Zoosyst. Evol. 97 (2) 2021, 483-495 | DO! 10.3897/zse.97.73630

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BERLIN

A fantastic new species of secretive forest frog discovered from forest
fragments near Andasibe, Madagascar

Carl R. Hutter’’*, Zo F. Andriampenomanana', Ginah Tsiorisoa Andrianasolo°, Kerry A. Cobb’,
Jary H. Razafindraibe*, Robin K. Abraham‘, Shea M. Lambert?

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
Département de Biologie Animale, Universit d’Antananarivo, BP 906, Antananarivo 101, Madagascar

Department of Biological Sciences & Museum of Natural History, Auburn University, Auburn, Alabama 36849, USA

oO BR WDNY FP

Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA
http://zoobank.org/4DC62CDB-A6F B-472D-9E6D-F DBF 4E 101785

Corresponding author: Carl R. Hutter (carl.hutter@gmail.com)

Academic editor: Johannes Penner # Received 29 August 2021 # Accepted 18 October 2021 Published 2 December 2021

Abstract

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 rep-
etition 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.

Key Words

Amphibia, Anura, Andasibe, Gephyromantis marokoroko, Mantadia, new species, taxonomy

Introduction

Madagascar hosts an impressively diverse and almost
entirely endemic amphibian fauna, diversified into a
multitude of different habitats and micro-habitats (Glaw
and Vences 2007). Amongst the small, brown leaf litter
frogs, members of the genus Gephyromantis Methuen,
1920 are well represented in Madagascar. Most small,
brown, leaf litter frogs in Madagascar are members of

Microhylidae, but Mantellidae has some representatives
in the genus Gephyromantis Methuen, 1920. Gephyro-
mantis 1s a genus within the Malagasy-Comoran endem-
ic family Mantellidae (Glaw and Vences 2007). Within
Gephyromantis, there are 47 recognised species belong-
ing to six subgenera (Glaw and Vences 2006; Vences et
al. 2017; AmphibiaWeb 2021), which are supported by
molecular and morphological criteria (Glaw and Vences
2006; Wollenberg et al. 2011; Kaffenberger et al. 2012).

Copyright Carl R. Hutter et al. 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.

484

Recently, Madagascar’s unique biota has been the fo-
cus of intensive field surveys (e.g. Goodman and Benstead
2003; D’Cruze et al. 2009; Hutter et al. 2015; Scherz et al.
2017a), with many previously unknown species discov-
ered, typically through extensive molecular identification
of candidate species lineages (Vieites et al. 2009; Perl et al.
2014). This is true for Gephyromantis, which has several
candidate lineages identified (Scherz et al. 2017a, 2017b;
Vences et al. 2017), some of which have recently been de-
scribed (Scherz et al. 2017b, 2018). Despite these barcod-
ing efforts, entirely new species, not previously identified
from molecular barcoding, are still being discovered and
described (Scherz et al. 2017a; Scherz et al. 2018), sug-
gesting that many new species to science remain elusive.

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 dis-
covery in recent expeditions to remote high-elevation for-
ests surrounding the village of Andasibe that lack biodiver-
sity surveys. Despite the Andasibe region being one of the
most well-studied areas in Madagascar (Vieites et al. 2009),
this study shows that clearly recognisable new species that
have not been barcoded are still being discovered by recent
surveys. As a result of these discoveries, we emphasise that
continued exploration and surveys in Madagascar are need-
ed, even in relatively well-studied regions. Conservation of
small forest fragments 1s especially important, as many un-
discovered species may remain undetected.

Materials and methods
Terminology

We follow the unified concept of species (1.e. general
lineage concept), which defines a species as a separately
evolving lineage (Simpson 1961; Wiley 1978; de Queiroz
1998, 2005, 2007). We use multiple lines of evidence (i.e.
secondary criteria) in assessing species boundaries, com-
bining data from morphology, phylogenetics, bioacous-
tics and biogeography (de Queiroz 2007; Padial et al.
2010; Vences et al. 2013). This evidence is then consid-
ered equally and used as support for the hypothesis that
a given population is an independently evolving lineage
and, thus, a distinct species. Family and generic names
follow the taxonomy proposed by Glaw and Vences
(2006). Geographic regions for biogeographic analyses
are defined according to Boumans et al. (2007). Accord-
ing to this zonation, the Andasibe area is within a region
named the “Northern Central East” of Madagascar.

