Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 June 2020 | 12(9): 16136–16142
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
doi: https://doi.org/10.11609/jott.6031.12.9.16136-16142
#6031 | Received 21 April 2020 | Final
received 24 May 2020 | Finally accepted 11 June 2020
Osteological description of
Indian Skipper Frog Euphlyctis cyanophlyctis (Anura: Dicroglossidae) from the Western Ghats of India
Pankaj A. Gorule
1, Sachin M. Gosavi 2, Sanjay S. Kharat 3 & Chandani
R. Verma 4
1 Department of Life and
Environmental Sciences, University of Cagliari, Via Tommaso Fiorelli
1, 09126, Cagliari, Italy
2 Department of Zoology,
Maharashtra College of Arts, Science and Commerce, 246-A, JBB Road, Opp.
Alexandra Cinema, Nagpada, Mumbai Central,
Maharashtra 400008, India
1,3,4Department of Zoology, Modern
College of Arts, Science and Commerce, Ganeshkhind,
Pune, Maharashtra 400016, India.
1 pankajgorule7@gmail.com, 2 schn.gosavi@gmail.com,
3 kharat.sanjay@gmail.com, 4 verma.chandani25@gmail.com
(corresponding author)
1 & 2 authors contributed equally.
Editor: Anonymity requested. Date
of publication: 26 June 2020 (online & print)
Citation: Gorule,
P.A, S.M. Gosavi, S.S. Kharat
& C.R. Verma (2020). Osteological description of
Indian Skipper Frog Euphlyctis cyanophlyctis (Anura: Dicroglossidae) from the Western Ghats of India. Journal of Threatened Taxa 12(9): 16136–16142. https://doi.org/10.11609/jott.6031.12.9.16136-16142
Copyright: © Gorule
et al. 2020. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: Department of Science
and Technology (DST), Government
of India, under DST-FIST scheme.
Competing interests: The authors
declare no competing interests.
Acknowledgements: We would like to thank anonymous
reviewers for their valuable comments and inputs on the earlier version of the
manuscript. We also thank Neelesh Dahanukar, Indian
Institute of Science Education and Research (IISER), Pune for his constant
support, encouragement and help in specimen identification. We thank Nitin Sawant, Priyanka Gore, Manoj Pise, and Pradeep Kumkar for
field assistance. Authors are grateful
to Deepak Apte and Rahul Khot
for their support in the registration of specimens in BNHS, Mumbai.
Abstract: The present study provides
description of the osteology of Skipper Frog Euphlyctis
cyanophlyctis.
Seven adult specimens of E. cyanophlyctis
from northern Western Ghats of India were cleared and double stained for
studying osteological characteristics.
The baseline description of osteological characters of cranial and
post-cranial elements (paired nasals, tubular sphenethmoid,
well-developed vomerine teeth, arciferal pectoral girdle, fan-shaped omosternum, cartilaginous W-shaped xiphisternum,
hind limb with longest cylindrical humerus, V-shaped
pectoral girdle and phalangeal appendages) provided in present study will help
in further taxonomic investigations of the genus Euphlyctis. Further, the baseline information on
osteology of Skipper frog will serve as a reference material for investigations
related to malformations, either in this or related species. We also provide first observation on sacro-pelvic malformation in one of the studied specimens.
Keywords: Skeletal morphology, amphibian
decline, anthropogenic stressors, malformation, conservation.
Amphibians are declining globally with highest number
of species at risk of extinction than any other vertebrate group (Stuart et al.
2004). Limited information on the basic
biology, population status, distribution, life-history and potential threats to
the anurans is one of the major hurdles in amphibian conservation (Dinesh &
Radhakrishnan 2011; Dahanukar et al. 2013). In addition to population declines, amphibian
malformations has become a major conservation concern and studies reporting and
analyzing amphibian deformities have gained momentum
(Johnson et al. 1999; Schoff et al. 2003; Peloso 2016).
Investigations into the basic biology, including the osteology of a
species, will not only help in understanding its taxonomy, but also form a
baseline for comparative osteology of malformed individuals. Therefore, the fundamental objective of the
present study was to describe detailed osteology of the Indian Skipper Frog Euphlyctis cyanophlyctis
(Schneider, 1799) to serve as a baseline data for further investigations.
