Xenopus itombwensis, Evans, Ben J., Carter, Timothy F., Tobias, Martha L. & Kelley, Darcy B., 2008

Evans, Ben J., Carter, Timothy F., Tobias, Martha L. & Kelley, Darcy B., 2008, A new species of clawed frog (genus Xenopus) from the Itombwe Massif, Democratic Republic of the Congo: implications for DNA barcodes and biodiversity conservation., Zootaxa 1780, pp. 55-68 : 59-64

Xenopus itombwensis , new species

Itombwe Massif clawed frog ( Fig. 2 )

Holotype: MCZ A- 138192 (field number BJE 0275), adult male, Democratic Republic of the Congo, South Kivu Province, Miki Town, 3.35679º S, 023.69011ºE, approximately 2200 m above sea level. 21 April, 2006, Ben J. Evans.

Paratypes: Three adult males: MCZ A- 138195 (BJE 0278), MCZ A- 138196 (BJE 0283), MCZ A- 138197 (BJE 0284), two juveniles, sex undetermined: MCZ A- 138193 (BJE 0276), MCZ A- 138194 (BJE 0277), same data as holotype.

Diagnosis: Xenopus itombwensis is a member of the vestitus-wittei subgroup of clawed frogs (Kobel et al. 1996) and can be distinguished from other members of this group by (1) unique but variable morphological coloration and smaller size (Table 1, Fig. 2 ), by (2) numerous temporal and spectral characteristics of the male advertisement call (Table 2, Fig. 3 ), and by (3) divergent mitochondrial and autosomal genes ( Fig. 4 ). Xenopus wittei and X. vestitus are both medium sized clawed frogs, with female snout-vent length (SVL) typically 46 and 47 mm, respectively, and a maximum SVL of 61 and 55 mm, respectively (Kobel et al. 1996; Tinsley et al. 1979). Based on a small sample size of two females, X. itombwensis appears smaller, averaging approximately 35 mm (Table 1). Dorsal coloration of X. wittei is a uniform dark brown to chocolate with no spots and of X. vestitus is a marbeling of light silver-golden to bronze chromatophores over a brown background (Kobel et al. 1996). In contrast, some X. itombwensis individuals have a mottled pattern of brown spots that are slightly darker than the brown background ( Fig. 2 B,C).

The male advertisement call of X. itombwensis differs from the male advertisement calls of X. wittei and X. vestitus in that the call of the new species is much shorter (~600 milliseconds), and consists of two distinct components including a “fast trill” and a “slow trill” (Table 2, Fig. 3 ; Kobel et al. 1996; Vigny 1979). The dominant frequency of the fast trill component is similar to that of X. wittei but other acoustic characteristics of this part of the vocalization are different, and all parameters we measured from the slow trill component are distinct (Table 2). The most obvious difference between these vocalizations is the slow trill that follows vocalizations of the new species but not X. wittei . Other aspects of the slow trill are distinct from call parameters of X. wittei including the lower dominant frequencies, longer interclick interval, and lower number of clicks (Table 2). The fast portion of the call of the new species is also unique in that the interclick interval is shorter than that of X. wittei and there are fewer clicks per call.

Evolutionary relationships to other clawed frogs; DNA barcodes. X. itombwensis and X. wittei are probably sister species derived from the same allo-octoploid ancestor. The paternal ancestor of X. vestitus , in contrast, shares recent common ancestry with the maternal ancestor of ( X. itombwensis + X. wittei ), but the maternal ancestor of X. vestitus is not as closely related to the paternal ancestor of ( X. itombwensis + X. wittei ). As a result, mitochondrial DNA of the vestitus-wittei group is not a clade ( Fig. 4 ). On a molecular level, X. itombwensis is diverged from X. wittei and from X. vestitus . Because of the polyphyletic origin of this group, molecular divergence of mitochondrial DNA data (~3000bp) between X. itombwensis and X. vestitus is large – on the order of about 9% (uncorrected pairwise distance; hereafter p -distance). Mitochondrial divergence between X. itombwensis and X. wittei is about 4% p -distance. When only the “barcode” region of the mitochondria (CO1) is considered, interspecific divergence between X. itombwensis and X. vestitus and X. itombwensis and X. wittei is even higher – 17.2% and 8.3% p -distance, respectively. Paralogs of RAG1 and of RAG2 are also diverged among these species. Over both genes, the average divergence between the most closely related orthologous paralogs of X. vestitus and X. itombwensis is 1.6% p -distance and between orthologous paralogs of X. wittei and X. itombwensis is also 1.6% p -distance. These levels of divergence are typical for closely related species of clawed frog ( Fig. 4 ; Evans 2007; Evans et al. 2005; Evans et al. 2004). The Genbank accession number of the barcode of the holotype is EU594660 .

