Indirana chiravasi, a new species of
Leaping Frog (Anura: Ranixalidae) from Western Ghats of India
Anand D. Padhye 1,
Nikhil Modak 2 & Neelesh Dahanukar 3
1 Department of Zoology,
Abasaheb Garware College, Karve Road, Pune, Maharashtra 411004, India
2 Department of Biodiversity,
Abasaheb Garware College, Karve Road, Pune, Maharashtra 411004, India
3 Indian Institute of Science
Education and Research (IISER), G1 Block, Dr. Homi Bhabha Road, Pashan, Pune,
Maharashtra 411008, India
3 Systematics, Ecology and
Conservation Laboratory, Zoo Outreach Organization (ZOO), 96 Kumudham Nagar,
Vilankurichi Road, Coimbatore, Tamil Nadu 641035, India
1 anand.padhye@mesagc.org (corresponding author), 2 nikhilsmodak@gmail.com,3 n.dahanukar@iiserpune.ac.in
Abstract: Indirana chiravasi, a new species of leaping
frog is described from the northern Western Ghats around Amboli, Sindhudurg
District, Maharashtra, India. It differs from all its congeners based on a
combination of characters including presence of median single internal vocal
sac, head longer than wide, distinct canthus rostralis, tympanum 2/3rdto 3/4th the diameter of eye, vomerine teeth in two oblique series
at the posterior border of choanae, long midventral lingual papilla, first
finger longer than or equal to second, presence of double outer palmer
tubercle, thin and elongated inner metatarsal tubercle, absence of outer
metatarsal tubercle, webbing moderate, dorsal skin with glandular folds but
without warts, ventral skin smooth without mottling and palms and soles dark
brown. Molecular analysis based on mitochondrial 12S and 16S genes and nuclear
rhodopsin and rag1 genes suggests that the species is genetically distinct from
other species for which genetic data is available. Preliminary observations on
the development of the species are also provided. We also provide genetic data and images
for Indirana gundia collected from the type locality.
Keywords: Endemic frogs, Indirana gundia, molecular
phylogeny, new species, taxonomy.
doi: http://dx.doi.org/10.11609/JoTT.o4068.6293-312 | ZooBank: urn:lsid:zoobank.org:pub:89196447-08BF-4F56-B37F-78A617DCCFB2
Editor: Annemarie Ohler, MusŽum national dÕHistoire
naturelle, Paris, France. Date
of publication: 26 September 2014 (online & print)
Manuscript details: Ms # o4068 | Received 23 June
2014 | Final received 16 September 2014 | Finally accepted 17 September 2014
Citation: Padhye, A.D., N. Modak &
N. Dahanukar (2014). Indirana chiravasi, a new species of Leaping Frog
(Anura: Ranixalidae) from Western Ghats of India. Journal of Threatened Taxa6(10): 6293–6312; http://dx.doi.org/10.11609/JoTT.o4068.6293-312
Copyright: © Padhye et al. 2014. Creative Commons
Attribution 4.0 International License. JoTT allows unrestricted use of this
article in any medium, reproduction and distribution by providing adequate
credit to the authors and the source of publication.
Funding: Nikhil Modak is supported by DST-INSPIRE Student Fellowship [IF
120398]. Nikhil Modak was supported by Ernst Mayr Travel Grant, MCZ, Harvard
University, US, for examination of specimens at BMNH, London and MNHN, Paris.
Neelesh Dahanukar is supported by DST-INSPIRE Faculty Fellowship and Research
Grant [IFA12- LSBM-21].
Conflict of Interest: The authors declare no competing interests. Funding sources had no
role in study design, data collection, results interpretation and manuscript
writing.
Author Contribution: ADP diagnosed the species. ADP, NM and ND collected specimens. NM
studied the type and comparative material. NM and ND performed molecular
analysis. ND performed statistical analysis. ADP, NM and ND wrote the paper.
Author Details: Anand D. Padhye is an Associate Professor in
Zoology. He works on systematics, ecology, diversity, distribution and
evolution of amphibians. Nikhil Modakis a doctoral student. He is interested in ecology, systematics and evolution
of amphibians of the Western Ghats. Neelesh
Dahanukar works in ecology and evolution with an
emphasis on mathematical and statistical analysis. He is also interested in
taxonomy, distribution patterns and molecular phylogeny of fish and frogs.
Acknowledgements: We thank Head of the Zoology and Biodiversity Departments and
Principal, MES Abasaheb Garware College, Pune and Indian Institute of Science
Education and Research, Pune, for providing infrastructural facilities. We are
thankful to Dr. H.V. Ghate for helpful discussions and valuable suggestions. We
are grateful to Dr. Asad Rahmani, Director; Dr. Deepak Apte, COO; Rahul Khot,
incharge Natural History Collection; and Vithoba Hegde, senior field assistant,
for their help during study of the museum specimens and registration of
specimens at Bombay Natural History Society (BNHS), Mumbai. NM wishes to
specially thank Dr. Barry Clarke, Curator; Dr. David Gower and Dr. Ralph Britz
for their help and advice while working at Natural History Museum (BMNH),
London. NM is indebted to Dr. Annemarie Ohler of Amphibians and Reptiles
section, MusŽum national dÕHistoire naturelle (MNHN) for her help and guiding
discussions about taxonomy. NM is also thankful to the staff at BMNH and MNHN
for their kind help. We also thank Dr. P.S. Bhatnagar, officer-in-charge, and
Dr. Shrikant Jadhav, Zoological Survey of India, Western Regional Center
(ZSI-WRC), Pune, for their help in registering specimens in ZSI-WRC. We are
thankful to Dr. Sanjay Molur and Keerthi Krutha for helping in registration of
specimens in the Wildlife Information Liaison Development (WILD), Coimbatore.
