Research Article |
Corresponding author: Carl R. Hutter ( carl.hutter@gmail.com ) Academic editor: Johannes Penner
© 2021 Carl R. Hutter, Zo F. Andriampenomanana, Ginah Tsiorisoa Andrianasolo, Kerry A. Cobb, Jary H. Razafindraibe, Robin K. Abraham, Shea M. Lambert.
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:
Hutter CR, Andriampenomanana ZF, Andrianasolo GT, Cobb KA, Razafindraibe JH, Abraham RK, Lambert SM (2021) A fantastic new species of secretive forest frog discovered from forest fragments near Andasibe, Madagascar. Zoosystematics and Evolution 97(2): 483-495. https://doi.org/10.3897/zse.97.73630
|
We describe a fantastic new species of forest frog (Mantellidae: Gephyromantis: subgenus: Laurentomantis) from moderately high elevations in the vicinity of Andasibe, Madagascar. This region has been surveyed extensively and has a remarkably high anuran diversity with many undocumented species still being discovered. Surprisingly, by exploring areas around Andasibe that lacked biodiversity surveys, we discovered a spectacular and clearly morphologically distinct species, previously unknown to science, Gephyromantis marokoroko sp. nov., documented for the first time in 2015. The new species is well characterised by a very rugose and granular dorsum, dark brown skin with bright red mottling, sparse light orange to white spots on the ventre, vibrant red eyes and femoral glands present only in males that consist of eight medium-sized granules. Bioacoustically, the new species has a quiet advertisement call that differs from related species by having a moderate call duration, 2–4 strongly pulsed notes and a slow note repetition rate. Furthermore, it has substantial differentiation in mitochondrial DNA, with pairwise distances of 7–9% to all other related species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given through a combined four mitochondrial markers and four nuclear exons concatenated species tree, strongly supporting G. striatus as the sister species of the new species in both analyses. The discovery of this new species highlights the need for continued inventory work in high elevation rainforests of Madagascar, even in relatively well-studied regions.
Amphibia, Anura, Andasibe, Gephyromantis marokoroko, Mantadia, new species, taxonomy
Madagascar hosts an impressively diverse and almost entirely endemic amphibian fauna, diversified into a multitude of different habitats and micro-habitats (
Recently, Madagascar’s unique biota has been the focus of intensive field surveys (e.g.
Herein, we describe another Gephyromantis species new to science from the subgenus Laurentomantis that has not been previously identified through molecular barcoding studies. This new species is not morphologically cryptic and was immediately recognisable as a new species upon discovery in recent expeditions to remote high-elevation forests surrounding the village of Andasibe that lack biodiversity surveys. Despite the Andasibe region being one of the most well-studied areas in Madagascar (
We follow the unified concept of species (i.e. general lineage concept), which defines a species as a separately evolving lineage (
Specimens were collected at night through targeted searches of the new species’ microhabitat. Specimens were euthanised using Tricaine, fixed in ~ 10% formalin (buffered with sodium phosphate to ~ 7.0 pH) for 24 hours and then stored in 70% ethanol for long-term preservation. We deposited and examined alcohol-preserved specimens from the amphibian collections at the Biodiversity Institute of the University of Kansas (
Morphological measurements were taken by ZFA with a Mituyo digital caliper (precision 0.01 mm) rounded to 0.1 mm. Terminology and measurements largely follow Glaw et al. (2001) and we used the following: (1) snout-vent length (SVL); (2) head width at the greatest point (HW); (3) head length (= rostrum) from snout tip to posterior edge of tympanum (HL); (4) horizontal eye diameter (ED); (5) interorbital distance (IOD); (6) eye-snout tip distance (ESD); (7) eye-nostril distance (END); (8) distance from nostril-snout tip (NSD); (9) distance between nostrils (NND); (10) horizontal tympanum diameter (TD); (11) upper arm length (humerus), from the articulation of the arm with the trunk to the elbow (UAL); (12) lower arm length (= radioulna), from the humerus-radioulna articulation point (elbow) to carpal-metacarpal articulation (LAL); (13) hand length from carpal-metacarpal articulation to tip of longest finger (HAL); (14) forelimb length, sum of UAL, LAL and HAL (FORL); (15) forearm length, summed from and UAL and LAL (FARL); (16) Finger I length from outer margin of palmar tubercle to tip of Finger I (FIL); (17) Finger II length from outer margin of palmar tubercle to tip of Finger II (FIIL); (18) femur length from femur-tibia articulation (knee) to cloaca (FEML); (19) tibia length from femur-tibia articulation to heel, measured along the shank (TIBL); (20) tarsus length from heel to base of foot (TARL); (21) foot length from tarsal-metatarsal articulation to tip of longest toe (FOL); (22) length of femoral gland, horizontal across the thigh (FGL); (23) width of femoral gland (FGW); and (24) the number of femoral gland clusters on each thigh (FGC).
