A fantastic new species of secretive forest frog discovered from forest fragments near Andasibe, Madagascar Author Hutter, Carl R. Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045 - 7561, USA & Museum of Natural Sciences and Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA carl.hutter@gmail.com Author Andriampenomanana, Zo F. Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045 - 7561, USA & Departement de Biologie Animale, Universit d'Antananarivo, BP 906, Antananarivo 101, Madagascar Author Andrianasolo, Ginah Tsiorisoa Departement de Biologie Animale, Universit d'Antananarivo, BP 906, Antananarivo 101, Madagascar Author Cobb, Kerry A. Department of Biological Sciences & Museum of Natural History, Auburn University, Auburn, Alabama 36849, USA Author Razafindraibe, Jary H. Departement de Biologie Animale, Universit d'Antananarivo, BP 906, Antananarivo 101, Madagascar Author Abraham, Robin K. Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045 - 7561, USA Author Lambert, Shea M. Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA text Zoosystematics and Evolution 2021 2021-12-02 97 2 483 495 http://dx.doi.org/10.3897/zse.97.73630 journal article http://dx.doi.org/10.3897/zse.97.73630 1860-0743-2-483 4DC62CDBA6FB472D9E6DFDBF4E101785 A35E13FBED76532F9DAFD0A2AC6487B4 Gephyromantis marokoroko sp. nov. Holotype. KU 343230 (field number CRH 1110), an adult male collected by Carl R. Hutter, Shea M. Lambert and Zo F. Andriampenomanana collected on 5 January 2016, at Vohidrazana Forest ( 18.976°S , 48.499°E ; ca. 1150 m a.s.l.) in mid-altitude rainforest near Andasibe in Northern Central East Madagascar (Fig. 1 ). Paratypes. Adult male KU 343229 (CRH 1108), adult female UADBA-CRH 472 and adult male UADBA-CRH 1061 collected on 5 January 2016, with same collection data as holotype. Adult female KU 343218 (CRH 1397) collected on 18 January 2016, at Tavolobe ( 19.005°S , 48.461°E ; ca. 1100 m a.s.l.) by Carl R. Hutter, Shea M. Lambert, Ginah Tsiorisoa Andrianasolo and Kerry A. Cobb. Adult male UADBA-CRH 1626, Adult female UADBA-CRH 1819 collected on 6 January 2017 at Vohidrazana Forest, adult female KU 347328 (CRH 1923) collected on 14 January 2017 at Andasibe-Mantadia National Park (Belakato: 18.821°S , 48.439°E ; ca. 1050 m a.s.l.) and adult female KU 347329 (CRH 2019) collected on 21 January 2017 at Vohimana ( 18.926°S , 48.489°E ; ca. 1050 m a.s.l.), collected by Carl R. Hutter, Jary Harinarivo and Robin K. Abraham. Available names. There are no junior synonyms available that could be assigned to the new species from the subgenus Laurentomantis . Etymology. 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. Diagnosis. Gephyromantis marokoroko (Fig. 2 ) is a member of the family Mantellidae , subfamily Mantellinae , as diagnosed by Glaw and Vences (2006) . The new species can be diagnosed to the genus Gephyromantis morphologically through its granular dorsum, moderately enlarged fingertips, absence of foot webbing, bifid tongue and small femoral glands present only in males as a small number of large granules (type 2; Glaw et al. 2000 ). Within Gephyromantis , the new species can be diagnosed to the subgenus Laurentomantis through its irregular and rough granular dorsum, single subgular vocal sac in males, completely connected lateral metatarsalia, inner and outer metatarsal tubercle present and tympanum is the same size in male and female. 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. 3 ). Gephyromantis marokoroko can be distinguished from other members of Laurentomantis morphologically (Table 1 ; Fig. 5 ). The rugose and granular dorsal texture with prominent ridge elements and red mottled colouration and the larger number of eight prominent femoral gland granules per femur readily characterise this species from other Laurentomantis (Figs 3 and 5 ). The new species is easily distinguished from G. horridus ( Boettger 1880 ), G. malagasius ( Methuen and Hewitt 1913 ) and G. ranjomavo ( Glaw and Vences 2011 ) by lacking tibial glands, its larger number of femoral gland granules and its rugose and granular dorsal texture with prominent ridge elements. Furthermore, the new species is easily distinguished from G. ventrimaculatus , where G. marokoroko has eight distinct femoral gland granules on each thigh (eight irregularly-shaped femoral gland granules in G. ventrimaculatus ), by the dark grey and red dorsal colouration (light brown in G. ventrimaculatus ) and by lacking blue marbling on the ventral surfaces (Fig. 5 ). The most similar species morphologically is G. striatus ( Vences et al. 2002 ), but the new species differs from G. striatus through its larger number of femoral gland granules (8 vs. 3-6), the vertebral stripe is absent or indistinct and short (always distinct in G. striatus ), bright red eye (orange-brown in G. striatus ) and its prominent and strong ridge elements, as well as the dark grey and red colouration on the dorsum (weak ridge elements and brown and orange colouration on the dorsum in G. striatus ). Figure 5. Ex-situ dorsal-lateral, dorsal and ventral photographs of A. Male Gephyromantis marokoroko sp. nov. (holotype, KU 343230); B. Gephyromantis striatus (Marojejy, ZCMV 15140; photographs by Mark D. Scherz); and C. Gephyromantis ventrimaculatus (Ranomanfana, KU 340917). 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 1 ). Despite these similarities, the clearly pulsed notes alone distinguish the new species from all other Laurentomantis , except G. ventrimaculatus ( Angel 1935 ), which has ca. 5-6 pulses/note; however, G. ventrimaculatus differs by having the shortest call duration non-overlapping with other Laurentomantis species at 407-455 ms and a slightly faster note repetition rate of 21-24 notes/s. Temperature is not likely to be an important factor in the characteristic differences described here, as structural characters, such as clearly defined pulses, would not be affected by temperature ( Schneider 1974 ). 