Adult specimens (n = 7; SVL= 25.50–55.67 mm) of E. cyanophlyctis consists of both male and female
individuals were collected from temporary pool situated at Sangrun
(18.404°N & 73.687°E) Haveli, Pune and Nere
(18.619°N & 73.702°E) Mulshi, Pune, Maharashtra,
India on 23 July 2017 and 2 August 2017, respectively. The specimens were cleared and double stained
differentially for bone and cartilage as per Potthoff
(1984). The osteological terminology
follows Trueb (1973), Duellman
& Trueb (1986) and Pugener
& Maglia (1997).
Cleared and double stained specimens are deposited in the museum
collection of Bombay Natural History Society (BNHS) under the accession numbers
BNHS 6031–BNHS 6037.
Irrespective of the sex, no significant differences
were observed in the osteology of seven specimens. The osteological representation of E. cyanophlyctis is shown in Figure 1. The detailed description of the osteology is
provided below.
Cranium (Figure 1A & B)
It is well-developed, triangular and dorso-ventrally flat structure. The frontoparietals
are paired, fully ossified and flat structure forming the roof of the
skull. They are separated throughout
their length, cover the sphenethmoid anteriorly and
extend till the exoccipitals posteriorly.
They fail to articulate with nasals anteriorly but firmly attached with
the prootic and exoccipitals posteriorly. Sphenethmoid is
located anterior to the cranial cavity and forms the posterior boundary of the
olfactory chambers. It is a
tubular-shaped bone formed by a combination of anterior ethmoidal and posterior
sphenoidal regions separated from each other by a transverse partition. Ethmoidal region separates into the right and
left halves by longitudinal partition, which further encloses the olfactory
sac. A small portion of the bone is
visible particularly at the posterior boundary (sphenoidal region), as it is
covered ventrally by parasphenoid and dorsally by
nasals and frontoparietals bones. Parasphenoid is
single, elongated, large dagger-like or inverted ‘T’ shaped bone forming the
cranial floor. It shows a pointed long
shaft directed anteriorly and its cross piece handle lies across the auditory
capsule. The occipitals form
posterior-most boundary of the cranium.
Presence of a large hole, foramen magnum is noticeable, which serves as
the entry point for the spinal cord. Postero-laterally, the foramen is surrounded by two roughly
oval-shaped exoccipitals. The
exoccipitals posteriorly have cartilaginous occipital condyles, which further
articulates with the first vertebra (atlas) anteriorly. The outer margin of each exoccipital is
firmly attached to the cartilaginous auditory capsule. The anterior wall and partly the roof and
floor of each capsule are formed by a fully ossified, roughly rectangular prootic. It is
partly covered by squamosal on its dorsal side.
The occipital region is enclosed by frontoparietals,
while the floor is occupied by a dagger-shaped parasphenoid. Supraoccipital and basioccipital are absent.
Sense capsule, consisting of an auditory capsule, an
olfactory capsule and an optical capsule, encloses the organs of the hearing,
the organs of the smell and the eye, respectively. The auditory and olfactory capsules are
firmly attached to the cranium, while the eyes are not fused with the
skull. Nasals are paired, fully ossified
bones that form the roof of the olfactory capsule covering the anterior dorsal
region of the skull and are not medially fused.
Their anterior ends extend to the dorsal processes of the premaxillae
and thus partially form the boundary of external nares. Septomaxillaries
are small, irregular-shaped bones present close to the anterior part of each
nasal bone. Vomers are paired,
completely ossified, roughly triangular bones, located ventrally to the nasal
capsule floor. The presence of a row of
vomerine teeth on the posterolateral margin is noticeable. Anterolateral and lateral margin bears pointed
projections.
Upper jaw (Figure 1A & B)
Premaxillae are paired bones, fuse medially,
well-ossified and form the anterior boundary of the maxillary arch. Each premaxilla has two rows (16 + 14) of
teeth. On the outer side, each
premaxilla articulates a maxilla of its respective side. Maxillae are quite long, sharply curved bone
and bears numerous, sharply pointed teeth arranged in two rows (34 + 51). At the middle of its length, it is
articulated with the palatine and pterygoid.