Following euthanasia, we extracted whole livers and left hind limb muscles and stored the tissues in 95% ethanol. We obtained new genetic data for four specimens of the new species and one specimen from five other species in Laurentomantis from the 3’ fragment of the 16S rRNA mitochondrial marker widely used for molecular comparisons and species barcoding in Mantellidae (e.g.
We aligned the new sequences with 16S sequences from
The 16S rRNA sequence data were first aligned with MAFFT v.7.3 using the RNA alignment algorithm Q-ins-I (
For Bayesian Inference (BI), we used MrBayes 3.2 (
Advertisement calls were recorded in the field with a Marantz PMD 661 MKII Field Recorder and a Sennheiser MKH 8060 shotgun microphone. The calls were recorded in WAV format with a sampling rate of 44.1 kHz/s with 16 bits/sample. Advertisement calls analysed here have been deposited on FigShare (10.6084/m9.figshare.16728994). Calling males were recorded while inside plastic collecting bags at ~ 100 cm because we could not approach them close enough to record them in the field (we did not perceive a difference between the captive and in situ advertisement calls). We measured call parameters using RavenPro 1.5 (
We chose the following relevant call variables, generally following the call-centred definitions of
Comparison of distinguishing characters used to differentiate species within Laurentomantis. Table adapted from
Character | Species | |||||
G. marokoroko sp. nov. | G. striatus | G. ventrimaculatus | G. horridus | G. malagasius | G. ranjomavo | |
Male SVL (mm) | 24.0–27.0 | 22.2–23.8 | 23.0–25.0 | 26.0–28.1 | 20.2–24.0 | 23.5–25.8 |
Female SVL (mm) | 23.9–24.6 | 23.9–26.9 | 29.1 | 35.4 | 23.2–25.7 | n/a |
Tibial gland | Absent | Absent | Absent | Present | Present | Present |
Granules in femoral glands (per femur) | 8 | 3–6 | 9 | 5–6 | 1–4 | 1 |
Dorsal skin texture | Strongly granular; strong ridge elements | Moderately granular; weak ridge elements | Strongly granular; strong ridge elements | Strongly granular; weak ridge elements | Strongly granular; weak ridge elements | Moderately granular, weak ridge elements |
Ventral skin texture | Slightly granular | Smooth | Smooth | Granular | Slightly granular | Smooth |
Red colour on hind-limbs | Absent | Absent | Absent | Absent | Present | Absent |
Dorsal colour | Dark grey with bright red marbling | Dark grey with brown and orange marbling | Dark grey with brown marbling | Dark grey with brown marbling | Brown with lighter brown marbling | Brown with yellow mottling, orange limbs |
Ventral colour | Dark-grey brown with light spotting | Dark grey-brown with few light spots | Brown with blue marbling | Dark with grey marbling | Brown with light marbling | Light brown with yellow, light spots |
Vertebral stripe | Absent or indistinct | Present | Absent | Absent or indistinct | Absent | Absent |
Advertisement call duration (ms) | 1095–1431 | 440–1266 | 407–455 | 1271–2521 | 768–1468 | n/a |
Advertisement call note repetition rate /s | 14–20 | 29–40 | 21–24 | 13 | 18–36 | n/a |
Genetic distance (from G. marokoroko) | 0.25–1.5% | 7–9% | 6–9% | 10–11% | 8–11% | 12% |
Finally, we evaluated the amount of bioacoustic differences between species following
We discovered a morphologically distinct new species belonging to the subgenus Laurentomantis from Gephyromantis in the Andasibe area of Northern Central East Madagascar (Fig.
The distribution of Gephyromantis marokoroko sp. nov. in east-central Madagascar, view from above (A.) and from a profile view (B.). The black star marker indicates the type locality at Vohidrazana Forest where the black circle “locality” markers indicate other confirmed localities for the new species. Gephyromantis marokoroko sp. nov. is also found at high elevations and, thus, is likely distributed at other high elevation sites not surveyed. Elevational and satellite imagery data acquired from the USGS Earth Explorer (http://earthexplorer.usgs.gov).