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). Description of the holotype. 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. 2 ), dorsal colouration is a dark grey ground colour with thick, bright red mottling distributed on the dorsum. Many of the raised ridges are dark grey with bright red edges. Lighter red stripe present short distance up the dorsum. Lateral head the same as dorsum, tympanum a lighter brown. Flanks are also dark grey, but have less bright red colouration, typically only found on ridges. Forelimbs have same colouration as dorsum, except bright red colouration is more spotted, with a few lighter red spots. Hind-limbs have same colouration as forelimbs, except with red crossbands present on the dorsal surface. A whitish annulus is present before the terminal disc on fingers and toes, fingers and toes light brown. Ventral surfaces brown, with no red present. White and light-yellow spots are present and scattered moderately along the ventre. Ventral sides of arms and hind-limbs brownish-grey, with light red spotting. Femoral glands lighter brown than surrounding limb surfaces. Single subgular vocal sac is light grey, with some light-yellow spotting down the centre. Jaw has scattered light-red spots along the lip. The pupil is black with a bright red iris, with black reticulations around the outer margin of the iris. Variation. 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. Morphometry of type series. Measurements of the holotype and paratypes are shown in Table 2 . Table 2. 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 KU 343230 KU 343229 KU 343218 UADBA CRH1061 KU 347328 KU 347329
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 - -
Bioacoustics. 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. 6A-C ) with a duration of 1095.1-1431.9 (1221.5 +/- 183.5; n = 3) ms. Each call consisted of a series of 22-28 (24 +/- 3.46; n = 72) short notes with a duration of 12-29 (19.9 +/- 4.3; n = 72) ms and an inter-note duration of 15.5-43.7 (32.3 +/- 5.6; n = 71) ms. Note rate within each call was 14.4-20.1 (17.9 +/- 3.1; n = 3) note/s. Each note was strongly pulsed, with 2-4 (3.1 +/- 0.783; n = 72) pulses per note and a pulse rate of 111.1-235.3 (156.3 +/- 25.8) pulses/s (Fig. 6D-F ). The call was strongly amplitude-modulated, beginning at a lower amplitude and increasing to the middle of the call, where the amplitude then decreased until the end of the call. The dominant frequency measured at peak amplitude of the call was 2390-2672 (2483 +/- 162; n = 3) Hz, while the dominant frequency at the peak amplitude of the note was 2250-2813 (2458 +/- 149; n = 72) Hz. For notes, the 90% bandwidth was from 1453-2297 (1942 +/- 177; n = 72) Hz to 3000-4125 (3749 +/- 290; n = 72) Hz. No harmonic frequencies were visible on the spectrogram (Fig. 6 ). Figure 6. Oscillograms and spectrograms of the call of Gephyromantis marokoroko sp. nov. (Holotype: KU 343230). A. The entire call spectrogram and B. Entire call oscillogram; C. Power spectra/frequency spectrogram of a single note; D. A close-up spectrogram of four notes and E. Corresponding oscillogram; and F. an individual note taken from the middle of the call. Phylogenetics. 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. 3 ). Uncorrected p-distances, using the 16S fragment, indicate that G. ventrimaculatus has the lowest distance to the new species, at ~ 6-9%. The combined nine marker multi-locus dataset places the new species sister to G. striatus with strong support (BS = 98; PP = 1.00) in both BI and ML analyses (Fig. 4 ). Overall, these results provide strong evidence that the species is a separately evolving lineage and strong evidence for the new species phylogenetic placement. Distribution. 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. 1 ). The new species is known from the following localities: Vohidrazana Forest ( 18.976°S , 48.499°E ), Tavalobe ( 19.005°S , 48.461°E ), Vohimana ( 18.926°S , 48.489°E ) and Andasibe-Mantadia National Park (Belakato: 18.821°S , 48.439°E ). Natural history. 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. 7 ). The species was infrequently encountered, always after moderate to heavy rain, with multiple individuals occasionally grouped in small clusters (~ 20 m2). The species' call is very quiet and irregular and is barely audible to a human observer, even within three metres of a calling individual. Individuals of the new species were often found syntopically with another Laurentomantis , G. sp. Ca13, which is a candidate species identified in Vieites et al. (2009) . Other syntopic Gephyromantis include G. eiselti , G. salegy , G. sp. aff. plicifer (not yet assessed for a candidate species number) and G. cornutus . Figure 7. In-situ photograph of Gephyromantis marokoroko sp. nov. (UADBA-CRH1629). Conservation status. 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. 1 ). Many other high elevation sites in the region have been surveyed by the authors over three field seasons. Furthermore, Vohidrazana Forest and Tavalobe face ongoing threats that result in the reduction of quality and extent of habitat. For example, slash-and-burn agriculture and forest products are frequently extracted directly from this species' habitats that are outside protected areas. Given this information, we categorise this species as "Endangered" [B1ab(iii-iv)] following IUCN Criteria ( IUCN 2001 ).