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An integrative approach to infer systematic relationships and define species groups in the shrub frog genus Raorchestes , with description of five new species from the Western Ghats, India

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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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An integrative approach to infer systematic
relationships and dene species groups in
the shrub frog genus Raorchestes, with
description of ve new species from the
Western Ghats, India
Sonali Garg
1
,
*, Robin Suyesh
1,2
,
*, Sandeep Das
3,4
, Mark A. Bee
5
and
S. D. Biju
1
1Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi, India
2Department of Environmental Studies, Sri Venkateswara College, University of Delhi, Delhi,
India
3Forest Ecology and Biodiversity Conservation Division, Kerala Forest Research Institute,
Peechi, Kerala, India
4EDGE of Existence programme, Conservation and Policy, Zoological Society of London,
London, UK
5Department of Ecology, Evolution, and Behavior, University of Minnesota - Twin Cities, St. Paul,
MN, USA
*These authors contributed equally to this work.
ABSTRACT
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 diversication.
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 ve 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
afnities of all known Raorchestes species and dene 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 eld identication, 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.
How to cite this article Garg S, Suyesh R, Das S, Bee MA, Biju SD. 2021. An integrative approach to infer systematic relationships and
dene species groups in the shrub frog genus Raorchestes, with description of ve new species from the Western Ghats, India. PeerJ 9:e10791
DOI 10.7717/peerj.10791
Submitted 22 September 2020
Accepted 26 December 2020
Published 3 March 2021
Corresponding author
S. D. Biju, sdbiju@es.du.ac.in
Academic editor
Diogo Provete
Additional Information and
Declarations can be found on
page 70
DOI 10.7717/peerj.10791
Copyright
2021 Garg et al.
Distributed under
Creative Commons CC-BY 4.0
Subjects Biodiversity, Ecology, Evolutionary Studies, Taxonomy, Zoology
Keywords Anura, Bioacoustics, Biodiversity hotspot, Diagnostic characters, Eye colour,
Integrative taxonomy, Molecular phylogeny, Morphology, Peninsular India, Rhacophoridae
INTRODUCTION
The genus Raorchestes Biju, Shouche, Dubois, Dutta, and Bossuyt, 2010 was described to
accommodate a large radiation of Asian Shrub frogs currently comprising 67 species with
distributions right from the Western Ghats in Peninsular India, up to central, eastern
and northeastern India, Nepal, Bangladesh, Myanmar, southern China, Thailand,
Malaysia, Laos, Cambodia, and Vietnam (Biju et al., 2010;AmphibiaWeb, 2020;Frost,
2020). The genus is closely related to Pseudophilautus Laurent, 1943, another radiation of
nearly 80 species chiey restricted to Sri Lanka with only three recognised members from
southern India (Biju et al., 2010;Meegaskumbura et al., 2019). Phylogenetically, the two
genera have shown a sister-group relationship (e.g., Li et al., 2009;Yu et al., 2009;Biju
et al., 2010;Pyron & Wiens, 2011;Vijayakumar et al., 2016) that has become debatable,
especially with recent descriptions of new closely related taxa (e.g., Abraham et al., 2013;Li
et al., 2013;Meegaskumbura et al., 2015,2019;Chan, Grismer & Brown, 2018). Until a few
decades ago, Raorchestes and Pseudophilautus members were included in a single genus
Philautus Gistel, 1848, which has now mostly been restricted to the Sunda Shelf and
Philippines (Biju et al., 2010;Li et al., 2013;Wostl et al., 2017;AmphibiaWeb, 2020;Frost,
2020). The presumed occurrence of genus Philautus in India based on literature prior to
Biju et al. (2010) (such as, Dubois, 1987;Bossuyt & Dubois, 2001;Delorme et al., 2006), and
the inclusion of at least six Indian taxa in Philautus thus far (Philautus dubius (Boulenger,
1882), Philautus garo (Boulenger, 1919), Philautus kempiae (Boulenger, 1919), Philautus
kempii (Annandale, 1912), Philautus microdiscus (Annandale, 1912), and Philautus
namdaphaensis Sarkar & Sanyal, 1985)(Frost, 2020) is erroneous and should be
considered uncertain until conrmed by future evidence.
The Western Ghats mountain range in Peninsular India is a major center for the origin
and diversication of Raorchestes frogs (Biju et al., 2010;Vijayakumar et al., 2016), and it is
here that the genus reaches its highest diversity (~80%) (Jiang et al., 2020) with near
absolute endemism. Until the end of twentieth century, the diversity of shrub frogs in
the Western Ghats comprised only 10 of the presently recognised Raorchestes species,
which were primarily described by colonial researchers (Jerdon, 1853;Günther, 1876;
Boulenger, 1882,1891;Annandale, 1919) followed by limited post-colonial descriptions
(Rao, 1937). It was also common belief that the Western Ghats and Sri Lanka, which
together form a single globally recognised biodiversity hotspot unit (Myers et al., 2000;
Mittermeier et al., 2004), share several known shrub frogs (Kirtisinghe, 1957;Inger et al.,
1984;Dutta & Manamendra-Arachchi, 1996). However, based on extensive eld
explorations in the Western Ghats, Biju (2001) not only doubted the occurrence of
shared species between these regions, suggesting that several confused members likely
represent undescribed taxa, but also showed the presence of an unprecedentedly high
number of previously undiscovered and new tree frog taxa within the Western Ghats.
At the same time, Bossuyt & Dubois (2001) taxonomically reviewed the genus
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 2/78
Philautus sensu lato resulting in nomenclatural stability and the transfer of several
species formerly attributed to various other genera such as Ixalus,Phyllomedusa, and
Rhacophorus (Jerdon, 1853,1870;Günther, 1876;Boulenger, 1882,1891;Annandale,
1919;Ahl, 1931). Subsequent studies also provided evidence for the fact that the shrub
frogs of the Western Ghats and Sri Lanka are endemic to the respective regions, with
considerably high undescribed diversity in both regions (Pethiyagoda & Manamendra-
Arachchi, 1998;Biju, 2001;Meegaskumbura et al., 2002;Bossuyt et al., 2004). Altogether,
what transpired was a spate of new species descriptions from yet unexplored as well as
previously explored regions across the Western Ghats, with an ever-increasing estimate of
its known shrub frog diversity (Bossuyt, 2002;Kuramoto & Joshy, 2003;Biju & Bossuyt,
2005a,2006,2009;Gururaja et al., 2007;Biju et al., 2010). The recognition of Raorchestes
(Biju et al., 2010) provided further stability to the generic allocations of Asian shrub
frogs, with frequent new discoveries holding up the genus as one of the most actively
researched anuran groups of the Western Ghats during the following decade (Zachariah
et al., 2011,2016;Seshadri, Gururaja & Aravind, 2012;Padhye et al., 2013;Vijayakumar
et al., 2014,2016;Priti et al., 2016;Gowande, Ganesh & Mirza, 2020). It is no surprise
that since the turn of the century 43 new species have been formally described, resulting in
a four-fold increase in the number of Raorchestes frogs known from this region within just
two decades.
Despite active research and frequent descriptions of new species, there has been a lack of
integrative understanding of species in this large and rather morphologically conserved
group of frogs ever-since the formal description of the genus. Although integrative
approaches have increasingly been employed to delimit and describe new species during
the past decade (e.g., Vijayakumar et al., 2014;Priti et al., 2016;Zachariah et al., 2016),
such studies largely rely on older works based on genus Philautus (e.g., Bossuyt & Dubois,
2001;Biju & Bossuyt, 2009) for comparisons with previously known taxa. Vijayakumar
et al. (2014,2016) provided comprehensive phylogenies of Western Ghats Raorchestes
frogs with lineage-based grouping of species; however, the diagnosis of these
phylogenetically identied species assemblages based on morphological, acoustic, or
behavioral characters remains unattempted.
Vocalisation in anurans has long been a subject of interest to behavioral ecologists,
evolutionary biologists, physiologists (Gerhardt & Huber, 2002;Wells, 2007), and more
recently to taxonomists as discussed elaborately in a review by Köhler et al. (2017).
Acoustic characters are known to be useful in identication and delimitation of species,
and vocalisations all the more conspicuous since they serve as premating isolation
mechanisms carrying useful evolutionary and systematic information (Ryan, 2001;Bee,
Suyesh & Biju, 2013a;Köhler et al., 2017). As taxonomic studies are increasingly
becoming integrative in nature, call characters have gained importance in Indian anuran
systematics (e.g., Kanamadi, Kadadevaru & Schneider, 2001;Kuramoto et al., 2007;
Grosjean & Dubois, 2011;Bee, Suyesh & Biju, 2013a,2013b;Garg et al., 2017,2019).
Specically in the case of genus Raorchestes, out of the 55 species known from Peninsular
India (prior to the present study), the call structure was previously known only for eleven
species, namely Raorchestes (as Philautus)tuberohumerus,Raorchestes (as Philautus)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 3/78
luteolus,R. kakachi,R. graminirupes,R. aviocularis,R. chalazodes,R. honnametti,
R. kollimalai,R. sanctisilvaticus,R. silentvalley,andR. lechiya (Kuramoto & Joshy, 2001;
Seshadri, Gururaja & Aravind, 2012;Bee, Suyesh & Biju, 2013b;Vijayakumar et al., 2014;
Priti et al., 2016;Zachariah et al., 2016;Mirza et al., 2019;Gowande, Ganesh & Mirza, 2020).
Due to lack of available acoustic data for a majority of Raorchestes species, vocalisation has
not been effectively utilized for integrative systematic studies on this taxonomically
challenging genus, and has become imperative for strengthening our understanding of
systematic relationships particularly among several morphologically cryptic species.
Several anuran studies have emphasized on the usefulness of eye colour and pattern as a
character for species level identication (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
eld identication 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.
This group is also notorious for lacking distinct morphological characters between
closely related species and high intraspecic variability in body colour and patterns in
some cases, together with the relatively small adult size of its members, which makes sole
reliance on morphology-based identication and systematic studies rather challenging
(Bossuyt & Dubois, 2001;Biju & Bossuyt, 2009;Vijayakumar et al., 2014). In this backdrop,
eye colouration as a character for species-level identication as well as interspecic and
group-level comparisons comprehensively across the genus remains overlooked, other
than a few new species descriptions (e.g., Gururaja et al., 2007;Biju & Bossuyt, 2009;
Vijayakumar et al., 2014;Zachariah et al., 2016).
In this study, we investigate the intrageneric systematic relationships among Raorchestes
frogs and characterise the phylogenetically identied 16 major species groups (largely
congruent with previous studies such as Biju & Bossuyt, 2009;Vijayakumar et al., 2014,
2016), based on morphological (including eye colouration and patterns), acoustic, and
associated behavioural traits (such as calling height). Our ongoing research has also
revealed another ve new species in this genus, all from the State of Kerala in southern
Western Ghats, which are formally described on the basis of integrative evidence.
In addition, we provide remarks on the taxonomic status of certain poorly known taxa.
New insights from this study aim to facilitate a better working taxonomy for this rather
large and taxonomically challenging genus, as well as guide future research on ecology,
biogeography, evolution, and conservation of its members.
