC3 were mainly loaded by features describing head morphology (Table 3B).In females, PC 2 unequivocally distinguished P. nilotica from the other taxa, and PC 3 unequivocally distinguished P. chrysogaster from P. anchietae, P. porosissima, and P. uzungwensis (Figure 3A).Also, P. anchietae and P. uzungwensis could be distinguished from each other.However, both species were represented by only few individuals (three and one, respectively) in the analysis.The two species did not differ significantly in shape from P. porosissima (Figure 3A).A similar pattern was observed in the analysis of the males (Figure 3B) but males of all five species were generally more similar to each other in shape.Males of P. nilotica could be distinguished unequivocally from males of P. chrysogaster and P. uzungwensis but not from some of the males of P. anchietae and P. porosissima (Figure 3B).Males of P. chrysogaster could be distinguished from males of all other species but some of the males of P. anchietae and P. uzungwensis were very similar in shape (Figure 3B).Males of P. anchietae did not differ significantly in shape from males of P. nilotica, P. porosissima, and P. uzungwensis.Gender-specific discriminant analyses based on the residuals of 17 SVL-adjusted morphometric variables had a classification success of 100 % among the five species in both males and females (Table 4A, B, C, Figure 4A, B).
The five Rwandan species can be distinguished unequivocally from each other using a combination of qualitative morphological characters (Table 1, Figure 5; see also Dehling and Sinsch in press).An identification key based on these characters is given below.Detailed morphological descriptions of the species are in Appendix 2.   toe webbing (Fig. 5)       in comparison with specimens assigned to "P. aff.uzungwensis" (4.2 %), "P.porosissima" from South Africa and Rwanda (4.7-4.9 %), "P.aff.porosissima" from Tanzania (6.0-6.9 %) , "P. mahnerti" (6.2 %), and "P.aff.bibroni" from Gabon (6.9 %).The consensus tree yielded by Maximum-Likelihood analysis indicated that P. chrysogaster is most closely related to the aforementioned species (Figure 6).The clade consisting of these species is well supported by bootstraping (value 0.90; Hillis and Bull 1993), whereas the relationships within the clade are not resolved (bootstrap values <50 %).

Discussion
Among the examined material we identified five distinct species of Ptychadena: P. anchietae, P. chrysogaster, P. nilotica, P. porosissima, and P. uzungwensis.The five species are distinguishable from each other unambiguously using quantitative morphometric as well as qualitative morphological characters.The comparison of the partial 16S rRNA sequence of a specimen of P. chrysogaster with sequences from congeners corroborated its distinct specific status.Sequences from specimens of P. anchietae, P. nilotica, and P. porosissima from Rwanda differ from each other considerably by an uncorrected p distance of more than 10 % (Sinsch et al. 2012, Dehling and Sinsch in press).Unfortunately, no homologous sequence of P. uzungwensis from Rwanda and only a sequence of a specimen with doubtful identity from Tanzania (P.aff.uzungwensis, GenBank# DQ525945) were available for comparison.We did not find any Ptychadena individual collected in Rwanda which was assignable to P. oxyrhynchus in the collections of the RMCA and the ZFMK.Three specimens from Kisenyi (= Gisenyi, nowadays Rubavu; RMCA 51565-67), Rwanda, had been deposited under the name P. oxyrhynchus but were re-identified as males of P. anchietae.Nieden (1913) reported on several specimens of P. oxyrhynchus (as Rana oxyrhyncha) which Schubotz had collected in what today is northwestern Tanzania, at Kifumbiro and in the Mpororo area, close to the present border with Rwanda.We have not examined Schubotz' material but if his specimens are indeed P. oxyrhynchus it is possible that the species can be found in Rwanda as well, given its vast distribution in eastern Africa and the fact that the herpetofauna of the northeastern part of Rwanda has been poorly sampled so far.