We thank Avishkar Munje, Shauri Sulakhe, Abhijeet Bayani and Unmesh Katwate for
photographying the material. NM would like to thank Hemant Ogle for providing
stay and help during field work at Amboli. We would also like to thank Abhijeet
Bayani, Anushree Jadhav, Anil Pujari, Chandrakant Redican, Keerti Krutha,
Sameer Padhye, Sayali Sheth, Shauri Sulakhe, Shriraj Jakhalekar, Shruti
Paripatyadar, Siddharth Kulkarni, and Unmesh Katwate for assisting in field
work.
For figures, images, tables -- click here
INTRODUCTION
The
genus Indirana Laurent, 1986, is one of the 10 endemic anuran genera of
the Western Ghats of India and the sole member of the family Ranixalidae
Dubois, 1987. Genus Indirana,
which has terrestrial tadpoles, is characterized by the presence of vomerine
teeth, mid-ventral lingual papilla, keratodont formula of 3–5/3–4,
T or Y-shaped terminal phalanges and absence of terminal intercalary cartilage
(Laurent 1986; Frost et al. 2006). Currently, genus Indirana comprises of 10 valid species, namely Indirana
beddomii (GŸnther, 1876), I. brachytarsus (GŸnther, 1876), I.
diplosticta (GŸnther, 1876), I. leptodactyla (Boulenger, 1882), I.
phrynoderma (Boulenger, 1882), I. semipalmata (Boulenger, 1882), I.
leithii (Boulenger, 1888), I. longicrus (Rao, 1937), I.
tenuilingua (Rao, 1937) and I. gundia (Dubois, 1986).
The
true species diversity of Indirana within the Western Ghats, however, is
poorly understood as several species are suggested to be in species complexes
(Nair et al. 2012a,b; Modak et al. 2014). With the presence of undescribed species (Nair et al. 2012a,b; Modak et
al. 2014) and poor knowledge on the distribution of known species (Modak et al.
2014), the knowledge on Indirana is plagued by both Linnean and
Wallacean shortfalls (Bini et al. 2006). Because several species of Indirana are under threatened category
(Stuart et al. 2008), taxonomic studies on this group are of immediate concern.
While
studying the diversity and distribution of species under the genus Indirana,
we came across a population showing marked difference in morphology from its
congeners and genetically distinct from species for which genetic data is
available. This population is described as a new species of Indirana from Amboli, in Western Ghats of southern Maharashtra, India.
MATERIALS AND METHODS
Study site and specimen
collection
Specimens
of the new species were collected from Amboli (15.9560N & 73.9970E;
744m), Sindhudurg District, Maharashtra, India. Five male and two female specimens were
collected and preserved in absolute alcohol for further analysis. Two specimen of Indirana gundiawere collected from non-protected area of Gundia, Karnataka, for genetic
analysis. Collection of specimens
was kept at the minimum and all collections were made following the IUCN (2008)
guidelines for use of threatened species in research.
Museum details
Specimens
studied in this paper are deposited in the museum collection of Natural History
Museum, London (BMNH); MusŽum National de histoire Naturelle, Paris (MNHN),
Bombay Natural History Society, Mumbai (BNHS); Wildlife information Liaison
Development Society, Coimbatore (WILD) and Zoological Survey of India, Western
Regional Center, Pune (ZSI-WRC). Inger et al. (1984) designated BMNH
1947.2.27.92 as a lectotype of Indirana brachytarsus. Therefore, we have
considered the specimen BMNH 1947.2.2.85, currently a syntype of I.
brachytarsus, as paralectotype.
Morphometry
Morphometry
was done with the help of a digital caliper (Ocean Premium measuring
instruments) to the nearest 0.1mm. Totally, 27 characters were selected
following Biju et al. (2011) and Dubois & Ohler (1999) with some
modifications for morphometry, viz.—SUL (Length of specimen from snout to
the visible tip of urostyle); HL (head length: from the posterior border of
tympanum to the tip of snout); HW (head width: width of head between to
posterior borders of tympanum); SL (Snout Length: from the anterior orbital
border to the tip of snout); EL (Eye Length: length of eye between two orbital
borders); TYL (maximum tympanum length); UEW (upper eyelid width); SNL (snout
to nostril distance); ENL (eye to nostril distance); INL (inter-narial
distance); IOL (inter-orbital distance: minimum distance between two eyelids);
UAL (Upper arm length); FoAL (Fore-arm Length); F1 to F4 (Finger 1 to Finger 4
length from the base of the sub-articular tubercle); F3D (finger three disc
width); F3W (finger three width at the base of disc); THL (thigh length); TL
(Tibia/shank length); ACL (Astragalo-calcaneal length); FOL (Foot length: from
the base of the inner metatarsal tubercle to the tip of the fourth toe); TFOL
(Total foot length: from the tibio-tarsal articulation to the tip of fourth
toe) and T1 to T5 (Toe1 to Toe5 length from the base of the respective
sub-articular tubercle). Webbing formula was determined following the method
provided by Savage & Heyer (1967) with modifications by Myers &
Duellman (1982).
