Research Article |
Corresponding author: Sandra Goutte ( sg5533@nyu.edu ) Corresponding author: Stéphane Boissinot ( stephane.boissinot@nyu.edu ) Academic editor: Angelica Crottini
© 2022 Sandra Goutte, Jacobo Reyes-Velasco, Abeje Kassie, Stéphane Boissinot.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Goutte S, Reyes-Velasco J, Kassie A, Boissinot S (2022) Genetic and morphometric analyses of historical type specimens clarify the taxonomy of the Ethiopian Leptopelis gramineus species complex (Anura, Arthroleptidae). ZooKeys 1128: 63-97. https://doi.org/10.3897/zookeys.1128.82176
|
Frogs of the genus Leptopelis have diversified in the Ethiopian Highlands to occupy forests and montane grasslands both east and west of the Great Rift Valley. Genetic studies revealed that the endemic species Leptopelis gramineus (Boulenger, 1898) comprises multiple unnamed taxa. A careful examination of historical type specimens is, however, needed to fully resolve the taxonomy of the group. Here we use mitochondrial DNA and morphological analyses on a large sample of recently-collected Ethiopian Leptopelis, as well as century-old type specimens to demonstrate that the recently resurrected L. montanus Tiutenko & Zinenko, 2021 (previously Pseudocassina ocellata Ahl, 1924) is a junior synonym of L. rugosus (Ahl, 1924) and corresponds to the taxon found west of the Great Rift Valley, not east as previously thought. Our results show that populations inhabiting the mountains and plateaus east of the Rift constitute a distinct and undescribed species. We provide a re-description of L. rugosus and describe two new species inhabiting the Highlands east of the Great Rift Valley. We provide an identification key, as well as a description of the calls of the members of the Leptopelis gramineus species complex.
African treefrogs, Afromontane, historical DNA, integrative taxonomy, Leptopelis shebellensis sp. nov., Leptopelis xeniae sp. nov., museomics, new species
Leptopelis is a genus of sub-Saharan treefrogs, currently counting 54 species (
The Ethiopian burrowing treefrog Leptopelis gramineus was described as Megalixalus gramineus by
Distribution ranges of six species of the Leptopelis gramineus species complex. Small, more arboreal forms are represented by green (L. diffidens), yellow (L. gramineus) and purple (L. xeniae sp. nov.) triangles. Larger, semi-fossorial forms are represented by red (L. rugosus) and white (L. shebellensis sp. nov.) circles. The large arboreal Leptopelis susanae is represented by a light blue triangle. Stars indicate type localities given in the original descriptions of L. gramineus (yellow; dashed line between Badditù and Dimé), Pseudocassina rugosa (red) and Pseudocassina ocellata (grey), both synonymised here with L. rugosus (see the discussion in the main text).
Here, we use mitochondrial DNA and morphological data of individuals of the Leptopelis gramineus complex, including the holotypes of L. gramineus and P. rugosa and the lectotype of Pseudocassina ocellata to clarify the taxonomic status of multiple species and populations from the Ethiopian Highlands. Our results show that Pseudocassina rugosa and P. ocellata are conspecific and distinct from L. gramineus. Furthermore, the lectotype of P. ocellata is not conspecific with the population occurring in the Bale Mountains and considered as L. montanus (= P. ocellata) by
Methods of sampling are discussed in detail in
We obtained the authorisation from the
Museum für Naturkunde Berlin (
We used a high sensitivity kit in a Qubit fluorometer (Life Technologies) to measure DNA concentration, while DNA fragment size distribution and concentration was estimated on a Bioanalyzer 7500 high sensitivity DNA chip (Agilent, Santa Clara, CA, USA). We used a NEBNext FFPE DNA Repair Mix (New England Biolabs) to repair damaged bases prior to library preparation. Library preparation was performed with the use of a NEB library preparation kit. During library preparation we skipped the shredding step due to the fragmented nature of historical DNA. All libraries were pooled and sequenced on an Illumina NextSeq 550 (75 bp paired-end) at the Genome Core Facility of New York University Abu Dhabi, UAE. We used the FASTx Toolkit (
Whole mitochondrial genomes of the type specimens of Pseudocassina ocellata and Pseudocassina rugosa were assembled from the Illumina reads using the programme MITObim (
To assess the relationships of the holotypes of Pseudocassina ocellata and P. rugosa and the validity of the names, we reconstructed phylogenetic relationships within the Leptopelis gramineus species complex using all the mitochondrial protein-coding genes, as well as the ribosomal RNA 12s and 16s of the individuals for which we had full mitochondrial genomes. We performed alignments using MAFFT version 7 (
We also performed a phylogenetic analysis using only sequences of the mitochondrial protein coding gene Cytochrome c oxidase I (COX1). The reason for choosing this gene is that many more sequences of COX1 are available for Ethiopian Leptopelis and this gene has been shown to be informative for estimating relationships in this group of frogs (
In certain studies of Ethiopian Leptopelis, species relationships were inferred using the ribosomal RNA 12s and 16s and no COX1 sequences were available for these specimens (
We estimated pairwise genetic distances (uncorrected P distances) of the mitochondrial data (whole mitogenome and COX1), including all codon positions, both transitions and transversions and Gamma distributed rates amongst sites in the programme MEGA X (
We measured 116 individuals that were collected in recent years, as well as type specimens of Leptopelis gramineus (lectotype Genoa-28564; paralectotypes Genoa-49850-1 & Genoa-49850-2), Pseudocassina ocellata (
Summary of the linear morphometric measurement for the Leptopelis gramineus species complex.
