Research Article |
Corresponding author: Arthur Tiutenko ( arthur.tiutenko@fau.de ) Academic editor: Angelica Crottini
© 2021 Arthur Tiutenko, Oleksandr Zinenko.
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:
Tiutenko A, Zinenko O (2021) A new species of Leptopelis (Anura, Arthroleptidae) from the south-eastern slope of the Ethiopian Highlands, with notes on the Leptopelis gramineus species complex and the revalidation of a previously synonymised species. ZooKeys 1023: 119-150. https://doi.org/10.3897/zookeys.1023.53404
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A new ground-dwelling species of treefrog in the genus Leptopelis is described from the Harenna Forest in south-eastern Ethiopia. The description is based on morphology and acoustics and is supported by molecular data. The new species has a small body size, and the digital discs on fingers and toes are significantly more conspicuous than in other semi-fossorial members of the L. gramineus complex. It occupies forest habitats at lower altitudes and is separated ecologically and geographically from high-altitude species of the complex. One of them, a parapatric cryptic species from Bale and Arsi Mountains, is resurrected from synonymy of L. gramineus and given a new name, L. montanus. Genetic barcoding of specimens from both populations showed that they belong to two distinct lineages that had been revealed by recent phylogenetic research. To confirm the geographic separation of the studied populations, the collection area of L. gramineus types was verified through analysis of the diary and the final report of the 2nd expedition of V. Bottego, and through matching of the route described in it with modern maps. The type locality of L. gramineus sensu stricto is restricted to Gamo Gofa, Ethiopia. Following the results of recent phylogenetic studies, the range of L. gramineus is limited to west of the Great Rift Valley. An identification key to the named Ethiopian species of the genus is provided.
Bale Mountains, Ethiopia, Harenna Forest, Leptopelis diffidens sp. nov., Leptopelis montanus nom. nov., Leptopelis rugosus, Pseudocassina ocellata, Pseudocassina rugosa
The genus Leptopelis Günther, 1859 is usually referred by the vernacular name ‘Forest Treefrogs’, or just ‘Tree Frogs’. Indeed, the majority of its currently recognised ca. 50 members are arboreal or semi-arboreal frogs, found on tall grass, scrubs, and trees in various habitats across sub-Saharan Africa (
For L. gramineus, the evolutionary relationships between populations east and west of the Great Rift Valley (GRV) were outlined in the recent works dealing with phylogeography and evolution of amphibians in the Ethiopian Highlands (
The examined and compared material in museum collections comprises 105 specimens identified as L. gramineus and 86 specimens of six other Leptopelis species from the Horn of Africa. The list of these specimens is provided in Suppl. material
Morphometric measurements of 45 preserved specimens of three lineages of L. gramineus complex were performed with digital callipers with ± 0.1 mm accuracy by the first author. For measured values see Suppl. material
The following abbreviations (mainly adopted from
Statistical analyses were performed with Statistica 8.0. To increase the statistical sample size, we pooled specimens of all ages and sexes in a single sample. To minimise the differences in measurements that are associated with the size of specimens, we divided these values by SVL. Characters with high proportion of missing values in the dataset were excluded. We performed principal component analysis first, to check if any strong signal exists in the study material. Subsequently, a multivariate canonical discriminant analysis was applied in order to assess the morphological distinctiveness and our ability to separate the species on a basis of external characters only. This analysis was done with specimens assigned to three groups according to their geographic origin within the clades determined in recent phylogenetic studies.
The metamorphosis stage of juvenile specimens and larvae we determined according to the table of
Localities of collection vouchers and of historical records were geo-referenced with Garmin BaseCamp 4.8.8 and QGIS 3.6. Geographic coordinates at our own study sites were taken with Garmin GPSmap 66st. For geographic coordinates and elevation of the localities mentioned in the text see Suppl. material
To ensure that the collection localities of our vouchers in the Harenna Forest do not coincide with the type locality and range of L. gramineus sensu stricto, we determined the collection area of L. gramineus types through analysis of the diary included in the final report of the 2nd expedition of Vittorio Bottego, and through matching of the route described in it with modern maps.