Specimen collection and morphological
measurement

Specimens were collected at night through targeted
searches of the new species’ microhabitat. Specimens

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Hutter, C.R. et al.: A new fantastic frog from Madagascar

were euthanised using Tricaine, fixed in ~ 10% forma-
lin (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-pre-
served specimens from the amphibian collections at
the Biodiversity Institute of the University of Kansas
(KU) and Département de Biologie Animale, Antanana-
tivo (UADBA) (Appendix I). Additional collection ac-
ronyms used herein are FAZC, ZCMV, FGZC and LR
(field number series of F. Andreone, M. Vences, F. Glaw
and L. Raharivololoniaina, respectively), FGMV (field
number series shared between M. Vences and F. Glaw)
and ZSM (Zoologische Staatssammlung Munchen, Ger-
many). All photographs were taken by CRH, unless oth-
erwise noted.

Morphological measurements were taken by ZFA
with a Mituyo digital caliper (precision 0.01 mm) round-
ed 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 great-
est 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 tym-
panum diameter (TD); (11) upper arm length (humer-
us), 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 fin-
ger (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 IT (FIIL); (18) femur length from femur-tib-
ia 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).

DNA sequencing and phylogenetics

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 compar-
isons and species barcoding in Mantellidae (e.g. Vieites
et al. 2009). The methods for DNA extraction, primers
used, PCR amplification and sequencing are described

Zoosyst. Evol. 97 (2) 2021, 483-495

in Hutter et al. (2018). Finally, additional mitochondrial
and nuclear markers from one specimen of the new spe-
cies (KU 343230) were acquired by extracting the tar-
get markers from samples sequenced using the FrogCap
Ranoidea-V1 probe-set (Hutter et al. 2021; available at:
https://github.com/chutter/FrogCap-Sequence-Capture).
Probe design, sequencing and analytical methods are de-
scribed in Hutter et al. (2021) in detail. After sequencing,
DNA data were manually edited for quality in Geneious
R9 (Biomatters 2016). Sequences were deposited in Gen-
Bank and their associated voucher specimens and acces-
sion numbers are provided in Appendix I.

We aligned the new sequences with 16S sequences
from Kaffenberger et al. (2012) to confirm the subgener-
ic relationship of the new species (tree not shown). We
next chose sequences for 16S from all Laurentomantis
and several representatives from other species in Gephy-
romantis and the distantly-related Mantella madagascar-
iensis as outgroups. In total, we supplemented these new
data with 182 published sequences of Gephyromantis
specimens from GenBank. The distantly-related Mantel-
la madagascariensis was used as an outgroup to root the
phylogeny. GenBank accession numbers and their associ-
ated specimen data are included in Appendix I.

The 16S rRNA sequence data were first aligned
with MAFFT v.7.3 using the RNA alignment algorithm
Q-ins-I (Katoh and Stanley 2013). We used Maximum
Likelihood (ML) in IQ-Tree v.1.5.5 (Nguyen et al. 2015)
to conduct phylogenetic tree reconstruction with default
options selected. We used ModelFinder (Kalyaanamoor-
thy et al. 2017) to find a best-fit partitioning scheme and
selected models of molecular evolution for each parti-
tion considering all models. We assessed support using
1000 ultrafast bootstrap replicates (Minh et al. 2013).
Strongly supported nodes are those with 95 or higher
bootstrap (BS).