Species | sex | N | SVL | HW | HL | SL | NS | IND | EN | IOD | ETD | TD |
---|---|---|---|---|---|---|---|---|---|---|---|---|
L. diffidens | F | 8 | 42 ± 5.5 | 15.6 ± 1.6 | 12.7 ± 1 | 5.5 ± 0.7 | 2.7 ± 0.5 | 3.3 ± 0.6 | 2.5 ± 0.3 | 3.9 ± 0.7 | 1.2 ± 0.3 | 1.8 ± 0.2 |
M | 20 | 27.5 ± 2.5 | 10.3 ± 1.1 | 9 ± 0.9 | 3.8 ± 0.3 | 2 ± 0.3 | 2.4 ± 0.3 | 1.7 ± 0.3 | 3.3 ± 0.4 | 0.7 ± 0.2 | 1.6 ± 0.4 | |
L. gramineus | F | 3 | 34.5 ± 8.8 | 13.9 ± 3.6 | 11.2 ± 2.5 | 5 ± 1.6 | 1.9 ± 0.9 | 2.5 ± 0.5 | 2 ± 0.6 | 4.2 ± 1.1 | 0.8 ± 0.6 | 1.8 ± 0.5 |
M | 8 | 30.3 ± 5.8 | 11.4 ± 2.7 | 9.5 ± 1.5 | 4.2 ± 0.6 | 2 ± 0.2 | 2.6 ± 0.2 | 1.7 ± 0.3 | 3.4 ± 0.8 | 0.7 ± 0.2 | 1.7 ± 0.4 | |
L. rugosus | F | 4 | 47.8 ± 5.3 | 16.4 ± 0.7 | 14.3 ± 0.7 | 6.7 ± 0.7 | 3.6 ± 0.4 | 4.4 ± 0.5 | 2.8 ± 0.4 | 4.3 ± 0.6 | 1.4 ± 0.6 | 2 ± 0.4 |
M | 22 | 38.7 ± 2.7 | 14.3 ± 1.4 | 11.8 ± 1.1 | 5.2 ± 0.4 | 2.9 ± 0.2 | 3.4 ± 0.3 | 2.1 ± 0.3 | 4.2 ± 0.5 | 0.9 ± 0.3 | 2.3 ± 0.4 | |
L. shebellensis sp. nov. | F | 8 | 53.4 ± 5.3 | 18.9 ± 2.3 | 15.8 ± 2.5 | 7 ± 1 | 3.8 ± 0.3 | 4.6 ± 0.5 | 3.3 ± 0.8 | 5.5 ± 0.8 | 1.2 ± 0.6 | 3.1 ± 0.5 |
M | 24 | 36.2 ± 3.3 | 13.2 ± 1.5 | 11.8 ± 1.3 | 5 ± 0.5 | 2.7 ± 0.3 | 3.1 ± 0.4 | 2.3 ± 0.3 | 3.8 ± 0.5 | 0.8 ± 0.3 | 2.1 ± 0.2 | |
L. susanae | F | 3 | 49.6 ± 6.6 | 18.1 ± 2.4 | 16.4 ± 2.2 | 7.4 ± 0.7 | 4.2 ± 0.2 | 4.6 ± 0.2 | 3.7 ± 0.4 | 5.9 ± 0.3 | 1.3 ± 0.4 | 2.6 ± 0.8 |
M | 12 | 33.2 ± 3.7 | 11.8 ± 1.3 | 11 ± 0.7 | 5 ± 0.4 | 2.6 ± 0.3 | 3.3 ± 0.3 | 2.4 ± 0.2 | 3.7 ± 0.3 | 0.9 ± 0.2 | 1.9 ± 0.3 | |
L. xeniae sp. nov. | F | 2 | 43.5 ± 5.7 | 15.9 ± 1.4 | 12.7 ± 0.4 | 5.1 ± 0.7 | 2.9 ± 0.4 | 3.2 ± 0 | 1.7 ± 0.3 | 3.3 ± 0.3 | 0.7 ± 0.1 | 2.1 ± 0.2 |
M | 20 | 27.6 ± 2.0 | 10.2 ± 0.6 | 9.1 ± 0.5 | 3.9 ± 0.3 | 2.2 ± 0.3 | 2.5 ± 0.3 | 1.7 ± 0.4 | 2.9 ± 0.4 | 0.7 ± 0.2 | 1.9 ± 0.3 | |
ED | UEW | FLL | HAL | FinDW | THL | TL | FL | Toe4DW | MTL | |||
L. diffidens | F | 8 | 4.5 ± 0.4 | 3 ± 0.4 | 9.6 ± 1.3 | 12.6 ± 1.9 | 1.5 ± 0.2 | 15.9 ± 3 | 15.6 ± 1.5 | 20.5 ± 2.6 | 1.4 ± 0.3 | 2.8 ± 0.5 |
M | 20 | 3.2 ± 0.4 | 2.5 ± 0.3 | 6.2 ± 0.9 | 8.1 ± 0.9 | 0.9 ± 0.1 | 10.9 ± 1.5 | 10.2 ± 0.9 | 12.5 ± 0.9 | 0.9 ± 0.1 | 1.8 ± 0.2 | |
L. gramineus | F | 3 | 4.2 ± 0.4 | 2.6 ± 0.5 | 8.5 ± 1.5 | NaN ± NA | NaN ± NA | 11.2 ± 2.1 | 11 ± 2.6 | NaN ± NA | 1.1 ± 0.2 | 1.8 ± 0.7 |
M | 8 | 3.6 ± 0.9 | 2.6 ± 0.5 | 6.3 ± 1.2 | 7.4 ± 0.5 | 0.8 ± 0.1 | 10.9 ± 1.2 | 10.3 ± 1.2 | 11.5 ± 1.2 | 0.9 ± 0.2 | 2 ± 0.7 | |
L. rugosus | F | 4 | 4.4 ± 0.3 | 3.5 ± 0.3 | 10.4 ± 1.3 | 13.5 ± 1 | 1.3 ± 0.3 | 20.5 ± 1.2 | 17.8 ± 1.4 | 21.3 ± 0.7 | 1.2 ± 0.4 | 3.7 ± 0.4 |