The maxilla is connected to the quadratojugal posteriorly. Quadratojugals are paired, short rod-like
bones, completely ossified. The
posterior portion of the quadratojugal underlies the margin of the
squamosal. Squamosals
are irregular shaped-bones located just above the pterygoid. Anteriorly it remains free, whereas
posteriorly it forms a connection to the auditory and prootic
capsules. Pterygoids are paired, fully
ossified, ‘Y’-shaped bone with three limbs, laterally positioned on either side
of cranium and ventrally positioned to the squamosal. The anterior limb is linked to the
maxilla. The inner limb is connected to
the pterygoid and the auditory capsule, whereas the posterior limb joins with
the quadratojugal. Pterygoids also
contribute for the formation of the posteroventral
margin of the orbit of respective side.
Palatines are paired, elongated, rod-shaped bones that are located
ventrally and form the anterior side of each orbit. They serve as a transverse link between the
anterior side of the cranium and the maxilla.
Lower jaw or mandible (Figure 1C)
It is semi-circular and consists of two halves or rami
which are connected to each other by a ligament at the anterior end. Teeth are absent. Each ramus consists of a core of Meckel’s
cartilage, surrounded by mento-meckelian, angulosplenial and dentary.
Mento-meckelian is small, entirely
cartilaginous and located at the extreme anterior end of the Meckel’s
cartilage, where both rami unite together.
Angulosplenial is a long, strongly curved
prominent bone forming most of the inner and posterior portion of each ramus of
the mandible. Its anterior end is
tapering, whereas the posterior end has articulating process/condyle for the quadrate
cartilage of the skull. Just close to
the articulating condyle, it also has a small knob-like coronary process. Dentary is an elongated rod-like bone
covering almost 50% of the outer surface of the anterior portion of Meckel’s
cartilage. Anteriorly, it extends up to
the Mento-meckelian, whereas posteriorly up to the
outer side of the coronary process.
Hyoid skeleton (Figure 1D)
It consists of thin cartilaginous shield-shaped hyoid
plate (=corpus), U-shaped hyoglossal sinus,
anterolateral (cartilaginous), anteromedial (longest), posterolateral processes
(cartilaginous and almost the same size as anterolateral), posteromedial (fully
ossified) processes and cartilaginous, thin, long, curved hyales
allowing the hyoid apparatus to be attached to the cranium. A small, thin, rounded medial element is
present on the anterior process of the hyale. Stalk
of anterolateral processes is comparatively thicker than its distal portion,
which bears the alary process. Hyoglossal sinus is deeply grooved. Posterolateral processes are long and nearly
50% of the total length of the posteromedial process.
Vertebral column (Figure 1L)
The presacral region consists of eight vertebrae, a
sacral region with one sacral vertebra and a caudal region with the urostyle. The atlas
(1st vertebra) anteriorly articulates with the posterior-most
regions of exoccipitals. Centrum of the atlas is comparatively larger than
other presacral vertebrae (visible from the ventral view). The presacral II–VII are procoelous,
while the presacral VIII is amphicoelous. All vertebrae connected to each other through
post-zygapophyses of one vertebra with the pre-zygapophyses of the next vertebra. The neural arches of each vertebra have a
well-developed dorsal ridge and a pair of transverse processes extending
laterally with terminal parasagital processes. The first four pairs of transverse processes
(presacrals II–IV) are relatively more robust than
those of last four pairs (presacrals V–VIII). The neural arches of I–V vertebrae are
imbricate. The relative lengths of
transverse processes are as follows: III > IV > V > VI=VII=VIII <
II. The transverse process of vertebrae
IV, V and VI are directed posteriorly, the VIII vertebra is directed
anteriorly, whereas III and VII are approximately perpendicular to the
notochordal axis. Sacral diapophyses are
laterally oriented, positioned perpendicular to the notochordal axis and are of
similar size to transverse processes of VI, VII, VIII vertebrae. Urostyle is
slender, shorter than the presacral length and articulates the sacrum through a
bicondylar articulation. It has a
prominent dorsal crest most of its length.