For Bayesian Inference (BI), we used MrBayes 3.2
(Ronquist et al. 2012) and the best partitions and models
selected above. We used reversible jump Markov Chain
Monte Carlo to accommodate uncertainty in model se-
lection (parameter set: nst=mixed). The analysis was run
for two independent runs of 50 million generations sam-
pling every 1000 generations. Chain mixing and station-
arity were assessed by examining the standard deviation
of split frequencies and by plotting the -InL per genera-
tion using Tracer 1.5 software (Rambaut and Drummond
2007), where we discarded 25% of the generations as
burn-in. Finally, results were combined using logCom-
biner 1.10 software (Rambaut and Drummond 2007) to
obtain a 50% majority rule consensus tree and node pos-
terior probabilities. Strongly supported nodes are those
with a posterior probability (PP) of 0.95 or higher.

Bioacoustics

Advertisement calls were recorded in the field with a Ma-
rantz PMD 661 MKII Field Recorder and a Sennheiser
MKH 8060 shotgun microphone. The calls were recorded

485

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 col-
lecting 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 us-
ing RavenPro 1.5 (K. Lisa Yang Center for Conservation
Bioacoustics 2014). Frequency information was obtained
through Fast Fourier Transformation (FFT; width 1012
points). A Hanning window (512 bands) was used to cre-
ate the spectrogram. Measures are reported as the range
followed by the mean + two standard deviations from
the mean. Terminology generally follows Kohler et al.
(2017), with a call defined as the entire assemblage of
acoustic signals emitted in sequence and notes are sub-
units separated by temporally distinct segments of back-
ground noise between each note.

We chose the following relevant call variables, gener-
ally following the call-centred definitions of Kohler et al.
(2017) and Hutter et al. (2013; Table 1): (1) number of
notes per call; (2) call duration (ms); (3) call interval du-
ration (ms); (4) note duration (ms); (5) inter-note interval
duration (ms); (6) note repetition rate within call (notes/s);
(7) pulse rate (/s); (8) dominant frequency, measured at
peak amplitude (Hz); and (9) frequency bandwidth (Hz),
measured as 90% of the sound energy.

Finally, we evaluated the amount of bioacoustic dif-
ferences between species following Vieites et al. (2009).
We considered differences in general call structure (e.g.
pulsed/tonal notes, consistent note arrangements, am-
plitude envelope shape; Ryan and Rand 1990) and such
temporal variables that are putatively less influenced by
temperature, body size and behaviour (e.g. note duration,
pulse rate; Gerhardt et al. 2000) to be important traits for
distinguishing species.

Results

We discovered a morphologically distinct new species
belonging to the subgenus Laurentomantis from Gephy-
romantis in the Andasibe area of Northern Central East
Madagascar (Fig. 1), present at high elevations in several
small forest fragments. The new species can be readily
identified morphologically through its rugose and granular
dorsal texture with prominent ridge elements, red dorsal
colouration on a dark brown background, bright red eyes,
the relatively large number of eight granules within each
femoral gland and absence (or indistinction) of vertebral
stripe (Fig. 2). Finally, comparisons of the uncorrected
raw genetic distances give a minimum distance of 6—9%
with G. ventrimaculatus in the mitochondrial marker 16S
rRNA and greater distances with other species in Lauren-
tomantis (Fig. 3). Phylogenetically, the new species posi-
tion is poorly supported in 16S rRNA, but strongly sup-
ported sister to G. striatus in the combined nine marker
mitochondrial and nuclear phylogeny (BS = 98; PP = 1.00;

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486

Fig. 4). Furthermore, the genetic distances between G.
striatus and the new species are 7—9%. The new species
also has an advertisement call similar to that of other Lau-
rentomantis, but can be distinguished through the combi-
nation of a moderate call duration, differing note structure
with 2-4 clearly defined pulses and slower note repetition
rate when compared to related species. Given the strong
evidence, we describe the new species as follows:

Gephyromantis marokoroko sp. nov.
http://zoobank.org/3A22A655-D3B9-4C69-BF6F-C8F 1E9595D84
Common English name: The Rugose Forest Frog.

Common Malagasy name: Ny sahon’ala marokoroko.

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.1.) in mid-altitude rainforest near Andasibe in North-
ern Central East Madagascar (Fig. 1).