The authors of phylogenetic studies of Ethiopian Leptopelis (
We performed genetic barcoding of specimens from populations that we were studying in order to confirm their identity and to connect our results to the published work of other authors. Total genomic DNA was extracted with NeoPrep DNA kit (NeoGene, Kyiv, Ukraine) from muscle tissue samples obtained from Leptopelis specimens housed in the Zoological State Collection Munich (
We compared our sequences with already available GenBank records and assigned the populations of our interest to the lineages and haplogroups from previous studies (
We recorded vocalisations of two Leptopelis species at 12 localities in the Harenna Forest (Gola, Hacho, Harawa, Haro Alati, Hordoba, Kaffa Guasaa, Manyate, Megano, Segoba, Sire, Woraba, Yagana) and of one parapatric species at one locality in Bale Mountains (Gaysay Grasslands). The taxonomic identity of these populations, hence of calling individuals, had been established through aforementioned genetic barcoding. The sound recordings were performed in May and June 2019 with a TASCAM DR-100MKII stereo recorder, processed with Adobe Audition CC 2019, analysed and visualised with Sonic Visualiser 4.0.1 (University of London). In the Harenna Forest the advertisement calls were recorded in the morning (9–11 a.m.), in the afternoon (2–4 p.m.) and after sunset (9–11 p.m.), at air temperatures of 19–26 °C and at no wind. At the high-altitude locality (Gaysay Grasslands) our activities in dark hours were not allowed for security reasons. We recorded there in the morning and in the afternoon at similar temperatures, however, at wind speed of ca. 30 km/h.
Initially we became aware of a possibly new species of Leptopelis when we heard advertisement calls during our first trip and stays on glades in the Harenna Forest in March 2012: They differed from vocalisations that we had heard previously in a high-altitude population of L. gramineus in the Web Valley, Bale Mountains. We made the same observations during our next trips and recorded the calls in May–June 2019. Comparative examination of specimens from the Harenna Forest and from other populations distributed across Ethiopian Highlands increased our confidence that two species of burrowing Leptopelis live in parapatry north and south of the Harenna Escarpment and occupy different habitats. Genetic barcoding of individuals from both populations showed that they belong to two distinct lineages that had been revealed by recent phylogenetic research (
Any further analyses and taxonomic decisions require clarity about the geographic range of L. gramineus sensu stricto. Since no species in this complex are sympatric, one of them with the range that includes the type locality can be considered as ‘true’ L. gramineus. Like many species of African herpetofauna described in the 19th and early 20th century, the type locality of L. gramineus was imprecisely given between two places separated from each other by several mountain ranges and by 180 km straight line. Fortunately, we know the exact route of Bottego’s expedition and can approximately determine the position of collection sites along it, thus restrict the type locality to a more concrete geographic area.
The often-used English vernacular name ‘Badditu Forest Treefrog’ seems to have been coined by
Since no specimens tagged only with the locality ‘Badditu’ exist, it should be safe for us to assume that L. gramineus was not found by Bottego’s expedition at the eastern shores of the lakes Abaya and Chamo, i.e., where the Badditu tribal territory is situated. This place cannot be found on modern maps because it is a historical tribe name that was used as toponym mainly by Italians. The members of this tribe called themselves ‘Koyra’ (
Bottego and his people came to Burji on 17 March 1896 (see Fig.
In the second half of June the Bottego expedition went further west, to explore the lower Omo River, and traversed mountain ranges of over 3000 m in altitude. Finally, they reached Dime – another tribal area around Mount Smith, today situated in northern part of Selamago district in Southern Nation, Nationalities and Peoples Region. After that they never went back to Badditu: In October 1896 the expedition returned to the north corner of Lake Rudolf (now Turkana) and went from there again north where Bottego was killed in a battle in Daga Roba on 17 March 1897.
In his revision of Ethiopian Leptopelis
With the lectotype series,
Since L. gramineus has never been reported from the Badditu territory, the other vernacular name of this species (found, for instance, in
The confidence that our study sites do not lie in the distribution area of L. gramineus sensu stricto and available material allow a description of a new species.
Between rivers Welmel and Yadot, Harenna Forest, Bale Zone, Oromia Region, Ethiopia (6°35'N, 39°45'E).