Results of phylogenetic analysis of the mitochondrial 16S rRNA barcode 3’ marker for Maximum Likelihood (ML) and Bayesian Inference (BI). Topology is a consensus tree from IQ-Tree. The support values are shown as Bootstrap on top and Posterior Probability on the bottom only for nodes that were not perfectly supported. Note that Gephyromantis marokoroko sp. nov. placement in the clade is weakly supported in both analyses.
Results of phylogenetic analyses of the concatenated alignment of five mitochondrial and four nuclear markers for Maximum Likelihood (ML) and Bayesian Inference (BI). Topology is a consensus tree from IQ-Tree. On the right, the dots represent markers that were present in blue and absent in orange for each sample. The notes marked with a circle are those that did not receive perfect support (Bootstrap = 100; Posterior Probability = 1.00) from ML and BI, with the support values as BS on top and PP on the bottom. Note that Gephyromantis marokoroko sp. nov. has strong support in both analyses for a sister relationship to G. striatus.
Adult male
There are no junior synonyms available that could be assigned to the new species from the subgenus Laurentomantis.
The specific epithet marokoroko is a Malagasy word meaning “rugose” or “rugged”. The name was chosen to describe the rugose skin texture of this species. The name is to be treated as an invariable noun in apposition.
Gephyromantis marokoroko (Fig.
Gephyromantis marokoroko is characterised by bright red eyes, prominent ridge elements on dorsum, life colouration with a dark brown ground colour with mottled red and grey, hind-limbs dark brown containing red crossbands, absence of red colouration on the sides of thighs and ventre, white spots on grey-coloured ventre and males with bulbous type 2 femoral glands with eight granules in two rows of four on each thigh. Furthermore, the new species is characterised by an advertisement call with a moderately long call duration (1095–1431 ms), 22–28 notes/call, 2–4 strong amplitude-modulated pulses per note and a dominant frequency of 2250–2812 Hz. Finally, Gephyromantis marokoroko has a large genetic distance of 6% or greater amongst related species in the 16S rRNA marker and has strongly supported reciprocal monophyly to all other species in Laurentomantis (Fig.
Gephyromantis marokoroko can be distinguished from other members of Laurentomantis morphologically (Table
Bioacoustically, the advertisement call of Gephyromantis marokoroko is similar to other species in Laurentomantis and can be distinguished from all other species in this subgenus through the following combination of continuous call characters: (1) moderately long call duration (1095–1431 ms); (2) 2–4 strongly amplitude-modulated pulses per note; and (3) a note repetition rate of 14–20 notes/s. Gephyromantis striatus, G. malagasius and G. horridus have overlapping call durations with the new species and overlapping note repetition rates, except for G. striatus, which has the fastest note repetition rate (Table
Motivation might affect number of notes emitted and, thus, call duration; however, the recording of G. ventrimaculatus is of a highly motivated male (i.e. many calls emitted in a short time) while the call of the new species was recorded from males which did not appear to be very motivated, emitting only 1–2 calls within an hour. Finally, comparisons could not be made to G. ranjomavo as calls were not available; however, the new species is clearly morphologically distinct (see above).
Fixed in 10% buffered formalin solution, preserved in 70% ethanol, in good state of preservation, except for skin loss near the anterior dorsum, with left thigh muscle removed for tissue sample. Adult male, SVL 26.0 mm. Body very slender; head longer than wide HL 33.4% of SVL; slightly wider than body, HW 33.7% of SVL; snout of moderate length, ESD 16.2% of SVL; snout rounded in dorsal and lateral view; nostrils directed laterally, slightly protuberant, nearer to snout tip than eye; ED larger than END; canthus rostralis indistinct, concave; loreal region slightly concave; single subgular vocal sac; gular glands absent. Tympanic annulus distinct and round, small, TD 64.5% of ED; supratympanic fold indistinct and irregular, tympanic membrane lighter than ground colouration. Vomerine teeth not visible on the buccal roof, present under mucosal skin; choanae small, rounded. Tongue longer than wide; ovoid in shape, posteriorly bifid. Dermal fold along lower jaw absent. Arms slender, subarticular tubercles single; outer and inner metacarpal tubercles present, indistinct. Fingers without webbing; nuptial pads absent; relative finger length 2 < 1 < 4 < 3; second finger distinctly shorter than fourth finger, only slightly shorter than finger one; finger discs distinctly enlarged, larger on third and fourth finger. Hind limbs slender; lateral metatarsalia connected; inner metatarsal tubercle distinct, outer metatarsal tubercle small, but recognisable; TIBL 55.2% of SVL; FOL 45.2% of SVL. Tibial glands absent. Toes without webbing; relative toe length 1 < 2 < 5 < 3 < 4; toe three distinctly longer and broader than toe five; toe discs distinctly enlarged. Femoral glands large, well delimited, having eight distinct clusters on each femur of almost the same size, in two rows of four. Skin coarsely granular and heavily rugose on dorsal surfaces; large and sharply elevated tubercles and ridges irregularly distributed across dorsal surfaces, with less distinct ridges on the lower back; some ridges are symmetrical, larger tubercles and short ridges present on head and anterior dorsal region. Ventral skin granular on stomach, throat and limbs.