M | 22 | 4.1 ± 0.5 | 3.1 ± 0.2 | 7.8 ± 1.1 | 10.9 ± 0.8 | 1.2 ± 0.2 | 14 ± 1.5 | 13.2 ± 0.9 | 16.6 ± 1.3 | 1.1 ± 0.2 | 2.8 ± 0.4 | |
L. shebellensis sp. nov. | F | 8 | 5.8 ± 1.6 | 4.1 ± 1.1 | 10.8 ± 1.8 | 14.8 ± 1.8 | 1.3 ± 0.4 | 16.7 ± 2 | 16.1 ± 1.5 | 22.8 ± 2.5 | 1 ± 0.2 | 3.7 ± 0.6 |
M | 24 | 4.2 ± 0.7 | 2.8 ± 0.5 | 8 ± 1.2 | 10.2 ± 1 | 0.9 ± 0.2 | 12.7 ± 1.4 | 11.8 ± 1 | 14.8 ± 1.2 | 0.8 ± 0.2 | 2.4 ± 0.3 | |
L. susanae | F | 3 | 5.7 ± 1.3 | 3.9 ± 0.8 | 12.1 ± 1.3 | 16 ± 0.8 | 2.7 ± 0.2 | 22.5 ± 1 | 21.2 ± 2.1 | 24.6 ± 2.2 | 2.5 ± 0.3 | 2.9 ± 0.6 |
M | 12 | 4.2 ± 0.5 | 3.1 ± 0.6 | 7.4 ± 1.1 | 10.6 ± 0.8 | 1.8 ± 0.2 | 14.9 ± 0.9 | 14.8 ± 1 | 16.5 ± 1.4 | 1.6 ± 0.2 | 1.7 ± 0.2 | |
L. xeniae sp. nov. | F | 2 | 4.8 ± 0.4 | 3.3 ± 1.1 | 9.9 ± 1.8 | 12.8 ± 1.1 | 1.4 ± 0.1 | 16.8 ± 2.8 | 16 ± 3.4 | 20.2 ± 3.4 | 1.4 ± 0.1 | 2.4 ± 0.3 |
M | 20 | 3.3 ± 0.4 | 2.4 ± 0.3 | 6.1 ± 0.7 | 8.2 ± 0.7 | 0.8 ± 0.2 | 10.2 ± 1.4 | 9.9 ± 0.9 | 12 ± 1 | 0.8 ± 0.2 | 1.8 ± 0.3 |
List of abbreviations: ED eye diameter; EN eye-nostril distance; ETD eye-tympanum distance; FinDW longest finger disc width; FL foot length; FLL forearm length; HAL hand length; HL head length; HW head width; IND inter-nares distance; IOD inter-orbital distance; MTL metatarsal tubercle length; NS snout-nostril distance; SL snout length; SVL snout-vent length; THL thigh length; TD tympanum diameter; TL tibia length; Toe4DW fourth toe disc width; UEW upper eyelid width.
We analysed males and females separately due to sexual dimorphism (Fig.
Morphometric analysis of the Leptopelis gramineus species complex. Scatter plots of principal component analyses, based on 16 morphometric measurements in 107 males (top) and 25 females (bottom). Type specimens of Leptopelis gramineus, Pseudocassina rugosa and Pseudocassina ocellata are represented by stars on the plot, but were not included in the analyses because of the important degree of shrinkage during their preservation.
Morphometric measurements were all log-transformed prior to analysis in order to approach normality. To correct for body size in our measurements, we used ratios of measurements over snout-vent length. We did not use other adjustment method such as the one proposed by
Spontaneously calling males were located acoustically or visually between 20:00 and 02:00 h. We recorded advertisement calls in situ at a distance between 0.5–2 m to avoid near-field effects (
Advertisement calls were analysed using Avisoft SAS (
Summary of call acoustic characters for the Leptopelis gramineus species complex.