Holotype
: Ethiopia • ♀; Segoba glade, Harenna Forest, Bale Zone, Oromia Region; 6°35'10.5"N, 39°44'30.7"E, 1770 m a.s.l.; 1 June 2019; A. Tiutenko leg.; “Found in a tussock near calling male on a flooded forest glade during rainy season”;
Ethiopia • 1 larva; Woraba glade, Harenna Forest, Bale Zone, Oromia Region; 6°35'39.0"N, 39°45'14.6"E, 1800 m a.s.l.; 5 June 2019; A. Tiutenko leg.; “temporary puddle”;
Medium-sized (SVL of males ca. 24–29 mm, females ca. 35–40 mm) ground-dwelling and burrowing frog with robust body, relatively wide and short head, and short limbs. Terminal phalanges of toes and fingers expanded to small, but distinct discs. Only base phalanges of toes II–IV with broad web; on toe V the web extends along phalanges 1 and 2. The rest of toe phalanges with feeble fringe. Fingers are free of web or fringe. Light-brown or light olive-green from above; either no dorsal pattern, or three indistinct broad longitudinal bands (one vertebral and two dorsolateral) present that are slightly darker than the ground colour. Males with pectoral glands. Advertisement call: quiet, high-pitched singleton ‘quack’, repeated at intervals of ca. 20 seconds.
Colouration in life pale green-brown or grey-green from above. Feeble one vertebral and two dorsolateral bands, a little darker than the ground colour, bordered with small irregular tubercles. The dorsal colour transits to light green and blue at thighs and shoulders. Venter off white, feebly mottled with grey. Gular area without pattern. Black band along canthus rostralis on each side of the head extending over the nostril to the eye. Behind the eye it continues over the tympanum and above the shoulder to approximately the middle of the flank. This band is not outlined. Iris dark bronze.
Colouration in preservative : Dorsum grey. Venter pale, grey mottled. Dorsolateral pattern as in life.
The paratype series comprises 27 specimens of various ages and sexes.
Paratype 1
(Fig.
Paratype 2
(Fig.
Paratype 3
(Fig.
Adult females of three species of the Leptopelis gramineus complex in life, shown to the same scale A L. diffidens sp. nov., Harenna Forest (holotype,
Paratypes 4–27
(Fig.
Leptopelis diffidens sp. nov. shares the following traits with the rest of the members of the genus Leptopelis: The terminal phalanx of finger kinked (out of alignment with the rest of phalanges); no fingers opposing each other; digital discs on fingers present; outer metatarsal tubercle absent; singleton subarticular tubercles; tympanum visible; pupil vertically elliptic; vomerine teeth arranged in two groups between choanae.
The digital discs both, on fingers and toes, are in the new species significantly more conspicuous than L. sp. ‘Shewa’ and in the parapartic high-altitude population in Bale and Arsi Mountains – L. montanus nom. nov. (Figs
The examined males of both, L. sp. ‘Shewa’ and L. montanus nom. nov., are much larger than in the new species: SVL 27.2–40.6 mm (mean 35.6 mm). Females in these two species are even larger, with SVL 47.5–56.6 mm (mean 51.5 mm). Generally, females of L. diffidens sp. nov. are sized like males of the montane species in this species complex (see comparison photographs in Suppl. material
Pedal webbing of five members of the Leptopelis gramineus complex A L. diffidens sp. nov.,
All examined males of L. gramineus sensu stricto (three specimens, including paralectotype
A principal component analysis showed that the low-altitude geographic group from the Harenna Forest that we describe here as L. diffidens sp. nov. is separated from the rest of the groups along the first axis accumulating 44.99% of morphological variation. The other groups are partially separated along the second axis accumulating 10.85% of variation while the most divergent groups are L. gramineus sensu stricto and the highland populations from the east of the GRV (L. montanus nom. nov.). The specimens from ‘Borana/Sidamo’ population are placed between them and partially overlap with both. See this plot in Suppl. material
A canonical discriminant analysis separated the three groups L. diffidens sp. nov., L. montanus nom. nov., and L. gramineus sensu stricto with high confidence (see Fig.