There is no specimen of P. grandisonae among the material Laurent collected in Rwanda.Laurent (1954) described the species based on type specimens from Muita in Angola, from Kanzenze and Kansenia in Katanga (DRC), and from Bitare in "Urundi" [= Burundi].The latter is a town in central Burundi, about 20 km north of Gitega at 3°15'S, 29°54'E.Several authors, however, have stated that P. grandisonae occurs in Rwanda (Poynton and Broadley 1985, Channing 2001, Poynton and Channing 2004), and Poynton and Channing (2004) even stated that there is no record from Burundi.This misinformation was very likely caused by Laurent himself in a paper which was cited by all above mentioned authors, an account on the "Reptiles et Amphibiens de l'Angola" (Laurent 1964).Therein, Laurent (1964: 139) cited one of the type localities of P. grandisonae wrongly as "Bitare (Ruanda)".On the same page, the locality is correctly given as "Bitare […] (Urundi)" in the account on Ptychadena uzungwensis.Thus, the often cited record of P. grandisonae from Rwanda in fact refers to specimens from Burundi (RMCA 109036-37; Appendix 1).So far, there is no evidence for the occurrence of P. grandisonae in Rwanda.The available evidence indicates that only five species of Ptychadena occur in Rwanda.At present, three of these species (P.anchietae, P. nilotica, and P. porosissima) are widespread and can be found abundantly in both wetlands of the eastern lowland between 1300 and 2000 m elevation which drains into the Nile River and the western lowland on the shore of Lake Kivu which drains into the Congo River.The species inhabit higher elevations of up to 2300 m in deforested, cultivated areas, but are absent from dense forest habitats at similar elevations which at present only remain in the Volcano and Nyungwe National Parks and in the Gishwati Forest.P. uzungwensis is known from Rwanda from only few specimens, five males from "Kumunini" [= Munini, South Province, 2°42'S, 29°32'E] and a female from "Astrida" [= Butare/Huye, South Province, 2°36'S, 29°44'E], collected in 1952 and 1951, respectively, and has not been found since.Assuming the species is still extant in Rwanda, its distribution is apparently restricted to the south of the country.
There are large series of P. chrysogaster from various localities in Rwanda in the collection of the RMCA (Appendix 1), collected by Laurent in 1951Laurent in -1952, indicating that the species was abundant at that time.We repeatedly conducted surveys at several of these localities including the type locality at Lac Karago (1°37'S, 29°30'E) but did not encounter individuals of P. chrysogaster.Our survey periods (February to April, September to October) were at similar times of the year to those of Laurent (Janurary, February, and October).Species of Ptychadena are among the most conspicuous frogs in areas they inhabit, usually occurring in high numbers and easy to detect.Although the absence of a species from a certain area cannot be proven ultimately, our observations indicate that P. chrysogaster has disappeared from these areas or at least is much less common than it used to be.The human population in Rwanda has grown from little more than 2 million people in 1950 to approximately 11 million in 2011 (United Nations, Population Division 2011).Nowadays almost every cultivatable area except the three national parks and few small forest patches has been altered to farmland (pers.observation).Gishwati Forest has been reduced to a small patch of a few square kilometres, but until the mid-1990s it had covered a large area in northwestern Rwanda and its extensions reached the shores of Lac Karago.The former presence of forest habitat at the lake is still indicated by the occurrence of two forest-dwelling frog species, Hyperolius castaneus and Leptopelis kivuensis, which call from bushes and groups of small trees at the shore of the river (own unpublished data; see also Sinsch et al. 2011).Judging from its collection sites, P. chrysogaster appears to occur primarily in wetlands within or at the edge of forest.Instead of P. chrysogaster, we found P. nilotica at Lac Karago and P. nilotica and P. anchietae in Huye (formerly Astrida and Butare) and in the vicinity of Muzanze (formerly Ruhengeri) during our recent surveys, two species that Laurent had not collected in Rwanda.Both species are known to be able to cope with habitat alteration and are often found in disturbed habitats and in human settlements (Spawls et al. 2006, pers. observation).It is possible that habitat alteration promoted population decline in P. chrysogaster and its replacement by other species.The distribution of P. chrysogaster in Rwanda is currently under study.If our preliminary observations are affirmed, the Red List classification of P. chrysogaster would have to be changed to a "threatened" category and it would call for conservation measures.
Our recent efforts to untangle the diversity of Ptychadena in Rwanda are a first step to clarify the complicated taxonomy of the genus in sub-Saharan Africa.The results of our studies show that species of Ptychadena can be easily distinguished, if standardized

Ptychadena anchietae
Body moderately sturdy, widest at temporal region, slightly tapering to groin; head large (HL/SUL 0.36-0.43 in males, 0.37-0.41 in females; HW/SUL 0.31-0.39 in males, 0.33-0.36 in females), longer than wide (HL/HW 1.01-1.26 in males, 1.11-1.13 in females); snout long (SL/HL 0.43-0.53 in males, 0.49-0.53 in females), pointed in dorsal view, rounded in profile, considerably projecting beyond lower jaw, longer than wide (SL/EE 1.08-1.24 in males, 1.30-1.33 in females); canthus rostralis distinct between eye and nostril, straight-lined; loreal region oblique, strongly concave; nostrils rounded, directed dorsolaterally; situated half-way between tip of snout and eye or closer to tip of snout than to eye (EN/NS 0.98-1.33 in males, 1.14-1.27 in females), separated from each other by distance subequal to distance between eye and nostril (NN/EN 0.87-1.05 in males, 0.85-0.96 in females); eyes directed anterolaterally, moderately protruding, relatively small (ED/HL 0.24-0.29 in males, 0.26-0.29 in females), its diameter much shorter than snout (ED/SL 0.50-0.63 in males, 0.53-0.58 in females); interorbital distance more or less equalling upper eyelid width (IO/ EW 0.83-1.10 in males, 0.89-1.06 in females) and smaller than internarial distance (IO/NN 0.59-0.83 in males, 0.63-0.67 in females); tympanum and its annulus distinctly visible, separated from eye by about one-fifth to one-third of its diameter (ET/ TD 0.21-0.36 in males, 0.19-0.25 in females); tympanum diameter 0.62-0.92(in males) and 0.74-0.83(in females) of eye diameter; upper jaw with dentition; choanae small, rounded, located far anterolaterally at margins of roof of mouth; vomer teeth in two short rows, separated from each other by distance about three times length of individual row; tongue long and narrow, bilobed for about one-sixth of its length, free distally for one-fourths its length; median lingual process absent; vocal sac in males paired, lateral; external vocal sac aperture as a longitudinal, posterolaterally orientated slit, inferior, terminating at level of ventral edge of insertion of arms; internal vocal sac apertures rounded, situated close to corner of mouth.
