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Eye and webbing colouration as predictors of specific distinctness: a genetically isolated new treefrog species of the Boophis albilabris group from the Masoala peninsula, northeastern Madagascar


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Eye and webbing colouration as predictors of specific distinctness: a genetically isolated new treefrog species of the Boophis albilabris group from the Masoala peninsula, northeastern Madagascar Abstract. We describe a large and distinctive new treefrog species with blue webbing from the west coast of the Masoala peninsula in northeastern Madagascar. Boophis masoala sp. n. is morphologically similar to the other species of the Boophis albilabris group but can be distinguished from them easily by several chromatic characters of the eyes. Despite its similar morphology, it is genetically highly differentiated (10.5-13.3% pairwise p-distance in a segment of the 16S rRNA gene) from all other species in the B. albilabris group including the morphologically most similar Boophis praedictus. Both species share the blue webbing between toes and are distributed on the Masoala peninsula, but so far were not found in close sympatry. Although we recorded the new species only from the unprotected areas near the coast, we are confident that it also occurs within the adjacent Masoala National Park. We discuss the importance of eye colouration as a predictor of specific distinctness in the genus Boophis and that of webbing colouration as taxonomic characters of large treefrogs. Based on a micro-CT scan we provide a comprehensive description of the osteology of the new species, which is the first for any Boophis species, and furthermore describe its distress call which consists of three distinct sections corresponding to (1) the starting phase with closed mouth, (2) the opening of the mouth and (3) the final section with an open mouth.
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New Boophis from Masoala
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© 2018 Deutsche Gesellscha für Herpetologie und Terrarienkunde e.V. (DGHT), Mannheim, Germany
SALAMANDRA 54(3) 163–177 15 August 2018 ISSN 0036–3375
Eye and webbing colouration as predictors of specic distinctness:
a genetically isolated new treefrog species of the Boophis albilabris group
from the Masoala peninsula, northeastern Madagascar
F G, M D. S,, D P  M V
1) Zoologische Staatssammlung München (ZSM-SNSB), Münchhausenstr. 21, 81247 München, Germany
2) Division of Evolutionary Biology, Zoological Institute, Technische Universität Braunschweig, Mendelssohnstr. 4,
38106 Braunschweig, Germany
Corresponding author: F G, e-mail:
Manuscript received: 1 July 2018
Accepted: 1 August 2018 by J K
Abstract. We describe a large and distinctive new treefrog species with blue webbing from the west coast of the Masoa-
la peninsula in northeastern Madagascar. Boophis masoala sp. n. is morphologically similar to the other species of the
Boophis albilabris group but can be distinguished from them easily by several chromatic characters of the eyes. Despite its
similar morphology, it is genetically highly dierentiated (.–. pairwise p-distance in a segment of the S rRNA
gene) from all other species in the B. albilabris group including the morphologically most similar Boophis praedictus. Both
species share the blue webbing between toes and are distributed on the Masoala peninsula, but so far were not found in
close sympatry. Although we recorded the new species only from the unprotected areas near the coast, we are condent
that it also occurs within the adjacent Masoala National Park. We discuss the importance of eye colouration as a predictor
of specic distinctness in the genus Boophis and that of webbing colouration as taxonomic characters of large treefrogs.
Based on a micro-CT scan we provide a comprehensive description of the osteology of the new species, which is the rst
for any Boophis species, and furthermore describe its distress call which consists of three distinct sections corresponding
to () the starting phase with closed mouth, () the opening of the mouth and () the nal section with an open mouth.
Key words. Amphibia, Anura, Mantellidae, Boophis masoala sp. n., biogeography, distress call, osteology.
Madagascar harbours an enormous diversity of amphi-
bians. Currently  endemic species have been described
from this island (AmphibiaWeb ) and numerous addi-
tional new species and candidate species are currently un-
der study (e.g. L et al. ). Although arid western
Madagascar also harbours several frog species (e.g. G
et al. , G , R , B et al.
) the species diversity hotspots are clearly in the rain-
forests of humid eastern Madagascar, e.g. in the regions of
Andasibe and Ranomafana, where more than  species
can occur in an area of just a few square kilometres (V
et al. , B et al. , R et al. ).
Due to its relatively large expanses of remaining prima-
ry rainforest, the Masoala peninsula in northeastern Mada-
gascar is assumed to represent another hotspot of herpeto-
logical biodiversity, yet the amphibians and reptiles of this
region remain poorly studied. A number of new species de-
scribed in the last decade (e.g. A  G ,
A et al. , F et al. ) and recent dis-
coveries indicate that the percentage of still unrecognized
species might be high in this region. A substantial portion
of the peninsula’s rainforest is now protected as Masoala Na-
tional Park (K et al. , K ), but there is
also some ecotourist infrastructure in the unprotected areas
along the coast, especially in the region around Tampolo
(southeast of Maroantsetra) which we used for our study.
e treefrog genus Boophis Tschudi,  forms the sub-
family Boophinae in the family Mantellidae (V 
G ). Most Boophis species have been described
since the early s (e.g. A , G  V-
 , G et al. , , K et al. , ,
, V et al. , V et al. , , P
et al. , H et al. ). B-S
B () distinguished only  species, whereas to-
day  species are scientically named and numerous ad-
ditional candidate species have been identied (V et
al. , P et al. , H et al. ). e genus is
divided into the two subgenera Boophis and Sahona (G
V ), and the former is currently subdivided
into nine species groups (H et al. ).
F G et al.
Currently, the Boophis albilabris group includes four
nominal species: Boophis albilabris (B, ),
B.occidentalis G  V, , B. praedictus G
et al., , and B. tsilomaro V et al., . A can-
didate species from the Masoala peninsula, preliminarily
listed as Boophis sp. a. albilabris “reticulated lip” by G
 V (), turned out to be genetically identical to
B. praedictus (V et al. ). However, photographs
of Boophis praedictusfrom low elevation of Masoala in-
dicate that these populations have distinct characteristics
delimiting them from other B. praedictus populations. e
most distinctive characters are those of the eye: B. prae-
dictus from the east (from Makira, Ambatovaky and Ve-
vembe) have a red iris periphery whereas those from low
elevation in Masoala are purple, and the areas around the
pupil dier between both populations as well.
Boophis are notorious for lacking distinct morphologi-
cal dierences between closely related species and their
intraspecic variability in body colour and pattern can be
substantial (e.g. in B. picturatus, see G et al. ). In
contrast, the colouration of the eyes turned out to be spe-
cies-specic for numerous Boophis species and therefore is
a crucial character for their taxonomy (G V
, A et al. ). Similarly, the colour of the web-
bing between toes and ngers can be a reliable character to
distinguish closely related species (e.g. G et al. ).
e relevance of eye colouration and webbing colouration
for intraspecic species recognition and as prezygotic iso-
lation mechanisms are still poorly understood given that
Boophis are largely nocturnal. However, recent studies re-
vealed that frogs have the unexpected ability to see colour
at night (Y et al. ), thereby shedding new
light on these unresolved questions. In this paper we de-
scribe a new species of the Boophis albilabris group with
distinct eye colouration and blue webbing and discuss the
possible roles of these remarkable colour traits. Further-
more, we study its genetic relationships to other Boophis
species and provide a detailed description of its skeleton
and its distress call.
Materials and methods
Frogs of the Boophis albilabris group were captured most-
ly at night and located by opportunistic searching, using
torches and head lamps. Photographs were taken in life,
either in the habitat or the next morning aer capture.
Specimens were euthanized and subsequently tissue sam-
ples for genetic analyses were preserved in  ethanol.
Specimens were xed in  ethanol, preserved in 
ethanol, and deposited in the collections of the Université
d’Antananarivo, Département de Biologie Animale, An-
tananarivo, Madagascar (UADBA), and the Zoologische
Staatssammlung München, Germany (ZSM). Other in-
stitutional abbreviations used are: MRSN (Museo Regio-
nale di Scienze Naturali, Torino, Italy), UMMZ (University
of Michigan, Museum of Zoology, Ann Arbor, Michigan,
USA), ZFMK (Zoologisches Forschungsmuseum Alex-
an der Koenig, Bonn, Germany). DRV, FGZC, RAX, and
ZCMV refer to eld numbers of D. R. V, F. G,
C. J. R and M. V, respectively. One speci-
men of the new species was collected, and tissue samples
of two additional, dierently coloured specimens were tak-
en (ZSM-DNA , green specimen, and ZSM-DNA
, brown specimen, both found on Masoala, around
.°S, .°E,  m a.s.l.).
