Rhacophorus malabaricus Jerdon, 1870

Sandeep, Sreedharan, Joelin, Joseph, Sanil, George & Antony, Mano Mohan, 2022, Ontogenetic systematic characterisation of an endemic frog Rhacophorus malabaricus Jerdon, 1870 (Anura: Rhacophoridae) from Western Ghats, Kerala, India, Zoosystema 44 (6), pp. 159-176 : 162-168

Rhacophorus malabaricus Jerdon, 1870

Rhacophorus malabaricus Jerdon, 1870: 66 .

MOLECULAR IDENTIFICATION

The 16S rRNA gene similarity search using BLAST confirmed the organism as Rhacophorus malabaricus . The sequences obtained were submitted to GenBank (Accession No. MW130836 and MW130837 ). Phylogenetic analysis was done based on a dataset of 26 sequences from the family Rhacophoridae including four sequences of R. malabaricus (two from present study and two obtained from GenBank). All the four sequences of R. malabaricus were recovered in a single cluster. The genetic distance calculated was significantly low (≤ 1.8%) which was in congruence with the obtained tree topology. The closest relative inferred for R. malabaricus from the BI tree was R. pseudomalabaricus Vasudevan & Dutta, 2000 as anticipated (genetic distance ≤ 7.61 %). The other sister species which had close genetic association with R. malabaricus was Rhacophorus catamitus Harvey, Pemberton & Smith, 2002 (genetic distance ≤ 13.2%; Fig. 12 ).

DEVELOPMENT OF TADPOLE

We observed that the life cycle of R. malabaricus was completed in 44 days. The suspended foam nests of R. malabaricus were formed by connecting leaves ( Fig. 2 ). Nest was observed with approximately 235 eggs (diameters of 2.17 ± 0.41, n = 15). Gosner stage 1- 21 was completed within the foam nest. The average temperature within the nest was 21.4 ± 0.1°C. They dropped down into the water body at Gosner stage 22, and their aquatic life then lasted up to Gosner stage 43. Metamorphosis was completed at Gosner stage 46 on 44 day during which the larva got adapted to terrestrial habitat.

The following description of different stages of tadpoles was based on the age, size and external morphological characters. Development and metamorphosis of Rhacophorus malabaricus has been briefly recorded below:

Fertilized egg (Gosner stage 1)

The spherical shaped fertilized egg (0 hr) measured was about 2.1 ± 0.2 mm diameter (n = 6). The animal pole was dark brown coloured and vegetal pole yellowish white coloured ( Fig. 3A ). The eggs were macrolecithal.

Cleavage and blastulation (Gosner stage 2 to 12)

Gosner stages 2 to 12 were completed within 10.30 hrs. After fertilisation, the first cell division took place in about 1 hr. The diameter was about 2.26 ± 0.11 mm (n =6). The embryo became 64 to 128 celled morula in 8 hours (Gosner stage 8) and later reached at blastula stage by the repeated cleavage in 8.20 hrs. The pigmented region on the animal pole slightly extended to vegetal pole. Later, the embryo entered gastrulation (Gosner stage 10) in 9 hrs with a diameter of about 3.2± 0.4 mm (n=4). The crescent shaped dorsal lip was visible. The pigmented area was extending to the vegetal pole and the unpigmented area was highly reduced. Yolk plug appeared at the end of 10.30 hrs (Gosner stage 12).

Neuralation (Gosner stage 13 to 16)

The duration for neuralation was 11 hrs to 2 days. The neural plate appeared in Gosner stage 13 ( Fig. 3B ) which showed a distinct broader cerebral region, followed by a narrow spinal code region. The rest of the embryo was light brown coloured except the neural plate which was sandy yellow coloured. The diameter of the embryo in Gosner stage 15 was 4.0 ± 0.31 mm (n = 5; Fig. 3C ). Neural tube was formed by the fusion of neural fold in both cerebral and spinal code regions. Gosner stage 16 was completed on the 2nd day.

Tail bud, external gills, operculum and pigmentation (Gosner stage 17 to 25)

On the 3rd day, the tail bud appeared at the posterior end (Gosner stage 17). The total length of the embryo was about 4.5 ±0.33 (n= 5) in tail bud stage. The head developed with characteristic optic and gill plate bulges.