Dorsal surfaces of head, trunk and limbs finely shagreened with many scattered small tubercles; dorsum with five or six longitudinal dermal ridges on each side; median ridge extending from interorbital region almost to vent, postpalpebral and external ridges from level just behind posterior edge of upper eyelid to insertion of leg; laterodorsal ridge extending from level about one snout length posterior to tympanum to groin; dorsal ridges interrupted in few specimens; sacral ridge extending from about one head length anterior to vent either medially to median ridges or between median and postpalpebral ridges to vent, in few specimens absent; external ridge forming anterior part of supratympanic fold; posterior part of supratympanic fold less distinct, branching off from external dorsal ridge posterior to tympanum in wide angle and extending posterolaterally to insertion of arm; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold; ventral side of limbs and body smooth except slightly areolate postaxial side of thigh; distinct transverse fold between arms on ventral side; supratympanic fold moderately distinct, angled, extending from posterior corner of eye to point dorsal from arm insertion; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold, continued after a small gap in form of large oval tubercle dorsally of posterior end of arm insertion.
Forelimbs moderately sturdy; hand relatively small (HND/SUL 0.23-0.27 in males, 0.25-0.26 in females); tips of fingers rounded, not enlarged into disks but slightly swollen volarly; transverse dorsal skin ridge separating ultimate from other phalanges on each finger; relative length of fingers: I ≤ II < IV < III; subarticular tubercles rounded, well developed, numbering one on Fingers I and II, two on Fingers III and IV, proximal tubercles on Fingers III and IV larger and more prominent than distal ones; finger webbing absent; thenar tubercle distinct, large, flat, oval, slightly more than half as long as metacarpal of Finger I; inner palmar tubercle on proximal half of metacarpal region of Fingers II and III, rounded, flat; outer palmar tubercle on proximal half of metacarpal region of Finger IV, oval, slightly more prominent than inner palmar tubercle; one supernumerary metacarpal tubercle between palmar or thenar tubercles and proximal subarticular tubercles on each finger; callous longitudinal ridges between subarticular tubercles on Fingers III and IV and between subarticular tubercles and finger tips on all fingers; nuptial pads in males covering almost entire dorsal surfaces of Fingers I and II, and proximal portion of dorsal side of Finger III.
Hindlimbs sturdy, very long (LEG/SUL 1.87-2.11 in males, 1.96-2.13 in females); knee reaching slightly beyond insertion of forelimbs and tibio-tarsal articulation reaching almost a head length beyond tip of snout when legs are adpressed forwardly to body; tibiofibula very long (TFL/SUL 0.61-0.68 in males, 0.65-0.72 in females), longer than thigh (TFL/THL 1.09-1.19 in males, 1.13-1.16 in females); heels overlapping each other considerably when knees flexed and thighs held perpendicularly to median plane; two low longitudinal ridges on plantar side of tarsus between heel and metatarsal tubercles; foot shorter than or equal in length to tibiofibula (FOT/TFL 0.85-1.00 in males, 0.91-0.93 in females); relative length of toes: I < II < III < V < IV; toe tips rounded, not enlarged into disks but slightly swollen plantarly; transverse dorsal skin ridge separating ultimate from other phalanges on each toe; subarticular tubercles numbering one on Toes I and II, two on Toes III and V, and three on Toe IV; low callous ridges between subarticular tubercles, and between subarticular tubercles and toe tips; pedal webbing formula I0.5-2II0.5-2III(0.5-1)-2IV2-0.5V;inner metatarsal tubercle moderately large, half as long as metatarsus of Toe I, oval, prominent; outer metatarsal tubercle rounded, flat, faintly visible, callous tissue weakly developed.