Description of the holotype: Holotype an adult male, small subocular tentacle present, comprising less than one third of the length of the eye. Claws present on toes I-III, prehallux prominent but without a claw. Size of new species slightly smaller than X. wittei and X. vestitus (Table 1; Kobel et al. 1996). Like X. wittei and X. vestitus , new species is octoploid with 8x =72 chromosomes ( Fig. 5). Ventral surface of forelimbs and forearm with scattered black nuptial pads.

Color of the holotype in preservative: Dorsum homogeneous dark brown, transitioning laterally on flanks to a cream-colored venter; dorsal surface of head dark brown; dorsal surface of limbs dark brown; underside of head speckled with gray; venter cream colored with sparse small brown spots on ventral surface of hind limbs; ventral surface of hind feet cream-colored; holotype color in life unrecorded.

Variation and color in life: There are two dorsal color patterns evident in our sample of X. itombwensis from the type locality; the difference between these patterns is more subtle in preserved than in live specimens. The first pattern, which is present in the holotype, is a uniform brown to dark brown coloration ( Fig. 2 A) that is similar to X. wittei . The second pattern is a brown dorsal pattern with darker brown spots, sometimes with a dark dorsal band that is perpendicular to the body axis and situated caudal to the eyes but rostral with respect to the forearms ( Fig. 2 B, C). The second color pattern is evident in three of the paratypes including one adult male: MCZ A-138194 (BJE 0277), and both of the juveniles: MCZ A-138193 (BJE 0276) and MCZ A-138195 (BJE 0278). In life, both the gular region and belly are cream colored and the leg and inguinal region are yellowish; sometimes there are small brown spots on the leg and inguinal region ( Fig. 2 C). Coloration at the margins of the lower jaw tends to be slightly darker than the venter, and varies from a dull gray under the head to a thin gray/brown line near the mouth. The barcode sequence of one of the paratypes (museum accession number MCZ A-138193, field number BJE 0 276, barcode accession number EU566832 ) is the same as the holotype.

Size dimorphism: Females are larger than males; we suspect that the females we measured are not fully grown and that size dimorphism is greater than our measurements would suggest (Table 1).

Ecology and distribution: Xenopus itombwensis was collected only at the type locality and the extent of its distribution is unknown beyond this locality. These animals were locally abundant in standing water associated with mineral extraction in a region that was surrounded by mature forest and also mixed use agricultural areas.

Etymology: The new species is named after the plateau where it occurs – the Itombwe Massif of South Kivu Province, Democratic Republic of the Congo.

Figures

FIGURE 2. Type specimen and variation of X. itombwensis. (A) Holotype specimen MCZ A- 138192 (field number BJE 0275), (B) Paratype MCZ A- 138193 (field no. BJE 0276), and (C) a live unvouchered male individual. Arrows indicate dorsal spots that are not found in X. wittei. Scale bars are 5 mm. Photo credit for B: Jon Woodward.

FIGURE 3. Male advertisement vocalization of (A) X. wittei, (B) X. vestitus, and (C) X. itombwensis. The slow trill portion of the X. itombwensis call (beginning at about 400 milliseconds) is a unique feature within the “ vestitus-wittei ” group.

FIGURE 4. Evolutionary relationships of (A) combined data from two tightly linked nuclear loci (RAG 1 and RAG 2) and (B) mitochondrial DNA illustrate a divergent but sister relationship of X. itombwensis and X. wittei. Nuclear loci but not mitochondrial loci illustrate a close relationship between (X. itombwensis + X. wittei) to half of the allopolyploid genome of X. vestitus. For clarity most posterior probabilities are omitted because they are similar or identical to those found elsewhere (Evans 2007; Evans et al. 2004). However, with reference to X. itombwensis, in (A and B) the red clades have over 95 % posterior probability and the blue clade has over 80 % posterior probability. (C) A species phylogeny illustrating bifurcating and reticulating evolutionary relationships in clawed frogs. The most recent common ancestor of X. wittei and X. itombwensis evolved through allopolyploidization of two tetraploid species. The number of chromosomes in each species is indicated in parentheses after each species name. (A) and (C) are modified from (Evans 2007).