MATERIALS AND METHODS
Field sampling
Field surveys, sampling, and call recordings were carried out primarily during the breeding
season of shrub frogs in the Western Ghats (May/JuneSeptember) between 2009 and
2019. Adults were found through opportunistic searches or by locating calling males.
Sampled individuals were photographed in life followed by euthanisation in Tricaine
methanesulphonate (MS-222). Tissue samples were extracted from the thigh muscle,
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 4/78
preserved in absolute ethanol, and eventually stored at 20 C for molecular studies.
The specimens were xed in 4% formalin and transferred to 70% ethanol for preservation.
Type specimens are deposited in the Bombay Natural History Society (BNHS),
Mumbai, and referred specimens are available at the Systematics Lab, University of Delhi
(SDBDU), India. Geographical coordinates of the sampling localities were recorded using a
Garmin 76CSx GPS with the WGS84 datum system. Distribution maps were prepared
in QGIS version 2.6.1 (http://www.qgis.org).
Fieldwork, including collection of animals in the eld, was conducted with
permissions and following guidelines from the responsible authorities in the State Forest
Departments (Field permits: Nos. WL12-1830/2009, WL10-2606/12, WL10-25421/2014,
67254/2001/WL5, D-22 (8)/Research/4543/2012-13; PCCF(WL)/E2/CR/13/201617
and WL10-43756/2015). Research received ethical approval from Department of
Environmental Studies, University of Delhi (DES/1020 dated 9 February 2015), India.
Phylogenetic study
Genomic DNA was extracted from populations sampled from the State of Kerala that were
suspected to represent undescribed species, using the Qiagen DNeasy blood and tissue kit
(Qiagen, Valencia, CA, USA). The following six gene fragments were PCR-amplied using
previously published primers: four mitochondrial16SrRNA (Simon et al., 1994),
12SrRNA + tRNA
VAL
(Richards & Moore, 1996), and Cytochrome b (Che et al., 2012); two
nuclearRhodopsin and Tyrosinase (Bossuyt & Milinkovitch, 2000). The fragments were
sequenced on both strands using the BigDye Terminator v3.1 Cycle Sequencing kit
(Applied Biosystems) on ABI 3730 automated DNA sequencer (Applied Biosystems).
Newly generated sequences were checked and assembled in ChromasPro v1.34
(Technelysium Pty Ltd., St. South Brisbane, QLD, Australia), and deposited in the National
Centre for Biotechnology Information (NCBI) GenBank under accession numbers
MW020034MW020035,MW020166MW020171 and MW023233MW023244
(Table S1).
Further taxon sampling for phylogenetic studies was carried out by retrieving previously
published DNA sequences for vouchers with maximum availability of the analysed
genes and as much as possible those representing typical exemplars of all the currently
recognised Raorchestes species (except R. thodai) available in the NCBI GenBank.
Additionally, one member (the type species, if available) from all known rhacophorid
genera and an outgroup taxon were included in the dataset (Table S1). The datasets for
different gene sequences were prepared and aligned for 94 taxa using the ClustalW tool
in MEGA 7.0 (Kumar, Stecher & Tamura, 2016). Alignments for coding DNA were
checked by comparison with amino acid sequences; non-coding fragments were manually
optimised and ambiguous sites were excluded from phylogenetic analyses. The tRNA
VAL
gene was also excluded from the 12S fragment, due to its non-availability for most of
the Genbank sequences. The resultant character set of total 2,327 basepairs was partitioned
by genes for the ve studied gene fragments and the best-t models of DNA evolution
determined individually by implementing the Akaike Information Criterion in ModelTest
3.4 (Posada & Crandall, 1998) were used for analyses.
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 5/78
Phylogenetic inferences were made under the Maximum Likelihood (ML) criteria. ML
searches were performed for 100 independent runs with GTRGAMMA model for each
gene partition along with 1,000 thorough bootstrap replicates for assessing the clade
support, using RAxML 7.3.0 (Stamatakis, 2006;Stamatakis et al., 2008) in raxmlGUI 1.1
(Silvestro & Michalak, 2012). Further, Bayesian analyses were performed in MrBayes 3.1.2
(Ronquist & Huelsenbeck, 2003) using the best determined model (GTR + I + G) for
each gene partition, with two parallel runs of four Markov chain Monte Carlo (MCMC)
chains executed for 10 million generations. Trees were sampled after every 1,000
generations and the Bayesian Posterior Probabilities (BPP) for clades were summarised
after discarding the rst 2.5 million generations as burn-in. Convergence of the parallel
runs was conrmed by split frequency standard deviations of less than 0.01 as well as
the nearing of potential scale reduction factors to 1.0 for all model parameters using Tracer
v1.3 (Rambaut et al., 2014).
Morphological study
Morphological studies were carried out to compare the populations suspected to represent
new species with all previously known Raorchestes species and available names, based on
examination of available types and other museum specimens, original descriptions, or
new topotypic material. All the Raorchestes species known from Peninsular India were
also comprehensively studied in order to identify shared morphological characters for
grouping of species. Sex and maturity were determined by the presence of secondary sexual
characters (such as nuptial pads and vocal sacs in males) or examination of gonads.
Only adult specimens were used for morphometric studies.
Measurements and associated terminologies follow Biju & Bossuyt (2009). The following
measurements were taken to the nearest 0.1 mm byusing a digital slide-caliper or a binocular
microscope with a micrometer ocular: snoutvent length (SVL), head width (HW, at the
angle of the jaws), head length (HL, from rear of mandible to tip of snout), MN
(distance from the rear of the mandible to the nostril), MFE (distance from the rear of the
mandible to the anterior orbital border), MBE (distance from the rear of the mandible to
the posterior orbital border), snout length (SL, from tip of snout to anterior orbital
border), eye length (EL, horizontal distance between bony orbital borders), inter upper
eyelid width (IUE, the shortest distance between the upper eyelids), maximum upper
eyelid width (UEW), internarial distance (IN), internal front of the eyes (IFE, shortest
distance between the anterior orbital borders), internal back of the eyes (IBE, shortest
distance between the posterior orbital borders), NS (distance from the nostril to the tip
of the snout), EN (distance from the front of the eye to the nostril), TYD (greatest
tympanum diameter), TYE (distance from the tympanum to the back of the eye),
forearm length (FAL, from exed elbow to base of outer palmar tubercle), hand length
(HAL, from base of outer palmar tubercle to tip of third nger), FL
IIV
(nger length),
thigh length (TL, from vent to knee), shank length (SHL, from knee to heel), foot
length (FOL, from base of inner metatarsal tubercle to tip of fourth toe), total foot length
(TFOL, from heel to tip of fourth toe), FD (maximum disc width of nger), width of
nger (FW, measured at the base of the disc), TD (maximum disc width of toe), width of
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 6/78
toe (TW, measured at the base of the disc). Digit number is represented by roman
numerals IV in subscript. All measurements and photographs were taken for the
right side of the specimen, except when a character was damaged, in which case the
measurement was taken on the left side. All measurements provided in the taxonomy
section are in millimetres.
For the convenience of discussion, Raorchestes species of the Western Ghats are
grouped based on their body size as small (male SVL 17.025.0 mm), medium (male
SVL 25.145.0 mm) and large (male SVL 45.165.0 mm). Terminologies for the snout
shape follow Heyer et al. (1990). The webbing formulae follow Savage & Heyer (1967) as
modied by Myers & Duellman (1982) and followed by Biju et al. (2014), and the degree of
webbing relative to subarticular tubercles is described by numbering the tubercles 13,
starting from the base. Further, the webbing is categorised as basal (slightly above or
beyond the basal subarticular tubercles on all toes), small (webbing on toe IV beyond the
third subarticular tubercle but below the second subarticular tubercle on either side),
medium (webbing on toe IV beyond the second subarticular tubercle but below the
rst subarticular tubercle on either side), and large (webbing on toe IV extending beyond
the rst subarticular tubercle on either side), following Garg & Biju (2017). Finger and toe
disc morphology types follow Biju et al. (2011).
Using the statistical software Statistica v7.1 (StatSoft Inc.), Principal Component
Analysis (PCA) and Discriminant Function Analysis (DFA) were performed to specically
assess the degree of morphological differentiation among the six recognised members
of the Raorchestes bombayensis group. PCA was performed using 20 morphometric
parameters taken from adult males. Factor scores of the rst two Principal Components
(PC) were observed on a scatterplot. Furthermore, sets of 20 predictor variables were
generated from the PCA and all the factor scores were used as input variables for
performing a DFA, in order to also determine the classication success of the
studied samples.
Eye colouration and pattern
We made a dedicated effort to photograph and document the colour and pattern of the
eyes of all the Raorchestes species in the Western Ghats from various angles. All the
photographs were taken with the aid of external ashlight, either during the day or
night. The interpretations for eye colour and pattern in this study are solely based on
photographs. The possibility of variations in eye colour were also observed under captivity
before photography. However, 19 randomly tested species did not show signicant
variation in eye colour, unlike the changes usually observed in the case of dorsal skin
colouration. The following parameters were described: (1) eye colour of individual species;
(2) comparisons with morphologically and phylogenetically related species. Terminologies
for the eye structures (Fig. 1) are adopted and modied from Glaw & Vences (1997).
Call recordings and acoustic analyses
The sound recordings of 43 species (representing 14 species groups) were made at night
when the animals were actively calling (18:0004:00 h). Calls were recorded in the eld
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 7/78
using a Sennheiser microphone ME 66 connected or MKH 416 directional microphone
connected to a digital solid-state recorder, such as Marantz PMD620, Marantz PMD670,
Fostex FR2LE, Zoom H6 and Zoom H4n (44.1 kHz sampling rate, 16-bit resolution) and
monitored in real time using Sony MDR V500 headphones. The gain settings of the
recorder were adjusted prior to each recording to avoid clipping the amplitude envelopes
of recorded calls and maintained throughout to ensure a constant signal to noise ratio
within a recording.
Acoustic properties were measured using Raven Pro v1.4 (Charif, Waack & Strickman,
2010). Our use of terminology to describe species-specic vocal repertoires follows our
earlier reports describing the vocalizations of Pseudophilautus kani and Raorchestes
graminirupes (Bee, Suyesh & Biju, 2013a,2013b), and readers are referred to those studies
for additional details not described here. Briey, the members of genus Raorchestes
produced one to three types of call, which either had pulsatile or non-pulsatile
temporal structures. Calls could be produced singly or organised into longer call groups
(a series of calls delivered in quick succession separated by a short time interval from a
Figure 1 Terminologies for eye structure and the types of eye colours and patterns discussed in the
text for Raorchestes members. Pupil is rounded and horizontal in all known Raorchestes species. (A and B)
R. resplendens eye marked with sclera, iris periphery, and iris. (CJ) Major types of eye colours and patterns.
(C) Iris with horizontal and vertical bands. (D) Black iris with dense metallic silver mosaic pattern. (E) Black
iris with golden yellow patches. (F) Brown iris with silver white or golden radiating lines. (G) Brown iris
with metallic greenish-yellow reticulations. (H) Iris horizontally divided into light upper and dark lower
halves. (I) Yellow iris with an inner reddish-brown ring. (J) Brown iris with dense golden yellow speckling.
(K) Reddish-brown iris with yellowish-green spots and blotches on the palpebral membrane.