Ptychadena chrysogaster Laurent, 1954
Body moderately sturdy, widest at temporal region, slightly tapering to groin; head large (HL/SUL 0.34-0.38 in males, 0.33-0.37 in females; HW/SUL 0.29-0.34 in males, 0.28-0.34 in females), longer than wide (HL/HW 1.04-1.22 in males, 1.05-1.23 in females); snout long (SL/HL 0.45-0.50 in males, 0.45-0.51 in females), pointed in dorsal view, rounded in profile, considerably projecting beyond lower jaw, longer than wide (SL/EE 1.11-1.28 in males, 1.11-1.37 in females); canthus rostralis distinct between eye and nostril, straight-lined; loreal region oblique, strongly concave; nostrils rounded, directed dorsolaterally; situated more or less half-way between tip of snout and eye (EN/NS 0.87-1.14 in males, 0.93-1.24 in females), separated from each other by distance subequal to or larger than distance between eye and nostril (NN/EN 1.02-1.22 in males, 0.94-1.24 in females); eyes directed anterolaterally, moderately protruding, relatively small (ED/HL 0.27-0.32 in males, 0.26-0.30 in females), its diameter much shorter than snout (ED/SL 0.57-0.66 in males, 0.51-0.66 in females); interorbital distance larger than upper eyelid width (IO/EW 1.12-1.48 in males, 1.09-1.44 in females) and smaller than internarial distance (IO/NN 0.75-0.89 in males, 0.78-0.91 in females); tympanum and its annulus distinctly visible, separated from eye by about one-fourth to two-fifths of its diameter (ET/TD 0.24-0.38 in males, 0.26-0.40 in females); tympanum diameter slightly smaller to subequal to eye diameter (TD/ED 0.76-0.98 in males, 0.85-1.01 in females); upper jaw with dentition; choanae small, rounded, located far anterolaterally at margins of roof of mouth; vomer teeth in two short rows, separated from each other by distance about three times length of individual row; tongue long and narrow, bilobed for about one-fifth of its length, free distally for one-fourth its length; median lingual process absent; vocal sac in males paired, lateral; external vocal sac aperture as a longitudinal, posterolaterally orientated slit, inferior, terminating at level of ventral edge of ventral insertion of arms; internal vocal sac apertures rounded, situated close to corner of mouth.Dorsal surfaces of head, trunk and limbs finely shagreened; dorsum with five or six longitudinal dermal ridges on each side, median one extending from interorbital region almost to vent, postpalpebral and external ones from level just behind posterior edge of upper eyelid to insertion of leg; laterodorsal ridge extending from level about one snout length posterior to tympanum to groin; sacral ridge extending from about one head length anterior to vent either medially to median ridges or between median and postpalpebral ridges to vent; in few specimens additional sacromedial ridge extending between sacral ridge and median ridge to vent for about half length of sacral ridge; external ridge forming anterior part of supratympanic fold; posterior part of supratympanic fold less distinct, branching off from external dorsal ridge posterior to tympanum in wide angle and extending posterolaterally to insertion of arm; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold; ventral side of limbs and body smooth except areolate proximal postaxial-ventral part of thigh; distinct transverse fold between arms on ventral side; ventral side of trunk and head densely covered with more or less evenly scattered tiny, pointed tubercles in males.
Forelimbs moderately sturdy; hand relatively small (HND/SUL 0.22-0.26 in males, 0.21-0.25 in females); tips of fingers rounded, not enlarged into disks but slightly swollen volarly; transverse dorsal skin ridge separating ultimate from other phalanges on each finger; relative length of fingers: I = II < IV < III; subarticular tubercles rounded, well developed, numbering one on Fingers I and II, two on Fingers III and IV, proximal tubercles on Fingers III and IV larger and more prominent than distal ones; finger webbing absent; thenar tubercle prominent, elongated, large, twothirds length of metacarpal of Finger I; inner palmar tubercle on proximal third of metacarpal region of Fingers II and III, oval, flat; outer palmar tubercle on proximal half of metacarpal region of Finger IV, elongated, slightly more prominent than inner palmar tubercle; one supernumerary metacarpal tubercle between palmar or thenar tubercles and proximal subarticular tubercles on all fingers; low callous longitudinal ridges between subarticular tubercles on Fingers III and IV and between subarticular tubercles and finger tips on all fingers; nuptial pads in males covering almost entire dorsal surfaces of Fingers I and II except distal phalanx, and preaxial half of dorsal side of metacarpal of Finger III.