Statistical analysis
Statistical
analysis of the morphometric data was performed on size adjusted measurements
by taking all measurements as percent of SUL. The null hypothesis that the data is
multivariate normal was checked using Doornik & Hansen (2008) omnibus. MANOVA/CVA was performed to understand
whether related species of Indirana form significantly different
clusters (Huberty & Olejnik 2006). PillaiÕs trace statistic was performed to find the significant
difference between the clusters (Harris 2001). Statistical analysis was performed in
PAST (Hammer et al. 2001).
Molecular analysis
Two
specimens of the new species (BNHS 5890 and WILD-14-AMP-489) and two specimens
of topotypic Indirana gundia (WILD-14-AMP-499 and WILD-14-AMP-500) were
used for molecular work. Genomic DNA was extracted from thigh muscle tissue.
The tissue was digested at 550C using STE buffer (50 mM Tris-HCl, 20
mM EDTA and 50μl of 10%SDS) with 10μl of 20mg/ml Proteinase K. RNase
treatment was given for two hours at 370C. Final extraction process was done using
phenol-chloroform method. Polymerase Chain Reaction was performed for amplification of two
mitochondrial genes (12S and 16S) and two nuclear genes (rho and rag 1) (Table
1). In addition to the DNA
extracted in the current study, we also used DNA extracted in a previous study
(Modak et al. 2014) for amplification of Rag1 gene from two topotypes of Indirana
leithii BNHS 5590 and BNHS 5591. PCR reaction was performed in a 25μl reaction volume containing
5μl of template DNA (~200ng), 2.5μl of 10X reaction buffer (100 mM
Tris pH 9.0, 500 mM KCl, 15 mM MgCl2, 0.1% Gelatin), 2μl of 25 mM MgCl2,
1μl of 10 mM dNTPs, 1μl of each primer, 1μl Taq polymerase and
16.5μl nuclease free water. The thermal profile was 10 minutes at 950C,
and 35 cycles of 1 minute at 940C, 1 minute at respective annealing temperature
for 12S, 16S, rho and rag1 primers (Table 1) and 2 minutes at 720C,
followed by extension of 10 minutes at 720C. Amplified DNA fragments
were purified using the ÔPromega Wizard Gel and PCR clean upÕ system and
sequenced. The purified PCR products were sequenced using ABI prism 3730
sequencer (Applied Biosystems, USA) and Big dye terminator sequencing kit (ABI
Prism, USA). Sequences were
analyzed by BLAST tool (Altschul et al. 1990). These sequences have been deposited in
GenBank (Accession numbers KM386526–KM386543).
Additional
sequences of related species were retrieved from NCBI GenBank database
(http://www.ncbi.nlm.nih.gov/). GenBank accession numbers for the sequences used for the analysis are
provided in Appendix A. Gene
sequences were aligned separately using MUSCLE (Edgar 2004) implemented in MEGA
6 (Tamura et al. 2013) and were concatenated to make a combined matrix of 1342
nucleotides. Best fit model for
nucleotide substitution was selected in TOPALi v2.5 (Milne et al. 2004) based
on minimum Akaike Information Criterion (AIC) value (Posada & Crandall
2001). The phylogenetic
relationships were inferred by maximum likelihood analysis using RAxML
(Stamatakis 2006) implemented in TOPALi v2.5 (Milne et al. 2004). Reliability of the phylogenetic tree was
estimated using bootstrap values run for 1000 iterations.
RESULTS
Indirana chiravasi sp. nov.
(Images 1, 2, 3a–c,
4a–b, 5, 6, 8, 9, 10)
urn:lsid:zoobank.org:act:21DA778F-2F2A-4FD8-9551-85D734A5BB45
Holotype
BNHS
5888, male, 11.vi.2013, 27.3mm SUL, Amboli, Sindhudurg District, Maharashtra,
India, coll. Nikhil Modak, Neelesh Dahanukar, Keerthi Krutha and Unmesh
Katwate.
Paratypes
BNHS
5889, female, 9.vi.2014, 39.2mm SUL, Amboli, Sindhudurg District, Maharashtra,
India, coll. Nikhil Modak; BNHS 5890, male, 11.vi.2013, 25.0mm SUL, Amboli,
Sindhudurg District, Maharashtra, India, coll. Nikhil Modak, Neelesh Dahanukar,
Keerthi Krutha and Unmesh Katwate; WILD-14-AMP-489, male, 11.vi.2013, 24.7mm
SUL, Amboli, Sindhudurg District, Maharashtra, India, coll. Nikhil Modak,
Neelesh Dahanukar, Keerthi Krutha and Unmesh Katwate; WILD-14-AMP-490, 31.7mm
SUL, female, 9.vi.2014, Amboli, Sindhudurg District, Maharashtra, India, coll.
Nikhil Modak; WILD-14-AMP-491, male, 19.vi.2013, 25.6mm SUL, Amboli, Sindhudurg
District, Maharashtra, India, coll. Nikhil Modak, Neelesh Dahanukar, Keerthi
Krutha and Unmesh Katwate; ZSI-WRC A/1541, male, 11.vi.2013, 25.2mm SUL, Amboli,
Sindhudurg District, Maharashtra, India, coll. Nikhil Modak, Neelesh Dahanukar,
Keerthi Krutha and Unmesh Katwate.