Species | N individuals | N notes | Note duration (ms) | Inter-note interval (s) | Call rate | Pulses per note | Inter-pulse duration (ms) |
---|---|---|---|---|---|---|---|
L. diffidens | 8 | 188 | 130 ± 21 | 12.08 ± 6.18 | 0.10 ± 0.04 | 7 ± 2 | 22 ± 4 |
L. gramineus | 2 | 19 | 93 ± 6 | 16.46 ± 3.17 | 0.07 ± 0.01 | 6 ± 1 | 19 ± 2 |
L. rugosus | 6 | 108 | 65 ± 21 | 13.12 ± 7.47 | 0.10 ± 0.04 | 4 ± 1 | 20 ± 5 |
L. shebellensis sp. nov. | 4 | 89 | 57 ± 5 | 8.28 ± 1.34 | 0.13 ± 0.02 | 5 ± 0 | 11 ± 2 |
L. susanae | 2 | 50 | 25.6 ± 2.1 | 11.21 ± 3.27 | 0.10 ± 0.02 | 5.2 ± 0 | 11 ± 2 |
L. xeniae sp. nov. | 4 | 51 | 175 ± 25 | 14.16 ± 6.50 | 0.09 ± 0.03 | 7 ± 1 | 30 ± 5 |
Pulse rate (s-1) | Peak frequency (Hz) | Min frequency (Hz) | Max frequency (Hz) | Frequency bandwidth (Hz) | Relative time of peak amplitude (ms) | ||
L. diffidens | 53 ± 8 | 1928 ± 235 | 1489 ± 218 | 2880 ± 286 | 1386 ± 203 | 39 ± 10 | |
L. gramineus | 62 ± 9 | 1863 ± 154 | 1445 ± 173 | 2832 ± 201 | 1385 ± 375 | 21 ± 9 | |
L. rugosus | 61 ± 14 | 1769 ± 60 | 1448 ± 140 | 2192 ± 64 | 742 ± 82 | 9 ± 5 | |
L. shebellensis sp. nov. | 88 ± 3 | 1616 ± 265 | 1211 ± 253 | 2192 ± 132 | 974 ± 163 | 21 ± 15 | |
L. susanae | 209 ± 20 | 1986 ± 130 | 1611 ± 66 | 2445 ± 201 | 832 ± 133 | 7 ± 6 | |
L. xeniae sp. nov. | 39 ± 7 | 2231 ± 585 | 1546 ± 73 | 3562 ± 505 | 2013 ± 573 | 44 ± 24 |
Results of both the Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the full COX1 dataset were largely congruent with each other and with previous analyses by
The uncorrected pairwise genetic distances between lineages in the mitogenome dataset ranged from 5.8% to 6.6% (Suppl. material
The phylogenetic analysis of the whole mitogenomes placed the types of Pseudocassina ocellata and P. rugosa as sister to one another and together sister to an individual from Mehal Meda (Semien Shewa Zone, Amhara Region; Suppl. material
In both COX1 and 16s phylogenies, the type specimens of Pseudocassina ocellata and P. rugosa grouped with individuals from northwest of the GRV (“Northern” clade of
Due to sexual dimorphism, we analysed male and female morphological measurements separately (Fig.
Within the arboreal group, head shape is most discriminant between species. The Kibre Mengist clade can be distinguished from the other small species of the complex (L. gramineus and L. diffidens) by a longer snout-nostril (SN) and a shorter inter-orbital distance (IOD). Leptopelis gramineus has a greater inter-nares (IND) and shorter snout-nostril (SN) distance than L. diffidens and the Kibre Mengist clade (Suppl. material
Advertisement calls of the Leptopelis gramineus species complex sound like a short rattle and species are difficult to distinguish by an untrained ear, except for L. susanae, which produces a call shorter in duration than the other species (Fig.
In 1924, Ahl described Pseudocassina rugosa and Pseudocassina ocellata as distinct species in the same article. Both names were subsequently synonymised with Leptopelis gramineus by Largen in 1977. In 2021, Tiutenko and Zinenko resurrected L. ocellatus from synonymy with L. gramineus, but because a senior homonym, Leptopelis ocellatus (Mocquard, 1902), existed, they assigned a new name to the species, Leptopelis montanus
Pseudocassina rugosa Ahl, 1924.
Pseudocassina ocellata Ahl, 1924.
Leptopelis montanus Tiutenko & Zinenko, 2021.
Holotype. Adult female (
In addition to the holotype, we examined one female collected at the end of July 1900 during O. Neumann’s and C. von Erlanger’s expedition in “Hochebene Didda” (
A large (male (n = 22) SVL 38.7 ± 2.7 mm, female (n = 2) SVL 52.4 ± 0.2 mm) species of the Leptopelis gramineus species complex (Figs
Larger body size, longer head and snout and greater snout-nostril distance and larger tympanum and metatarsal tubercle than L. gramineus, L. diffidens and L. sp. Kibre Mengist (Table
Relatively large adult female (SVL 44.7 mm) in good condition of preservation (Fig.
Dorsal ground colour and canthal region dark olive brown with no visible pattern, except for a large light brown blotch covering about a third of the dorsum (Fig.
Leptopelis rugosus presents less colour polymorphism than the smaller members of the L. gramineus species complex. Dorsum is green to dark green and can be completely uniform or have a few to many irregular brown blotches (Fig.
Leptopelis rugosus is found in grassy meadows of the Ethiopian Highlands north of the GRV at mid- to high elevations (2,339–3,337 m a.s.l.). This species occurs notably near Debre Birhan, Debre Sina, Fiche, Addis Ababa, Holeta and Ambo (Fig.