Paratypes. Adult male KU 343229 (CRH 1108), adult
female VADBA-CRH 472 and adult male VADBA-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.

Legend

Type Locality
Other Locality
Village

1 @@ ) a

« Protected Area

Hutter, C.R. et al.: A new fantastic frog from Madagascar

Lambert, Ginah Tsiorisoa Andrianasolo and Kerry A.
Cobb. Adult male UADBA-CRH 1626, Adult female
UADBA-CRH 1819 collected on 6 January 2017 at Vo-
hidrazana Forest, adult female KU 347328 (CRH 1923)
collected on 14 January 2017 at Andasibe-Mantadia Na-
tional Park (Belakato: 18.821°S, 48.439°E; ca. 1050 m
a.s.1.) and adult female KU 347329 (CRH 2019) collected
on 21 January 2017 at Vohimana (18.926°S, 48.489°E;
ca. 1050 maz.s.1.), collected by Carl R. Hutter, Jary Hari-
narivo and Robin K. Abraham.

Available names. There are no junior synonyms avail-
able that could be assigned to the new species from the
subgenus Laurentomantis.

Etymology. The specific epithet marokoroko is a Mal-
agasy 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 Mantelli-
nae, 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). With-
in Gephyromantis, the new species can be diagnosed to

< Belakato

4 Vohimana

q Vohidrazana Forest
<q Tavalobe

= es > ameters = seas
2025000. 10000 =

Tse Ww OOOL

Figure 1. 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).

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Zoosyst. Evol. 97 (2) 2021, 483-495

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 1s characterised by bright
red eyes, prominent ridge elements on dorsum, life co-
louration with a dark brown ground colour with mottled
red and grey, hind-limbs dark brown containing red cross-
bands, 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 1s 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 coloura-
tion 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 spe-
cies 1s easily distinguished from G. horridus (Boettger

A

487

1880), G. malagasius (Methuen and Hewitt 1913) and
G. ranjomavo (Glaw and Vences 2011) by lacking tib-
ial glands, its larger number of femoral gland granules
and its rugose and granular dorsal texture with prom-
inent 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 gran-
ules in G. ventrimaculatus), by the dark grey and red dor-
sal 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 gran-
ules (8 vs. 3-6), the vertebral stripe is absent or indis-
tinct 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 co-
louration on the dorsum (weak ridge elements and brown
and orange colouration on the dorsum in G. striatus).
Bioacoustically, the advertisement call of Gephyro-
mantis marokoroko is similar to other species in Lauren-
tomantis and can be distinguished from all other species
in this subgenus through the following combination of
continuous call characters: (1) moderately long call dura-
tion (1095-1431 ms); (2) 24 strongly amplitude-modu-
lated 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

scale: ~10 mm

Figure 2. Ex-situ dorsal-lateral, dorsal and ventral photographs of A. Male Gephyromantis marokoroko sp. nov. (holotype, KU

343230) and B. Female (paratype, KU 343218) in life.

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488

Hutter, C.R. et al.: A new fantastic frog from Madagascar

Gephyromantis silvanus

Gephyromantis rivicola

78/
1.00

70/
0.99

0.94

57/
0.53

64/
0.93

20/
0.51

72/
0.97

68/
0.77

0.05 substitutions / site
Figure 3. 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.