Hindlimbs sturdy, very long (LEG/SUL 1.94-2.27 in males, 1.93-2.14 in females); knee reaching to insertion of forelimbs and tibio-tarsal articulation reaching slightly more than a snout length beyond tip of snout when legs adpressed forwardly to body; tibiofibula very long (TFL/SUL 0.61-0.73 in males, 0.62-0.70 in females), longer than thigh (TFL/THL 1.13-1.22 in males, 1.13-1.25 in females); heels overlapping each other considerably when knees flexed and thighs held perpendicularly to median body plane; two low longitudinalridges on plantar side of tarsus between heel and metatarsal tubercles; foot subequal in length to tibiofibula (FOT/TFL 0.97-1.07 in males, 0.94-1.03 in females); relative length of toes: I < II < III < V < IV; toe tips rounded, not enlarged into disks but slightly swollen plantarly; transverse dorsal skin ridge separating ultimate from other phalanges on each toe; subarticular tubercles numbering one on Toes I and II, two on Toes III and V, and three on Toe IV; low callous ridges between subarticular tubercles, and between subarticular tubercles and toe tips; pedal webbing formula I2-2.5II(1.5-1.75)-3III(2-2-)(3.25-3+ )IV3-(1.5-2)V;inner metatarsal tubercle elongated, prominent, small, less than half as long as metatarsus of Toe I; outer metatarsal tubercle very faintly visible, rarely prominent, small and pointed.

Ptychadena nilotica
Body moderately sturdy, widest at temporal region, slightly tapering to groin; head large (HL/SUL 0.35-0.43 in males, 0.35-0.39 in females; HW/SUL 0.30-0.37 in males, 0.32-0.35 in females), longer than wide (HL/HW 1.10-1.27 in males, 1.06-1.15 in females); snout long (SL/HL 0.38-0.46 in males, 0.40-0.45 in females), pointed in dorsal view, rounded in profile, considerably projecting beyond lower jaw, longer than wide (SL/EE 1.20-1.40 in males, 1.15-1.36 in females); canthus rostralis distinct between eye and nostril, straight-lined; loreal region oblique, moderately concave; nostrils rounded, directed dorsolaterally; situated half-way between tip of snout and eye or closer to tip of snout than to eye (EN/NS 0.99-1.18 in males, 1.05-1.34 in females), separated from each other by distance slightly less than or subequal to distance between eye and nostril (NN/EN 0.82-0.99 in males, 0.87-0.97 in females); eyes directed anterolaterally, moderately protruding, relatively small (ED/HL 0.25-0.32 in males, 0.26-0.29 in females), its diameter shorter than snout (ED/SL 0.60-0.69 in males, 0.59-0.69 in females); interorbital distance smaller to equalling upper eyelid width (IO/EW 0.64-1.03 in males, 0.72-0.90 in females) and smaller than internarial distance (IO/NN 0.55-0.74 in males, 0.57-0.72 in females); tympanum and its annulus distinctly visible, separated from eye by about one-fourth to onethird of its diameter (ET/TD 0.28-0.36 in males, 0.26-0.37 in females); tympanum diameter 0.75-0.88(in males) and 0.77-0.89(in females) of eye diameter; upper jaw with dentition; choanae small, rounded, located far anterolaterally at margins of roof of mouth; vomer teeth in two short rows, separated from each other by distance about three times length of individual row; tongue long and narrow, bilobed for about onesixth of its length, free distally for one-third its length; median lingual process absent; vocal sac in males paired, lateral; external vocal sac aperture as a longitudinal, posteriorly orientated slit, situated dorsally of level of insertion of arms, parallel to mandibel and infratympanic fold, covered by narrow dermal flap on ventral edge of slit; internal vocal sac apertures rounded, situated close to corner of mouth.
Dorsal surfaces of head, trunk and limbs shagreened; dorsum with four or five longitudinal dermal ridges on each side, median one extending from interorbital region almost to vent, postpalpebral and external ones from level just behind posterior edge of upper eyelid to insertion of leg; laterodorsal ridge extending from level about one snout length posterior to tympanum to groin; in few specimens additional very short ridge present between median and postpalpebral ridge from about one snout length anterior to vent extending almost to vent; external ridge forming anterior part of supratympanic fold; posterior part of supratympanic fold less distinct, branching off from external dorsal ridge posterior to tympanum in wide angle and extending posterolaterally to insertion of arm; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold; ventral side of limbs and body smooth except areolate postaxial side of thigh; distinct transverse fold between arms on ventral side.