Pectoral girdle (Figure 1E)
It is arciferal in structure. Suprascapulae are
paired, completly ossified, with the larger partion forming the distal edge of the pectoral girdle and
articulates with scapula medially. Cleithrum is cartilaginous and covers most of the suprascapula.
Scapula is rectangular and articulates with the clavicle anterolaterally
and coracoid medially at the glenoid cavity.
Laterally it joins with supra scapula and the cleithrum. Clavicle is slender, completely ossified,
slightly curled, with a shape of bow that forms the anterior part of the
pectoral girdle. Procoracoids
are present, separated medially, extend along the dorsal posterior of
clavicles, and articulate with the scapula at the distal end. The coracoids are rectangular shaped, fully
ossified and distally expanded extremities which articulate with the epicoracoid medially and scapula ventrolaterally. Epicorocoids is
cartilaginous, separated throughout their length and forms arciferal
arrangement. Omosternum
is cartilaginous, fan-shaped expanded distal end and comparatively smaller than
‘W’ like shaped cartilaginous xiphisternum. Episternum is fully ossified, inverted ‘Y’
shaped structure, articulates with omosternum
anteriorly and procoracoids posteriorly. Mesosternum is
ossified with the anterior cartilaginous end and comparatively wider than the
episternum.
Forelimbs (Figure 1F and 1G)
Humerus is the longest bone of forelimb with well-defined
humeral crest, cylindrical in appearance, articulates into glenoid cavity
proximally and radio-ulna distally. Six
carpel elements (ulnare, distal carpel 3–4–5, distal
carpel 2, element ‘Y’, radial, and prepollex) are
present, representing ‘Type-C’ morphology of Fabrezi
(1992). The sesamoid is cartilaginous,
rounded and positioned above the radial.
The relative length of metacarpals is as follows:
II=IV>III>V. The phalangeal
formula is 2–2–3–3.
Pelvic girdle (Figure 1K and 1L)
It is ‘V’ shaped and consists of pair of ilia, ischia
and pubis. The ilial
shafts are round in cross-section and articulate with the sacral diapophysis.
Ischia and pubis are fused together, forming the acetabulum.
Hind limbs (Figure 1H, 1I, 1J)
Femur is the longest bone (slightly larger than
tibia-fibulae) of the body, articulates with the acetabulum proximally and with
tibia-fibula distally. Tibia-fibulae
distally articulate with tibiale and fibulare, which are separated throughout their length,
except for proximal and distal ends. Tibiale and fibulare are about
half of the length of tibia-fibula. A sesamoid is positioned proximally to tibiale. Pes
consists of fused distal tarsals 2–3, element ‘Y’ prehallical
elements (cartilaginous elements; I, II, III and IV), metatarsals and
phalanges. The relative length of the
metatarsals is as follows: V=IV=III>II>I.
The phalangeal formula is 2–2–3–4–3.
Sacro-pelvic malformation
In one of the specimens collected from Sangrun, we observed two sacral vertebrae with transverse
processes (Image 1A; malformed
individual) in contrast to the one sacral vertebra (Image 1B; normal individual).
Knowledge of the basic osteology of anurans is
critically important in taxonomic investigations (Lynch 1971). At present, the genus Euphlyctis
is represented by eight extant species (Priti et al.
2016). For the first time, using
skeletal characters, Deckert (1938) provided brief
generic description for Dicroglossus Günther
(1860) and placed cyanophlyctis under Dicroglossus (= Euphlyctis
Fitzinger 1843 according to Dubois 1980) other than Rana.
However, detailed osteological characters for any species of the genus Euphlyctis is not available, which limits
comparative analysis. Therefore, the
osteology of E. cyanophlyctis was compared
with the Quasipaa robertingeri
(= Quasipaa boulengeri)
and Nannophrys marmorata which belongs
to the family Dicroglossidae (Senevirathne
& Meegaskumbura 2015; Zhang et al. 2016). The comparative descriptions of the prominent
and distinctive osteological characters between E. cyanophlyctis,
Q. robertingeri and N. marmorata are
given in Table 1. Although the
characters presented in this study are generic rather than species-specific,
they could be used as a reference data for more thorough species-specific
osteological investigations of the genus Euphlyctis.