Gephyromantis plicifer
Gephyromantis sculpturatus
Gephyromantis luteus
Gephyromantis granulatus
Gephyromantis webbi
Gephyromanitis spinifer
Gephyromantis runewsweeki
Gephyromantis decaryi
Gephyromantis klemmeri FGZC2727
Gephyromantis klemmeri ZCMV2063
Gephyromantis klemmeri UMMZ213033
Gephyromantis lomorina ZCMV15273
Gephyromantis lomorina FGZC3714
Gephyromantis lomorina FGZC3716
Gephyromantis lomorina FGZC3734 Pie
Gephyromantis sp Ca12 UMMZ201404 ae
Gephyromantis horridus UADBA10002 -~
Gephyromantis ranjomavo FGZC2843 == = = == === 55"
Gephyromantis marokoroko KU343218 __
Gephyromantis marokoroko KU343225 _
Gephyromantis marokoroko UADBA-CRH4
Gephyromantis marokoroko KU343230
Gephyromantis striatus FGZC 2774
Gephyromantis striatus MRSNA193€
Gephyromantis striatus UMMZ21 1685
Gephyromantis ventrimaculatus UADBA-CRH119 —
Gephyromantis ventrimaculatus ZCMV10655
Gephyromantis ventrimaculatus ZCMV4096 _
Gephyromantis ventrimaculatus ZCMV4927 _
Gephyromantis ventrimaculatus ZCMV4591 na
Gephyromantis ventrimaculatus ZCMV3362 _
Gephyromantis sp Ca14 MRSNA1991

Femoral glands

93/

Gephyromantis malagasius FAZC1

Gephyromantis malagasius ZCMV730!
Gephyromantis malagasius P$G1015

Gephyromantis malagasius PSG49

Table 1. Comparison of distinguishing characters used to differentiate species within Laurentomantis. Table adapted from Vences et
al. (2002) combined with new data. Genetic distances are uncorrected and taken from the 16S rRNA mitochondrial marker.

Character
Male SVL (mm)
Female SVL (mm)
Tibial gland

Granules in femoral
glands (per femur)

Dorsal skin texture

Ventral skin texture

Red colour on hind-
limbs

Dorsal colour

Ventral colour

Vertebral stripe

Advertisement call
duration (ms)

Advertisement call
note repetition rate /s

Genetic distance
(from G. marokoroko)

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Species
G. marokoroko sp. nov. G. striatus G. ventrimaculatus G. horridus G. malagasius G. ranjomavo
24.0-27.0 22.2-23.8 23.0-25.0 26.0-28.1 20.2-24.0 23.5-25.8
23.9-24.6 23.9-26.9 29.1 35.4 23.2-25.7 n/a
Absent Absent Absent Present Present Present
8 3-6 9 5-6 1-4 1
; Moderately ; Strongly granular; Strongly Moderately
ieee Bevan granular; weak as pola weak ridge granular; weak granular, weak
8 ridge elements 8 Mes elements ridge elements _ ridge elements
Slightly granular Smooth Smooth Granular Slightly granular Smooth
Absent Absent Absent Absent Present Absent
; ; Dark grey with Brown with Brown with yellow
Dark grey with bright red Dark grey with brown Dark grey with ;
marbling brown and orange marbling brown marbling lighter brown mottling, orange
marbling marbling limbs
Dark-grey brown with light Dark grey-brown Brown with blue Dark with grey Brown with light = Light brown with
spotting with few light spots marbling marbling marbling yellow, light spots
Absent or indistinct Present Absent Absent or indistinct Absent Absent
1095-1431 440-1266 407-455 1271-2521 768-1468 n/a
14-20 29-40 21-24 13 18-36 n/a
0.25-1.5% 7-9% 6-9% 10-11% 8-11% 12%

Zoosyst. Evol. 97 (2) 2021, 483-495

species and overlapping note repetition rates, except for
G. striatus, which has the fastest note repetition rate (Ta-
ble 1). Despite these similarities, the clearly pulsed notes
alone distinguish the new species from all other Lauren-
tomantis, except G. ventrimaculatus (Angel 1935), which
has ca. 5—6 pulses/note; however, G. ventrimaculatus dif-
fers 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. Tem-
perature is not likely to be an important factor in the char-
acteristic differences described here, as structural charac-
ters, 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. ventri-
maculatus is of a highly motivated male (i.e. many calls
emitted in a short time) while the call of the new spe-
cies 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 1s
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 ante-
rior 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 later-

Gephyromantis granulatus
Gephyromantis plicifer
Gephyromantis luteus
Gephyromantis sculpturatus
Gephyromantis spinifer
0.64 Gephyromantis decaryi
Gephyromantis runewsweeki

| 48/ Gephyromantis webbi

0.95 Gephyromantis rivicola

Gephyromantis silvanus

0.78 Gephyromantis klemmeri

61/ Gephyromantis sp Ca12
89/ au Gephyromantis ranjomavo

Gephyromantis ventrimaculatus

0.86 Gephyromantis sp Ca14
38] Gephyromantis sp Ca13

0.05 substitutions / site

Gephyromantis lomorina

Gephyromantis horridus

Gephyromantis marokoroko sp. nov.