Compared to all other frogs currently assigned to L. gramineus, the new species appears to have smoother skin on dorsum, with just some small singleton warts. L. diffidens sp. nov. differs from other members of the complex also in overall dorsal colouration: It is rather grey-green or pale beige while L. gramineus and other high-altitude species of this complex have more bright green or yellow-brown colouration. Furthermore, the new species has no such dorsal pattern of scattered blotches and ocelli as in the parapatric L. montanus nom. nov. The latter, in turn, usually lacks continuous black lateral bands that seems to be typical for L. diffidens sp. nov. (Fig.
From non-sympatric, but also ground-dwelling, L. bocagii the new species differs by less robust body form, smaller head, rounded canthus rostralis (versus strongly angled in L. bocagii). The digital discs in L. bocagii are similarly expanded except on toes IV and V where they seem to be larger. The inner metatarsal tubercle is similarly large and compressed. Unlike in L. bocagii that has strong vomerine teeth, they are feeble in L. diffidens sp. nov. and additionally, the colouration of L. bocagii is very different, see
Leptopelis diffidens sp. nov. is generally ground-dwelling and does not occur on trees or scrubs, although males and juveniles may occasionally climb grass. From all scansorial members of the genus distributed at the Horn of Africa, including sympatric L. ragazzii (Boulenger, 1896), it differs by much more robust body (particularly in females), comparatively small head, shorter limbs, reduced digital discs. Moreover, L. ragazzii has a flatter head with less curved snout and comparatively larger eyes. This applies also to non-sympatric L. vannutellii (Boulenger, 1898), which does not differ morphologically from L. ragazzii. Both, L. ragazzii and L. vannutellii, have small inner metatarsal tubercle and well-developed webbing on feet, extending on toes III and IV well beyond the joint of the phalanges 1 and 2. Similarly to L. ragazzii that has two clearly different colour phases, bright green and brown, occurring together. Leptopelis diffidens sp. nov. exhibits such dichromatism as well, although not that distinct: unlike in L. ragazzii, the brown phase in the new species is not purely brown, but rather olive-green or grey-brown. No plain green phase was ever recorded, but grey-green or pale green individuals occur in populations of L. vannutellii. Overall, the colour of L. vannutellii is not like that of L. diffidens sp. nov.
The non-sympatric species L. susanae and L. yaldeni Largen, 1977 have much broader digital discs, small inner metatarsal tubercles, and more extensive webbing on toes III and IV than the new species. Both are scansorial forms with more slender bodies. Leptopelis yaldeni has two colour phases which are clearly distinct, green and brown, like in L. ragazzii. Similar to L. diffidens sp. nov., L. susanae does not show such clear dichromatism, but its general colouration clearly differs very much from the new species (compare photographs and description in
Another species, L. concolor Ahl, 1929, recorded from the Juba river basin, south-east of the distribution area of L. diffidens sp. nov., is a scansorial form with a similar body size that occupies coastal savanna habitats. Similar to the new species, it has reduced pedal webbing but larger discs, particularly on toes III, IV, and V. Like other arboreal Leptopelis, it has a slender body and a large head. The colouration of L. concolor is rather constant: light brown from above, with dark interorbital bar and an indistinct reversed ‘Y’ on dorsum.
Males usually call from tussocks surrounded by water, but also from tall and dense grass at margins of pools and puddles, and occasionally from shrubs surrounding the glade. The animal sits near the roots of the grass, but not on grass and not in water. When approached, it stops calling and becomes silent for many minutes. The calls are rather weak and may be difficult to perceive in places with background chorus of other frogs, particularly of Ptychadena, or with loudly shouting birds. During a rainy season, in May–June, we did not notice any significant difference in the intensity of vocalisations between day and night. However, in the same season we observed more calling individuals and more frequent calls in the parapatric population of L. montanus nom. nov. in the Bale Mountains.
Like other Ethiopian members of this species complex (
The vocalisation of L. ragazzii partially resembles that of L. diffidens sp. nov. in containing a ‘quack’ sound of similar pitch and frequency. However, it is normally preceded by a screech, lasting for ten or more seconds, that consists of 4 or 5 notes with increasing pitch and frequency (Fig.