Males are heard calling at night and sometimes during the day for extended periods of time. Advertisement calls are emitted from the ground, either on the grass or from a cavity in the ground or under a rock, generally near a stream or a flooded area. Several males were found calling from the banks of a completely dried-out stream, although it is unknown to us whether the reproduction period extends to the dry season or whether males keep calling only during shorter dry periods.
The call of Leptopelis rugosus is a short rattle composed of a single note of 65 ± 21 ms in duration, containing 4 ± 1 pulses (Fig.
The call of Leptopelis rugosus is distinguishable from the calls of L. gramineus, L. diffidens and the Kibre Mengist and Bale/Assela clades by its lower number of pulses per note and narrower frequency band width. It is further distinguished from the call of L. gramineus, L. diffidens and L. sp. Kibre Mengist by its shorter note duration and from L. sp. Kibre Mengist by its lower peak frequency and higher pulse rate. Finally, it is distinguished from the call of L. susanae by its longer duration and lower pulse rate (Table
Diagnostic characters used in the original description.
Ahl described Pseudocassina rugosa, based on a single female and provided three main diagnostic characters when compared with Pseudocassina ocellata, which he described in the same article, based on one female and one male: (1) the texture of the skin (rugose for P. rugosa and smooth for P. ocellata), (2) the length of the tibia (P. rugosa TL/SVL 1/3.5 and P. ocellata TL/SVL 1/3), (3) the visibility of the tympanum (hidden for P. rugosa and visible for P. ocellata). Ahl also named P. ocellata after the presence of ocelli on the flanks of the individuals he examined, which reflects on its specificity, even though he did not use this trait as a diagnostic character.
We found individuals of L. rugosus and the Bale/Assela clade with either a completely smooth, slightly rugose or coarsely rugose dorsum. The rugosity of the skin thus seems to be variable amongst individuals and, perhaps, age or season. Additionally, we have noticed that the rugosity of the skin may disappear after euthanasia and/or fixation of the specimen. Finally, even though almost all individuals of L. diffidens, L. gramineus and the L. sp. Kibre Mengist examined had smooth skin, we found two females L. diffidens and one female L. sp. Kibre Mengist with slightly rugose skin. The visibility of the tympanum seems to be variable across the individuals as well, perhaps linked to the size of the individual and the rugosity of the skin. While some female L. rugosus have a partially hidden tympanum, all males examined had a visible tympanum. The difference in tibia length between the specimens described by Ahl reflects individual variations as shown in our dataset. Finally, ocelli on the flanks and/or dorsum are present in certain individuals of L. rugosus and the Bale/Assela clade and is not a diagnostic character.
Ahl described Pseudocassina ocellata, based on one female and one male, collected at different localities during Oscar Neumann’s and Carlo von Erlanger’s 1900 expedition. The information given for the female is “Hochebene, Didda, end of July 1900”, while for the male, only “Somaliland” is given. Although imprecise, both localities are clearly situated east of the Great Rift Valley (GRV). In contrast, the specimen used as holotype for the original description of P. rugosa and all recent L. rugosus specimens were collected west of the GRV. We propose two, non-exclusive explanations for this discrepancy.
First, based on Neumann’s report and map of the expedition (
Second, although the distribution range of L. rugosus is mostly west of the GRV, it is possible that a population occupies the west Arussi Plateau (or did in 1900). We found individuals of the grass frog Ptychadena beka Goutte, Reyes-Velasco, Freilich, Kassie and Boissinot 2021 at the north-western edge of the Arussi Plateau, even though the distribution range of this species is otherwise exclusively west of the GRV. We also found in the same area individuals of the river frog Amietia nutti (
English: Shebelle River burrowing African treefrog.
Holotype. Adult male (SB482), collected on 26 June 2018 by S. Goutte and Y. Bourgeois near the town of Ch’ange, Oromia, Ethiopia (8.1263°N, 39.4360°E, 2429 m a.s.l.). Paratypes. One male (15–46), collected on 5 August 2015 by X. Freilich, J. Reyes-Velasco and S. Boissinot, south of Assela (7.9068°N, 39.1238°E, 2520 m a.s.l.), one male (15–79) and one female (15–83), collected on 6 August 2015 by X. Freilich, J. Reyes-Velasco and S. Boissinot, southwest of Dinsho (7.1156°N, 39.7390°E, 3029 m a.s.l.), one male (15–84), collected on 6 August 2015 by X. Freilich, J. Reyes-Velasco and S. Boissinot southwest of Dinsho (7.1105°N, 39.7461°E, 3042 m a.s.l.), one male (15–143), collected on 8 August 2015 by X. Freilich, J. Reyes-Velasco and S. Boissinot between Robe and Ali (7.1720°N, 39.9722°E, 2431 m a.s.l.), one female (15–152), collected on 8 August 2015 by X. Freilich, J. Reyes-Velasco and S. Boissinot in Goba (7.0110°N, 39.9677°E, 2699 m a.s.l.), two females (16–8, 16–9), collected on 5 July 2016 by J. Reyes-Velasco and S. Boissinot south of Assela (7.8656°N, 39.1305°E, 2605 m a.s.l.), one females (16–25), collected on 6 July 2016 by J. Reyes-Velasco and S. Boissinot south of Assela (7.8836°N, 39.1245°E, 2531 m a.s.l.), two males (16–26, 16–28), collected on 6 July 2016 by J. Reyes-Velasco and S. Boissinot north of Bekoji (7.5585°N, 39.2520°E, 2721 m a.s.l.), one male (16–88), collected on 10 July 2016 by J. Reyes-Velasco and S. Boissinot east of Dinsho (7.1065°N, 39.8184°E, 3065 m a.s.l.), one male (16–93), collected on 10 July 2016 by J. Reyes-Velasco and S. Boissinot west of Dinsho (7.1204°N, 39.7358°E, 3048 m a.s.l.), three males (SB61, SB62, SB63), collected on 26 June 2018 by S. Goutte and J. Reyes-Velasco south of Dinsho (7.0915°N, 39.7834°E, 3079 m a.s.l.), two males (SB483, SB484), collected on 26 June 2018 by S. Goutte and Y. Bourgeois near Ch’ange (8.1263°N, 39.4360°E, 2429 m a.s.l.), three males (SB502, SB504, SB505), collected on 28 June 2018 by S. Goutte and Y. Bourgeois north of Arussi Robe (7.9190°N, 39.6091°E, 2433 m a.s.l.).