Full-size
DOI: 10.7717/peerj.10791/g-1
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 8/78
subsequent call group). Call groups consisted of either repetitions of the same call type or a
mixture of different call types. We labelled calls as Type 1 (Type 1calls were the
most frequently delivered call type or if two different call types were delivered together as
groups then the rst delivered call in a call group was named as Type 1) and Type 2
and Type 3 for the species producing more than one call type. For species with pulsatile
calls, we analysed ve temporal properties (call duration, call rise time, call fall time,
number of pulses per call, and pulse rate) and one spectral property (overall dominant
frequency). Three temporal properties (call duration, call rise time, and call fall time) and
one spectral property (overall dominant frequency) were used for analyses for species
with non-pulsatile calls. We would note that in addition to among-species variation, the
temporal and spectral properties of anuran vocalisations also vary among individuals within
species and within individuals, for example, as a function of differences in temperature and
social context (Gerhardt & Huber, 2002). The values reported below do not take into
consideration these sources of call variation, which are to be expected to operate within each
species.
For visual representations of calls, oscillograms showing the amplitude versus time
waveform were prepared using a time frame of 1 s for species groups (n= 8) that produce
calls/call groups longer than 0.1 s and a time frame of 0.1 s for species groups (n=6)
that produce calls shorter than 0.1 s. The overall dominant frequency information for
the calls of each species was obtained using Ravens spectrogram function after selecting
the entire duration of the call (1,024-point fast Fourier transform, Hann window, 50%
overlap, 43.1 Hz resolution). Spectrograms were prepared for graphical representation of
the call spectrum at similar time frames as the oscillograms.
New species names
The electronic version of this article in Portable Document Format (PDF) will represent a
published work according to the International Commission on Zoological Nomenclature
(ICZN), and hence the new names contained in the electronic version are effectively
published under that Code from the electronic edition alone. This published work and the
nomenclatural acts it contains have been registered in ZooBank, the online registration
system for the ICZN. The ZooBank LSIDs (Life Science Identiers) can be resolved
and the associated information viewed through any standard web browser by appending
the LSID to the prexhttp://zoobank.org/. The LSID for this publication is: urn:lsid:
zoobank.org:pub:7021B266-C54A-4E64-8645-AACCBFBB1A72. The online version of
this work is archived and available from the following digital repositories: PeerJ, PubMed
Central and CLOCKSS.
RESULTS
Phylogenetic relationships
Our Maximum Likelihood (ML) and Bayesian phylogenetic analyses (Fig. 2) recovered
genus Raorchestes as a well-supported monophyletic clade, showing a sister-group
relationship with the genus Pseudophilautus (e.g., Li et al., 2009;Yu et al., 2009;Biju et al.,
2010;Pyron & Wiens, 2011;Vijayakumar et al., 2016). The focal genus showed two major
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 9/78
Figure 2 Phylogenetic relationships in the genus Raorchestes and State-wise gures for species in the
Western Ghats. (A) Maximum Likelihood phylogram, based on 2,327 bp partitioned dataset for three
mitochondrial and two nuclear gene fragments from 94 taxa, showing phylogenetic relationships among
60 previously recognised and ve new Raorchestes species along with representatives of other known
rhacophorid genera. The focal genus Raorchestes comprises 16 major species-groups discussed in the
study. New species described in the study are indicated in bold letters. The values above and below the
branches indicate Bayesian Posterior Probabilities (BPP) and RAxML Bootstrap support (BS), respec-
tively. (B) Number of Raorchestes species reported from the Indian States encompassing the Western
Ghats. (C) Proportion of the currently recognised species originally described from each state.
Descriptions from regions with colonial names that may include more than one State are categorised
separately. Full-size
DOI: 10.7717/peerj.10791/g-2
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 10/78
radiations, the northern clade and the southern clade (Vijayakumar et al., 2016), that were
further divided into 16 major sub-clades largely congruent with Vijayakumar et al. (2014)
and hereafter referred to as species groups, as indicated with modications in Fig. 2.
Most of the recognised species groups were recovered with high (BPP 95, BS 70)
support, except for the Raorchestes charius group and R. aureus group that received
moderate support probably due to the phylogenetic position of R. marki (included in
R. charius species group in the present study; in R. aureus sub-clade as per Vijayakumar
et al., 2016; unresolved in Vijayakumar et al., 2014). The relationships of three species
(R. crustai,R. echinatus, and R. indigo) also remained unresolved, as shown previously by
Vijayakumar et al. (2014,2016). While R. crustai appreared to show a closer but
unsupported phylogenetic afnity to members of the R. graminirupes group, we
provisionally assign it to the R. nerostagona group based on additional morphological
and acoustic evidence (see Grouping of species using integrative approaches), until
further evidence proves otherwise. On the other hand R. echinatus and R. indigo are treated
as ungrouped species (see Grouping of species using integrative approachesfor discussion
on morphological afnities). Although the phylogenetic relationships at the species-
level were often well-supported, several lineages were also either moderately to weakly
support or remained unresolved (Fig. 2).
In addition to the previously known Raorchestes species, our study also included ve
populations representing potential candidate species (Fig. 2). Our analyses concordantly
supported the distinct phylogenetic position of all these lineages in four recognised
species groups (one in R. bombayensis group; one in R. charius group; two in
R. chromasynchysi group; and one in R. nerostagona group) with well-supported sister-
group relationships. Based on additional integrative evidence, we conrm that these
putative lineages represent distinct new species and are formally described below.
Description of new species
Raorchestes drutaahu sp. nov.
http://zoobank.org/urn:lsid:zoobank.org:act:4B07924D-80B3-468B-8C35-C3A8C3001E23
Fast-calling Shrub Frog
(Figs. 23;Tables 13;Tables S1 and S2)
Etymology. The species name is derived from Sanskrit druta(meaning fast) and ahu
(meaning call), referring to the fast-pulsatile calls of the new species. The species epithet
drutaahu is treated as an invariable noun in apposition to the generic name.
Holotype. BNHS 6088, an adult male, from Kadalar (10.1311N, 77.0005E, 1,430 m asl),
Munnar, Idukki district, Kerala State, India, collected by SDB and SG on 17 August
2014. Paratypes. BNHS 6089, an adult male, collected by SDB and SG, along with the
holotype; BNHS 6090, an adult male, collected by RS and SDB, from the holotype locality
on 10 August 2012; and BNHS 6091, an adult male, from Siruvani (10.9587N, 76.6667E,
1,048 m asl), Palakkad district, Kerala State, India, collected by SDB, SG, and RS on
07 July 2015. Referred specimen. SDBDU 2015.3025, an adult female, from Siruvani,
Palakkad district, Kerala State, India, collected by SDB, SG, and RS on 07 July 2015.
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 11/78
Phylogenetic relationship. Raorchestes drutaahu sp. nov. is a member of the Raorchestes
charius group and shows a well-supported sister-group relationship with R. coonoorensis
(Fig. 2). For the mitochondrial 16S rRNA, Raorchestes drutaahu is divergent from
other members of the group as: 4.57.9% from R. charius; 4.35.2% from R. coonoorensis;
4.36.6% from R. griet; and 4.65.6% from R. honnametti.
Morphological diagnosis and comparison. Raorchestes drutaahu sp. nov. can be
distinguished from other known congeners, except members of the Raorchestes charius
group, by the combination of following morphological characters: a small-sized species
(male SVL 2023 mm); outline of the snout rounded to sub-ovoid in ventral view;
tympanum distinct, nearly half of the eye diameter; dorsum light to dark brown or straw
coloured with horny spinules and ridges; dorsum with two faint to prominent shaped
concave bands, extending from behind the eye to nearly the vent; lateral surfaces of the
head dark brown from tip of the snout and along the margins of the eye and
Figure 3 Type and referred specimens of Raorchestes drutaahu sp. nov. (AH) Holotype, in life
(BNHS 6088, adult male). (A) Dorsolateral view. (B) Dorsal view. (C) Ventral view. (D) Dorsal view of
body and thighs. (E) Lateral view. (F) Posterior view of thighs. (G) Ventral view of hand. (H) Ventral view
of foot. (IJ) Paratypes, in life. (I) Dorsolateral view (BNHS 6089, adult male). (J) Dorsolateral view
(SDBDU 2015.3025, adult female). (KP) Holotype, in preservation (BNHS 6088, adult male). (K) Dorsal
view. (L) Ventral view. (M) Lateral view of head. (N) Ventral view of hand. (O) Ventral view of foot.
(P) Schematic illustration of webbing on foot. Full-size
DOI: 10.7717/peerj.10791/g-3
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 12/78
Table 1 Dorsal colouration and eye characters in 59 Raorchestes species of Peninsular India.
Group/Species Dorsum Eye colouration and markings
Colour Markings Iris Iris periphery Sclera
Raorchestes anili
group
Raorchestes anili Light to dark brown Dark brown inverted V-shaped mark Light golden brown with
reddish tinge
Dark brown Light
blue
Raorchestes
kaikatti
Greyish or reddish-brown With or without inconspicuous dark
spots and markings
Brown or reddish-brown to
orange
Dark brown Light
blue
Raorchestes
kakachi
Light to dark brown or
light grey
With or without dark irregular spots and
markings
Dark brown or reddish-brown Dark brown Light
blue
Raorchestes sushili Brown, greyish or
reddish-brown
Dark inverted V-shaped mark and
irregular spots
Brown, copper, or reddish-
brown
Dark brown Light
blue
Raorchestes
aureus group
Raorchestes
aureus
Light brown or pale
yellow
Absence of prominent markings Golden brown Black Light
blue
Raorchestes
lechiya
Light brown or greyish-
brown
With or without dark bands, spots or
markings
Golden brown Black Light
blue
Raorchestes
beddomii group
Raorchestes
beddomii
Bright green or yellowish-
green
Absence of prominent markings Red, brick red, or orange Black Light
blue
Raorchestes
dubois
Highly variable from
white, grey, green,
brown, yellow to red
With or without contrasting and variable
spots, streaks, bands or markings
Light golden brown with
reddish tinge
Black Scarlet
blue
Raorchestes
munnarensis
Brown to yellowish-grey Dark inverted Vor X-shaped mark Light brown to coffee brown Black Scarlet
blue
Raorchestes
resplendens
Reddish-orange
interspersed with black
Multiple prominent bright orange
macroglands
Red or brick red Black Scarlet
blue
Raorchestes
theuerkau
Brown or reddish brown Irregular dark mottling and scattered
patches
Light golden brown or copper Black Light
blue
Raorchestes
bombayensis
group
Raorchestes
bombayensis
Brown or greyish-brown With or without inverted Vor X-
shaped mark
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
Raorchestes ghatei Brown or greyish-brown With or without inconspicuous dark
bands (Vor X-shaped), spots or
markings
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
Raorchestes
kakkayamensis
sp. nov.