The 16S mitochondrial gene sequence (GenBank no. MN909554) of the specimen
As
In order to align our material to the already defined groups we have constructed a statistical parsimony network of 16S haplotypes (showed in Fig.
Network of haplotypes and a map showing the distribution of the Leptopelis gramineus species complex members: L. gramineus (filled circle), L. sp. ‘Shewa’ (empty circle), L. montanus nom. nov. (empty triangle), L. diffidens sp. nov. (filled square), L. sp. ‘Borana/Sidamo’ (empty diamond). Type localities of the described species are marked with a cross. The colours on the haplotypes chart correspond to various groups recognised by
The Latin adjective diffidens (used here in nominative singular) means diffident, anxious, shy, mistrustful. This name refers to the behaviour of this frog that appears even more cryptic and defensive than L. gramineus from other populations. The vocalisation of a male is so quiet that the animal is very difficult to locate and to spot. When approached, at a distance of only 5 m, it stops calling and remains silent until the disturber has gone away or has not moved for at least 10 minutes. As a vernacular name for this species, we propose ‘Harenna Burrowing Treefrog’.
According to our current knowledge, the new species is endemic to the Harenna Forest where it occurs at elevations of 1400–2300 m a.s.l. We observed these frogs and recorded vocalisations at the following localities, which are glades in a tall forest: Gola, Hacho, Harawa, Haro Alati, Hordoba, Kaffa Guasaa, Manyate, Megano, Segoba, Sire, Woraba, and Yagana (Fig.
Leptopelis diffidens sp. nov. seems to be confined to glades in the forest that are temporarily flooded with rain water during rainy season. These frogs do not live in areas of dense high trees, where they are replaced by L. ragazzii, typically found along streams. These two species never occur syntopically. We also did not encounter L. diffidens sp. nov. in man-made open areas in the forest, such as clearings, plantations, or settlements. However, we found two males of L. ragazzii (brown and green phase) on Aboye, a large clearing regularly used for barley plantation. Moreover, unlike L. ragazzii and the highland members of the L. gramineus complex whose larvae live in fast-flowing water, the new species uses lentic or slow-flowing waterbodies for breeding and does not occur at fast flowing streams (Fig.
Leptopelis diffidens sp. nov. shares breeding sites with Phrynobatrachus inexpectatus, Ptychadena sp., and Xenopus clivii. We observed this even in the middle of Manyate, a large village in the south of the Harenna Forest, because it has been built around a natural glade that retained its flora and hydrological characteristics.
An altitudinal and ecological barrier exists between L. diffidens sp. nov. and the not-yet-named montane grassland-dwelling L. montanus nom. nov.: L. diffidens sp. nov. does not ascend the Harenna Escarpment and is absent already in the sub-alpine heathlands, i.e., above 2500 m a.s.l. On the other hand, L. montanus nom. nov. that is distributed in the high-altitude areas, including the Sanetti Plateau, was not recorded in the areas directly adjacent to the Harenna Escarpment that have elevations around 4000 m a.s.l.; hence, no contact zone seems to exist here (Fig.
Males can be distinguished from females by a much smaller body size, less robust body, larger head, and the presence of pectoral glands.
Leptopelis diffidens sp. nov. is a ground-dwelling and semi-fossorial frog that spends most of its life hidden. In the dry season it is nocturnal, but in the rainy season we did not notice a significant difference between diurnal and nocturnal activity patterns at least in males.
Since we found a gravid female of L. diffidens sp. nov. and individuals at the last stages of metamorphosis on the same day and at the same locality, we assume that this species breeds more than once a year. Its breeding seasons appear to be out of sync with that of the parapatric high-altitude population of L. montanus nom. nov. in the Bale Mountains. We found metamorphs of L. diffidens sp. nov. (
The tadpole of L. gramineus has been described by
The population at the high-altitude lake Wonchi that Largen also mentions as an example of small-sized L. gramineus requires an additional study. Unfortunately, we did not find the specimens that Largen refers to (AAU H.548/1–2 and AAU H.718/1–4) during our visit in the
For resolving the taxonomic status of the western populations of L. gramineus, new material and DNA samples from the type locality or from nearby highlands in Ethiopian administrative zones Gamo Gofa, Keffa, Wolayita, Kenta, and Dawuro are required together with further studies of morphological differences, bioacoustics, ecology, and contact zones.