Diagnosis
Indirana chiravasi sp. nov. differs from all other congeners based
on the following combination of characters: medium-sized (24.7–39.2 mm
SUL) frog, with median single internal vocal sac, head longer than wide,
distinct canthus rostralis, tympanum 3/4th the diameter of eye in
males and 2/3rd in females, vomerine teeth in two oblique series at
the posterior border of choanae, long midventral lingual papilla, first finger
longer than or equal to second, presence of double outer palmer tubercle, thin
and elongated inner metatarsal tubercle present, outer metatarsal tubercle
absent, webbing moderate and differs in male (I1-2II1-3III1¼-3IV3-1¼V)
and female (I1-2II1-2½III1¼-3IV3-1¼V) by having reduced
webbing on the third toe in males as compared to females, dorsally skin with
glandular folds but without warts, ventrally skin smooth without mottling and
palms and soles dark brown.
Description
General
appearance of holotype as in Image 1 and of female paratype as in Image 2.
Morphometric details as in Table 2.
Description of the holotype
(BNHS 5888; male) (all measurements in mm)
Medium-sized
(SUL 27.3); head longer than wide (HL 11.4 > HW 9.8); snout longer than
horizontal diameter of eye (SL 4.8 > EL 3.7); pupil horizontal; outline of
snout rounded in shape dorsally (Image 1a), truncated laterally (Image 3a);
ventrally snout protruding beyond the mouth (Image 3c); nostrils nearer to snout
than to the eye (SNL 2.0 < ENL 2.7); tympanum about 3/4th the
diameter of eye, very close to eye; supra-tympanic fold distinct; upper eyelid
width slightly more than half the horizontal diameter of eye; upper eyelids
bear very few granulations; inter-narial distance almost equal to the
inter-orbital distance (INL 2.6 Å IOL 2.6); canthus rostralis obtuse; loreal
region slightly concave and oblique; vomerine teeth in two slightly oblique
rows at the posterior border of choanae; tongue thin, bifid; bear a long mid
ventral papilla.
Upper
arm shorter than fore arm (UAL 5.3 < FoAL 6.0); hand long (PAL 6.7); finger
lengths from shortest to longest – F2 (1.9) < F1 (2.4) < F4 (2.6)
< F3 (3.5); palmer tubercles present, outer palmer tubercle double,
subarticular tubercles moderate, supernumerary tubercles present, single;
nuptial pads on the sides of first finger, distinct, flat, granular; finger
discs moderate in shape, broad, truncate, bearing semicircular groove; fingers
without web or fringe of skin (Image 4a).
Hindlimbs
long; thigh shorter than shank (tibia) (THL 13.4 < TL 13.9); thigh bearing
distinct femoral glands, occupying posterior-ventral side of thigh;
astragalus-calcaneum about half the length of foot; total foot length
(including astragalus-calcaneum) longer than tibia (TFOL 18.0); toe lengths
from shortest to longest are- T1 (1.6) < T2 (2.7) < T5 (4.2) ² T3 (4.2)
< T4 (6.5); toe discs moderate; bear semicircular groove; inner metatarsal
tubercle thin, elongated and shovel shaped; outer metatarsal tubercle absent;
supernumerary tubercles absent; subarticular tubercles moderate; tarsal fold
and outer phalangeal fringe absent; webbing formula
I1-2II1-3III1¼-3IV3-1¼V (Image 5a).
Dorsal
and ventral skin smooth; few longitudinal folds on dorsal side; lateral side
granular.
Description of female
paratype (BNHS 5889) (all measurements in mm)
Medium-sized
(SUL 39.2); with head longer than wide (HL 14.9 > HW 13.9); snout longer
than eye (SL 6.5 > EL 5.0); outline of snout rounded in shape dorsally
(Image 2a); truncated laterally (Image 3b); ventrally protruding beyond the
mouth (Image 2b); nostrils nearer to snout than to the eye (SNL 2.6 < ENL
3.9); tympanum about 2/3rd the diameter of eye, slightly more apart
from eye than in male; supra-tympanic fold distinct; upper eyelid width
slightly more than half the horizontal diameter of eye; upper eyelid bearing
very few granulations; inter-narial width slightly narrower than inter-orbital
distance (INL 4.1 < IOL 4.2); canthus rostralis obtuse; loreal region slightly
concave and oblique; vomerine teeth in two slightly oblique rows at the
posterior border of choanae; tongue thin, bifid; bearing a long mid-ventral
papilla.
Upper
arm shorter than fore arm (UAL 7.1 < FoAL 8.4); hand about 1/5thof SUL; finger lengths from shortest to longest - F2 (3.1) < F1 (3.5) <
F4 (4.1) < F3 (5.6); palmer tubercles present, outer palmar tubercle double;
subarticular tubercles moderate; all supernumerary tubercles present, single;
finger discs moderate in shape, broad, truncate, bearing semicircular groove;
fingers without web or fringe of skin (Image 4b).
Hindlimb
long, thigh shorter than tibia (THL 20.0 < TL 23.2); astragalus-calcaneum
about half the length of foot; total foot length (including
astragalus-calcaneum) longer than tibia; toe lengths in order of T1 (2.9) <
T2 (4.7) < T5 (7.4) ² T3 (7.4) < T4 (12.8); toe discs moderate; bear
semicircular groove; inner metatarsal tubercle thin, long; outer metatarsal
tubercle absent; supernumerary tubercles absent; subarticular tubercles moderate;
tarsal fold and outer phalangeal fringe absent; webbing formula
I1-2II1-2½III1¼-3IV3-1¼V (Image 5b).