Morphological measurements (in millimetres) were
taken by FG with a digital calliper to the nearest . mm.
e denition of measurements, terminology and the de-
scription scheme follow G et al. () and V et
al. (), and G  V () for eye colouration.
Webbing formulae follow B-S ().
A micro-CT scan of the skeleton of the new species
was produced following methods established in previous
work (S et al. ). Scanning was performed in a
nanotom|m cone-beam micro-CT scanner (GE Measure-
ment & Control, Wunstorf, Germany), using a tungsten
target and a . mm Cu lter. e specimen was mount-
ed on polystyrene in a closed plastic vessel and anchored
in place with small wooden struts to minimise move-
ment. A small volume of  ethanol was added to the
vessel to prevent desiccation. Scanning was performed at
kV and  µA with exposure times of  ms for a to-
tal of  minutes ( images). Scans were reconstruct-
ed in datos|x reconstruct (GE Measurement & Control),
and visualised and rened in VG Studio Max . (Volume
Graphics GmbH, Heidelberg, Germany). DICOM images
stacks of the scans and rotational videos were deposited
in MorphoSource at
jectDetail/Show/project_id/. Osteological terminology
follows T (, ).
Distress calls were recorded as a video with a Lumix
DMC-FT digital camera with built-in microphone, saved
in MTS format, converted to wav format and analysed us-
ing the soware Cool Edit version  (Syntrillium Soware
Corporation). We used the Hanning windowing function at
a resolution of  bands and a range of  dB to create the
spectrogram. Call terminology follows K et al. ().
For the genetic analysis, we used the S rRNA align-
ment of the Boophis albilabris group from V et al.
() and added newly obtained sequences of three indi-
viduals of the new Boophis species and several individuals
of Boophis albilabris, as well as further sequences down-
loaded from GenBank. We extracted DNA from tissue
samples preserved in  ethanol and amplied and se-
quenced a fragment of the mitochondrial S rRNA gene
using standard protocols (G et al. , V et al.
), and with primers Sar-L and Sbr-H of P
et al. (). Sequences were resolved on an ABI xl cap-
illary sequencer and aligned using MEGA (K et al.
); the alignment required only a small number of in-
dels. We carried out Maximum Likelihood phylogenet-
ic inference under a GTR+I+G model as in V et al.
(), in MEGA, testing robustness of nodes with 
bootstrap replicates. Sequence divergences were calculated
as uncorrected pairwise distances (p-distances) in MEGA.
New Boophis from Masoala
All new sequences were submitted to GenBank (accession
numbers MH–MH).
Nomenclatural acts
e electronic edition of this article conforms to the re-
quirements of the amended International Code of Zoo-
logical Nomenclature, and hence the new names contained
herein are available under that Code from the electronic
edition of this article. is published work and the nomen-
clatural acts it contains have been registered in ZooBank,
the online registration system for the ICZN. e LSID (Life
Science Identier) for this publication is: urn:lsid:zoobank.
org:pub:C-D-B--BBDF. e
elec tronic edition of this work was published in a journal
with an ISSN, and has been archived and is available from
the following digital repositories: www.salamandra-jour-,
Molecular phylogeny and genetic divergences
e three newly sequenced samples of the new species
from Masoala, representing three dierent colour morphs,
had identical S sequences. ey diered by two substi-
tutions (. p-distance) from a previously published se-
quence from the Antalaha Province originally obtained by
R et al. () and available from GenBank (spec-
imen UMMZ ; accession number AF). All
included sequences of B. albilabris are remarkably similar
to each other (.–. p-distance, Fig. ) throughout the
large distribution range of the species (Fig. ).
e inferred phylogenetic tree (Fig. ) based on a total
of  aligned nucleotides reconstructed the following re-
lationships among species of the Boophis albilabris group:
B. occidentalis and B. tsilomaro were sister species, together
forming the sister clade of B. praedictus, and the clade of
these three species was sister to B. albilabris. is poorly
supported branching pattern is congruent with a previous
Figure 1. Maximum Likelihood tree of the Boophis albilabris group, based on an alignment of 505 bp of the mitochondrial 16S rRNA
gene. Values at nodes are bootstrap support values in percent (2000 replicates). Boophis tephraeomystax (subgenus Sahona) and
B.goudoti were used as the outgroup. For each individual, as far as available, the tree gives information on sampling locality, voucher
specimen, and GenBank accession number.
F G et al.
study based on the S gene (V et al. ), but dif-
fers from those of multigene phylogenies (W et
al. , H et al. ) in which the branching pattern
(((B. occidentalis, B. tsilomaro) B. albilabris) B. praedictus)
was found.
e tree placed the new species from Masoala sister to
all remaining species of the B. albilabris group, with a re-
markably long branch indicating its extremely strong ge-
netic dierentiation. e genetic divergences of the new
species to all other species of the B. albilabris group were re-
markably high: .–. pairwise uncorrected p-distance
to B. albilabris, .–. to B. occidentalis, .–. to
B. tsilomaro and .–. to B. praedictus. A comparison
with homologous sequences of all other nominal species
of Boophis, belonging to dierent species groups, revealed
even higher genetic divergences to these. ese results in-
Figure 2. Map of Madagascar showing reliably identied locality records of the species in the Boophis albilabris group largely based
on sequences used in Fig. 1 and on unambiguously identied voucher specimens or photographic records.
New Boophis from Masoala
dicate a phylogenetically isolated position of these frogs
from coastal Masoala, and strongly suggest that they repre-
sent a new species, which will be described below.
Updated distribution of species
in the Boophis albilabris group
Compared to the distribution map presented in V et
al. () our updated map (Fig. ) provides additional lo-
calities of B. albilabris (from near Ankaramy, Betampona,
Tarzanville, Tsaratanana and Vohimana) which are con-
rmed by our own genetic sequences or others taken from
GenBank (Fig. ), and a few other records which were reli-
ably identied by photographs or voucher specimens (from
Benavony, Anjanaharibe-Sud and Tsinjoarivo). Records
of B. albilabris from eastern and northern Madagascar in-
clude Vohimana (ZSM / = FGZC ; .°S,
.°E, ca.  m a.s.l.), Tarzanville near Anosibe AnAla
(ZSM / = FGZC ; .°S, .°E,  m
a.s.l.), Tsinjoarivo (ZSM / = FGZC ; .°S,
.°E, , m a.s.l., apparently the highest elevation re-
corded for this species), Betampona (Betakonana and Sa-
haïndrana,  and  m a.s.l., see R et al. ), An-
janaharibe-Sud (several records, including ZFMK ,
see A et al. ), Tsaratanana Reserve (RAX ,
Antsahatelo Camp, .°S, .°E, ca.  m a.s.l.,
GenBank: DQ), and Andohahela (ZSM / =
FGZC , ca. .°S, .°E, ca.  m a.s.l.). Records
from the Sambirano region in the northwest include
Manongarivo Camp  (UADBA-FGMV .; .°S,
.°E,  m a.s.l.), a forest near Benavony (ZFMK ,
.°S, .°E, ca.  m a.s.l., the lowest reliable alti-
tudinal record for the species), and the Maromiandra for-
est fragment near Ankaramy (ZSM / = DRV ;
.°S, .°E,  m a.s.l.). e straight-line distance
between the Maromiandra forest fragment and the type lo-
cality of Boophis tsilomaro (given as °.’S, °.’E
in the original description, but here corrected to °.’S,
°.’E = .°S, .°E) is only km, yet no in-
dications of potential mitochondrial introgression were de-
tected. We also slightly correct the position of the locali-
ty of a juvenile Boophis praedictus (MRSN A) which
was reported by V et al. () from the west coast of
Masoala close to the type locality of Boophis masoala and
would have resulted in sympatric occurrence of B. prae-
dictus and B. masoala. However, this juvenile B. praedic-
tus used for the DNA sequencing was actually collected
in the northwest of Masoala, at a campsite locally known
as Andasin’i Governera Campsite (ca. °’S, °’E =
.°S, .°E; coordinates according to A 
G ). An apparently adult B. praedictus with typi-
cally red iris peri phery was photographed by F. A
in a nearby region, at Mont Beanjada (ca. °’S, °’E
= .°S, .°E; coordinates according to A 
G ), conrming that ‘typicalB. praedictus occur
in the north of Masoala. On the other hand, the DNA se-
quence of ‘Boophis albilabris’ published by R et al.