Medium to large (male (n = 21) SVL 35.9 ± 3.5 mm, female (n = 5) SVL 53.4 ± 5.3 mm), robust semi-fossorial species of the Leptopelis gramineus species complex (Fig.
Larger body size, longer head and snout and greater snout-nostril distance and larger tympanum and metatarsal tubercle than L. gramineus, L. diffidens and L. sp. Kibre Mengist (Table
Medium size adult male (SVL 40.6 mm). Body robust (Fig.
Fingers and toes robust with ovoid discs not expanded, but distinct. Finger formula: I <II < IV < III. Hand free of webbing. Hind-limbs short and robust (TL/SVL 0.31 and THL/SVL 0.38). Foot 1.32× tibia length. Inner metatarsal tubercle present, oval in shape, 0.16× foot length. Outer metatarsal tubercle absent. Toe formula: I < II < III < V < IV. Foot webbing minimal, except between toe III and toe IV where webbing extends to half-way between the first and the second phalanges. Skin of the dorsum, flanks and ventrum slightly rugose.
Dorsal ground colour sand, slightly iridescent, with green hues in the lower two-thirds (Fig.
Dorsal ground colour bluish-grey with large irregular black bands and spots outlined by a white line (Fig.
Dorsum can be green to dark green, sand or brown. All examined specimens had light or dark brown to black irregular markings, variable in size and number, on the dorsum. In many individuals, those markings are bi- or tricolour (yellowish-cream, light and dark brown) and form lateral and dorso-lateral ocelli in some animals. A thin yellowish line is present from the tip of the snout to behind the tympanum in all examined individuals. The canthal stripe can be light brown to black and can be underlined by a second yellowish line from the snout to the eye in some individuals. Flanks can be the same colour or a lighter version of the dorsal ground colour or green while the dorsum is brown or vice versa. Larger versions of the dorsal blotches are found on the flanks, sometime merging into an irregular band. Limbs are the same colouration as the dorsum and sometimes have irregular brown markings. Upper lip may be light brown or the same colour as the dorsum without any markings. Iris golden to brown. Tympanum partially or completely covered with a brown blotch, either joining the brown bar behind the eye or as a separate blotch. Throat and chest uniformly white to pale yellow. Ventrum white to orange-yellow, sometimes with yellow to orange zones on the sides and extending to the ventral side of the thighs. In gravid females, yellow eggs are visible through the thick ventral skin. Palms of the hands, ventral sides of feet and tibia more or less heavily dusted with dark grey. Dorsal skin may be smooth, slightly or very rugose.
The specific name refers to the Shebelle River, as the distribution range of the species appears restricted to the Shebelle River Basin, with populations found both north and south of the source of the river (Fig.
Leptopelis shebellensis sp. nov. inhabits the grassy meadows of the Didda Plateau and the northern Bale Mountains at mid- to high elevations (2,429–3,296 m a.s.l.). This species is notably found near Assela, Huruta, Dinsho, Adaba, Dodola, Goba and Chole (Fig.
The call of Leptopelis shebellensis sp. nov. is a very short rattle. It is composed of one or two identical notes at 704 ± 85 ms interval (Fig.
The call of Leptopelis shebellensis sp. nov. is distinguishable from the calls of L. gramineus, L. diffidens, L. sp. Kibre Mengist and L. rugosus by its higher pulse rate. It is further distinguished from the call of L. gramineus, L. diffidens and L. sp. Kibre Mengist by its shorter note duration and narrower frequency band width and from L. susanae by its longer note duration and lower pulse rate. Finally, it can be distinguished from the call of L. rugosus by its greater number of pulses per note.
English: Xenia’s African treefrog.
Holotype. Adult male (SB183), collected on 18 April 2018 by S. Goutte and J. Reyes-Velasco east of Kibre Mengist (5.8782°N, 39.1330°E, 1832 m a.s.l.). Paratypes. One female (SB151) and two males (SB152, SB153), collected on 17 April 2018 by S. Goutte and J. Reyes-Velasco northwest of Kibre Mengist (5.9988°N, 38.8798°E, 2097 m a.s.l.), four males (SB167, SB168, SB169, SB170), collected on 17 April 2018 by S. Goutte and J. Reyes-Velasco northwest of Kibre Mengist (6.0093°N, 38.8576°E, 2105 m a.s.l.) and one male (SB184) collected on 18 April 2018 by S. Goutte and J. Reyes-Velasco east of Kibre Mengist (5.8782°N, 39.1330°E, 1832 m a.s.l.).