After four years in preservative, dorsal ground colouration is a uniform dull brown including forelimbs and hind-limbs. The red colouration has faded to become light brown. Lighter coloured spots on ventral surfaces are still present.
In life (Fig.
All paratypes resemble the holotype in morphology and colouration. In life, dorsal colouration varies slightly in the amount and intensity of red present. Spotting on the ventral surfaces varies in the colouration of the spots being white, light-yellow, light-orange or light-red. The vertebral stripe varies from being absent in some individuals to indistinct in others. Females lack femoral glands and have a granular texture on the femur.
Measurements of the holotype and paratypes are shown in Table
Morphometric measurements (in mm) of the holotype and paratypes of Gephyromantis marokoroko sp. nov. Femoral Gland Clusters (FGC) shown as “left, right” count.
Type status | Specimen | |||||
Holotype | Paratype | Paratype | Paratype | Paratype | Paratype | |
Field Number | CRH 1110 | CRH 1108 | CRH 1397 | CRH 1061 | CRH 1923 | CRH 2019 |
Museum Number |
|
|
|
|
|
|
Sex | M | M | M | M | F | F |
SVL | 26.0 | 25.3 | 27.0 | 24.0 | 24.6 | 23.9 |
HW | 8.8 | 9.4 | 8.6 | 8.6 | 8.4 | 8.6 |
HL | 8.7 | 10.0 | 8.8 | 8.2 | 8.7 | 8.6 |
ED | 3.8 | 3.6 | 3.5 | 3.8 | 3.6 | 3.9 |
IOD | 2.5 | 2.7 | 2.7 | 2.5 | 2.8 | 2.5 |
ESD | 4.2 | 4.0 | 4.4 | 4.4 | 4.1 | 4.3 |
END | 3.0 | 3.5 | 3.3 | 3.1 | 2.3 | 2.9 |
NSD | 1.2 | 1.5 | 1.7 | 1.5 | 1.9 | 1.4 |
NND | 2.4 | 2.6 | 2.5 | 2.3 | 2.4 | 2.2 |
TD | 2.5 | 1.6 | 2.4 | 2.5 | 2.0 | 2.2 |
FIL | 5.1 | 14.9 | 6.3 | 6.2 | 4.9 | 5.0 |
FIIL | 6.8 | 5.7 | 8.1 | 8.2 | 6.4 | 6.6 |
FEML | 13.9 | 14.1 | 14.3 | 13.4 | 14.1 | 14.1 |
TIBL | 14.4 | 14.3 | 14.4 | 13.3 | 14.6 | 13.8 |
FOL | 11.8 | 11.3 | 12.2 | 11.2 | 12.1 | 11.3 |
TARL | 8.1 | 8.2 | 8.4 | 7.6 | 8.4 | 8.0 |
HAL | 8.4 | 8.3 | 7.9 | 8.2 | 8.3 | 7.6 |
LAL | 7.7 | 7.4 | 8.3 | 7.4 | 8.1 | 8.2 |
UAL | 6.1 | 6.2 | 6.2 | 6.0 | 6.2 | 6.0 |
FORL | 22.2 | 21.8 | 22.3 | 21.6 | 22.6 | 21.7 |
FGL | 6.4 | 5.8 | 10.1 | 5.7 | - | - |
FGW | 2.8 | 2.4 | 3.3 | 3.2 | - | - |
FGC | 8; 8 | 8; 8 | 8; 8 | 8; 8 | - | - |
We recorded three calls from two males at Vohidrazana Forest after collection at ca. 02:00 hr on 6 January 2016. Males call infrequently with extremely quiet calls from the upper surfaces of leaves up to 50 cm above the ground. The recorded male was captured and placed in a separate plastic collecting bag. Males would not call when we were within recording distance, so we placed the microphone 100 cm away from the bag near where it was captured and moved several metres away. Calls were recorded during light rain at a temperature of 20.4 °C.