() is almost identical to the new species described be-
low (Fig.), strongly suggesting that it actually belongs to
this new species rather than to B. albilabris or B. praedictus.
e corresponding specimen (UMMZ ) was collect-
ed at the Ankavanana river (.°S, .°E,  m a.s.l.,
G. S pers. comm.). ese data demonstrate that
both B. praedictus, and the new species described below,
inhabit the Masoala peninsula, but so far are not known to
occur in close sympatry. Locality records of B. occidentalis
added to the map (Fig.) are Analavelona forest (based on
a photograph of Boophis albilabris’ by H. S in C-
 : ), Makay (based on A et al. :
), and Tsaranoro Valley, near Andringitra massif (D.
A pers. comm.).
Boophis masoala sp. n.
Figs 3 and 4
ZooBank LSID:FDDD-D-
Holotype: ZSM / (FGZC ), adult female, col-
lected between ‘Eco-Lodge chez Arol’ and ‘Tampolo
Lodge’, coastal Masoala peninsula, Maroantsetra district,
Analanjirofo Region, northeastern Madagascar, .°S,
.°E,  m a.s.l., on  August  by F. G, D.
P, J. F, K. G  T. G. No paratypes.
Diagnosis: Assigned to the genus Boophis based on the
presence of an intercalary element between ultimate and
penultimate phalanges of ngers and toes (veried by ex-
ternal and osteological examination), enlarged terminal
discs of ngers and toes, lateral metatarsalia separated by
webbing, absence of outer metatarsal tubercle, molecular
phylogenetic relationships (Fig. ), and overall similarity to
other Boophis species. Assigned to the Boophis albilabris
group based on the following combination of characters:
large size (snout–vent length of holotype . mm); well
developed webbing between ngers; presence of vomerine
teeth; presence of a white line along upper lip; molecular
phylogenetic relationships; and overall morphological sim-
ilarity to B. praedictus and B. albilabris.
Boophis masoala diers from all other Boophis species
including all species of the B. albilabris group by its distinc-
tive eye colouration (Fig. ). It furthermore diers from all
other Boophis species except B. praedictus by bluish (rare-
ly yellowish) webbing between ngers and toes. It diers
from most other Boophis species by the white line along
the upper lip and distinctly larger size. It mostly resembles
B.praedictus from which it diers by the colour of the iris
periphery and by iris colour. An overview of diagnostic
characters of the species in the Boophis albilabris group is
provided in Table . In addition, Boophis masoala diers
from the other species in the Boophis albilabris group by
remarkable genetic dierentiation, with pairwise S diver-
gences of .–. to all other species of the group (and
even higher divergences to all other species of Boophis).
F G et al.
Description of the holotype: Adult female (veried by dis-
section and presence of white oviducts, though without
any oocytes), snout–vent length . mm. Body moder-
ately slender; head length (. mm) slightly shorter than
width (.mm), slightly wider than body; snout rounded
in dorsal view, obtuse in lateral view, nostrils directed later-
ally, nearer to tip of snout than to eye (eye–nostril distance
.mm, nostril–snout tip distance . mm), canthus ros-
tralis moderately distinct, slightly concave in dorsal view,
loreal region slightly concave; tympanum distinct (hori-
zontal diameter . mm), rounded, tympanum diameter
 of horizontal eye diameter (. mm); supratympan-
ic fold thin, distinct; vomerine odontophores prominent,
well separated in two elongated patches, positioned pos-
teromedial to choanae; choanae medium-sized, elongated.
Tongue posteriorly bid, free (le tip removed as tissue
sample). Arms slender, with a poorly developed white der-
mal edge from elbow to the lateral base of the nger. Sub-
articular tubercles single, round; metacarpal tubercles not
recognizable; ngers broadly webbed; webbing formula
(), i(.), e(), i(.), e(), (); relative length of n-
gers <<< (nger  distinctly shorter than nger ); n-
ger discs strongly enlarged. No bony prepollex at the base
of the rst nger, nor any black keratinized nuptial pads on
the base of the inner sides of ngers –. Hindlimbs slen-
der; tibiotarsal articulation reaching nostril when hind-
limb is pressed along body; lateral metatarsalia separat-
ed by webbing; inner metatarsal tubercle small, distinct,
elongated; no outer metatarsal tubercle; toes almost fully
webbed; webbing formula (), i(), e(), i(), e(),
i(.), e(.), (); relative length of toes <<<<; toe
discs enlarged. Skin smooth on dorsal surfaces. Cloaca dis-
tinct, slightly concealed by a skin fold. Skin partly folded
on the anks, almost smooth on throat and chest, slightly
granular on belly and ventral surfaces of thighs.
Aer . years in preservative, ground colour of head
and dorsum brown, marbled with few black spots on the
posterior back and poorly delimited grey spots mostly on
head and neck. Tympanic region grey. Iris grey with the
dark pattern around the horizontal pupil still recognis-
able. Posterior iris periphery purple. A distinct and nar-
row white line along the entire upper lip and one (right) or
Figure 3. Boophis masoala sp. n.: (A) Holotype in dorsolateral and (B) ventral view. Two additional individuals were sampled and
sequenced but not collected: (C) ZSM-DNA 00289 and (D) ZSM-DNA 00290, both representing dierent colour morphs.
New Boophis from Masoala
two (le) small white spots below the eye. Dorsal surfaces
of hindlimbs with – alternating brown and grey cross-
bands, which largely extend from thighs to shanks and tar-
sus if limbs are placed in a ‘sitting position. A white lateral
line along tarsus and distal edge of outer toe. Dorsal sur-
faces of four inner toes light grey, outer toe brown-grey.
Webbing between toes purple-grey. Hidden dorsal sur-
faces of thighs uniformly magenta without spots or dots.
Forelimbs dorsally with three brown spots on the arm and
grey colour in-between. A poorly developed whitish lat-
eral line along lower arm and distal edge of outer nger.
Dorsal surface of the three inner ngers dirty white, outer
nger grey with indistinct brown spots. Webbing between
ngers grey. Flanks are marbled with strongly contrasting
grey and white. All ventral surfaces cream except the grey
Colouration in life (Fig. A, B) is generally similar to
that in preservative, but the brown colour on the back was
lighter, the grey spots were beige and the webbing and the
hidden parts of the hindlimbs were blue. e iris was yel-
low with a brown area around the pupil and a purple poste-
rior iris periphery. e ventral surfaces were largely white
(Fig. B).
Osteology of the holotype (Fig. ): Skull containing all of
the typical anuran elements and no new elements; gener-
ally well ossied.
Cranium. Cranium widest near the posterior-most ex-
tension of the maxilla, at the level of the tip of the zygo-
matic ramus of the squamosal, highest at the level of the
exoccipital-frontoparietal junction. Orbits relatively spa-
cious, snout of moderate length, over one third of total
Table 1. Diagnostic characters to distinguish the species of the Boophis albilabris group.