Dorsal
and ventral skin smooth; few longitudinal folds on dorsal side; lateral side
granular.
Colouration
Male: Dorsal colour grey to brown in preservation (Image 1) and olive
brown with scattered yellow markings in living specimen (Image 6) with W-shaped
marking comprising of densely organized black spots; black strip running from
tip of snout to shoulder through eye and tympanum; creamy white band across
head between upper eyelids usually present followed posteriorly by a dark band;
in life, olive green to brown band running from above shoulder to groin (Image
6); forelimb bearing transverse bands also on fingers; palm dark brown in
colour at least in live condition; brown bars on thigh, tibia, outer side of
foot and dorsal surface of toes; ventral and inner side of foot dark brown in
colour up to tibiotarsal articulation; ventrally white with few melanophores
visible only under magnification.
Female: General appearance as in male. Dorsally more pale than male without
W-shaped marking (Image 2); darker flanks; limbs with dark cross bars in life,
faded in preservation.
Sexual Dimorphism
Tympanum
about 3/4th the diameter of eye in male and 2/3rd the diameter
of eye in female. Tympanum very
close to eye in male and slightly farther apart in female (Image 3). Inner metatarsal tubercle thin and
shovel-shaped in male while slightly thicker and long in female (Image 5). Webbing formula I1-2II1-3III1¼-3IV3-1¼V
in male and I1-2II1-2½III1¼-3IV3-1¼V in female. Breeding males show single internal
vocal sac that is visible only during calling (Appendix B); bearing nuptial pad
on the outer side of first finger and femoral glands on thighs; mature eggs
visible from transparent latero-ventral skin of breeding females.
Etymology
The
specific epithet, a combination of words ÔchirÕ (singular) or ÔchiraÕ (plural)
which means crevice or crevices in Marathi and ÔvasiÕ in Sanskrit means
Ôinhabitant ofÕ, which emphasizes crevice dwelling habit of this species. The specific name is noun in apposition.
Distribution
The
species is currently known only from its type locality at Amboli (15.9560N
& 73.9970E; 744m), which is a small hill station in the
southwestern Maharashtra, India (Fig. 1).
Habitat
The
species occupies lateritic rocky outcrops (Image 7a). It is often found in the crevices of the
laterite boulders (Image 7b). Males
were mostly seen while calling from the wet rocks or boulders covered with
mosses. Females were collected from
under the log in the forest and from under the roadside stone.
Natural history and
description of tadpoles
Eggs
were seen laid under the mosses on lateritic wet rocks and boulders (Image
8). Hatchlings remain at the egg
laying site (Image 9a). Embryos,
hatchlings and tadpoles of two different stages were observed in the same
habitat. Unhatched eggs (Images
8a,b) show the embryos with external gills, parallel to stage 20 (Gosner
1960). Tadpoles were seen on wet
boulders feeding on algal matter (Image 9). Image 9a shows hatchlings, parallel to
stage 25 (Gosner 1960). Image 9b
shows tadpole in its terrestrial habitat with fully developed hind limbs
(without forelimb), parallel to stage 40 (Gosner 1960). Image 9c shows terrestrial tadpoles with
long, finless tail which is not under resorption, oral apparatus and fully
developed forelimbs as well as hind limbs, parallel to stage 42 (Gosner
1960). Image 9d shows stage 44
(Gosner 1960) with mouth beneath eye and greatly reduced tail, while image 9e
shows stage 46 (Gosner 1960) - a completely metamorphosed froglet.
Tadpoles
showed semi-condensed individual keratodont formula (Dubois 1994) as 4[A1-A4]/4[P1-P2].
The oral apparatus is divided into two lateral parts by large horny beak. The
first anterior keratodont ridge A1 is divided while three succeeding
anterior keratodont ridges A2–A4 are placed lateral
to the horny beak. On the posterior labia first keratodont ridge P1 is
marginal, present on the either sides, while the second one - P2 is
placed lateral to the horny beak. Third and fourth keratodont ridges - P3 and
P4 are continuous. Although, the ridge P3 appears to be
divided into four subunits the keratodont rows are continuous on it (Image 10).
Common name
Amboli
Leaping Frog.
Statistical analysis
Size
corrected morphometric data was not significantly different from multivariate
normal (Doornik & Hansen omnibus, Ep = 67.14, P = 0.0561). MANOVA suggested
that all species formed significantly distinct clusters (PillaiÕs trace =
5.001, F208,216 = 1.86, P < 0.0001). First three
canonical axes explained 86.81% of the total variation in the data where the
first axis explained 43.89%, second axis explained 25.39% and third axis
explained 17.54% of the total variation. First two canonical axes separated Indirana
chiravasi sp. nov. from I. beddomii, I. diplosticta, I.
leithii, I. leptodactyla and I. phrynoderma (Fig. 2a). Indirana
chiravasi sp. nov. was separated from I. brachytarsus, I. gundia andI. semipalmata on the third canonical axis (Fig. 2b). CVA loadings of morphometric characters
on the first three canonical axes are shown in Table 3. Characters such as ENL, INL, F3, F4, TL,
ACL, FOL, T1 and T2 separated Indirana chiravasi sp. nov. from other
related species.