One female (SB197) and 12 males (SB186–SB196, SB206), collected on 19 April 2018 by S. Goutte and J. Reyes-Velasco southeast of Kofele (7.0226°N, 38.8701°E, 2,561 m a.s.l.).
Small to medium-sized (male (n = 20) SVL 27.6 ± 2.0 mm, female (n = 2) SVL 43.5 ± 5.7 mm), robust arboreal species of the Leptopelis gramineus species complex (Fig.
Smaller body size, narrower head than L. rugosus, L. shebellensis sp. nov. and L. susanae (Table
Small-sized male (SVL 28.9 mm) adult (Fig.
Dorsal ground colour grey-brown with three dark, but very faint, wide bands; the central band forming a triangle pointing towards the snout and each eye and extending along the spine to about three-fourths of the dorsum (Fig.
Dorsal ground colour grey with a few small irregular dark grey spots (Fig.
As with other members of the Leptopelis gramineus species complex, L. xeniae sp. nov. shows significant colour polymorphism. Dorsal colouration varies from light grey (with or without some light green) or light brown to bright or dark green with important level of dark pigmentation. Most individuals examined display a similar dorsal pattern composed of three bands, which can be barely distinguishable to well-marked. The canthal stripe may extend as a wide dark brown to black stripe on the flank to four-fifths of the abdomen or be replaced by large blotches of the same colour. Limbs are the same colouration as the dorsum, with variable number of small to medium brown or black markings. Upper lip may be cream, light grey or green, with variable size and number of irregular brown or black markings. Iris golden to dark golden. Tympanum dark brown or black either entirely or on its upper half, with the lower half being the same colour as the dorsum. Rarely, the tympanum is entirely the same colour as the dorsum. Lower lip cream mottled with brown or grey or with a continuous brown blotch. In one female (SB151), the lower lip was light green with a few brown markings. Ventrum and throat white, with small brown blotches on either or both in most individuals. Inner thighs white or lacking any pigmentation, except dark brown and yellow spots towards the knee in some individuals. Cream-coloured eggs visible through the thick ventral skin of the gravid female SB151. Dorsal skin smooth in all specimens examined, except in the female SB197, which had a slightly rugose dorsal skin.
Leptopelis xeniae sp. nov. is named after Xenia Freilich, who conducted her doctoral research on Ethiopian anurans, including the Leptopelis gramineus complex.
Leptopelis xeniae sp. nov. is found in the forested areas around the towns of Kibre Mengist and Kofele, Oromia, Ethiopia (5.87–7.02°N, 38.80–39.13°E; Fig.
As for other members of the Leptopelis gramineus species complex, the call of Leptopelis xeniae sp. nov. is a short rattle (Fig.
The call of Leptopelis xeniae sp. nov. is distinguishable from the calls of L. gramineus, L. diffidens, L. shebellensis sp. nov., L. rugosus and L. susanae by its longer note and longer inter-pulse intervals (Fig.
Around the town of Kofele (7.0226°N, 38.8701°E, 2,561 m a.s.l.; Fig.
Leptopelis diffidens shows substantial colour polymorphism, with dorsal colouration ranging from light yellow, brown, sand, orange to bright or bluish-green. Many individuals have a more or less marked three-banded pattern also shared with other species of the genus. Contrary to what is stated in the original description, we found L. diffidens in syntopy with Leptopelis ragazzii in multiple forest clearings, with males calling simultaneously. Male L. diffidens were found calling on flooded grass or reeds, while males L. ragazzii called from trees at 1.5–3 m above the ground. We also found multiple L. diffidens males calling on vegetation and under rocks around a rivulet running through the small town of Rira. Several female L. diffidens were found on shrubs as high as 1.5 m above the ground.