The advertisement call of this species sounds like a heavily pulsed trill or ‘groan’ to the human observer, emitted irregularly. We define each groan as a call (Fig.
Oscillograms and spectrograms of the call of Gephyromantis marokoroko sp. nov. (Holotype:
The phylogenetic results support the morphological diagnosis by placing Gephyromantis marokoroko within the Laurentomantis subgenus with strong support. At the species level, G. marokoroko is monophyletic with strong support in ML and BI analyses (BS = 100, PP = 1.00; Fig.
Gephyromantis marokoroko is known from several sites in the forests in the vicinity of Andasibe, but has only been found at high elevation sites (~ 1000–1200 m a.s.l.; Fig.
Gephyromantis marokoroko is apparently locally rare and, thus far, only found within undisturbed, primary forests at highland elevations (ca. 1000–1200 m). Individuals of the species were perched on the surfaces of vegetation less than 50 cm in height (Fig.
The new species is known from Andasibe-Mantadia National Park and several other managed areas (e.g. Vohimana, the community managed Vohidrazana Forest and Tavalobe). However, as currently understood, the distribution of this species is severely fragmented and restricted to only four known high-elevation localities (~ 1000–1200 m), which are very small patches with no connectivity (Fig.
Gephyromantis (Laurentomantis) marokoroko sp. nov. is a clearly distinct species, as evidenced through morphology, bioacoustics and molecular phylogenetics. The new species can be readily distinguished from other members in Laurentomantis by its heavily rugose granular skin, vibrant red eyes, bright red body colouration and distinctive femoral glands. The call of G. marokoroko also differs from all other Laurentomantis through its moderately long call duration, clearly pulsed notes and slower note repetition rate. Phylogenetic analyses strongly support the new species as monophyletic in the 16S rRNA mitochondrial marker multi-sample dataset. Additionally, the single-sample per species dataset of nine-markers (five mitochondrial and four nuclear) and both phylogenetic analyses strongly supported G. striatus and G. marokoroko as sister species (Fig.
Gephyromantis marokoroko is a remarkable discovery that was immediately obvious as a new species in the field as its general appearance is very distinct and spectacular, with several clear morphological differences from related species. Distinctive new species are typically discovered in poorly unexplored areas and G. marokoroko was discovered in the well-explored vicinity of Andasibe. In addition, the species had never been barcoded before, eluding past herpetological surveys. This new species highlights the importance of continued fieldwork in Madagascar, as the discovery of previously undocumented new species is occurring frequently (
The discovery and conservation of these new and unique species is critically important as habitat loss continues, especially in the study area. The distribution of G. marokoroko is severely fragmented and restricted to only four locations and occurs in small habitat patches (Fig.
CRH and SML conceived the study. CRH wrote the first draft of the manuscript and the other authors provided input. ZFA collected phenotypic data. All the co-authors were involved with fieldwork, data collection and logistics.
We thank the Malagasy authorities for approving research permits (N°298/13/MEF/SG/DGF/DCB.SAP/SCBSE, N°303/14/MEEMF/SG/DGF/DAPT/SCBT, N°329/15/MEEMF/SG/DGF/DAPT/SCBT); specimens were exported under permits: N°017N-EV01/MG14, N°055N-EA02/MG15, N°041N-EA01/MG16. We also thank the organisation MICET for logistics assistance for acquiring permits, transportation and other logistics. This work was supported by the University of Kansas Graduate Studies (support to CRH); the USA National Science Foundation Graduate Research Fellowship (grant numbers 1540502, 1451148, 0907996 to CRH); and postdoctoral support for CRH from National Science Foundation grant to Jacob E. Esselstyn (grant number DEB-1754393). We are especially grateful to Centre ValBio, John Cadle and Eileen Larney for their logistical help and hospitality. We also thank various friends and colleagues for logistic, field and laboratory help: James Herrera, Russ Mittermeier, Molly Bletz, Asa Conover, Barbara Korten, Vincent Premel, Devin Edmonds, Gaga and Regis.
Appendix 1
Data type: csv