B. masoala sp. n. B. praedictus B. albilabris B. occidentalis B. tsilomaro
outer iris colour beige to whitish golden-yellow brown to yellow blue yellow to golden
inner iris colour pattern of brown,
almost vertical lines
as outer iris or
brown brown
iris periphery purple bright red whitish to greyish blue blue?
white band behind iris
present absent absent absent absent
green band above eye absent absent mostly present sometimes indistinct
yellow band
usually absent
tympanum moderately distinct distinct distinct usually distinct distinct
white ridge on lower
arm and elbow
less distinct in ob-
served specimens
very distinct less distinct in males,
distinct in females
distinct less distinct in
white ridge on lower
less distinct in ob-
served specimens
very distinct relatively indistinct distinct poorly developed
dorsal ground colour green or brown
with or without
green very variable: green,
brown or grey with or
without dark or light
spots or markings
bright green
(rarely brown)
green to brown
ank colour with distinct white
spots or white
marbling, no sharp
rather sharp
border between
green dorsal and
grey-white anks
with brown spots
variable not very sharp border
between green dorsal
and grey/reddish
anks; anks with or
without white spots
not very sharp
light dorsolateral stripe
from eye to midbody
absent absent absent usually distinct
relatively short,
sometimes absent
dorsal surface of upper
less pigmented at least partly
less pigmented less pigmented less pigmented?
supratympanic fold mostly distinct weak to moder-
ately distinct
distinct distinct mostly by
relatively distinct
webbing between toes
blue (rarely yellow) blue mostly yellowish reddish to purple reddish
ngertips (hand) grey-brown whitish to
variable: oen grey
or greenish
yellow to orange yellow
white line along
upper lip
distinct distinct distinct thin, oen indistinct
or absent
narrow, but distinct
dorsal colour
in ethanol
grey (n=1) purple purple or grey purple purple (with dense
black spicules in
breeding condition)
F G et al.
skull length. Braincase deepest at its posterior-most level,
narrowing anteriorly to the sphenethmoid by the upward
tendency of the parasphenoid (see below).
Neurocranium. e sphenethmoid is moderately ossi-
ed. It extends posteriorly under and the frontoparietals,
maintaining contact with them, and is ventrally in contact
with the parasphenoid. Medially it forms a septum anteri-
orly, and dorsally it is attened and acts to extend the ossi-
ed roof of the skull beyond the anterior tip of the fronto-
parietals and the neopalatine, but not reaching the nasals.
e otic capsule is rather well ossied. e prootic is fused
medially with the frontoparietal and laterally with the otic
ramus of the squamosal, ventrally in broad contact with
the parasphenoid alae. It possesses a long lateral arm that
extends along the ventral surface of the otic ramus but not
to the body of the squamosal. e exoccipital is strongly
ossied and in contact with its contralateral. It is bound to
the otic ramus of the squamosal through a poorly ossied,
probably cartilaginous shelf dorsally. ree foramina are
present in the lateral surface of the exoccipital, the lowest-
most and largest of which is the otic foramen. Ventrally a
further three foramina are present.
e septomaxilla is small and spiralled upwards coun-
ter-clockwise on the le and clockwise on the right. Its an-
terior ramus is thicker than the medial and lateral arms. It
is oriented obliquely, situated near to the lingual shelf of the
maxilla, roughly above the anterior-most extension of the
vomer. As we have warned elsewhere (S et al. )
we caution against the over-reliance on micro-CT data for
the structure of this very ne bone, unless the snout itself is
scanned specically; physical investigation may be neces-
sary to establish its detailed anatomy.
Figure 4. Osteology of the holotype of Boophis masoala sp. n. Full skeleton shown in (a) dorsal, (b) lateral, and (c) ventral view; skull
shown in (d) ventral, (e) dorsal, and (f ) lateral view; and (g) hand and (h) foot in ventral view. Scale bar indicates 5 mm. Abbrevia-
tions: angspl – angulosplenial; col – columella; cpl(s) – carpal(s); exoc – exoccipital; exoc.oc – occipital condyle of exoccipital; fpar –
frontoparietal; ice – intercalary element; max – maxilla; max.fp – pars fascialis of maxilla; mc – metacarpal; mmk – mentomeckelian;
mt – metatarsal; npl – neopalatine; pmax – premaxilla; – alary process of premaxilla; pmx.lp – lateral process of premaxilla; – medial process of premaxilla; proot – prootic; – parasphenoid alae; prsph.cp – cultriform process of parasphenoid; – anterior ramus of pterygoid; – medial ramus of pterygoid; – posterior ramus of pterygoid; qj – quadratojugal;
smax – septomaxilla; spheth – sphenethmoid; sq.or – otic ramus of squamosal; sq.vr – ventral ramus of squamosal; sq.zr – zygomatic
ramus of squamosal; tar(s) – tarsal(s); vom – vomer; vom.t – dentigerous process of vomer.
New Boophis from Masoala
e columella (stapes) is oriented perpendicular to the
snout–vent axis, and is only slightly sloped upwards. e
otic foramen is rather small, and the oval baseplate of the
columella occupies most of its area.
Dorsal investing bones. e nasal is roughly triangu-
lar, and sits well isolated from its contralateral. Its medial
area is not especially wide, and the posterolateral maxil-
lary process is rather long and tapering, extending ven-
trally as it proceeds posterolaterally, at its posterior-most
point reaching the anteroposterior level of the neopalatine,
where it is approached from ahead and below by the max-
illary pars facialis (described below), but the two are not
in contact.
e frontoparietal is fairly elaborate in shape. Anteriorly
it is broad, and it tapers posteriorly, at its narrowest (rough-
ly half of its breadth anteriorly) at its posterior-most point,
where it is in contact with the exoccipital. At the mid-level
of the otic capsule, it possesses a small dorsal process. e
posterior half of the lateral surface is in contact with the
prootic’s anterior ramus, so it lacks a ventral shelf itself. It
is in medial contact with its contralateral.
Ventral investing and palatal bones. e parasphenoid
is T-shaped. Its cultriform process is narrow, at its broadest
point (near its middle) merely one third of the width of the
frontoparietals. It broadens from the alae to the mid-point,
beyond which it tapers more strongly and becomes increas-
ingly thin and unmineralised. It does not reach the level of
the neopalatine or the vomer. e anterior half of the cul-
triform process is in dorsal contact with the sphenethmoid.
e parasphenoid alae are also narrow, narrowest medial-
ly and broadening laterally, becoming less mineralised to-
ward their ends; they are slightly posterolaterally oriented.
Anterodorsally they are contacted by the prootic, and pos-
teriorly they are contacted by the exoccipital. e poster-
omedial process of the parasphenoid is pronounced, but
does not reach the foramen magnum, being excluded by a
mineralisation between the exoccipitals.
e vomer is a large and robust element, possessing four
distinct rami: a thick anterior ramus extends towards the
lingual shelf of the maxilla; a thinner, more acuminate lat-
eral ramus is almost as long as the anterior ramus, but does
not approach the maxilla; a posterolateral triangular ramus
extends toward but does not achieve the level of the neo-
palatine; and a broadening posteromedial ramus bearing
at least ten, clearly dened teeth along its posteroventral
edge extends posteriorly to the level of the neopalatine, ly-
ing below but not in contact with this bone. It is broadly
separated from its contralateral.
e neopalatine is an arced bone that lies perpendicular
to the longitudinal body axis, running dorsomedially from
the maxilla, with which it articulates but is not in direct
contact (probably separated by cartilage), to a point just
shy of the midline of the skull; it does not approach its con-
tralateral. Around its midpoint, a strong ridge is present on
the ventral surface of this bone, and dorsally its surface is
Maxillary arcade. e maxillary arcade bears many
small teeth on the premaxilla and maxilla. e premax-
illae are separated medially, but juxtaposing the maxillae
laterally. e premaxilla bears a strong anterodorsal alary
process that is long and dorsolaterally oriented, narrow at
its proximal extent, broadening and then narrowing again
dorsally. e pars palatina bears two well-dened process-
es, of which the medial or palatine process is thin and pos-
terolaterally oriented, whereas the lateral process is thicker
and curving posterolaterally, but both are of roughly equal
e maxilla is long, with a narrow pars palatina along its
lingual margin, and a strongly developed, stark, long, tri-
angular pars facialis that approaches but does not contact
the posterolateral maxillary process of the nasal.
Suspensory apparatus. e tri-radiate pterygoid bears a
short medial ramus with a posteriorly sculpted surface, a
posterior ramus of moderate length that reaches the level
of the quadratojugal, but does not exceed it (it is however
exceeded by the articular surface of the mandible, see be-
low), and a long and strongly curved anterior ramus with a
strongly sculpted lateral surface, that articulates anteriorly
with the mandible. On the lateral surface of the medial ra-
mus, a large round are is present that appears to articulate
with the medial surface of the squamosal.
e quadratojugal appears reduced, and is not ossied
along its full length; anteriorly it contacts the posterior-
most tip of the maxilla weakly. It is robust at its posterior
junction with the squamosal, but does not have any bul-
bous processes.
e squamosal is robust, with a distinct crest along its
medial surface that articulates with the pterygoid, a short
and curving zygomatic ramus, and a long, medially orient-
ed otic ramus that comprises much of the dorsal surface of
the otic capsule.