Beside the advertisement call produced by males, we recorded three calls, which we identified as aggressive calls, from two different males (Suppl. material
1 | Medium to large body size (male SVL 26–38 mm, female SVL 44–57 mm), finger and toe discs very enlarged, elongated hind-limbs (male TL/SVL 0.40–0.49, female TL/SVL 0.41–0.45; male THL/SVL 0.40–0.49, female THL/SVL 0.42–0.49 | L. susanae |
– | Small to large body size (male SVL 21.5–45 mm, female SVL 35.9–61 mm), finger and toe discs barely expanded or not expanded at all, hind-limbs short to moderately elongated | 2 |
2 | Medium to large body size (male SVL 30–45 mm, female SVL 45–61 mm), large tympanum (male TD/ED > 0.51, female TD/ED > 0.54), metatarsal tubercle spade-shaped and well developed | 3 |
– | Small to medium body size (male SVL 21.5–38.8 mm, female SVL 35.9–50.2 mm), small tympanum (male TD/ED < 0.54, female TD/ED < 0.45), small ovoid metatarsal tubercle | 4 |
3 | Ventral area and inner limbs with very little to no melanisation | L. rugosus |
– | Palms of the hands, ventral sides of feet and tibia more or less heavily dusted with dark grey | L. shebellensis |
4 | Inter-orbital distance very short (male IOD/ED 0.88 ± 0.16, female IOD/ED 0.69 ± 0.12), snout narrow and elongate (male SN/SL 0.56 ± 0.05, female SN/SL 0.57 ± 0.00 | L. xeniae |
– | Inter-orbital distance not very short (male IOD/ED > 0.76, female IOD/ED > 0.77) snout less narrow (male and female SN/SL < 0.53 | 5 |
5 | Finger tips barely enlarged | L. gramineus |
– | Finger tips enlarged | L. diffidens |
In the present work, we resolved issues related to the taxonomy of the Leptopelis gramineus species complex, which consists of at least six species. We described two new species found east of the GRV and clarified the status of montane populations west of the GRV. Using genetic and morphometric analyses of type specimens, we demonstrated that Leptopelis rugosus (= Pseudocassina rugosa) and L. montanus (= Pseudocassina ocellata) are, in fact, conspecific with the population northwest of the GRV and that the mountain populations east of the GRV belong to a new species, L. shebellensis sp. nov. In this species complex, including historical type specimens was necessary to assign the correct names to newly-discovered and previously-synonymised taxa. The present case is reminiscent of the situation reported in Ethiopian Ptychadena, where the analysis of type specimens allowed us to clarify the convoluted taxonomy of the group (
The Leptopelis gramineus species complex comprises species living in most of the biotopes present in the Ethiopian Highlands, from mid-elevation forests (~ 1,800 m a.s.l.) to high-elevation montane grasslands (> 3,200 m a.s.l.). Members of this clade show traits associated either with a semi-fossorial lifestyle (e.g. well-developed metatarsal tubercles, robust limbs and body shape) in species living in high elevation grasslands (L. rugosus and L. shebellensis sp. nov.) or with a more arboreal lifestyle (e.g. elongated limbs, slender body) in species inhabiting forests and forest edges (L. diffidens, L. susanae and L. xeniae sp. nov.). The L. gramineus species complex thus adds to a growing list of groups that have diversified in the Ethiopian Highlands (e.g.
The restriction of Leptopelis gramineus to the south-eastern population near Chencha and description of several species previously thought to be L. gramineus should impact the conservation status of Ethiopian Leptopelis. Currently, Leptopelis susanae is listed as “Endangered” by the IUCN because of its limited distribution range, while L. gramineus, considered widespread in Ethiopia, is listed as “Least concern” (
We would like to thank the Ethiopian Wildlife Conservation Authority and the Ethiopian Biodiversity Institute for providing us with collecting and export permits for the samples. We are very grateful to Yann Bourgeois, Marcin Falis, Yejie Yun and Paula Mora Rojas who helped with the acoustic recordings in the field. Fieldwork in Ethiopia would not have been possible if not for the invaluable assistance of Megersa Kelbessa, Itbarek Kibret and Samuel Woldeyes of Rock Hewn Tours. This work could not have been possible without the assistance of many curators and collection managers from numerous institutions, including Bezawork Afework Bogale and M. Ketema (Zoological Natural History Museum, Addis Ababa University, Ethiopia), Jeff Streicher (The Natural History Museum, London, UK), Giuliano Doria and Enrico Borgo (Natural History Museum of Genova, Italy) and Mark-Oliver Rödel and Frank Tillack (Museum für Naturkunde Berlin, Germany). We thank Yann Bourgeois and Sebastian Kirchhof who helped with measuring specimens and Marcin Falis and Kole Utzinger who helped with lab work. We are indebted to Marc Arnoux and Nizar Drou from the Genome Core Facility and the Bioinformatics group at NYUAD. The NYUAD Sequencing Core is supported by NYUAD Research Institute grant G1205A to the NYUAD Center for Genomics and Systems Biology. This research was supported by New York University Abu Dhabi Research Funds AD180 (to SB).
Maximum Likelihood phylogenetic inference
Data type: jpg file
Explanation note: Figure S1. A Maximum Likelihood phylogenetic inference, based on 13 mitochondrial protein coding genes and the two ribosomal RNAs (12s and 16s) for members of the Leptopelis gramineus complex. Names in bold represent historical type specimens of Pseudocassina rugosa (
Bayesian phylogenetic inference of the Leptopelis gramineus species complex, based on COX1
Data type: jpg file
Explanation note: Posterior probabilities are given at the nodes. Names in bold represent historical type specimens of Pseudocassina rugosa (
Maximum Likelihood phylogenetic inference, based on the ribosomal RNAs 16s for members of the Leptopelis gramineus complex
Data type:jpg file
Explanation note: Names in bold represent historical type specimens of Pseudocassina rugosa (
Aggressive call of Leptopelis diffidens and aggressive or warm-up call of Leptopelis suanae
Data type: jpg file
Explanation note: A Aggressive call of Leptopelis diffidens (SB128) and B aggressive or warm-up call of Leptopelis suanae (SB223).
Tables
Data type: localities, genetic, and morphometric data
Explanation note: Tables: S1. Localities of sampled specimens; S2. Number and average length of raw reads extracted from historical type specimens; S3. GenBank accession numbers; S4. PartitionFinder models; S5. Pairwise genetic distances (uncorrected P distances) of the whole mtDNA dataset; S6. airwise genetic distances (uncorrected P distances) of the COX1 dataset; S7. Linear morphometric measurements; S8. PCA loadings and scores for males and females of the Leptopelis gramineus complex; S9. Tukey HSD pairwise tests for linear morphometric measurements of males and females of the Leptopelis gramineus complex; S10. Acoustic characteristics of advertisement calls, averaged per individual.