Forelimbs moderately sturdy; hand relatively small (HND/SUL 0.21-0.27 in males, 0.22-0.26 in females); tips of fingers rounded, not enlarged into disks but slightly swollen volarly; transverse dorsal skin ridge separating ultimate from other phalanges on each finger; relative length of fingers: I ≤ II < IV < III; subarticular tubercles rounded, well developed, numbering one on Fingers I and II, two on Fingers III and IV; proximal tubercles on Fingers III and IV larger and more prominent than distal ones; finger webbing absent; thenar tubercle prominent, elongated and narrow, comparatively small, less than half as long as metacarpal of Finger I; inner palmar tubercle small, flat, less conspicuous, on proximal one-third to one-fourth of metacarpal region of Fingers II and III, rounded, flat; outer palmar tubercle on proximal half of metacarpal region of Finger IV, prominent, elongate and narrow; single supernumerary metacarpal tubercle between outer palmar tubercle and proximal subarticular tu-bercle of Finger IV, often indistinct; callous longitudinal ridges between subarticular tubercles on Fingers III and IV and between subarticular tubercles and finger tips on all fingers; nuptial pads in males covering almost entire dorsal surfaces of Fingers I and II and preaxial half of dorsal surface of Finger III.

Ptychadena porosissima
Body moderately sturdy, widest at temporal region, slightly tapering to groin; head large (HL/SUL 0.35-0.41 in males, 0.35-0.40 in females; HW/SUL 0.31-0.36 in males, 0.30-0.34 in females), longer than wide (HL/HW 1.02-1.19 in males, 1.03-1.21 in females); snout moderately long (SL/HL 0.38-0.49 in males, 0.43-0.51 in females), pointed in dorsal view, rounded in profile, considerably projecting beyond lower jaw, longer than wide (SL/EE 1.21-1.27 in males, 1.11-1.61 in females); canthus rostralis distinct between eye and nostril, straight-lined; loreal region oblique, strongly concave; nostrils rounded, directed dorsolaterally; situated more or less halfway between tip of snout and eye (EN/NS 0.89-1.15 in males, 0.87-1.29 in females), separated from each other by distance subequal to or larger than distance between eye and nostril (NN/EN 0.96-1.15 in males, 0.96-1.11 in females); eye directed anterolaterally, moderately protruding, relatively small (ED/HL 0.24-0.30 in males, 0.25-0.29 in females), its diameter much shorter than snout (ED/SL 0.54-0.67 in males, 0.51-0.63 in females); interorbital distance subequal to upper eyelid width (IO/EW 0.80-1.09 in males, 0.81-1.10 in females) and smaller than internarial distance (IO/ NN 0.65-0.74 in males, 0.60-0.82 in females); tympanum and its annulus distinctly visible, separated from eye by about one-fourth to two-fifths of its diameter (ET/TD 0.27-0.39 in males, 0.29-0.39 in females); tympanum diameter smaller than eye diameter (TD/ED 0.68-0.81 in males, 0.70-0.84 in females); upper jaw with dentition; choanae small, rounded, located far anterolaterally at margins of roof of mouth; vomer teeth in two short rows, separated from each other by distance about three times length of individual row; tongue long and narrow, bilobed for about one-fifth of its length, free distally for one-third its length; median lingual process absent; vocal sac in males paired, lateral; external vocal sac aperture as a longitudinal, posterolaterally orientated slit, inferior, terminating at level of ventral edge of insertion of arms; internal vocal sac apertures rounded, situated close to corner of mouth.Dorsal surfaces of head, trunk and limbs finely shagreened; dorsum with four or five longitudinal dermal ridges on each side; median ridge extending from interorbital region almost to vent, postpalpebral and external ridges from posterior edge of upper eyelid to insertion of leg; laterodorsal ridge extending from level of insertion of arm to groin; sacral ridge extending from about one head length anterior to vent to vent, in some specimens absent; external ridge forming anterior part of supratympanic fold; posterior part of supratympanic fold less distinct, branching off from external dorsal ridge posterior to tympanum in wide angle and extending posterolaterally to insertion of arm; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold; ventral side of limbs and body smooth except areolate postaxial side of thigh; distinct transverse fold between arms on ventral side; ventral side of trunk and head densely covered with more or less evenly scattered small, pointed tubercles in males.
Forelimbs moderately sturdy; hand relatively small (HND/SUL 0.22-0.26 in males, 0.22-0.24 in females); tips of fingers rounded, not enlarged into disks but slightly swollen volarly; transverse dorsal skin ridge separating ultimate from other phalanges on each finger; relative length of fingers: I ≤ II < IV < III; subarticular tubercles rounded, well developed, numbering one on Fingers I and II, two on Fingers III and IV, proximal tubercles on Fingers III and IV larger and more prominent than distal ones; finger webbing absent; thenar tubercle distinct, elongated, large, twothirds length of metacarpal of Finger I; inner palmar tubercle on proximal third of metacarpal region of Fingers II and III, oval, flat; outer palmar tubercle on proximal half of metacarpal region of Finger IV, elongated, slightly more prominent than inner palmar tubercle; one supernumerary metacarpal tubercle between palmar or thenar tubercles and proximal subarticular tubercles on Fingers I, II, and IV, two on Finger III; callous longitudinal ridges between subarticular tubercles on Fingers III and IV and between subarticular tubercles and finger tips on all fingers; nuptial pads in males covering almost entire dorsal surfaces of Fingers I and II and preaxial half of dorsal side of Finger III.