Mandible. e mandible is quite robust and edentate.
e mentomeckelians are thin and arcuate, anteriorly in
lateral contact with the dentary. e dentary is long and
laminar, running along the lateral surface of the angulo-
splenial, and at least partly in contact with this bone.
e angulosplenial is long and arcuate, strongly laterally
sculpted where Meckel’s cartilage runs along its length. e
coronoid process is quite weak, at the level of the posterior-
most extent of the maxilla. e articulatory surface is very
long, dorsally at, and smooth, extending beyond any oth-
er element of the skull.
Hyoid. e posteromedial processes of the hyoid are
quite thin, spade-shaped bones, with their proximal edges
broadened to be spatulate. ey do not possess any strong
crests. No ossied parahyoid is present.
Postcranium. Vertebral column. e vertebral column
is diplasiocoelous with the rst seven vertebrae being pro-
coelous but the eighth presacral being apparently bicon-
cave. No vertebrae are fused. e neural arch of the atlas
(presacral I) is complete. e neural arches of presacrals
II–IV bear weak neural spines. e transverse processes of
presacrals II–IV are thick; those of presacral II are laterally
but not ventrally oriented and rather short; those of presac-
ral III are thick and oriented more ventrally; those of pre-
sacral IV are oriented more posteriorly and somewhat dor-
F G et al.
sally. e thinner transverse processes of presacrals V–VIII
are also oriented somewhat dorsally, each becoming less
posteriorly-oriented to the point of presacral VIII, which
has laterally oriented transverse processes.
e sacrum bears weakly expanded, posteriorly orient-
ed diapophyses. e leading and trailing edges are largely
straight, and are not strongly sculpted. e urostyle is long,
and possesses a moderately strong dorsal ridge along the
anterior two thirds of its length. It lacks any major orna-
mentation at its head. Its articulation with the sacrum is
Pectoral girdle. e pectoral girdle possesses a well-
ossied omosternum, clavicle, coracoid, and sternum, as
well as an unossied xiphisternum. e omosternum is
small and only very weakly bifurcated posteriorly without
any real denition to the posterior arms. Its anterior neck
broadens slightly anteriorly. It contacts but is not fused to
the clavicles.
e clavicle is straight, medially strongly fused to the
coracoid and in contact with its contralateral, lateral-
ly strongly fused with the scapula. e lateral scapula is
broadened but its exact shape is dicult to distinguish in
micro-CT scans due to the extent of mineralisation of this
e coracoid is robust, strongly ared at either end with
a rather narrow middle. e medial end is broader than the
lateral end, anteriorly attened at its fusion with the clavi-
cle, medially broadly fused to its contralateral, and posteri-
orly in weak contact with the sternum. Together the cora-
coid and clavicle form a robust D-shaped foramen.
e sternum is anteriorly ared, tapering posteriorly to
a square tip, itself as long as the prezonal and zonal ele-
ments of the girdle combined.
e scapula is long, with a short pars glenoidalis and
longer and broader pars acromialis, forming a strongly an-
gular notch in the glenoid socket. It borders and is contact-
ed by the cleithrum dorsally.
e cleithrum is thin and laminar, thickest along its an-
terior edge and fading indistinctly posteriorly. e supras-
capula is broad and poorly ossied, the greatest ossication
being along the border with the suprascapula.
Forelimb and manus. e humerus bears moderate ven-
tral and lateral cristae, and lacks a medial crista. e ven-
tral crista is continuous with the caput humeri. e radio-
ulna is fairly slender, with a distinct sulcus intermedius.
e carpus is composed of a prepollex, a centrale, Ele-
ment Y, carpal , and a large post-axial element formed by
the fused carpals –. e prepollex is half as long as the
rst metacarpal. e phalangeal formula is ---. ere
are very distinct intercalary elements between the ulti-
mate and penultimate phalanges of each digit. e distal
phalanges are long and have a small paired protuberance at
the middle of their length, beyond which they are slightly
arced and become weakly distally bilobed.
Pelvic girdle. e pelvic girdle is long. e iliac shas
pass ventral to the distal ends of the sacral diapophyses,
but do not extend beyond them, making the iliosacral ar-
ticulation type IIB sensu Emerson (). eir shas pos-
sess strong dorsal crests arising at the strong dorsal prom-
inence, and becoming lower anteriorly, disappearing just
before the articulation with the sacrum. ere is no oblique
groove. e ilia are posteriorly fused with the ischium, and
ventrally with the pubis. e pubis is ossied.
Hindlimb and pes. e femur is very weakly sigmoid.
It is shorter than the tibiobula, and bears a distinct pos-
terior crest near the pelvic articulation. e tibiobula has
a weak sulcus intermedius. e tibiale and bulare and
proximally and distally fused, but much weaker distally
than proximally. Two tarsals, T and T+T, a small cen-
trale and a small prehallux are present, articulating with
the rst through third toes. e phalangeal formula is --
--. Here too, intercalary elements are present. e dis-
tal phalanges of the toes are much the same as those of the
ngers; see above.
Variation and distribution: Since only one specimen was
collected, the variation described here is restricted to the
colouration in life based on photographs of several speci-
mens. e three dierent colour morphs observed by us
are shown in Fig. . e dorsal ground colour can vary
from green (Fig. C) to brown (Fig. D) with or without
additional spots. Additional individuals of this species
were gured on the Internet either as Boophis albilabris,
e.g. photographs by N. G (, www. and by A. H (www.alexhydepho- or as B. praedictus, e.g. photographs by D.
A ( and by A. N (https:// All these photographs share the
characteristic eye colouration of the species and all of them
show bluish webbing, except one individual with distinctly
reticulated anks, which has yellow webbing (https://nick-KPjyPvMpg, ac-
cessed on  July ). As far as data are available, all these
frogs were photographed at Masoala, suggesting that the
species might be endemic to this peninsula. As already dis-
cussed above, the DNA sequence published by R
et al. () suggests that B. masoala also occurs in the
lowlands of northern Masoala (Fig. ). e known eleva-
tional range of the species is within – m a.s.l.
Habitat and habits: e holotype was discovered at night
perching on branches ca.  meters above the ground, in
close proximity (ca.  m) to the sea shore, but not close to
any other water body. Another individual was discovered
at night in a tree, ca. – m above the ground at the edge
of the trail that leads from the shore to the EcoLodge, just
above a small stream and likewise not more than ca.  m
distance from the shore. e third individual was found in
the same area, but only ca.  m above the ground.
We did not notice any potential advertisement calls of
this species during our survey in the dry season (from –
August ). e eco-touristic region of the west coast of
Masoala (between the EcoLodge and the Tampolo Lodge)
appears to be well protected and we did not discover any
signicant traces of logging or other illegal activities in the
unprotected coastal region. However, we have seen only a
New Boophis from Masoala
small portion of the coast and did not enter the Masoala
National Park, which is well-known to be heavily aected
by illegal rosewood logging (B et al. , I
Distress call: Upon capture by hand, the holotype emitted
several loud distress calls (Fig. , Supplementary Material
). ree recorded distress calls had durations of ms,
 ms and  ms, respectively, and the intervals be-
tween two calls were  ms and  ms (n=). Each of
the three calls started with a section of increasing inten-
sity with a duration of – ms (n=) which was emit-
ted when the mouth was still closed and had a frequency
range between – Hz. is short rst section was fol-
lowed by an intensive sound peak of – ms duration
(n=), which was obviously produced by the opening of
the mouth. Aer this peak the long remaining section of
the call was emitted with the mouth opened and its in-
tensity remained relatively constant over most of the call
until its decrease at the end. e frequency range of this
section was mostly between – Hz. e three sec-
tions of the call were identied from a video (Supplemen-
tary Material , showing the second distress call of  ms
length) and are well recognizable in the audiospectrogram
(Fig. ).