U0 Gephyromantis striatus

Gephyromantis malagasius

489

al view; nostrils directed laterally, slightly protuberant,
nearer to snout tip than eye; ED larger than END; can-
thus 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, tym-
panic membrane lighter than ground colouration. Vom-
erine 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 recognis-
able; 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 broad-
er than toe five; toe discs distinctly enlarged. Femoral
glands large, well delimited, having eight distinct clus-
ters 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

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eb
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cs
S,
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0008000008000 08080880008
0008080000800 0888088
0000000008080 0888008
0000008008080 80800808008
0000008008000 80800800008
0008000808000 80008080008
00008808008 80800808080808008*
0000800008000 808008808008
0000000880000 0880088

Figure 4. Results of phylogenetic analyses of the concatenated alignment of five mitochondrial and four nuclear markers for Max-
imum 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.

zse.pensoft.net

490

Hutter, C.R. et al.: A new fantastic frog from Madagascar

scale: ~10 mm

Figure 5. Ex-situ dorsal-lateral, dorsal and ventral photographs of A. Male Gephyromantis marokoroko sp. nov. (holotype, KU
343230); B. Gephyromantis striatus (Marojeyy, ZCMV 15140; photographs by Mark D. Scherz); and C. Gephyromantis ventrimac-

ulatus (Ranomanfana, KU 340917).

head and anterior dorsal region. Ventral skin granular on
stomach, throat and limbs.

After four years in preservative, dorsal ground co-
louration 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, tympa-
num a lighter brown. Flanks are also dark grey, but have
less bright red colouration, typically only found on ridg-
es. Forelimbs have same colouration as dorsum, except
bright red colouration is more spotted, with a few lighter

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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 sur-
faces brown, with no red present. White and light-yellow
spots are present and scattered moderately along the ven-
tre. 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 cen-
tre. Jaw has scattered light-red spots along the lip. The
pupil is black with a bright red iris, with black reticula-
tions 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.

Zoosyst. Evol. 97 (2) 2021, 483-495

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.

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, emit-
ted 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; =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

491

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).

Phylogenetics. The phylogenetic results support the
morphological diagnosis by placing Gephyromantis ma-
rokoroko within the Laurentomantis subgenus with strong
support. At the species level, G. marokoroko 1s monophy-
letic 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 Anda-
sibe, but has only been found at high elevation sites (~

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.

Specimen

Type status 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 20.3 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
lOD 2.5 2.7 20 25 2.8 235
ESD 4.2 4.0 4.4 4.4 4.1 4.3
END 3.0 35 3.3 3.1 2.3 2.9
NSD ihe 1,5 Lee 15 1.9 1.4
NND 2.4 2.6 2.5 2.3 2.4 De?
TD 25) 1.6 2.4 25 2.0 22
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 1421 14.1
TIBL 14.4 14.3 14.4 13.3 14.6 13.8
FOL 11.8 11.3 12.2 11.2 I] 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 Pa. 7.4 5:3 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 22.6 217
FGL 6.4 5.8 10.1 oe)
FGW 2.8 2.4 oe, 3.2
FGC 8;8 8;8 8:8 8;8

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Hutter, C.R. et al.: A new fantastic frog from Madagascar

Relative
amplitude
1.0

0.10 0.15

Time (s)

0.02

Time (s)

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 spectro-
gram of four notes and E. Corresponding oscillogram; and F. an individual note taken from the middle of the call.