Brown or reddish-brown Dark discontinuous concave bands and
scattered darks streaks or markings
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
Raorchestes
leucolatus
Brown to reddish-brown Scattered orange spots or patches and
inconspicuous dark markings
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
(Continued)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 13/78
Table 1 (continued )
Group/Species Dorsum Eye colouration and markings
Colour Markings Iris Iris periphery Sclera
Raorchestes
sanctisilvaticus
Brown or greyish-brown With or without inverted Vor X-
shaped mark
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
Raorchestes
tuberohumerus
Light to dark brown Faint to prominent dark X-shaped mark
or irregular markings
Brown with dense golden
speckling, and dark brown
horizontal and vertical bands
Dark brown Light
silvery
blue
Raorchestes
chalazodes
group
Raorchestes
chalazodes
Green, yellowish or
bluish-green
Rarely with scattered spots Black with a golden yellow ring
that may or may not be
divided by a black cross mark
Black Scarlet
blue
Raorchestes
aviocularis
Green or reddish green Lichen pattern exposing reddish eshy
skin
Black with a golden yellow ring
that may or may not be
divided by a black cross mark
Black Indistinct
Raorchestes
ochlandrae
Brown or reddish-brown Light yellow dorsolateral bands with or
without elongate blotches or scattered
spots
Black with light yellow patches
in a radial pattern
Black Indistinct
Raorchestes
manohari
Bright yellow to greyish-
yellow
Scattered dark brown spots Black with dense metallic silver
mosaic pattern
Black Indistinct
Raorchestes
uthamani
Yellow with grey or red
tinge
With or without faint dark brown streaks
or spots
Black with dense metallic silver
mosaic pattern
Black Indistinct
Raorchestes
charius group
Raorchestes
charius
Brown or reddish-brown Contrasting concave bands and irregular
patches
Light or dark brown with
golden tinge, horizontally
divided into light upper and
dark lower halves
Blackish-
brown
Light
grey
Raorchestes
coonoorensis
Light brown or reddish-
brown
With or without, continuous or
discontinuous dark concave bands
Brown with golden tinge,
horizontally divided into
light upper and dark lower
halves
Blackish-
brown
Light
blue
Raorchestes
drutaahu sp.
nov.
Light to dark brown or
straw
With or without, continuous or
discontinuous grey bands or stripes
Brown with golden tinge,
horizontally divided into
light upper and dark lower
halves
Blackish-
brown
Light
grey
Raorchestes griet Brown with grey or
reddish tinge
Irregular black patches or dark concave
bands
Brown with golden tinge,
horizontally divided into
light upper and dark lower
halves
Black Light
grey
Raorchestes
honnametti
Brown or grey Faint to prominent contrasting concave
bands
Brown with golden tinge,
horizontally divided into
light upper and dark lower
halves
Black Light
blue
Raorchestes
kollimalai
Light or dark brown Faint to prominent, continuous or
discontinuous contrasting concave
bands
Brown with golden tinge,
horizontally divided into
light upper and dark lower
halves
Black Light
blue
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 14/78
Table 1 (continued )
Group/Species Dorsum Eye colouration and markings
Colour Markings Iris Iris periphery Sclera
Raorchestes marki Grey, brown or reddish-
brown
Dark X-shaped mark or a pair of concave
bands
Brown with dense golden
yellow speckling, and with
brown horizontal and vertical
bands
Black Light
bluish-
grey
Raorchestes
chotta group
Raorchestes
archeos
Greyish, reddish or
yellowish-brown
With or without dark broad median band Golden brown, with dark
vertical band
Brown Greyish-
white
Raorchestes
blandus
Greyish to reddish-brown Irregular dark brown and orange patches Golden brown Brown Greyish-
white
Raorchestes chotta Brown with yellow or
grey tinge
Irregular dark brown blotches or
scattered dark spots
Golden brown Brown Greyish-
white
Raorchestes
chromasynchysi
group
Raorchestes
chromasynchysi
Brown or Green With or without contrasting dark bands,
streaks or markings
Golden brown with reddish
tinge
Blackish-
brown
Scarlet
blue
Raorchestes ravii Brown or orangish-
brown
With or without dark median band, faint
X-shaped mark or scattered spots
Golden brown Dark brown Scarlet
blue
Raorchestes
sanjappai sp.
nov.
Green Without prominent dark markings,
occasionally with markings
Reddish-brown Dark brown Light
blue
Raorchestes
silentvalley
Green With or without yellow or bluish-black
spots
Brown or dark red Blackish-
brown
Light
blue
Raorchestes
vellikkannan sp.
nov.
Brown or pale yellow Dark brown X-shaped mark and
scattered spots
Silver grey with minute brown
speckling
Dark brown Light
blue
Raorchestes
aviventris
group
Raorchestes
aviventris
Green or yellowish-green With or without scattered pale yellow,
golden yellow, or white spots
Creamy white with minute
brown speckles
Dark brown Bluish-
grey
Raorchestes
ponmudi
Brown, reddish-brown or
light to dark grey
Dark brown concave bands or X-shaped
mark, with or without few scattered
white blotches and minute black spots
Light brown or golden brown
with minute dark speckles
Dark brown Bluish-
grey
Raorchestes
glandulosus
group
Raorchestes
akroparallagi
Shades of green, yellow,
brown, reddish-brown,
or light grey
With or without yellow dorsolateral
streaks, scattered small grey or large
blackish-brown spots
Light brown to reddish-brown
with golden speckles
Blackish-
brown
Light
blue
Raorchestes
bobingeri
Green Without prominent markings Light yellow or greyish-yellow
with an inner brown ring or
irregular spots
Blackish-
brown
Light
blue
Raorchestes
glandulosus
Green, greenish-yellow,
brown, reddish-brown,
or purplish
With or without uniformly scattered
contrasting spots and reticulations
Bright to dark red, or
reddish-brown with golden
speckles
Blackish-
brown
Light
blue
(Continued)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 15/78
Table 1 (continued )
Group/Species Dorsum Eye colouration and markings
Colour Markings Iris Iris periphery Sclera
Raorchestes
jayarami
Green, bluish-green or
yellow
With or without uniformly scattered
contrasting spots or irregular streaks
Bright yellow or greyish-yellow
with an inner reddish-brown
ring or spots
Blackish-
brown
Light
blue
Raorchestes
graminirupes
group
Raorchestes
graminirupes
Brown or yellow with
grey or reddish tinge
Dark irregular patches, streaks, or
longitudinal bands
Greyish-brown with dense
metallic silver or light brown
speckles, with scarlet blue
ring
Black Light
silvery
blue
Raorchestes
johnceei
Brown, grey, pale yellow,
or reddish
Dark irregular patches, broad median
band, pair of continuous or
discontinuous concave bands or
inverted V-shape mark
Light greyish-brown with
dense metallic silver or light
brown speckles, with scarlet
blue ring
Black Light
silvery
blue
Raorchestes
nerostagona
group
Raorchestes
crustai
Brown, greyish-brown or
green
Irregular dark brown or greenish-brown
blotches, continuous or discontinuous
concave bands, or inverted V-shape
mark
Light greyish-brown Dark brown Scarlet
blue
Raorchestes
keirasabinae sp.
nov.
Brown, greyish-brown or
greenish-brown
Irregular brown, black and green patches Reddish-grey with faint or
prominent horizontal brown
band
Dark brown Scarlet
blue
Raorchestes
nerostagona
Brown or greyish-green Irregular dark green, reddish-brown, or
bluish-black patches of various size
Reddish-grey with faint or
prominent horizontal brown
band
Dark brown Scarlet
blue
Raorchestes
signatus group
Raorchestes
signatus
Brown, grey, or red With or without, faint to prominent,
dark X-shape or inverted V-shape
mark, contrasting broad median band,
or scattered dark spots and patches
Reddish-brown with or
without silver white or
golden radiating lines and
golden speckling
Without
prominent
ring
Greyish-
brown
Raorchestes
tinniens group
Raorchestes
montanus
Light brown to chocolate
brown, pinkish or
reddish-brown, with
metallic tinge in all
morphs
With or without dark streaks, concave
bands, X-shape mark, or variable
mosaic patterns
Dark brown or golden brown Black Ash grey
Raorchestes
primarrump
Brown, grey, reddish-
brown, or greenish-
brown, with metallic
tinge in all morphs
With or without dark irregular patches or
contrasting yellowish spots
Dark brown or greyish-brown Black Ash grey
Raorchestes
tinniens
Brown, grey, reddish-
brown, or greenish-
brown, with metallic
tinge in all morphs
With or without dark irregular patches or
contrasting yellowish spots
Dark brown with golden
brown speckling
Black Ash grey
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 16/78
supratympanic fold; a pair of black irregular shaped spots near the groin on either side
of posterior dorsum; ank and groin grey or light brown without contrasting colour
blotches or markings; posterior surface of thighs dark to light brown without prominent
markings; iris brown with a golden tinge, horizontally divided into light upper and dark
lower halves; foot webbing small, below the second subarticular tubercle on either side
of toe IV (Fig. 3).
Within the Raorchestes charius group, R. drutaahu sp. nov. is more closely related to
R. charius,R. coonoorensis, and R. honnametti. However, R. drutaahu sp. nov. can be
differentiated from these three species by its groin with faint white blotches (vs. groin light
brown with pale yellow or greyish blotches in R. coonoorensis; groin deep brown with
yellow blotches in R. charius; groin light brown with minute white marbling in
R. honnametti); a pair of black irregular shaped spots on either side of the posterior
dorsum near the groin (vs. absent); and relatively reduced webbing on foot, third toe
Table 1 (continued )
Group/Species Dorsum Eye colouration and markings
Colour Markings Iris Iris periphery Sclera
Raorchestes
travancoricus
group
Raorchestes
agasthyaensis
Brown, greyish-brown, or
reddish-brown
Dark inverted V-shape mark with
scattered dark patches or dark brown
broad median band
Dark brown with dense golden
speckling, and horizontally
divided into light upper and
dark lower halves
Dark brown Ash grey
Raorchestes
chlorosomma
Brown or grey Continuous or discontinuous dark
concave bands or irregular streaks, or
dark broad median band
Metallic greyish-green or
greenish-yellow with dark
brown reticulations
Dark brown Scarlet
blue
Raorchestes
kadalarensis
Brown, greyish-brown,
reddish-brown, or
bluish-brown
Dark inverted V-shape mark Dark brown with dense golden
speckling, and horizontally
divided into light upper and
dark lower halves
Dark brown Ash grey
Raorchestes
luteolus
Yellow, reddish-yellow, or
yellowish-brown
Faint, continuous or discontinuous, dark
longitudinal lines or without prominent
markings
Golden yellow or light grey
with brown speckling with a
cobalt blue outsider ring
Black or
bluish-black
Indistinct
Raorchestes
travancoricus
Red or reddish-brown Prominent longitudinal dark lines Golden yellow or light grey
with brown speckling with a
cobalt blue outsider ring
Black or
bluish-black
Indistinct
Ungrouped
species
Raorchestes indigo Green or greenish-yellow With or without irregular and scattered
black, yellow or bluish-black spots
Golden brown Dark brown Light
blue
Raorchestes
echinatus
Brown, greyish-brown, or
reddish-brown
Thin middorsal line, irregular dark
streaks and scattered minute dark spots
Golden brown with dark
vertical band
Dark brown Indistinct
Note:
Two species are not assigned to any species group. Values are calculated for a typical single call of each species. Broad calling height categories: Ground (ground and
associated grass): 00.5 m; Low (low bushes and shrubs): 0.51.5 m; Medium 1 (high shrubs): 1.54 m; Medium 2 (lower canopy): 47 m; Canopy (high canopy): 7 m and
above (up to 40 m).
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 17/78
Table 2 Acoustic properties and calling height in 59 Raorchestes species of Peninsular India summarised in 15 species groups.