Etymology: e specic epithet masoala is used as a
noun in apposition and is composed of the Malagasy
words ‘maso (meaning eye) and ‘ala’ (meaning forest),
and is usually translated as ‘eye of the forest’ (e.g. R
). In contrast to many other new Boophis species (e.g.
B.feonnyala, meaning ‘voice of the forest’) which we no-
ticed rst by their distinctive advertisement calls, we dis-
covered B. masoala by its large eyes shining many meters
in the torchlight at night, and we did not hear its advertise-
ment calls (in the dry season). In addition, its eye colour
turned out to be species-specic, allowing us to distinguish
B. masoala from all other species. e specic name fur-
thermore refers to the known distribution of the new spe-
cies, which might be endemic to the Masoala peninsula.
Available names: e problems with the identity of the
Boophis albilabris holotype were extensively discussed in
Get al. () and since then, no new data have be-
come available that would challenge its attribution to the
widespread species which occurs mostly at higher eleva-
tions of eastern Madagascar. Since the type locality of
B.albi labris (‘eastern Imerina’) is in central eastern Mada-
gascar, where B. masoala most likely does not occur, con-
specicity of B. masoala and B. albilabris can be excluded
with very high probability.
Figure 5. Distress call of the holotype of Boophis masoala sp. n., spectrogram (above) and corresponding oscillogram (below). Note
that the call started with a short section (marked by red bar) that was emitted when the mouth was closed. is section was followed
by a short intensive sound peak (marked by green arrow) obviously produced by the opening of the mouth and a long remaining
section aer this peak (marked by blue bar) emitted with an open mouth. e corresponding video of this distress call is shown in
Supplementary Material 1.
F G et al.
Boophis masoala is a distinctive new species that has an
unexpectedly high genetic distance to any other Boophis
species in the sequenced mitochondrial S gene fragment
and also diers from almost all other species by distinct
colourations of its eyes and webbing. In the following we
discuss these traits in more detail.
Species-specic eye colouration: Adults of all ve species
of the B. albilabris group can be solely and reliably dis-
tinguished based on their eye colouration in life (Table )
which appears to be rather constant within the species. In
contrast, the body colouration of B. albilabris, B. praedic-
tus and B. tsilomaro is known to vary substantially within
and among populations. ese observations conrm pre-
vious studies, which found species-specic eye coloura-
tion in frogs and especially in treefrogs (G  V
, A et al. ). Boophis and other treefrog species
appear to be largely nocturnal, especially when breeding,
making it unlikely that the colourful eyes can be used as a
prezygotic isolation mechanism. Although recent studies
revealed the ability of nocturnal colour vision in frogs (Y-
 et al. ), the colourful iris of the frog is only
visible to the human eye during the day when light inten-
sity is high and the pupil of the frog is small. If the frog eye
is illuminated at night and the frog pupil is very large, only
a small iris ring is visible. us it might be more reason-
able to assume that the iris colour is used during the day
when the frogs are usually sleeping high up in the trees. On
days of heavy rainfalls, Boophis frogs sometimes start call-
ing during the day and it is plausible that they could also
use optical signals for mate recognition under such condi-
tions. Using optical instead of acoustical signals for mate or
competitor recognition during the day could help to avoid
being captured by diurnal predators and also may explain
why amplectant couples appear already at dusk when call-
ing activity has only just started.
Species-specic webbing colouration: e recent discov-
ery of nocturnal colour vision in frogs (Y et al.
) might also shed new light on a comparable phenom-
enon, i.e. the species specic colouration of webbing be-
tween toes (and ngers) which is also known to dier be-
tween several sister species pairs in Boophis.
All three individuals of Boophis masoala encountered by
us and several photographs in the internet displayed blue
webbing (Fig. ), although a single photograph of this spe-
cies on the internet with yellow webbing suggests possi-
ble sexual dichromatism or variation in this character. Blue
webbing is rarely found in frogs, and in Madagascar it is re-
stricted to Boophis praedictus and B. masoala in the B.albi-
labris group, both occurring on the Masoala peninsula.
Outside of Madagascar, partial or complete black-bluish
webbing can also occur in a few large Asian gliding tree-
frogs in the family Rhacophoridae (i.e, the Rhacophorus
nigro palmatus clade, containing species such as R.borne-
ensis, R. helenae, R. kio, R. nigropalmatus, R. norhayatii,
R.reinwardtii, and others; O  D , M-
 et al. ). Red webbing is known from the families
Rhacophoridae (Rhacophorus malabaricus, R. pardalis,
R. dulitensis), Hylidae (Hypsiboas rutelus), Hyperolii-
dae (Hyperolius) and Mantellidae (e.g. Boophis haemato-
pus, B. picturatus, B. occidentalis). e dierent coloura-
tion of the webbing is the most distinctive character to dis-
tinguish the closely related species Rhacophorus kio and
R. reinwardtii (O  D ) as well as other
species of this complex (M et al. ), and is gen-
erally an important feature to distinguish among Rhaco-
phorus species from mainland Southeast Asia (R et
al. ). Webbing colouration is also a good character to
distinguish Boophis roseipalmatus from the closely related
B.madagascariensis (pink versus greyish webbing; G et
al. ), B. pyrrhus from B.haematopus (yellowish-brown
versus red webbing; G et al. ), and to dierentiate
several species in the B. albilabris group (Table ). ese
observations conrm that webbing colour can be a species-
specic character in treefrogs, similar to the colouration of
and around the eye, and might also be an optical signals for
mate recognition during the day (and possibly at night).
Distress calls: e genus Boophis contains more than 
species and candidate species (H et al. ) and
thousands of Boophis individuals were captured or handled
by us over the last  years, yet only very few distress calls
were heard or recorded. ese are from Boophis entingae
(V et al.  under the name B. brachychir, G et
al. ), Boophis albilabris (F et al. : suppl. Table
S, but calls not found in Fonozoo), Boophis tsilomaro (un-
der the name B. occidentalis, interpretation as distress call
uncertain, see A et al. ) and Boophis masoala
(reported herein). ese species are among the largest Boo-
phis, conrming the results of previous authors (e.g. H
 G , T  H , F et al.
) who found that distress calls in anurans are more
common in large species. e general spectral structure of
the distress calls of B. masoala is similar to those report-
ed previously (e.g. H  G , T 
H ). However, in contrast to most other anuran
distress calls which appear homogeneous in their structure
and are entirely produced with an open mouth, the distress
calls of B. masoala are unusual in consisting of three dis-
tinct sections, including () the starting phase with closed
mouth, () the opening of the mouth and () the nal sec-
tion with an open mouth. is observation indicates that
distress calls might be more variable than oen assumed.
Biogeography of the B. albilabris group: Unlike most other
Boophis species groups, the B. albilabris group inhabits a
relatively wide variety of habitats, including both rainforest
of the east as well as dry forests and even remains of gal-
lery forests in western Madagascar. It also includes poten-
tial local or regional endemics (B. tsilomaro, B. masoala)
as well as very widespread species (B. albilabris, B. prae-
dictus, B.occidentalis). Boophis albilabris is one of only few
Malagasy frogs which are widespread over most of the hu-
New Boophis from Masoala
mid areas of Madagascar, ranging over more than  km
straight-line distance from Andohahela in the southeast to
Tsaratanana and the Sambirano region in the north (but
possibly excluding the Masoala peninsula) and with an el-
evational range of ca. – m above sea level. Remark-
ably, the species is very homogenous in the mitochondrial
S gene across its huge range (Fig. ), suggesting that it
might have evolved as a local endemic and then rapidly ex-
panded its range over most of Madagascar only recently.
However, the available data do not show any signal where
its original distribution could have been and also the re-
markable variability in its dorsal body colouration is not
obviously correlated with biogeography.
Osteology: Expanding on the brief summaries for the
whole genus given originally by G () and updated
by B-S  B (), in this paper
we have presented the rst comprehensive osteological de-
scription of a particular Boophis species, and indeed the
rst such description of any member of the family Man-
tellidae. Previous work on the osteology of the mantellids
has largely focussed on the roles of single bones – espe-
cially the elements of the pectoral girdle and characteris-
tics of the manus and pes – in supraspecic systematic in-
terpretations (e.g. L , G , B-
S  B, , , B-S
, G et al. , V et al. , M et
al. ). To our knowledge, only one publication has ever
published any images or illustrations of the articulated
skeleton of a mantellid frog (G  V , x-ray
images of Boophis goudoti and Mantidactylus guttulatus).