Genetic analysis
Concatenated
genetic sequences of mitochondrial 12S and 16S genes and nuclear rho and rag1
genes had total 1342 bases. Best
fit model for the nucleotide substitution was general time reversal model with
gamma distribution and invariant sites (GTR+G+I, AIC = 10516.66, lnL =
-5157.33, G = 0.39, I = 0.34, df = 101, n = 1342). Maximum likelihood analysis of the
genetic data (Fig. 3) suggested that Indirana chiravasi sp. nov. formed
a monophyletic group genetically distinct from the other Indiranaspecies for which genetic data are available.
Comparison with other species
of Indirana
Indirana chiravasi differs from I. diplosticta, I. leithii, I.
leptodactyla, I. longicrus and I. phrynoderma in having first
finger equal to or longer than second finger (vs. first finger shorter than
second) (Image 4). Furthermore, I.
chiravasi has moderate webbing (I1-2II1-3III1¼-3IV3-1¼V*) vs.
reduced webbing (I2-2II2-3III2½-4IV4-2½V in I. diplosticta;
I1-2II2-3III3-4IV4-3V in I. phrynoderma and I1-2¼II2-3III3-4IV4-3V
in I. leptodactyla); distinct canthus rostralis (vs. indistinct canthus
rostralis in I. phrynoderma); smooth skin with glandular folds (vs.
warty skin in I. phrynoderma), presence of double outer palmer tubercle
(vs. single outer palmer tubercle in I. leithii); vomerine teeth in two
oblique series at the posterior border of choanae and long midventral lingual
papilla (vs. vomerine teeth none and tongue without papilla in I. longicrus)
and tympanum 2/3rd (in female) to 3/4th (in male) the
diameter of eye (vs. tympanum half the diameter of eye in I. longicrus). Raw genetic distance between I.
chiravasi and I. leptodactyla (as identified by Nair et al. 2012b)
is 23.9% for 16S gene and 12.3–12.7 % for all the genes together; betweenI. chiravasi and I. diplosticta (as identified by Nair et al. 2012b)
is 9.1–11.0 % for 16S gene and 4.2–4.7 % for all the genes
together; and between I. chiravasi and I. leithii (topotypic
material from Modak et al. 2014) is 12.5–12.8 % for 16S gene and
6.9–7.3 % for all the genes together.
The
whereabouts of the type specimen of Indirana tenuilinguadescribed by are not known and is suggested to be lost (Dubois 1984). Therefore, for the comparison of the new
species with I. tenuilingua, we have relied on the original description
by Rao (1937). Indirana
chiravasi differs from I. tenuilingua in having head longer than
broad (vs. head slightly wider than long), inter-orbital distance equal to or
wider than inter-narial distance (vs. interorbital width more than twice the
distance between the nostrils) and toes and fingers with deep semicircular
groove (vs. semicircular groove in front of the toes and fingers absent, faint
or indistinct).
Indirana chiravasi differs from I. semipalmata in having broader head
(35.2–36.4 % SUL vs. 33.6–33.7 % SUL), tympanum 2/3rd(in female) to 3/4th (in male) the diameter of eye (vs. tympanum
equal to or larger than eye in male and slightly smaller in female) and
moderately webbed toes with the webbing formula
I1-2II1-3III1¼-3IV3-1¼V in male and
I1-2II1-2½III1¼-3IV3-1¼V in female (vs. half webbed toes
with the webbing formula I2-2II2-3III2-3¼IV3¼-2V I both the
sexes). Raw genetic distance
between I. chiravasi and I. semipalmata (as identified by Nair et
al. 2012b) is 7.4–8.4 % for 16S gene and 4.1–4.3 % for all the
genes together.
Indirana chiravasi differs from I. beddomii in having smaller finger 2 length
(7.0–7.9 % SUL vs. 8.3–10.5 % SUL), webbing formula
I1-2II1-3III1¼-3IV3-1¼V in male and
I1-2II1-2½III1¼-3IV3-1¼V in female (vs. webbing formula,
I1-2II1-2III1-3IV3-1V, in both males and females) and long, thin, shovel-shaped
inner metatarsal tubercle (vs. long and stout inner metatarsal tubercle).
Genetic difference between I. chiravasi and I. beddomii cannot be
determined as Nair et al. (2012b) have considered several genetically distinct
populations as members of I. beddomii species complex.
Indirana chiravasi can be distinguished from I. brachytarsus in having
few longitudinal folds on dorsal side (vs. numerous longitudinal folds on dorsal
side), thin, shovel-shaped inner metatarsal tubercle (vs. long and stout inner
metatarsal tubercle) moderate webbing (vs. extensive webbing, webbing formula,
I1-2II1-21/2III1-3IV3-1V) and having broader head (35.2-36.4% SUL vs.
33.1-34.6% SUL). Furthermore, if the identification of I. brachytarsusby Nair et al. (2012b) is correct then the two species are also genetically
quite distinct (Fig. 3). Raw
genetic distance between I. chiravasi sp. nov. and I. brachytarsus(as identified by Nair et al. 2012b) is 11.6% for 16S gene and 8.0–8.3 %
for all the genes together.