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
Raorchestes anili group 12
types
Pulsatile 51.2980.4 Nil960.2 Nil48.8 522 6.3250.8 Single 2.42.9 1.55
R. anili Delivered
in groups
Type 1 Pulsatile, widely
spaced pulses
980.4 960.2 20.2 7 6.3 Single 2.9 1.55
R. kaikatti Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile, more
closely packed
pulses than
Type 2
102.7 67.5 Not
signicant
22 250.8 Single 2.4 1.55
Type 2 Pulsatile, more
widely packed
pulses than
Type 1
68.9 Not
signicant
14.4 5 89.3 Single 2.4
R. sushili Uniform
intervals,
delivered
in groups
Type 1 Pulsatile, closely
spaced pulses
51.2 Not
signicant
48.8 11 242.8 Single 2.6 1.55
R. kakachi not studied 1.53
Raorchestes beddomii group 13
types
Pulsatile or
Non-pulsatile
13.2498.2 1.0475.3 Nil49.2 115 Nil406.5 13 2.22.7 020
R. beddomii Delivered
in groups
(only
Type 2
call)
Type 1 Pulsatile 150.0 140.2 11.2 4 29.2 Single 2.6 0.52
Type 2 Non-pulsatile 13.2 1.1 12.0 1 Single 2.6
R. dubois Generally
uniform
intervals,
not
delivered
in groups
Type 1 Non-pulsatile 10.6 1.0 8.3 1 Single 2.7 02
R. munnarensis Delivered
in
groups,
lack any
xed call
order
Type 1 Pulsatile, pulses
more widely
spaced than
Type 2
498.2 475.3 Not
signicant
3 6.4 Single 2.2 1.520
Type 2 Pulsatile, pulses
more closely
packed than
Type 1
22.8 7.7 14.4 5 406.5 Single 2.2
R. resplendens Delivered
in groups
Type 1 Non-pulsatile 51.3 16.7 49.2 1 Three 2.5
Type 2 Pulsatile 28.7 1.5 27.1 7 269.2 Two 2.7 01
Type 3 Non-pulsatile 64.7 41.6 24.7 1 Four 2.7
R. theuerkauNot studied 0.53
Raorchestes bombayensis
group
1 type Non-pulsatile 11.629.4 1.29.2 9.525.4 1 Single 2.94.1 0.55
R. bombayensis Not
delivered
in groups
Type 1 Generally single
pulse
11.6 1.2 9.5 1 Single 3.1 25
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 18/78
Table 2 (continued )
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
R. ghatei Not
delivered
in groups
Type 1 Generally single
pulse
17.7 1.5 16.0 1 Single 2.9 14
R. kakkayamensis
sp. nov.
Not
delivered
in groups
Type 1 Generally single
pulse
25.3 9.2 15.9 1 Single 3.8 13
R. leucolatus Not
delivered
in groups
Type 1 Generally single
pulse
29.4 2.7 25.4 1 Single 4.1 14
R. sanctisilvaticus Not
delivered
in groups
Type 1 Generally single
pulse
12.2 1.3 10.8 1 Single 3.1 13
R. tuberohumerus Not
delivered
in groups
Type 1 Generally single
pulse
14.6 1.3 13.3 1 Single 3.3 0.52
Raorchestes chalazodes group 1 type Non-pulsatile 18.436.0 1.24.1 11.032.3 1 Single 2.73.6 17
R. chalazodes Rapidly
delivered
in long
call
groups
Type 1 Non-pulsatile 18.4 2.9 15.2 1 Single 2.7 1.57
R. ochlandrae Rapidly
delivered
in long
call
groups
Type 1 Non-pulsatile 25.3 4.1 20.9 1 Single 2.7 1.57
R. manohari Rapidly
delivered
in long
call
groups
Type 1 Non-pulsatile 12.3 1.2 11.0 1 Single 3.6 17
R. uthamani Rapidly
delivered
in long
call
groups
Type 1 Non-pulsatile 36.0 3.2 32.3 1 Single 3.4 17
R. aviocularis Not studied 1.57
Raorchestes charius group 1 type Pulsatile 24.692.6 1.259.4 16.249.1 317 89.3266.6 Single 2.44.1 04
R. charius Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
92.6 49.2 37.2 17 226.6 Single 2.4 01.5
R. griet Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
75.6 59.4 16.2 10 151.3 Single 3.5 0.51.5
(Continued)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 19/78
Table 2 (continued )
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
R. honnametti Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
68.6 13.3 44.2 6 89.3 Single 2.6 0.51.5
R. coonoorensis Not
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
24.6 1.2 22.2 3 200 Single 2.9 0.51.5
R. drutaahu sp.
nov.
Not
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
50.6 1.2 49.1 6 134.5 Single 3.6 0.51.5
R. kollimalai Not studied 14
R. marki Not
delivered
in groups
Type 1 Pulsatile, closely
packed pulses
36.7 1.3 32.6 4 266.6 Single 4.1 1.53
Raorchestes chotta group 12
types
Pulsatile or
Non-pulsatile
17.171.2 1.138.3 15.018.8 119 Nil382.1 Single 3.13.6 04
R. archeos Uniform
intervals,
delivered
in groups
Type 1 Pulsatile,
relatively short
and closely
packed pulses
19.7 1.2 17.2 6 382.1 Single 3.1 02
R. blandus Delivered
in groups
(only
Type 1
call), lack
any xed
call order
Type 1 Pulsatile 17.1 1.5 15.8 3 370.3 Single 3.5 0.54
Type 2 Non-pulsatile 20.1 1.4 18.8 1 Single 3.5
R. chotta Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile 71.2 38.3 18.6 19 283.5 Single 3.6 02
Type 2 Non-pulsatile 17.2 1.1 15.0 1 Single 3.6
Raorchestes chromasynchysi
group
1 type Pulsatile 411.2716.0 345.2693.8 Nil35.1 26 2.77.2 Single 2.22.5 0.56
R. chromasynchysi Not
delivered
in groups
Type 1 Pulsatile, widely
spaced pulses
381.4 345.2 Not
signicant
3 5.8 Single 2.5 0.56
R. ravii Not
delivered
in groups
Type 1 Pulsatile, widely
spaced pulses
499.2 483.1 16.1 3 4.2 Single 0.55
R. sanjappai sp.
nov.
Not
delivered
in groups
Type 1 Pulsatile, widely
spaced pulses
411.2 376.2 35.1 2 2.7 Single 2.4 0.53
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 20/78
Table 2 (continued )
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
R. silentvalley Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile, widely
spaced pulses
716.0 693.8 Not
signicant
6 7.2 Single 2.2 14
R. vellikkannan
sp. nov.
Not studied 14
Raorchestes aviventris group 1 type Pulsatile 480.0721.7 458.7712.4 1215 21.126.4 Single 1.71.9 17
R. aviventris Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 721.7 712.4 Not
signicant
15 21.1 Single 1.9 16
R. ponmudi Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 480.0 458.7 Not
signicant
12 26.4 Single 1.7 17
Raorchestes glandulosus group 1 type Pulsatile 445.6813.3 429.4711.2 Nil99.1 611 10.913.7 Single 2.73.6 18
R. akroparallagi Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 445.6 429.4 Not
signicant
6 13.7 Single 3.4 14
R. bobingeri Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 565.6 550.1 Not
signicant
6 10.9 Single 3.6 1.57
R. glandulosus Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 609.6 588.1 Not
signicant
8 11.7 Single 2.7 1.58
R. jayarami Uniform
intervals,
not
delivered
in groups
Type 1 Pulsatile 813.3 711.2 99.1 11 12.5 Single 2.9 1.56
Raorchestes graminirupes
group
12
types
Pulsatile 27.891.3 Nil50.7 15.988.7 318 114.8222.5 Single 2.12.8 07
R. graminirupes Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile 91.3 1.7 88.7 18 222.5 Single 2.7 03
Type 2 Pulsatile 27.8 1.0 26.8 5 203.3 Single 2.8
(Continued)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 21/78
Table 2 (continued )
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
R. johnceei Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile 66.3 50.7 15.9 8 114.8 Single 2.1 0.57
Type 2 Pulsatile 28.8 not
signicant
28.0 3 171.4 Single 2.2
Raorchestes nerostagona
group
1 type Non-pulsatile 13.323.3 1.22.2 12.020.2 Nil Nil Single 2.02.2 340
R. crustai Not
delivered
in groups
Type 1 Non-pulsatile 13.3 1.2 12.0 1 Single 2.2 320
R. nerostagona Not
delivered
in groups
Type 1 Non-pulsatile 23.3 2.2 20.2 1 Single 2.0 540
R. keirasabinae sp.
nov.
Not studied 530
Raorchestes signatus group 1 type Non-pulsatile 20.2 2.0 18.2 Nil Nil Single 2.1 0.510
R. signatus Uniform
intervals,
delivered
in groups
Type 1 Non-pulsatile 20.2 2.0 18.2 1 Single 2.1 0.510
Raorchestes tinniens group 1 type Non-pulsatile 8.5 1.1 6.5 Nil Nil Single 2.6 02
R. tinniens Uniformly
intervals,
delivered
in groups
Type 1 Non-pulsatile 8.5 1.1 6.5 1 Single 2.6 01.5
R. montanus Not studied 02
R. primarrumpNot studied 01.5
Raorchestes travancoricus
group
2 types Pulsatile or
Non-pulsatile
15.6474.5 1.6355.7 13.0116.8 347 31.9238.1 Single 2.23.4 04
R. agasthyaensis Not studied 02
R. chlorosomma Delivered
in
groups,
lack any
xed call
order
Type 1 Pulsatile, closely
packed pulses
246.2 109.5 106.6 47 195.8 Single 2.2 1.54
Type 2 Pulsatile, closely
packed pulses
30.2 2.0 27.3 5 238.1 Single 2.2
R. kadalarensis Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Non-pulsatile 15.6 1.6 13.0 1 Single 3.4 02
Type 2 Pulsatile 52.6 33.7 18.9 3 66.9 Single 3.4
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 22/78
webbing just above the rst subarticular tubercle on the outside (vs. more extensive, nearly
up to the second subarticular tubercle). Specically, it also differs from R. charius and
R. honnametti by its relatively smaller adult size, male SVL 2023 mm, female SVL
24.5 mm (vs. larger, male SVL 2635 mm in R. charius; and male SVL 2328 mm in
R. honnametti). Further, it differs from R. griet by relatively larger adult size, male SVL
2023 mm, female SVL 24.5 mm (vs. smaller, male SVL 1822 mm, female SVL 22 mm);
and more extensive webbing on foot, well beyond the rst subarticular tubercle on
either side of toe IV (vs. reduced, up to or slightly above the rst subarticular tubercle on
either side). Raorchestes drutaahu sp. nov. also differs from the most recently described
species R. kollimalai by relatively smaller adult size, male SVL 2023 mm, female SVL
24.5 mm (vs. larger, male SVL 25.829.7 mm); head nearly as wide as long (vs. wider
than long); shank nearly equal or shorter than thigh (vs. longer) (Gowande, Ganesh &
Mirza, 2020); and webbing between toes III and IV rudimentary, well below the rst
subarticular tubercle on either side (vs. nearly up to the rst subarticular tubercle).