Detailed reference literature is important as it can provide
a foundation for individuals for whom osteology is unfa-
miliar, serve as an anchor point for understanding osteo-
logical variation, be a source of taxonomic characters, and
be crucial in interpreting large- and ne-scale evolutionary
patterns within groups. Evolution of ecology within Man-
tellidae, as a single, highly-diverse, insular radiation, is an
interesting topic of study (e.g. B  M
, W et al. , W V et al.
, H et al. ), and adding osteology to our in-
vestigation of this eld will greatly augment our ability to
understand the specic changes associated with ecological
shis and speciation among mantellid frogs, perhaps even-
tually shedding light on the functional underpinnings of
the evolutionary patterns within these frogs.
We are grateful to the other members of our eld team: our guide
A M (guide of the Ecolodge), J (rang-
er of Madagascar National Park, Maroantsetra), as well as J
F, K G, and T G who were all very
helpful in the eld. We furthermore thank O F
and his team of the EcoLodge for their hospitality, P L-
 (Wildlife Conservation Society, Maroantsetra) for impor-
tant informations, local authorities in Maroantsetra for issuing
permits and for logistic support, F A (MRSN)
for providing comparative pictures of B. praedictus, and G
S  (UMMZ) and D A for providing locality
data. J K provided many helpful comments which sig-
nicantly improved the manuscript. We are grateful to the Mala-
gasy Ministère de l’Environnement, des Eaux et des Forêts (Di-
rection des Eaux et Forêts, DEF) for approving the eld research,
collecting and export of specimens (autorisation de recherche
N°//MEEF/SG/DGF/DSAP/SCB, dated  July , autori-
sation de sortie N°N-EA/MG, dated September ).
Research was carried out in the framework of established collabo-
rations with the UADBA.
A, F., K. C. W  M. V (): Correlates of
eye colour and pattern in mantellid frogs. – Salamandra, :
AW (): – University of
California, Berkeley, CA, USA, accessed  June .
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Supplementary Material 1
Video showing the distress call of the holotype of Boophis masoa-
la. Available at
... Despite the high rate of new species discovery in this genus (Vieites et al. 2009;Hutter et al. 2015;Glaw et al. 2018Glaw et al. , 2019Glaw et al. , 2021, details on the life and natural history for most of these species remain unknown. Among the handful of records of reproductive behaviour are observations of Boophis occidentalis, of the B. albilabris group, which exhibits some shared traits with Sahona . ...
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The Red-fingered Bright-eyed Frog Boophis erythrodactylus is an endemic, range-restricted arboreal frog found only in the eastern rainforests of Madagascar. We report new localities for this species, along with locality records for its sister species B. tasymena, including localities where they occur in sympatry, which has never been reported before. We also document evidence for the existence of a colour variant of B. tasymena that is identical to B. erythrodactylus in appearance. We also observed never before documented breeding behaviour in the poorly known B. erythrodactylus, where the male and female in amplexus used a submerged site in a river potentially for oviposition. We also explore why this may be a predator-avoidance strategy considering the presence of aquatic invertebrate predators active near the surface and absence of fully aquatic vertebrate predators, such as fish in headwater stream habitats. Predator-avoidance strategies are a vital component of organismal survival, particularly for amphibians with complex life cycles. Natural history observations are key to revealing such strategies and behaviour, and they form the basis of evolutionary biology and is also fundamental for conservation management.
... Several anuran studies have emphasized on the usefulness of eye colour and pattern as a character for species level identification (e.g., Duellman, 1970;Glaw & Vences, 1997;Amat, Wollenberg & Vences, 2013;Glaw et al., 2018) or study of ontogenetic colour changes (e.g., Hoffman & Blouin, 2000;Biju et al., 2013); however, the application of this trait for field identification of frogs is seldom attempted (Glaw & Vences, 1997;Stuebing & Wong, 2000). Among the~230 known frog species of the Western Ghats, genus Raorchestes is the most remarkably diverse in terms of skin colouration as well as eye colours and patterns. ...
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The genus Raorchestes is a large radiation of Old World tree frogs for which the Western Ghats in Peninsular India is the major center for origin and diversification. Extensive studies on this group during the past two decades have resolved long-standing taxonomic confusions and uncovered several new species, resulting in a four-fold increase in the number of known Raorchestes frogs from this region. Our ongoing research has revealed another five new species in the genus, formally described as Raorchestes drutaahu sp. nov., Raorchestes kakkayamensis sp. nov., Raorchestes keirasabinae sp. nov., Raorchestes sanjappai sp. nov., and Raorchestes vellikkannan sp. nov., all from the State of Kerala in southern Western Ghats. Based on new collections, we also provide insights on the taxonomic identity of three previously known taxa. Furthermore, since attempts for an up-to-date comprehensive study of this taxonomically challenging genus using multiple integrative taxonomic approaches have been lacking, here we review the systematic affinities of all known Raorchestes species and define 16 species groups based on evidence from multi-gene (2,327 bp) phylogenetic analyses, several morphological characters (including eye colouration and pattern), and acoustic parameters (temporal and spectral properties, as well as calling height). The results of our study present novel insights to facilitate a better working taxonomy for this rather speciose and morphologically conserved radiation of shrub frogs. This will further enable proper field identification, provide momentum for multi-disciplinary studies, as well as assist conservation of one of the most colourful and acoustically diverse frog groups of the Western Ghats biodiversity hotspot.
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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.
The African reedfrog taxon Alexteroon consists of only three described species with rather restricted geographical ranges. Although the assignment to a distinct genus is supported by multiple evidence, its position within the larger African hyperoliid radiation has been disputed. Available molecular data are scarce and the geographic records are few and scattered. The partially formalin fixed type series were previously not accessible to molecular analyses. This changed only very recently with the advancement of Next Generation Sequencing and ancient DNA techniques. Here we provide a reassessment of the current distribution and identity of all known species in this taxon based on (a) historical and new records, (b) morphological reanalyses of the type material and newly collected specimens from Angola and Gabon, and (c) newly established, nearly complete mitochondrial genome data from historical type and modern non-type material. We also present a molecular phylogeny (five mitochondrial loci 12S, 16S, ND1, ND2, COI, Cytb) for 78 sequences from 75 different species of Hyperolius retrieved from GenBank and 14 newly established Alexteroon sequences. We demonstrate that Alexteroon is more widely distributed than previously thought with records from northern Angola representing major southern range extensions. Results of the quantitative morphometric analyses show that the group has a rather conserved general body plan. Therefore qualitative phenotypic features observable in live specimens appear to be more useful for ad hoc species delimitation. We found Alexteroon to be nested within Hyperolius, corroborating previous findings. However, the combination of molecular data and consistent differences observed in morphology and ecology provide strong support for the distinctiveness of this evolutionary lineage within Hyperolius sensu lato. We therefore treat Alexteroon as a subgenus of Hyperolius and argue that the large and diverse genus Hyperolius is in need of revision that may result in new generic arrangements.
Aim The diversity of brood size across animal species exceeds the diversity of most other life‐history traits. In some environments, reproductive success increases with brood size, whereas in others it increases with smaller broods. The dominant hypothesis explaining such diversity predicts that selection on brood size varies along climatic gradients, creating latitudinal fecundity patterns. Another hypothesis predicts that diversity in fecundity arises among species adapted to different microhabitats within assemblages. A more recent hypothesis concerned with the consequences of these evolutionary processes in the era of anthropogenic environmental change predicts that low‐fecundity species might fail to recover from demographic collapses caused by rapid environmental alterations, making them more susceptible to extinctions. These hypotheses have been addressed predominantly in endotherms and only rarely in other taxa. Here, we address all three hypotheses in amphibians globally. Location Global. Time period Present. Major taxa studied Class Amphibia. Methods Using a dataset spanning 2,045 species from all three amphibian orders, we adopt multiple phylogenetic approaches to investigate the association between brood size and climatic, ecological and phenotypic predictors, and according to species conservation status. Results Brood size increases with latitude. This tendency is much stronger in frogs, where temperature seasonality is the dominant driver, whereas salamander fecundity increases towards regions with more constant rainfall. These relationships vary across continents but confirm seasonality as the key driver of fecundity. Ecologically, nesting sites predict brood size in frogs, but not in salamanders. Finally, we show that extinction risk increases consistently with decreasing fecundity across amphibians, whereas body size is a “by‐product” correlate of extinction, given its relationship with fecundity. Main conclusions Climatic seasonality and microhabitats are primary drivers of fecundity evolution. Our finding that low fecundity increases extinction risk reinforces the need to refocus extinction hypotheses based on a suggested role for body size.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from free of charge.