Hindlimbs sturdy, very long (LEG/SUL 1.77-2.05 in males, 1.80-2.06 in females); knee reaching to insertion of forelimbs and tibio-tarsal articulation reaching slightly more than one snout length beyond tip of snout when legs adpressed forwardly to body; tibiofibula long (TFL/SUL 0.56-0.65 in males, 0.56-0.66 in females), longer than thigh (TFL/THL 1.08-1.18 in males, 1.05-1.26 in female); heels overlapping each other considerably when knees flexed and thighs held perpendicularly to median plane; two low longitudinal ridges on plantar side of tarsus between heel and meta-tarsal tubercles; foot slightly shorter than or equal in length to tibiofibula (FOT/TFL 0.94-1.01 in males, 0.92-1.01 in females); relative length of toes: I < II < III < V < IV; toe tips rounded, not enlarged into disks but slightly swollen plantarly; transverse dorsal skin ridge separating ultimate from other phalanges on each toe; subarticular tubercles numbering one on Toes I and II, two on Toes III and V, and three on Toe IV; low callous ridges between subarticular tubercles, and between subarticular tubercles and toe tips; pedal webbing formula I(1.75-2)-2.25II1.5-3III1.75-(3-3.25)IV3-(1-1.5)V;inner metatarsal tubercle very prominent, shovel-like, large, more than half as long as metatarsus of Toe I; outer metatarsal tubercle faintly visible, rarely prominent and pointed.

Ptychadena uzungwensis
Body moderately sturdy, widest at temporal region, slightly tapering to groin; head large (HL/SUL 0.36-0.39 in males, 0.37 in females; HW/SUL 0.32-0.35 in males, 0.32 in female), longer than wide (HL/HW 1.06-1.23 in males, 1.16 in female); snout long (SL/HL 0.50-0.52 in males, 0.50 in female), pointed in dorsal view, rounded in profile, considerably projecting beyond lower jaw, much longer than wide (SL/EE 1.30-1.53 in males, 1.56 in female); canthus rostralis distinct between eye and nostril, straight-lined; loreal region oblique, strongly concave; nostrils rounded, directed dorsolaterally; situated more or less half-way between tip of snout and eye (EN/NS 0.91-1.03 in males, 0.97 in female), separated from each other by distance subequal to or shorter than distance between eye and nostril (NN/EN 0.82-0.94 in males, 0.89 in female); eyes directed anterolaterally, moderately protruding, relatively small (ED/ HL 0.27-0.32 in males, 0.28 in female), its diameter much shorter than snout (ED/ SL 0.55-0.63 in males, 0.56 in female); interorbital distance subequal to or larger than upper eyelid width (IO/EW 0.97-1.24 in males, 1.19 in female) and smaller or subequal to internarial distance (IO/NN 0.80-1.06 in males, 0.78 in female); tympanum and its annulus distinctly visible, separated from eye by about two-fifths to half of its diameter (ET/TD 0.36-0.47 in males, 0.41 in female); tympanum diameter smaller than eye diameter (TD/ED 0.70-0.83 in males, 0.77 in female); upper jaw with dentition; choanae small, rounded, located far anterolaterally at margins of roof of mouth; vomer teeth in two short rows, separated from each other by distance about two and half times length of individual row; tongue long and narrow, bilobed for about onethird of its length, free distally for one-third its length; median lingual process absent; vocal sac in males paired, lateral; external vocal sac aperture as a longitudinal, posterolaterally orientated slit, semi-inferior, terminating at level of centre of insertion of arms; internal vocal sac apertures rounded, situated close to corner of mouth.Dorsal surfaces of head, trunk and limbs finely shagreened; dorsum with four longitudinal dermal ridges on each side; median ridge extending from dorsal side of snout between nostrils almost to vent; postpalpebral ridge extending from posterior portion of upper eyelid, external ridge from level of tympanum to insertion of leg; laterodorsal ridge extending from level of tympanum to groin; external ridge forming anterior part of supratympanic fold; posterior part of supratympanic fold less distinct, branching off from external dorsal ridge posterior to tympanum in wide angle and extending posterolaterally to insertion of arm; infratympanic fold thick and conspicuous, almost straight-lined, extending from ventral edge of eye to level of arm insertion, meeting with supratympanic fold; ventral side of limbs and body smooth except areolate postaxial side of thigh; distinct transverse fold between arms on ventral side; ventral side of trunk and head densely covered with more or less evenly scattered small, pointed, very small tubercles in males.