Table 2 (continued )
Group/species Call
delivery
pattern
Call
type
Temporal properties Spectral properties Calling
height
(meters)
Temporal
structure
Call
duration
(ms)
Call rise
time (ms)
Call fall
time (ms)
Pulses/call Pulse rate
(Pulses/sec)
Broad
frequency
peaks
Dominant
frequency
(kHz)
R. luteolus Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile 390.5 270.5 88.6 12 31.9 Single 2.7 0.51.5
Type 2 Non-pulsatile 27.2 1.6 25.6 1 Single 2.5
R. travancoricus Delivered
in
groups,
xed call
order
(Type 1
followed
by Type
2)
Type 1 Pulsatile 474.5 355.7 116.8 15 33.1 Single 3.3 0.51.5
Type 2 Non-pulsatile 17.2 1.8 15.3 1 Single 3.3
Raorchestes aureus group 1 type Pulsatile or
Non-pulsatile
180320 NA NA 18 NA Single 2.63.0 0.52
R. aureus*Not
studied
Type 1 Non-pulsatile 180 NA NA 1 NA Single 3.0 0.52
R. lechiya*Not
studied
Type 1 Pulsatile 320 NA NA 8 (average) NA Single 2.6 0.52
*Call properties based on data available in Zachariah et al., 2016
Ungrouped species
R. indigo Not studied 0.53
R. echinatus Not studied 01
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 23/78
Table 3 Distribution localities of Raorchestes samples examined in the present study. Localities are
arranged by State.
State/District Locality Species studied
Tamil Nadu
Coimbatore Grass Hills R. dubois,R. aviventris,R. griet,R. resplendens,R. sushili
Sholayar R. akroparallagi,R. anili,R. blandus, R ochlandrae,R. sushili
Valparai R. akroparallagi,R. beddomii,R. aviventris,R. griet,
R. jayarami,R. ochlandrae,R. sushili,R uthamani
Dindigal Anna Kodaikanal R. dubois
Kanyakumari Kiriparai R. akroparallagi
Namakkal Kolli Hills R. kollimalai
Nilgiris Avalanche R. signatus,R. tinniens
Bangitapal R. primarrump,R. signatus,R silentvalley
Coonoor R. charius, R.coonoorensis, R. signatus, R. tinniens
Kotagiri R. coonoorensis,R. signatus,R. tinniens
Mukurthi R. lechiya,R. primarrump,R. signatus,R. silentvalley,
R. tinniens
Naduvattam R. charius,R. coonoorensis,R. ravii,R. signatus,R. tinniens
Ooty R. signatus,R. tinniens
Parsons Valley R. signatus,R. tinniens
Pykara R. coonoorensis,R. signatus,R. tinniens
Salem Yercaud R. kollimalai
Theni Bodinayakkanur R. travancoricus
Meghamalai R. beddomii,R. chlorosomma,R. dubois,R. aviocularis,R
griet,R. cf. kaikatti,R. munnarensis,R. travancoricus
Tirunelveli Kakachi R. agasthyaensis,R. bobingeri,R. chalazodes,R. crustai,
R. graminirupes,R. johnceei,R. kakachi,R. manohari
Kannikatti R. akroparallagi,R. archeos
Kodayar R. agasthyaensis,R. bobingeri,R. chalazodes,R. crustai,
R. graminirupes,R. johnceei,R. kakachi, R. manohari
Sengaltheri R. bobingeri,R. johnceei
Singampatti R. beddomii
Kerala
Idukki Chinnar R. chlorosomma,R dubois,R griet,R. jayarami,
R. kadalarensis,R. munnarensis,R. resplendens
Devikulam R. beddomii,R chlorosomma,R. dubois,R. griet,R. jayarami,
R. kadalarensis,R. munnarensis
Eravangalar R. dubois,R. aviocularis,R uthamani
Eravikulam
National Park
R. beddomii,R. chlorosomma,R. dubois,R. aviventris,
R griet,R. kadalarensis,R. munnarensis,R. ochlandrae,
R. resplendens,R. sushili
Kozhikana R. akroparallagi,R. anili
Kadalar R. chlorosomma,R. dubois,R. drutaahu sp. nov.,
R. aviventris,R. jayarami,R. kadalarensis,R. keirasabinae
sp. nov., R. munnarensis,R. ochlandrae,R. sushili,
R. theuerkau
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 24/78
Table 3 (continued )
State/District Locality Species studied
Mathikettan R. beddomii, R. chlorosomma,R.jayarami,R munnarensis,
R.sushili
Mattupetti R. beddomii,R. chlorosomma,R. griet,R. jayarami,
R. kadalarensis,R. munnarensis
Meesapulimala Rdubois, R. resplendens
Munnar R. beddomii,R. cf. bobingeri,kadalarensis, R. munnarensis,
R. chlorosomma, R. griet, R. resplendens,R. kadalarensis
Painav R. akroparallagi
Thekkady R. anili,R. keirasabinae sp. nov.
Upper Manalar R. beddomii,R. chlorosomma,R. dubois,R. aviocularis,
R. cf. kaikatti,R. munnarensis,R. travancoricus,R. uthamani
Vandiperiyar R. anili,R. griet,R. keirasabinae sp. nov., R. ponmudi,
R. travancoricus
Vagamon R. akroparallagi,R. anili,R. cf. bobingeri,R. griet,
R. keirasabinae sp. nov., R. ponmudi,R. travancoricus
Vaguvarai R. beddomii,R chlorosomma,R. dubois,R griet,
R munnarensis
Vattavada R. beddomii,R dubois,R. griet
Kannur Paithal Mala R. charius,R luteolus,R. tuberohumerus
Aralam R. akroparallagi
Kasargod Anakallu R. akroparallagi
Ranipuram R. anili,R. charius,R. luteolus,R. ponmudi,R. tuberohumerus
Kollam Shendurney R. agasthyaensis,R. akroparallagi,R. anili,R. archeos,
R. bobingeri,R. beddomii,R. chalazodes,R. chotta,R. crustai,
R. johnceei,R. kakachi,R. keirasabinae sp. nov., R. manohari,
R. ponmudi
Thenmala R akroparallagi
Kozhikode Kakkayam R. akroparallagi,R. anili,R glandulosus,R. kakkayamensis sp.
nov., R. ochlandrae,R. ponmudi
Palakkad Nelliyampathi R. jayarami,R. kaikatti,R. marki,R ochlandrae
Parambikulam R. akroparallagi,R. anili,R. blandus,R. ochlandrae,R. sushili
Silent Valley
National Park
R. anili,R charius,R. glandulosus,R. lechiya,R. signatus,
R. silentvalley,R. tinniens, R. vellikannan sp. nov.
Siruvani R. anili,R. aureus,R. drutaahu sp. nov., R. leucolatus,
R. vellikannan sp. nov.
Pathanamthitta Gavi R. akroparallagi, R. anili,R. keirasabinae sp. nov,
R. ponmudi,R. uthamani
Pamba R. akroparallagi
Thrissur Chimmini R. kaikatti,R. keirasabinae sp. nov., R ochlandrae
Malakkappara
Sholayar
R. akroparallagi,R. anili,R. blandus,R. ochlandrae,R. sushili
Vazhachal R. akroparallagi,R. anili,R. blandus,R. ochlandrae
Thiruvananthapuram Athirimala R. agasthyaensis,R. archeos,R. beddomii,R. chalazodes,
R. crustai,R. graminirupes,R. johnceei,R. kakachi,
R. manohari
(Continued)
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 25/78
Table 3 (continued )
State/District Locality Species studied
Bonacaud R. akroparallagi,R. anili,R. chotta,R. keirasabinae sp. nov.,
R. ponmudi
Chathankod
Makki
R. akroparallagi,R. keirasabinae sp. nov.
Pandipath R. agasthyaensis,R. archeos,R. bobingeri,R. beddomii,
R. chalazodes,R. crustai,R. graminirupes,R. johnceei,
R. manohari
Ponkalapara R. archeos,R. bobingeri,R. beddomii,R. graminirupes
Ponmudi R. akroparallagi,R. anili,R. archeos,R. bobingeri,R. chotta,
R. graminirupes,R. keirasabinae sp. nov., R. ponmudi
Wayanad Banasura R chromasynchysi,R. glandulosus,R. ochlandrae,
R tuberohumerus
Kalpetta R. akroparallagi,R. anili,R. nerostagona,R. ponmudi,
R. tuberohumerus
Kurichiyarmala R. anili,R charius,R. chromasynchysi,R. glandulosus,
R. ponmudi
Mananthavady R. akroparallagi,R. anili,R. glandulosus,R. nerostagona,
R. ponmudi,R. tuberohumerus
Muthanga R. anili,R. tuberohumerus
Periya R. akroparallagi,R. anili,R. ponmudi,R. sanjappai sp. nov.
Pozhuthana R. akroparallagi,R. anili,R. ochlandrae,R. ponmudi
Sultan Bathery R. akroparallagi,R. anili,R. glandulosus,R. nerostagona,
R. ponmudi
Thirunelly R. anili,R. charius,R. chromasynchysi,R. ochlandrae,
R. tuberohumerus
Vellarimala R. charius,R. glandulosus,R. signatus
Vythiri R akroparallagi,R. anili,R. nerostagona,R. ochlandrae,
R. ponmudi,R.tuberohumerus
Karnataka
Belgaum Londa R. bombayensis
Chamrajnagar BR Hills R. honnametti
Chikmagalur Baba Budangiri R. charius,R. chromasynchysi,R. echinatus
Balehoonoor R. luteolus,R. tuberohumerus
Bygoor R. luteolus,R. tuberohumerus
Chikmagalur R. charius,R. tuberohumerus,R. luteolus
Kemmangundi R. charius,R. chromasynchysi,R. ochlandrae
Kudremukh NP R. charius,R. chromasynchysi,R. indigo,R. luteolus,
R. montanus,R. ochlandrae,R. tuberohumerus
Mudigere R. luteolus,R. tuberohumerus
Muthodi R. charius,R. chromasynchysi, R. luteolus,R. ochlandrae,
R. tuberohumerus
Dakshin Kannada Beluvai R. akroparallagi
Mangalore R. akroparallagi
Punacha R. akroparallagi
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 26/78
We specically also compare Raorchestes drutaahu sp. nov. with the type series of
R. ravii (see taxonomic remarks for that species), and show that it differs due to the
presence of black irregular shaped spots on either side of the posterior dorsum near the
groin (vs. absent); head nearly as wide as long, HW/HL ratio 0.991.01 (vs. wider than
long, HW/HL ratio 1.351.36); snout rounded to sub-ovoid (vs. pointed); tympanum
distinct (vs. indistinct); shank nearly equal or shorter than thigh, SHL/TL ratio
Table 3 (continued )
State/District Locality Species studied
Hassan Kempholay R. luteolus,R. tuberohumerus
Kottigehara R. charius,R. tuberohumerus
Sakleshpur R. luteolus,R. tuberohumerus
Kodagu Bhagamandala R. anili,R. ponmudi
Madikeri R. anili,R. charius,R. glandulosus,R. luteolus,
R. nerostagona,R. ponmudi,R. tuberohumerus
Nishanimotta R. chromasynchysi
Thalakaveri R. chromasynchysi,R. tuberohumerus
Yevakapadi R. anili,R. charius,R. chromasynchysi,R. glandulosus,
R. luteolus,R. ponmudi,R. tuberohumerus
Shimoga Agumbe R. tuberohumerus,R. nerostagona
Jog falls R. luteolus, R. tuberohumerus
Uttara Kannada Castle Rock R. bombayensis
Mavingundi R. luteolus
Goa
South Goa Netravali R. bombayensis
Maharashtra
Pune Bhimashankar R. ghatei
Lavasa R. ghatei
Raigad Matheran R. ghatei
Phansad R. bombayensis
Satara Kaas R. ghatei
Mahabaleshwar R. ghatei
Sawantadi Amboli R. bombayensis
Andhra Pradesh
East Godavari Maredumilli R. sanctisilvaticus
Vishakhapatnam Peddavalasa R. sanctisilvaticus
Chintapalli R. sanctisilvaticus
Borra Caves R. sanctisilvaticus
Araku Valley R. sanctisilvaticus
Odisha
Khurda Barbara R. sanctisilvaticus
Mayurbhanj Similipal R. sanctisilvaticus
Madhya Pradesh
Anuppur Amarkantak R. sanctisilvaticus
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 27/78
0.951.0 (vs. shank longer than thigh, SHL/TL ratio 1.061.07); relatively reduced foot
webbing, fourth toe webbing below the second subarticular tubercle on either side,
I12
+
II2
+
3III3
3
1
/
2
IV3
+
2
+
V (vs. more extensive, up to or above the second
subarticular tubercle on either side of toe IV, I1
+
2
+
II2
+
3
+
III23
+
IV3
2V; iris brown
with golden tinge, horizontally divided into light upper and dark lower halves (vs.
uniformly golden brown or reddish-brown); sclera light grey (vs. scarlet blue, based on the
holotype photograph); and short pulsatile male advertisement calls with closely packed
pulses and relatively faster pulse rate, characteristic for members of the R. charius group
(vs. widely spaced pulses, characteristic for members of the R. chromasynchysi group).