1000-1200 maz.s.1.; 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 1s ap-
parently 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, al-
ways after moderate to heavy rain, with multiple indi-
viduals occasionally grouped in small clusters (~ 20 m”).
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. Cal3, which is a candidate species identified in Vie-
ites 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.

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 current-
ly understood, the distribution of this species is severely
fragmented and restricted to only four known high-ele-
vation 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, Vohidra-

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zana Forest and Tavalobe face ongoing threats that result
in the reduction of quality and extent of habitat. For ex-
ample, 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” [Bl] ab(iil-1v) |
following IUCN Criteria (IUCN 2001).

Discussion

Gephyromantis (Laurentomantis) marokoroko sp. nov. 1s
a clearly distinct species, as evidenced through morphol-
ogy, bioacoustics and molecular phylogenetics. The new
Species can be readily distinguished from other members
in Laurentomantis by its heavily rugose granular skin, vi-
brant 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 repeti-
tion rate. Phylogenetic analyses strongly support the new
species as monophyletic in the 16S rRNA mitochondrial
marker multi-sample dataset. Additionally, the single-sam-
ple 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. 4). In addition, morphological similarity in the num-
ber and shape of femoral glands and the occasional pres-
ence of vertebral stripe support this relationship.
Gephyromantis marokoroko is a remarkable discovery
that was immediately obvious as a new species in the field

Zoosyst. Evol. 97 (2) 2021, 483-495

493

Figure 7. In-situ photograph of Gephyromantis marokoroko sp. nov. (UADBA-CRH1629).

as 1ts 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 dis-
covered in the well-explored vicinity of Andasibe. In addi-
tion, 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 1s
occurring frequently (Lambert et al. 2017; Scherz et al.
2017a; Scherz et al. 2018), despite the extensive past bar-
coding efforts for Malagasy frogs (e.g. Vieites et al. 2009;
Perl et al. 2014). Typically, such species have low popula-
tion densities, small geographic or elevational ranges and/
or are in areas that have not been extensively surveyed.
Many recent species descriptions of Malagasy frogs are
from previously-known candidate species and/or are
cryptic lineages that required molecular evidence to diag-
nose (e.g. Hutter et al. 2015; Scherz et al. 2017c; Vences
et al. 2017) and it is uncommon to find new species that
have not already been documented through barcoding ef-
forts (e.g. Vieites et al. 2009; Perl et al. 2014). These dis-
coveries are rare and there are only a few recent examples
of new discoveries that include G. /omorina (Scherz et
al. 2018) and Boophis masoala (Glaw et al. 2018). The
continuation of basic field inventories is, therefore, clear-
ly necessary to fully understand the patterns of species
richness and complete evolutionary histories of frogs in
Madagascar and other tropical regions.

The discovery and conservation of these new and
unique species 1s critically important as habitat loss con-

tinues, especially in the study area. The distribution of G.
marokoroko 1s severely fragmented and restricted to only
four locations and occurs in small habitat patches (Fig. 1).
While the species is protected within Andasibe-Mantadia
National Park and Vohimana Special Reserve, it has only
been found in low abundance in single, very small habitat
patches. The localities Vohidrazana Forest and Tavalobe
face ongoing threats from slash-and-burn agriculture and
forest products are frequently extracted directly from this
species’ habitat that are outside protected areas. Further-
more, climate change could exacerbate these risks reduc-
ing further the suitable habitat for this already micro-en-
demic species.

Acknowledgements

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/
MEEME/SG/DGE/DAPT/SCBT); specimens were ex-
ported under permits: N°017N-EVO1/MG14, N°O55N-
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 Stud-
ies (Support to CRH); the USA National Science Foun-
dation 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

zse.pensoft.net

494

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 Mittermei-
er, Molly Bletz, Asa Conover, Barbara Korten, Vincent
Premel, Devin Edmonds, Gaga and Regis.

Author contributions

CRH and SML conceived the study. CRH wrote the
first draft of the manuscript and the other authors pro-
vided input. ZFA collected phenotypic data. All the
co-authors were involved with fieldwork, data collec-
tion and logistics.

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