Forelimbs moderately sturdy; hand relatively small (HND/SUL 0.22-0.24 in males, 0.21 in female); tips of fingers rounded, not enlarged into disks but slightly swollen volarly; transverse dorsal skin ridge separating ultimate from other phalanges on each finger; relative length of fingers: II = IV ≤ I < III; subarticular tubercles rounded, well developed, numbering one on Fingers I and II, two on Fingers III and IV, proximal tubercles on Fingers III and IV larger and more prominent than distal ones; finger webbing absent; thenar tubercle distinct, elongated, large, half as long as metacarpal of Finger I; inner palmar tubercle on proximal third of metacarpal of Finger III, small, roundish, flat; outer palmar tubercle on proximal third of metacarpal of Finger IV, elongated, more prominent than inner palmar tubercle; one supernumerary metacarpal on Fingers I and IV, two on Finger II, and two to four on Finger III between palmar or thenar tubercles and proximal subarticular tubercles; callous longitudinal ridges between subarticular tubercles on Fingers III and IV and between subarticular tubercles and finger tips on all fingers; nuptial pads in males covering almost entire dorsal surfaces of metacarpal and proximal phalanx of Fingers I and II and preaxial halves of dorsal sides of metacarpal and [in Rwandan specimens but not in paratype and specimens from Kivu and Burundi] proximal portion of proximal phalanx of Finger III.
Hindlimbs sturdy, very long (LEG/SUL 1.84-2.04 in males, 1.87 in female); knee reaching to insertion of forelimbs and tibio-tarsal articulation reaching slightly more than one snout length beyond tip of snout when legs adpressed forwardly to body; tibiofibula long (TFL/SUL 0.59-0.66 in males, 0.62 in female), longer than thigh (TFL/THL 1.12-1.20 in males, 1.16 in female); heels overlapping each other considerably when knees flexed and thighs held perpendicularly to median body plane; two low longitudinal ridges on plantar side of tarsus between heel and metatarsal tubercles, postaxial one less distinct than preaxial one; foot shorter than or subequal in length to tibiofibula (FOT/TFL 0.94-1.03 in males, 1.07 in female); relative length of toes: I < II < III < V < IV; toe tips rounded, not enlarged into disks but slightly swollen plantarly; transverse dorsal skin ridge separating ultimate from other phalanges on each toe; subarticular tubercles numbering one on Toes I and II, two on Toes III and V, and three on Toe IV; low callous ridges between subarticular tubercles, and between subarticular tubercles and toe tips; pedal webbing formula I2-(2.25-2.5)II1.5-3-III(1.75-2- )-3IV3-(1 + -1.25)V; inner metatarsal tubercle prominent, elongated, large, half as long as metatarsus of Toe I; outer metatarsal tubercle faintly visible.

Figure 4 .Figure 5 .
Figure 4. Morphological shape differentiation among 89 specimens representing five Ptychadena species, as assessed by discriminant analyses (Statistical details are given in Table4).A Individual scores obtained for 31 females B Individual scores obtained for 58 males.

Figure 6 .
Figure 6.Maximum likelihood phylogram of species in the genus Ptychadena and Hildebrandtia ornata as outgroup, based on comparison of 548 base pairs of the mitochondrial 16S rRNA gene.Included are specimens from Rwanda and samples taken from GenBank (see Dehling and Sinsch in press for a complete list of sequences and accession numbers).Numbers above nodes are bootstrap support values (only values >0.50 are shown).

Table 2 .
Morphometric features of Ptychadena species from Rwanda.Data are given as arithmetic means and minimum and maximum values (in mm).

Table 3 .
Principal component Analysis based on 18 standardized morphometric features of 89 specimens belonging to five Ptychadena species from Rwanda.Morphometric parameters accounting strongly for discrimination among species are highlighted in bold.

Table 4A .
Gender-specific discriminant functions based on 17 SVL-adjusted morphometric features (residuals) to distinguish among five Ptychadena species from Rwanda.Statistical significance:

Table 4B .
Gender-specific discriminant functions based on 17 SVL-adjusted morphometric features (residuals) to distinguish among five Ptychadena species from Rwanda.Morphometric parameters accounting strongly to discrimination among species are highlighted in bold.

Table 4C .
Gender-specific discriminant functions based on 17 SVL-adjusted morphometric features (residuals) to distinguish among five Ptychadena species from Rwanda.Classification success.