Several stressors (environmental pollution, greater
exposure to the UV–B radiation, and parasitic overload) contribute to the
development of abnormal or bizarre morphological features, especially affecting
the limbs and vertebrae of amphibians, which are referred to as ‘malformation’
(Silva et al. 2019). The incidence of
occurrence of malformed anurans is increasing (Blaustein
& Johnson 2003; Pelaso 2016). Therefore, the osteological description
provided in present study will also serve as a baseline data of normal form
which could be effectively use to differentiate malformed individuals from
normal onces.
At present it is difficult to pinpoint the exact cause/s of observed
malformation in E. cyanophlyctis, however, the
malformed individuals are often easy target for the predators, failed to
reproduce and thereby compromise with their survival and fitness (Sower et al.
2000; Blaustein & Johnson 2003; Bowerman et al.
2010). Owing to the global amphibian
decline and ongoing controversy over the types of amphibian malformations
caused by various factors, the information presented in this study not only
have implications for the continued investigation of amphibian malformations
but also has a conservation implication.
Table 1. Comparative analysis of osteological
characters of three species belongs to the family Dicroglossidae
representing three different genera.
Characters |
Euphlyctis
cyanophlyctis |
Quasipaa
robertingeri |
Nannophrys
marmorata |
Frontoparietal
|
Thin and
long |
Rigid and
broad |
Rigid and
broad |
Episternum |
Y-shaped |
Funnel
shaped |
Cylindrical |
Xiphisternum
|
W-shaped |
W-shaped |
Fan-shaped |
Omosternum
|
Fan-shaped
expanded distally |
Fan-shaped
expanded distally |
Tube like
fused with the cartilaginous epicoracoids |
Metacarpal
length |
II=IV>III>V |
IV>V>
II>III |
IV>III>V>II |
Phalanges
(Palm) |
2–2–3–3 |
2–2–3–3 |
3–3–4–4 |
Metatarsal
length |
V=IV=III>II>I
|
V>III
>V >II >I |
IV>V>III>II>I |
Phalanges
(Toes) |
2–2–3–4–3 |
2–2–3–4–3 |
3–3–4–5–4 |
Reference |
Present
study |
Zhang et
al. (2016) |
Senevirathne
& Meegaskumbura (2015) |
For figure & image - - click here
References
Blaustein, A.R. & P.T. Johnson (2003). Explaining frog deformities. Scientific American
288(2): 60–65. https://doi.org/10.1038/scientificamerican0203-60
Bowerman, J., P.T. Johnson & T. Bowerman (2010). Sublethal predators and their injured prey: linking
aquatic predators and severe limb abnormalities in amphibians. Ecology 91(1):
242–251. https://doi.org/10.1890/08-1687.1
Dahanukar, N., K. Krutha, M.S. Paingankar, A.D. Padhye, N. Modak & S. Molur (2013). Endemic Asian chytrid strain infection in threatened
and endemic anurans of the northern Western Ghats, India. PLoS
ONE 8(10): e77528. https://doi.org/10.1371/journal.pone.0077528
Deckert, K. (1938). Beitrage zur Osteologie
und Systematik ranider Froschelurche. Sitzungsberichte
der Gesellschaft Naturforschender Freunde
zu Berlin: 127–184.
Dinesh, K.P. & C. Radhakrishnan (2011). Checklist of amphibians of Western Ghats. Frog leg
16: 15–21.
Dubois, A. (1980). L’influence de l’homme sur la répartition des Amphibiens dans l’Himalaya central et
occidental. Comptes Rendus
de la Société de Biogéographie
55: 155–178.
Duellman, W.E. & L. Trueb
(1986). Biology of amphibians. First
Edition Philippines, McGraw-Hill Book Company, Baltimore, Maryland, 670pp.
Fabrezi, M. (1992). El carpo de anuros. Alytes 10(1):
1–29.
Günther, A. (1860). Contributions to a knowledge of the Reptiles of the
Himalaya mountains. Proceedings of the Zoological Society 1860: 148–175.