Description of holotype (measurements in mm). Small-sized adult male (SVL 21.9) with a
slender body; head nearly as long as wide (HL 7.8; HW 7.7; MN 6.8; MFE 5.1; MBE 2.8);
outline of the snout rounded to sub-ovoid in dorsal and ventral view, rounded in
lateral view; snout length (SL 3.4) longer than horizontal diameter of eye (EL 2.6);
loreal region acutely at with rounded canthus rostralis; distance between posterior
margins of eyes (IBE 6.7) 1.7 times the distance between anterior margins of eyes (IFE 4.0);
tympanum rather distinct (TYD 1.2), 46.2% of eye diameter (EL 2.6); supratympanic
fold rather distinct; tongue with a lingual papilla. Forearm (FAL 4.9) shorter than hand
(HAL 5.7); ngers without lateral dermal fringe; webbing absent; subarticular tubercles
rather prominent, rounded, single, III2 and IV2 weakly-developed; prepollex rather
indistinct; palmar tubercle small, rounded; supernumerary tubercles present; nuptial pad
present, smooth. Hindlimbs moderately long, thigh (TL 10.5) nearly equal to shank
(SHL 10.4) and longer than foot (FOL 7.9); distance from heel to tip of toe IV (TFOL 14.0);
foot webbing small: I12
+
II2
+
3III3
3
1
/
2
IV3
+
2
+
V, below the second subarticular
tubercle on either side of toe IV; dermal fringe along toe V absent; subarticular tubercles
rather prominent, rounded, simple, IV2 and V2 weakly-developed; supernumerary
tubercles present (Fig. 3).
Skin of snout and between eyes shagreened with ne scattered and various sized
granular projections; a faint horny ridge extending from the tip of the snout to the
vent; a weakly-developed horny ridge between the eyes, arranged in a triangle directed
posteriorly; lateral surfaces of head shagreened; dorsal surface of limbs shagreened to
sparsely granular. Ventral skin on throat shagreened to granular; chest, belly, and posterior
surface of thighs granular.
Colour of holotype. In life. Dorsum brown; a faint dark grey stripe between the eyes;
dorsum with two dark brown )( shaped concave bands, extending from behind the eyes to
the level of the groin; a pair of black irregular shaped spots on either side of the posterior
dorsum near the groin; lateral surfaces of head dark brown; lateral abdominal surfaces
lighter than dorsum; groin light greyish-brown without blotches; anterior and posterior
surface of thighs brown with dark greyish-brown mottling; dark blackish-brown markings
around the cloacal opening; fore and hind limbs (including ngers and toes) brown or
light brown with a few scattered dark brown cross-bands; iris brown with golden tinge,
upper half lighter than lower half. Ventral surfaces light brown with minute dark brown
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 28/78
speckling; hand and foot greyish-brown (Fig. 3). In preservation. Dorsum dark grey
with blackish-brown )( shaped concave bands; lateral surfaces of head blackish-brown;
lateral abdominal surfaces greyish with small off-white spots; groin grey with light grey
blotches; posterior surface of thighs light brown with scattered creamish mottling; limbs
with dark cross-bands. Ventral surface of throat creamish-white with dense minute dark
brown speckles; chest, belly, fore and hind limbs creamish-white with scattered dark
brown speckles; limbs greyish-brown with creamish-white mottling (Fig. 3).
Variations. Morphometric data from ve specimens, including the holotype, is given in
Table S2. The dorsal colouration and markings are variable in life: BNHS 6089 and BNHS
6090: dark blackish-brown markings around the cloacal opening, surrounded with
white patches; BNHS 6091: dorsum greyish-brown with prominent dark brown markings;
stripe between eyes dark brown. BNHS 6089 and SDBDU 2015.3025: dorsum light
greyish-brown to straw coloured with faint and irregular dark brown dorsal markings.
Vocalisation. Raorchestes drutaahu sp. nov. males produce a single type of call. Calls
are not delivered in groups and have a pulsatile temporal structure, with relatively short
and closely packed pulses. A typical call shows a duration of 50.6 ms; the amplitude
envelope being characterised by a rise time of 1.2 ms and fall time of 49.1 ms; with six
pulses delivered at a rate of 134.5 pulses/second; and the spectrum showing a single broad
peak with mean dominant frequency of 3.6 kHz. For comparison see Table 2 and the group
denition, including the oscillogram and spectrogram gures cited therein.
Distribution and natural history. Raorchestes drutaahu sp. nov. is endemic to the
Western Ghats and currently known only from elevations ranging between 1,000 to
1,450 m asl at two localities: Kadalar in Idukki district (south of Palghat gap) and Siruvani
in Palakkad district (north of Palghat gap). The species has been observed in forest areas,
either on grassland-shola fringes or fragmented forest patches near plantations.
Individuals were located on leaves of short shrubs at heights of 0.51.5 m.
Raorchestes kakkayamensis sp. nov.
http://zoobank.org/urn:lsid:zoobank.org:act:BD7A17C6-A1E2-421A-B0E8-
2336931CD63F
Kakkayam Shrub Frog
(Figs. 2,4and 5;Tables 13;Tables S1S5)
Etymology. The species is named after the place Kakkayam, where the type series was
collected.
Holotype. BNHS 6092, an adult male, from Kakkayam (11.5542N, 75.9196E, 750 m
asl), Kozhikode district, Kerala State, India, collected by SDB and RS in June 2018.
Paratypes. BNHS 60936096, four adult males, collected along with holotype. Referred
specimen. SDBDU 2019.3423, an adult male, from the holotype locality, collected by RS
and SD on 28 July 2018.
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 29/78
Phylogenetic relationship. Raorchestes kakkayamensis sp. nov. is a member of the
Raorchestes bombayensis group and shows a well-supported sister-group relationship with
R. leucolatus (Fig. 2). For the mitochondrial 16S rRNA, Raorchestes kakkayamensis is
divergent from other members of the group as: 3.24.6% from R. bombayensis; 5.35.9%
from R. ghatei; 2.32.4% from R. leucolatus; 4.04.5% from R. sanctisilvaticus; and
3.13.6% from R. tuberohumerus.
Morphological diagnosis and comparison. Raorchestes kakkayamensis sp. nov. can
be distinguished from other known congeners, except members of the Raorchestes
bombayensis group, by the combination of the following morphological characters:
small-sized species (male SVL 1719 mm, female SVL 24 mm); a knobbed bony projection
on humerus in males (visible externally in preserved specimens); lateral surfaces of
abdomen and groin with contrasting white blotches on grey to dark grey background;
presence of horny spinules and horny ridges on dorsal skin; and nger and toe discs
yellowish-brown (Fig. 4).
Within the Raorchestes bombayensis group, Raorchestes kakkayamensis sp. nov. differs
from R. bombayensis and R. ghatei by its smaller adult size, male SVL 1719 mm (vs. male
SVL > 19 mm); differs from R. tuberohumerus by its relatively smaller adult size, male
SVL 1719 mm (vs. larger, male SVL 1822 mm); ank and groin with white blotches on
grey to dark grey background (vs. yellow blotches); and nger and toe discs
Figure 4 Holotype (BNHS 6092, adult male) of Raorchestes kakkayamensis sp. nov. (AH) In life.
(A) Dorsolateral view. (B) Dorsal view. (C) Ventral view. (D) Dorsal view of body and thighs. (E) Lateral
view. (F) Posterior view of thighs. (G) Ventral view of hand. (H) Ventral view of foot. (IN) In pre-
servation. (I) Dorsal view. (J) Ventral view. (K) Lateral view of head. (L) Ventral view of hand.
(M) Ventral view of foot. (N) Schematic illustration of webbing on foot.
Full-size
DOI: 10.7717/peerj.10791/g-4
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 30/78
yellowish-brown (vs. grey to brown); and differs from R. leucolatus by its head longer than
wide, HW/HL ratio 0.860.95 (vs. wider than long, HW/HL ratio 1.201.33); thigh
relatively longer than shank, TL/SHL ratio 1.031.12 (vs. nearly equal, TL/SHL ratio
0.991.04); supernumerary tubercles prominent on toes IIV (vs. weakly-developed); and
nger and toe discs yellowish-brown (vs. orange or orangish-red).
Morphometrically, the new species Raorchestes kakkayamensis was also well
differentiated from the other recognised members of the Raorchestes bombayensis group,
that is R. bombayensis,R. ghatei,R. leucolatus,R. sanctisilvaticus, and R. tuberohumerus.
All the six species formed distinct clusters when projected on the rst two PCA factor
planes that had eigenvalues >1.0 and explained 79.44% of variation among the species
(Fig. 5). The PCA factor loadings representing the composition of PCA factors, and
the parameters correlated with each PCA factor are shown in Table S3. PCA Factor 1
loaded heavily on all the morphometric parameters (total 20) except FW
III
, while PCA
Factor 2 loaded heavily on two morphometric parameters, FW
III
and TW
IV
. Furthermore,
our DFA resulted in 100% classication success with all the individual samples being
classied into their respective species (Table S5). The coefcients of canonical discriminant
function representing the composition of standardized canonical discriminant scores
are shown in Table S4. All the ve discriminant function roots showed eigenvalues >1.0
and explained 100% of the variations among these species. The PCA and DFA results
provide additional evidence for morphometric differentiation of Raorchestes
kakkayamensis sp. nov. from its closest congeners.
Figure 5 Projection of the rst two Principal Component (PC) factor planes explaining 79.45% of
the total variation among six species of the Raorchestes bombayensis group and showing
morphometric distinctness of the new species, R. kakkayamensis.
Full-size
DOI: 10.7717/peerj.10791/g-5
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 31/78
Description of holotype (measurements in mm). Small-sized adult male (SVL 18.8) with
a slender body; head longer than wide (HL 7.0; HW 6.5; MN 5.0; MFE 5.1; MBE 1.9);
outline of the snout nearly sub-ovoid in dorsal and ventral view, acute in lateral view; snout
length (SL 2.8) longer than horizontal diameter of eye (EL 2.5); loreal region acute with
indistinct canthus rostralis; distance between posterior margins of eyes (IBE 6.1) twice the
distance between anterior margins of eyes (IFE 3.1); tympanum (TYD 1.0) 40% of eye
diameter (EL 2.5); supratympanic fold rather indistinct; tongue with a lingual papilla.