Indirana chiravasi sp. nov. differs from I. gundia in the most prominent
feature of having a single internal vocal sac (vs. bilateral vocal sacs). The presence of bilateral vocal sac in I.
gundia not only appears in the original description (Dubois 1986) but they
can also be seen in two patches on the ventral side of the head (Image
3f). Furthermore, I. chiravasi sp.
nov. differs from I. gundia in having the webbing formula
I1-2II1-3III1¼-3IV3-1¼V in male and
I1-2II1-2½III1¼-3IV3-1¼V in female (vs. webbing formula,
I1-2II1-2⅟2III1-3IV3-1V, in both males and females) and inner metatarsal
tubercle thin shovel-shaped (vs. distinct and stout). Morphometrically (Fig. 2)
both the species form significantly distinct clusters (FisherÕs distance =
3.142, P = 0.004). Raw genetic
distance between I. chiravasi sp. nov. and I. gundia is
5.2–5.5 % for 16S gene and 2.8–3.1% for all the genes together.
DISCUSSION
Indirana chiravasi sp. nov. forms the eleventh species in the Western Ghats endemic
genera Indirana and monotypic family Ranixalidae. Phylogenetic analysis of Indiranaspecies based on two mitochondrial and two nuclear genes suggests that, I.
chiravasi forms a monophyletic group with topotypic I. gundia and
five specimens (IND/AA/DD/CC 200, 220, 227, 230 and 231) of I. beddomiispecies complex (as identified by Nair et al. 2012b).
Both
morphologically and genetically, Indirana gundia is one of the
close congeners of I. chiravasi. However, apart from the morphological
variations, there was a significant difference in the multivariate morphometric
analysis of the two species. Further, the two species are separated by a
genetic difference of 5.2–5.5 % for 16S gene. Based on the suggestions of Vences et
al. (2005) the high genetic divergence in 16S gene validates that I.
chiravasi is a distinct species. Further, based on the current distribution records, these two species
are separated by a geographical distance of more than 400km. Genetic data for I. gundia is
provided for the first time in the current study. This data is based on topotypic material
(Image 11), for which the species identity is confirmed by its morphological
comparison with type series as well as morphometric analysis. The two specimens of I. gundiadid not form a monophyletic group, although there was only 0.3% genetic
distance considering all genes together and 0.2% genetic distance in 16S
gene. This could be attributed to
small sample size (only two specimens) in the current study and further genetic
analysis with additional samples may reveal within species variation in I.
gundia.
Based
on the phylogenetic analysis (Fig. 3) five specimens (IND/AA/DD/CC 200, 220,
227, 230 and 231) of Indirana beddomii species complex (as identified by
Nair et al. 2012b) form a monophyletic group and fall between I. chiravasi andI. gundia. This cluster is separated from I. chiravasi by raw
genetic distance of 3.5–3.9 % and from I. gundia by 3.6–3.9
% in 16S gene. However, because of
the lack of information on the collection locality as well as details of museum
deposition, we could not compare these specimens with I. chiravasi.
Nevertheless, it should be noted that both I. chiravasi and I. gundiaare morphologically distinct from any of the syntypes of I. beddomii.
Despite
of the above mentioned lacunae, Nair et al. (2012b) have provided genetic data
for a wide distribution of Indirana populations in southern Karnataka,
Kerala and Tamil Nadu. Further,
Modak et al. (2014) have provided details for Indirana leithii based on
topotypic material and a wide distribution in the northern Western Ghats. As a result genetic data from Nair et
al. (2012b) and Modak et al. (2014) forms a good comparative material for
assessing the affinities between I. chiravasi and other populations of Indiranasp. nov. from a wide geographic range within the Western Ghats.
Recent
documentation of primitive breeding in Indirana species from Amboli by
Gaitonde & Giri (2014) actually refers to the breeding biology of I.
chiravasi based on the photographs provided by Gaitonde & Giri (2014).
Further,
according to our extensive survey records, there is no other species of Indiranapresent in Amboli. Gaitonde &
Giri (2014) have provided a detailed account of courtship and breeding
bahaviour of the species along with the fertilization success. However, they do not provide the
information on developmental stages.
Our
observations, therefore, complete the information on breeding and developmental
cycle of the species. Although we
have provided developmental stages of this species parallel to the stages
suggested by Gosner (1960), it is essential to note that the development and
metamorphosis of the genus Indirana is different from the general ranid
pattern, as the tadpoles do not have tail fins and are semi-terrestrial (Dubois
1986; Gaitonde & Giri 2014; Modak et al. 2014). The developmental stages of Indirana
chiravasi sp. nov. differs from the stages in Gosner (1960) as
follows. The unhatched eggs with embryo
of I. chiravasi sp. nov. showed presence of external gills. This stage is parallel to stage 20 of
Gosner (1960). However, hatching
does not occur at this stage. It should be noted that in I. chiravasi,
hatching does not occur until the development of operculum, which is a stage
parallel to stage 25 of Gosner (1960). Further, the semi terrestrial tadpole with oral apparatus, full tail
without any regression and fully developed forelimb (stage 42 of Gosner 1960)
persists for a long duration. In I.
chiravasi, tadpole of this stage continues to feed in its terrestrial
habitat for around a month after which metamorphosis completes. This is unlike the classical
metamorphosis described by Gosner (1960) where in on complete development of
fore limb there is onset of metamorphosis with changes such as the beginning of
tail regression, oral apparatus starts disappearing leading to formation of
adult like mouth and metamorphosis is essentially completed within a short
duration. These subtle differences
in the development of I. chiravasi from the pattern provided by Gosner
(1960), calls for the detailed study of developmental patterns in Western Ghats
endemic genera such as Indirana.