Johnson, P.T.J., K.B. Lunde, E.G. Ritchie & A.E. Launer (1999). The
effect of trematode infection on amphibian limb development and survivorship. Science
284(5415): 802–804. https://doi.org/10.1126/science.284.5415.802
Lynch, J.D. (1971). Evolutionary relationships, osteology, and
zoogeography of leptodactyloid frogs. Miscellaneous
Publication - University of Kansas, Museum of Natural History 53: 1–238.
Peloso, P.L. (2016). Osteological malformation in the tree frog Hypsiboas geographicus
(Anura: Hylidae). Phyllomedusa 15: 91–93. https://doi.org/10.11606/issn.2316-9079.v15i1p91-93
Potthoff, T. (1984). Clearing and staining techniques, pp. 35—37. In:
Moser, H.G., W.J. Richards, D.M. Cohen, M.P. Fahay,
A.W. Kendall, Jr., & S.L. Richardson (eds.). Ontogeny and Systematics of
Fishes. American Society of Ichthyologists and Herpetologists, Allen Press, Lawrence, KS. Special
Publication 1.
Priti, H., C.R. Naik, K.S. Seshadri, R. Singal,
M.K. Vidisha, G. Ravikanth
& K.V. Gururaja (2016). A new species of Euphlyctis
(Amphibia, Anura, Dicroglossidae)
from the west coastal plains of India. Asian Herpetological Research
7(4): 229–241. https://doi.org/10.16373/j.cnki.ahr.160020
Pugener, L.A. & A.M. Maglia
(1997). Osteology and skeletal
development of Discoglossus sardus (Anura:Discoglossidae).
Journal of Morphology 233(3): 267–286. https://doi.org/10.1002/(SICI)1097-4687(199709)233:3%3C267::AID-JMOR6%3E3.0.CO;2-0
Schneider, J.G. (1799). Historia Amphibiorum
Naturalis et Literarariae. Fasciculus Primus. Continens Ranas, Calamitas, Bufones, Salamandras et Hydros in Genera et Species Descriptos
Notisque suis Distinctos. Jena: Friederici Frommanni. 137–138.
Schoff, P.K., C.M. Johnson, A.M. Schotthoefer,
J.E. Murphy, C. Lieske, R.A. Cole, L.B. Johnson & V.R. Beasley (2003). Prevalence of skeletal and eye malformations in frogs
from north-central United States: estimations based on collections from
randomly selected sites. Journal of Wildlife Diseases 39(3): 510–521. https://doi.org/10.7589/0090-3558-39.3.510
Senevirathne, G. & M. Meegaskumbura
(2015). Life among crevices: Osteology of
Nannophrys marmorata (Anura: Dicroglossidae). Zootaxa 4032(2): 241–245. https://doi.org/10.11646/zootaxa.4032.2.12
Silva, G.K.V.P.T., W.A.D. Mahaulpatha
& A. de Silva (2019). Amphibian
abnormalities and threats in pristine ecosystems in Sri Lanka. Journal of
Threatened Taxa 11(15): 15004–15014. https://doi.org/10.11609/jott.5394.11.15.15004-15014
Sower, S.A., K.L. Reed & K.J. Babbitt (2000). Limb malformations and abnormal sex hormone
concentrations in frogs. Environmental Health Perspectives 108(11):
1085–1090. https://doi.org/10.1289/ehp.001081085
Stuart, S.N., J.S. Chanson, N.A. Cox, B.E. Young, A.S.
Rodrigues, D.L. Fischman & R.W. Waller (2004). Status and trends of amphibian declines and
extinctions worldwide. Science 306(5702): 1783–1786. https://doi.org/10.1126/science.1103538
Trueb, L. (1973). Bones, frogs and evolution: J.L. Vial. (Eds.).
Evolutionary Biology of the Anurans: Contemporary research on major
problems. University of Missouri Press, Columbia, 65–132.
Zhang, M., X. Chen & X. Chen (2016). Osteology of Quasipaa
robertingeri (Anura: Dicroglossidae). Asian Herpetological Research 7(4):
242–250. https://doi.org/16373/j.cnki.ahr.150067