Forearm (FAL 3.9) shorter than hand (HAL 4.9); ngers without lateral dermal fringe;
webbing absent; subarticular tubercles rather prominent, rounded, single, III2 and IV2
weakly-developed; prepollex rather indistinct; palmar tubercle small, rounded;
supernumerary tubercles absent; nuptial pad present, smooth. Hindlimbs moderately
long, thigh (TL 9.2) longer than shank (SHL 8.9) and foot (FOL 7.3); distance from heel to
tip of toe IV (TFOL 11.8); foot webbing small: I22II23III2
1
/
4
3
1
/
2
IV3
+
2V, below the
second subarticular tubercle on either side of toe IV; dermal fringe along toe V absent;
subarticular tubercles rather prominent, rounded, simple, IV2 and V2 weakly-developed;
supernumerary tubercles absent (Fig. 4).
Skin of snout and between eyes shagreened; upper eyelids and lateral surfaces of head
shagreened to sparsely glandular; anterior and posterior part of back shagreened with
sparsely scattered spinular projections; lateral surfaces of abdomen with scattered
granulations. Ventral surface of throat and chest shagreened to granular; belly and
posterior surface of thighs granular (Fig. 4).
Colour of holotype. In life. Dorsum brown to reddish-brown with a pair of dark
discontinuous concave bands extending from behind the eye to the level of the groin, with
a thin mid-dorsal line; a dark brown coloured horizontal band between the upper eyelids;
snout lighter in colour than the dorsum; lateral surfaces of head darker than dorsal
colouration; lateral abdominal surfaces light brown with small off-white spots; groin and
anterior surface of thighs dark brown with distinct white blotches; posterior surface of
thighs light and dark brown with scattered white spots; limbs with dark brown cross-
bands; nger and toe discs yellowish-brown; iris brown with dense golden yellow
speckling and faint dark brown horizontal and vertical bands. Ventral surface of throat
blackish-brown with scattered white speckles; chest, and belly, light brown with dense dark
brown speckling and irregular white spots; fore and hind limbs dark brown with white to
ash blue spots and minute light brown speckling (Fig. 4). In preservation. Dorsum
greyish-brown with blackish-brown discontinuous concave bands extending from behind
the eye to the level of the groin, with a thin light grey mid-dorsal line; snout light grey;
lateral surfaces of head darker than dorsal colouration; lateral abdominal surfaces
greyish-brown with small off-white spots; groin and anterior surface of thighs dark brown
with distinct white blotches; posterior surface of thighs light and greyish-brown with
scattered white spots; limbs with faint or prominent dark greyish-brown cross-bands.
Ventral surface of throat dark brown with scattered white speckles, especially along the
margins of lower jaw; chest, belly, fore and hind limbs light brown with white spots and
minute dark brown speckling (Fig. 4).
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 32/78
Variations. Morphometric data from six specimens, including the holotype, is given in
Table S2. The dorsal colouration and markings are slightly variable from light to dark
brown or reddish-brown, but there is a uniform triangular light brown colouration from
the snout tip to the anterior margins of the eyes. BNHS 6096: less prominent dorsal
markings and granulations.
Vocalisation. Raorchestes kakkayamensis sp. nov. males produce a single type of call.
The calls generally have a single pulse and are not delivered in groups. A typical call
has duration of 25.3 ms, the amplitude envelope being characterised by a rise time of
9.2 ms and fall time of 15.9 ms, with a dominant frequency of 3.8 kHz. For comparison see
Table 2 and the group denition, including the oscillogram and spectrogram gures cited
therein.
Distribution and natural history. Raorchestes kakkayamensis sp. nov. is endemic to
the Western Ghats and currently known only from its type locality (Kakkayam) in
Kozhikode district, north of Palghat gap in Kerala State. The species was observed inside a
primary forest patch and adjoining secondary forest areas at an elevation of 750 m asl.
Individuals were located on the ground leaf litter or found perching on vegetation
13 m high.
Raorchestes keirasabinae sp. nov.
http://zoobank.org/urn:lsid:zoobank.org:act:39BCB0CE-166E-4C90-AE1E-
564BD626ABA9
Keiras Shrub Frog
(Figs. 2 and 6;Tables 13;Tables S1 and S2)
Etymology. The species is named after a young nature lover Keira Sabin, in appreciation
of the long-time support and commitment of the Andrew Sabin Family Foundation
towards amphibian research and conservation around the world. The species epithet
keirasabinae is treated as a noun in the genitive case.
Holotype. BNHS 6097, an adult male, from Chathankod-Makki (8.6723N, 77.1301E,
230 m asl), Thiruvananthapuram district, Kerala State, India, collected by SDB and SG
on 29 April 2015. Paratype. BNHS 6098, an adult male, from Ponmudi (8.75N, 77.13E,
980 m asl), Thiruvananthapuram district, Kerala State, India, collected by SDB in
May 2006. Referred specimen. SDBDU 2019.3450, an adult male, from Vallakadavu
(9.5281N, 77.1144E, 834 m asl), Periyar Tiger Reserve, Idukki district, Kerala State,
India, collected by SD on 27 July 2016.
Phylogenetic relationship. Raorchestes keirasabinae sp. nov. is a member of the
Raorchestes nerostagona group and shows a well-supported sister-group relationship
with R. nerostagona (Fig. 2). For the mitochondrial 16S rRNA, Raorchestes keirasabinae sp.
nov. is divergent from other members of the group as: 6.16.6% from R. crustai and
3.13.7% from R. nerostagona.
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 33/78
Morphological diagnosis and comparison. Raorchestes keirasabinae sp. nov. can be
distinguished from other known congeners, except members of the Raorchestes
nerostagona group, by the combination of the following morphological characters:
medium-sized (male SVL 2931 mm) predominantly canopy dwelling species; snout
vertical in lateral view; webbed ngers; nearly fully webbed toes; and a distinct dermal
fringe along the outer margins of the fore and hind limbs (Fig. 6). Further, its distribution
is reportedly restricted to regions south of Palghat gap in the Western Ghats, whereas
its sister species R. nerostagona is found north of Palghat gap.
Within the Raorchestes nerostagona group, Raorchestes keirasabinae sp. nov. differs
from the closely related R. nerostagona by relatively reduced webbing between ngers,
I12
+
II23
-
III2
1
/
2
2IV (vs. more extensive, I12
+
II22
1
/
2
III22
-
IV) as well as toes,
I12II1
+
2III1
+
2IV21
+
V (vs. more extensive, I12II12
-
III12
-
IV21V); and its snout
length nearly equal to the eye diameter, SL/EL ratio 1.01.02 (vs. snout relatively longer
than eye, SL/EL ratio 1.321.47). Further, Raorchestes keirasabinae sp. nov. differs
from R. crustai by the outline of its snout nearly rounded in dorsal and ventral view
(vs. nearly pointed), vertical in lateral view (vs. obtuse); well-developed dermal fringe
along the outer margins of the fore and hind limbs (vs. weakly-developed); nearly fully
webbed toes, I12II1
+
2III1
+
2IV21
+
V (vs. relatively reduced webbing between toes,
I12
+
II23
-
III23IV21
2
/
3
V); and dorsal skin granular with prominent spinular
Figure 6 Raorchestes keirasabinae sp. nov. (AF) In life. (AC) Holotype (BNHS 6097, adult male),
dorsolateral view. (D) Enlarged view of spinular projections on the upper eyelid of the holotype.
(E) Dorsal view (not collected). (F) Frontolateral view (not collected). (GN) Holotype (BNHS 6097,
adult male), in preservation. (G) Dorsal view. (H) Enlarged view of spinular projections on the upper
eyelid. (I) Ventral view. (J) Lateral view of head. (K) Ventral view of hand. (L) Schematic illustration of
webbing on hand. (M) Ventral view of foot. (N) Schematic illustration of webbing on foot.
Full-size
DOI: 10.7717/peerj.10791/g-6
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 34/78
projections on lateral surfaces of head, between eyes, on upper eyelids, and on dorsum and
anks (vs. dorsal skin granular with a few spinular projections on upper eyelids).
Description of holotype (measurements in mm). Medium-sized adult male (SVL 29.6)
with a slender body; head wider than long (HL 11.3; HW 12.1; MN 9.7; MFE 7.8; MBE 4.1);
outline of the snout rounded in dorsal and ventral view, vertical in lateral view; snout
length (SL 4.5) nearly equal to the horizontal diameter of eye (EL 4.4); loreal region
obtusely concave, canthus rostralis sharp; tympanum (TYD 1.4) 31.8% of eye diameter
(EL 4.4); supratympanic fold rather indistinct; tongue emarginate with a lingual papilla.
Forearm (FAL 5.8) shorter than hand (HAL 8.9); ngers without prominent lateral
dermal fringe; webbing present, I12
+
II23
III2
1
/
2
2IV; subarticular tubercles rather
prominent, rounded, III1 and IV1 double, III2 and IV2 weakly-developed; prepollex rather
distinct and oval; supernumerary tubercles present. Hindlimbs moderately long, thigh
(TL 15.7) longer than shank (SHL 14.5) and foot (FOL 12.9); distance from heel to tip of
toe IV (TFOL 20.3); foot webbing: I12II1
+
2III1
+
2IV21
+
V; dermal fringe along toe V
present with serrated margins, ending with a well-developed spinular projection on the
heel; subarticular tubercles rather prominent, rounded, simple, IV1 and V1 weakly-
developed; supernumerary tubercles absent, foot ventral side granular (Fig. 6).
Dorsal skin on snout shagreened to granular; lateral surfaces of head, between eyes, and
upper eyelids, glandular with short spinular projections; anterior and posterior parts of
dorsum prominently glandular with spinular projections; dorsal surfaces of fore and
hind limbs shagreened with some scattered granules; a distinct dermal fringe along the
outer margin of the fore and hind limbs, ending with a well-developed spinular projection
on the heel and elbow. Ventral surfaces of throat, chest, belly, and posterior part of thighs
glandular (Fig. 6).
Colour of holotype. In life. Anterior part of dorsum brown with black and dark brown
patches, posterior half green with grey patches; snout light green dorsally; lateral surfaces
of head dark greyish-brown; dorsal surface of fore and hind limbs light greyish-brown
with irregular dark grey and light green cross-bands; lateral surfaces of belly with brown
and grey mottling; groin and anterior parts of thigh brown with white patches; posterior
margins of thigh and shank chocolate dark brown; hand and foot brownish-grey with
green tinge; iris reddish-grey with faint horizontal brown band. Ventral surfaces off-white,
with variable amounts of brown or grey spots forming a vermiculated pattern; throat
darker compared to the belly, with dark grey margins and bands along the lips (Fig. 6).
In preservation. Anterior part of dorsum dark greyish-brown; posterior half light brown
with light pink tinge; snout light pinkish-grey; lateral surfaces of head light brown; dorsal
surface of fore and hind limbs light pinkish-brown with dark brown cross-bands; lateral
surfaces of belly light brown vermiculated with cream white; groin brown with white
patches; posterior margins of thigh and shank brown. Ventral surfaces light greyish-brown
with white and brown spots; throat darker brown compared to the belly, with dark brown
margins and bands along the lips (Fig. 6).
Garg et al. (2021), PeerJ, DOI 10.7717/peerj.10791 35/78