In the same paper
The new specific name montanus (living in mountains, montane) refers to the fact that this species ranges into afromontane areas up to elevation of almost 4000 m a.s.l., thus being a Leptopelis with probably the highest altitudinal distribution. The name is an adjective in nominative singular.
This key covers only species of the genus that are currently known from Ethiopia and were formally described. Species candidates are not included due to lack of knowledge about their morphological traits.
1 | Metatarsal tubercle small, length not exceeding 6% of SVL | 2 |
– | Metatarsal tubercle large, length ca. 8% of SVL | 5 |
2 | Tibia long, ca. 50% of SVL or longer | 3 |
– | Tibia moderate, shorter than 50% of SVL | 4 |
3 | Blue-green colouration at axilla and groin | vannutellii |
– | No blue-green colouration at axilla and groin | ragazzii |
4 | Dorsal pattern (dark triangle) not confluent with interorbital bar | yaldeni |
– | Dorsal pattern (mid-dorsal stripe) confluent with interorbital bar | susanae |
5 | Canthus rostralis strongly angled | bocagii |
– | Canthus rostralis rounded | 6 |
6 | Digital discs distinct, at least 30% wider than terminal phalanx | 7 |
– | Digital discs indistinct, not wider than terminal phalanx | montanus nom. nov. |
7 | Dorsal skin smooth, with small singleton warts | diffidens sp. nov. |
– | Dorsal skin rough | gramineus |
This study was part of a Harenna Forest exploration project funded by the National Geographic Society (grant WW-243S-17 to Arthur Tiutenko). We thank the NGS Committee for Research and Exploration for this support. The fieldwork in four expeditions between 2012 and 2019 was carried out with support of representatives of a number of Ethiopian governmental authorities, including administrations of the Bale Zone, of districts Goba and Delo Mena (ref. no. 777970 from 22.10.2018), and the Ethiopian Wildlife Conservation Authority (ref. no. 31/196/2011 from 16.10.2018).
We are grateful to John James Wilson and Tony Parker (
Discriminant function analysis summary
Data type: statistics
Explanation note: Supplementary data of multivariate canonical discriminant analysis.
Factor Structure Matrix
Data type: statistics
Explanation note: Supplementary data of multivariate canonical discriminant analysis.
Standardized Coefficients
Data type: statistics
Explanation note: Supplimentary data of multivariate canonical discriminant analysis.
Mean plots of morphometric characters grouped by species
Data type: statistics
Explanation note: Supplimentary data of multivariate canonical discriminant analysis.
Table S1. Morphometric measurements of specimens from three populations of Leptopelis gramineus complex
Data type: morphological
Explanation note: The table contains morphometric data from 45 specimens found in collections., including the holotype and the paratypes of L. diffidens sp. nov. For meaning of abbreviations see Material and methods in the main article text.
Table S2. Examined material
Data type: specimen list
Explanation note: List of examined 105 specimens of Leptopelis gramineus complex and 86 specimens of six other Leptopelis species occuring sympatrically or in nearby geographic areas.
Table S3. Gazetteer
Data type: geographic
Explanation note: List of geographic locations mentioned in the text of the article with their geographic coordinates and elevation.
Table S4. GenBank sequences
Data type: phylogenetic
Explanation note: This table contains accession numbers of all mtDNA (16S) sequences used for this study that are published in GenBank along with names of clades (given by the authors) that these sequences belong to.
Data table of geographic locations
Data type: geographic
Explanation note: Gazetteer in a software readable format.
Principal component analysis plot
Data type: statistics
Explanation note: Projection of the cases on the factor-plane (1 × 2).
Phylogenetic tree
Data type: phylogenetic
Explanation note: Phylogenetic tree built from 16S mtDNA sequences.
Comparison of mature female Leptopelis diffidens sp. nov. and mature male L. montanus nom. nov.
Data type: multimedia
Explanation note: Adult females of the new speices have a similar size as adult males of the parapatric species from Bale Mountains. This plate allows visual comparison of two specimens.