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Over the last three years, three new species of saw-browed diamond frogs (Rhombophryne serratopalpebrosa species group)—a clade of cophyline microhylid frogs native to northern and eastern Madagascar—have been described. We here review the taxonomy of these frogs based on a new multi-gene phylogeny of the group, which confirms its monophyly but is insufficiently resolved to clarify most intra-group relationships. We confirm Rhombophryne guentherpetersi (Guibé, 1974) to be a member of this group, and we re-describe it based on its type series and newly collected material; the species is characterised by small superciliary spines (overlooked in its original description), as well as large tibial glands and an unusually laterally compressed pectoral girdle. We go on to describe two new species of this group from northern Madagascar: both R. diadema sp. nov. from the Sorata Massif and R. regalis sp. nov. from several sites in the northeast of the island possess three superciliary spines, but they are characterised by several subtle morphological and osteological differences. The new species are separated from all known congeners by an uncorrected pairwise distance of at least 5.1% in a ca. 550 bp fragment of the 16S rRNA gene. In order to highlight the significance of the skeleton in the taxonomy of this group, we provide a detailed description of its generalized osteology based on volume-rendered micro-CT scans of all described members, revisiting al- ready-described skeletons of some species, and describing the skeletons of R. guentherpetersi, R. coronata, and the new taxa for the first time. Use of volume rendering, instead of surface rendering of micro-CT data, resulted in some discrepancies due to the properties of each method. We discuss these inconsistencies and their bearing on the relative value of surface and volume rendering in the taxonomist’s toolkit. We argue that, while surface models are more practical for the reader, volumes are generally a more objective representation of the data. Thus, taxonomic description work should be based on volume rendering when possible, with surface models presented as an aid to the reader.
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Anuran amphibians undergo major morphological transitions during development, but the contribution of their markedly different life-history phases to macroevolution has rarely been analysed. Here we generate testable predictions for coupling versus uncoupling of phenotypic evolution of tadpole and adult life-history phases, and for the underlying expression of genes related to morphological feature formation. We test these predictions by combining evidence from gene expression in two distantly related frogs, Xenopus laevis and Mantidactylus betsi-leanus, with patterns of morphological evolution in the entire radiation of Madagascan mantellid frogs. Genes linked to morphological structure formation are expressed in a highly phase-specific pattern, suggesting uncoupling of phenotypic evolution across life-history phases. This gene expression pattern agrees with uncoupled rates of trait evolution among life-history phases in the mantellids, which we show to have undergone an adaptive radiation. Our results validate a prevalence of uncoupling in the evolution of tadpole and adult phenotypes of frogs.
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The presence of two spectrally different kinds of rod photoreceptors in amphibians has been hypothesized to enable purely rod-based colour vision at very low light levels. The hypothesis has never been properly tested, so we performed three behavioural experiments at different light intensities with toads (Bufo) and frogs (Rana) to determine the thresholds for colour discrimination. The thresholds of toads were different in mate choice and prey-catching tasks, suggesting that the differential sensitivities of different spectral cone types as well as task-specific factors set limits for the use of colour in these behavioural contexts. In neither task was there any indication of rod-based colour discrimination. By contrast, frogs performing phototactic jumping were able to distinguish blue from green light down to the absolute visual threshold, where vision relies only on rod signals. The remarkable sensitivity of this mechanism comparing signals from the two spectrally different rod types approaches theoretical limits set by photon fluctuations and intrinsic noise. Together, the results indicate that different pathways are involved in processing colour cues depending on the ecological relevance of this information for each task. This article is part of the themed issue ?Vision in dim light?.
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A new species of bright-eyed treefrog (Mantellidae) from Madagascar, with comments on call evolution and patterns of syntopy in the Boophis ankaratra complex Abstract We describe a new species of Boophis treefrog from Ranomafana National Park in the southern central east of Madagascar. This region has remarkably high anuran diversity, and along with neighbouring sites, hosts more than 35 Boophis species. Boophis boppa sp. nov. is part of the B. ankaratra sub-clade (herein named the B. ankaratra complex), previously identified within the monophyletic B. albipunctatus species group. It occurs sympatrically with two other species of the complex (B. ankaratra and B. schuboeae). Morphological differentiation of species within the B. ankaratra clade remains elusive, but species are well characterized by distinct advertisement calls, with B. boppa having the longest note duration and inter-note intervals when compared to closely related species. Furthermore, it has moderate differentiation in mitochondrial DNA, with pairwise distances of 1.9–3.7% to all other species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given by the lack of haplotype sharing with related species for the nuclear exon DNAH-3. All examples of syntopic occurrence in this complex involve species with strongly different advertisement calls, while allopatric species have more similar calls. Such a pattern might result from adaptive call co-evolution but could also be the result of non-adaptive processes. Thorough clarification of the systematics of the B. ankaratra sub-clade is required, and we outline future directions for both bioacoustic and genetic research.
We describe two new frog species of the endemic Malagasy-Comoroan genus Boophis. One species, described as Boophis baetkei sp. n., originates from Forêt d'Ambre Special Reserve in northernmost Madagascar, whereas Boophis lilianae sp. n. was discovered near Ifanadiana and Ranomafana in the Southern Central East of the island. Both new species have very deep genetic divergences in the 16S rRNA gene that complicate the assessment of their phylogenetic affinities but are here tentatively assigned to the recently defined Boophis ulftunni species group based on phenetic similarity and preliminary results of analyses of other genes. All three species known in this group share a green dorsum with translucent shade in life, a pigmented venter and, most characteristic, pink markings in life and in preservative. Boophis lilianae sp. n. is the smallest species of Boophis known so far (SVL of adult male 18.3 mm, ovigerous female 20.0 mm). Phylogenetic relationships, distribution and threat status of the new species are discussed.
We describe a new species of the Boophis albilabris group (Anura: Mantellidae) from north-western Madagascar. Boophis tsilomaro sp. nov. is most similar to B. occidentalis from the Isalo Massif, but differs by substantial genetic differentiation, larger size, absence of a turquoise iris colour, presence of a more distinct white stripe along upper lip in life, and longer note duration and lower pulse repetition rate in advertisement calls. Due to its small known range and continuing decline in the extent and quality of its habitat we propose to classify this new species as "Critically Endangered" according to the IUCN criteria. We also describe the advertisement calls of B. occidentalis for the first time.
We investigate the molecular phylogeny of Boophis, a group of arboreal frogs from the Malagasy-Comoroan family Mantellidae. Based on newly acquired DNA sequences of five mitochondrial and five nuclear markers (7444 base pairs), we infer a phylogeny of Boophis with complete species-level taxon sampling. We reconstruct the phylogeny using Bayesian inference and maximum likelihood and estimate divergence dates for the major clades of the genus. The phylogenetic analyses together support the monophyly of the two subgenera (Sahona and Boophis), and provide strong support for most previously identified species groups, except that the B. ulftunni group is nested within the B. majori group. We also erect a new species group related to the B. mandraka group, the B. blommersae group, composed of small-sized, brown stream-breeding frogs previously included within the B. majori group. Finally, we use the resulting phylogeny to illustrate striking examples of repeated evolution of coloration and ventral transparency and address the biogeographic history and broad pattern of species diversification in the genus. Ancestral area reconstructions provide evidence that Boophis diversified within the Eastern highland forests of Madagascar, and we suggest that adaptation to these highland areas was important in their diversification.