Description
of the new species Indirana chiravasi, and previous suggestions that
there are species complexes and undescribed species (Nair et al. 2012a, 2012b;
Modak et al. 2014), suggests that especially for an endemic and monotypic
family such as Ranixalidae, the Western Ghats are subject to Linnean shortfall
(Bini et al. 2006). Out of the 10
known species of this genus, Indirana gundia and I. phrynodermaare Critically Endangered; I. brachytarsus, I. leptodactyla and I.
diplosticta are Endangered; I. leithii is Vulnerable; I.
longicrus and I. tenuilingua are Data Deficient; and I. beddomiiand I. semipalmata are Least Concern (IUCN 2014). With high proportion of threatened
species within this endemic family, there is immediate concern for conservation
initiatives. Therefore, further
studies to resolve taxonomic status of species complexes and understanding
their distribution patters are essential. Moreover, additional information on ecology and natural history would
help in designing conservation strategies.
Comparative Material
Indirana beddomii: Syntype, BMNH 1947.2.27.72 (Female), Syntype, BMNH 1947.2.27.82
(female), Syntype, BMNH 1947.2.27.83 (Male), Syntype, BMNH 1947.2.27.85
(female), 4 exs., Malabar, coll. Col. Beddome; Syntypes, BMNH
1947.2.27.89–91 (females), 3 exs. Anamallays (=Annamalai), coll. Col.
Beddome; Syntype, BMNH 1947.2.4.86 and 87 (females), BMNH 1947.2.4.88 (Male), 3
exs., Sevagherry (=Sivagiri, Tamil Nadu), coll. Col. Beddome; Syntype, BMNH
1947.2.27.87 (female), Syntype BMNH 1947.2.27.88 (Male), 2 exs., Travancore,
coll. Col. Beddome.
Indirana brachytarsus: Lectotype, BMNH 1947.2.27.92 (Female), 1 ex., Anamallays
(=Annamalai), coll. Col. Beddome; Paralectotype, BMNH 1947.2.2.85 (female), 1
ex., Sevagherry (=Sivagiri, Tamil Nadu), coll. Col. Beddome.
Indirana diplosticta: Syntypes, BMNH 1947.2.2.21 and 23 (females), 2 exs., Malabar,
coll. Col. Beddome; Syntype, BMNH 1947.2.2.22 (male), 1 ex., Malabar, coll.
Col. Beddome.
Indirana gundia: Holotype, MNHN 1985.0633 (Male), 26.vii.1984, 1 ex., Gundia,
fort de Kemphole, ˆ lÕouest de Sakleshpur, Karnataka, Inde (Gundia, Kemphole
forest, west of Sakleshpur, Karnataka, India), coll. A. Dubois; Paratypes, MNHN
1985.0596 (male), 24.vii.1984, 1 ex., MNHN 1985.0599, MNHN 1985.0603, MNHN
1985.0605, MNHN 1985.0608, 1985.0610 and MNHN 1985.0628 (males), 26.vii.1984, 6
exs.; MNHN 1985.0637-0638 (females), 26.vii.1984, 2 exs., MNHN 1985.0611, MNHN
1985.0617–0620 and MNHN 1985.0622 (females), 27.vii.1984, 6 exs., Gundia,
fort de Kemphole, ˆ lÕouest de Sakleshpur, Karnataka, Inde (Gundia, Kemphole
forest, west of Sakleshpur, Karnataka, India), coll. A. Dubois. Topotype,
WILD-14-AMP-499 (Male), 1 ex., 29.vii.2014, Gundia, Karnataka (12.8250N
& 75.5690E, 128m), coll. A. Padhye, N. Modak and S. Sulakhe;
Topotype WILD-14-AMP-500 (female), 1 ex., 29.vii.2014, Gundia, Karnataka
(12.8290N & 75.6070E, 224m), coll. A. Padhye, N.
Modak and S. Sulakhe.
Indirana leithii: Topotypes, BNHS 2830–31, BNHS 2833, BNHS 2838–39
(females), 8.viii.1991, 5 exs., Matheran, Mumbai, India, coll. A.G. Sekar and
V. Hegde; Topotype, BNHS 5590 (Female), 30.ix.12, 1 ex., Matheran, Mumbai,
India, coll. N. Modak and A. Bayani.
Indirana leptodactyla: Syntype, BMNH 1947.2.29.39-40 (females), 2 exs., Malabar, coll.
Col. Beddome; Syntype BMNH 1947.2.29.41 (Male), 1 ex., Malabar, coll. Col.
Beddome; Non-Type, BMNH 1897.1.10.11 (female), 1 ex. Devicolum, Travancore,
1,219–2,133 m, coll. Fergusson.
Indirana phrynoderma: Syntypes, BMNH 1947.2.3.8–9 (males), 2 exs., Anamallays
(=Annamalai), coll. Col. Beddome.
Indirana semipalmata: Syntype, BMNH 1947.2.29.50 (female), 1 ex., Malabar, coll. Col.
Beddome; Syntype, BMNH 1947.2.29.51 (male), 1 ex., Malabar, coll. Col. Beddome.
Data
for I. longicrus and I. tenuilingua from Rao (1937) as the type
specimens are missing and are suggested to be lost (Dubois 1984)
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