The embryo got elongated in Gosner stage 18 and the tail began to be curved within the vitelline membrane. The head, abdomen and tail were completely differentiated on 5th day (Gosner stage 19). The length of the embryo was about 5.2± 0.06 mm (n= 6). The hatching of larva started on 3rd day and the hatched motile larvae stayed submerged in the foam (Gosner stage 21). The larva is 9.16 ±0.08 (n = 5) mm long. While hatching, the body was yellowish-brown coloured and tail was whitish-grey. At Gosner stage 22, they attained a total length of 11± 24 mm (n = 4) and mouth was slightly wider than internarial ditance ( Fig. 3D ). The external gills were covered by opercular foldings in Gosner stage 23. Pigmentation of the tail occurred on the 6th day. Operculum was closed on the right side by Gosner stage 24 (age 6 days) and closed on left side at Gosner stage 25 (age 7 days). The oral disc was well-developed and distinct. The anal tube was opened and the total length was measured as 14.5 ± 0.08 mm (n =6).

Hind limb bud development (Gosner stage 26 to 30)

The larva reached the 26th Gosner stage on 8th day having a body length of 17.23 ± 0.02 mm ( Fig. 3E ). A limb bud appeared at the rear part of the body near the vent. The pigmentation extended to tail and was later spread over the translucent tail. The upper and lower tail height was more or less the same. In 27th Gosner stage (age 9 days), the hind limb bud was equal to half of its height ( Fig. 3F ). The tail was about 60% of the total length. The eye diameter is about 0.50 ± 0.02 mm. In Gosner stage 28 (age 10 days) the length of the larva was about 21.33 ± 0.08 mm and the tail was 62% of total body length. The length of hind limb bud is equal to 1.5 times of its height at Gosner stage 29 (age 11 days) with total body length of 24.65 ± 0.23 mm ( Fig. 3G ). Body length was 34.5% of its total length. In Gosner stage 30 (age 12 days, total body length 28.36± 0.45 mm), the length of the limb bud was equal to twice its height.

Toe differentiation (Gosner stage 31 to 39)

Foot pads were visible in Gosner stage 31 (age 13 days, total body length 31.60± 0.30 mm). No pigmentation was seen in limb buds and tail length was more than half of the total body length ( Fig. 3H ). The first indentation between the fourth and fifth toes was perceivable in Gosner stage 32 (age 14-15 days). The larva became 32.49± 0.37 mm long. The second indentation between the third fourth toe appeared in Gosner stage 33 (age 16-17 days, total body length 33.66± 0.21 mm). The head and trunk were well developed. On the 18th day (Gosner stage 34) the third indentation between the second and third toe was visible (total body length 34.51 ± 0.43 mm) ( Fig. 4A ). The pigmentation was predominant. For fourth and last indentation, first and second toes appeared in Gosner stage 35 (age 19-20 days, total body length 37.70± 0.36 mm) ( Fig. 4B ). In this stage all toes were visible but they were not separated from each other. In Gosner stage 36 ( Fig. 6 ) (age 21-22 days, total body length 39.48± 0.45 mm) the third toe and the fifth toe was separated independently from fourth toe ( Fig. 4C ). All toes separated from each other in Gosner stage 37 (age 23-24 days, total body length 40.71± 0.34 mm) ( Fig. 4D ) and inner metatarsal tubercle appeared posterior to the first toe in Gosner stage 38 ( Fig. 4D ; age 25-27 days, total body length 43.44 ± 0.39 mm). The subarticular tubercles were visible in Gosner stage 39 (age 28-30 days, total body length 44.49 ± 0.39 mm; Fig. 4F ). Dark coloured pigmentation was seen in hind limbs except for first and second toes.

Well-developed hind limb (Gosner stage 40 to 42)

In Gosner stage 40 ( Fig. 4G ; age 31-33 days, total body length 46.44± 0.39 mm), outer metatarsal tubercle and foot subarticular tubercles were distinct. Mouth parts gradually degenerated. The vent tube was still present. Forelimb buds appeared in Gosner stage 41 (age 34-36 days, total body length 43.44 ± 0.39 mm) and the vent tube disappeared ( Fig. 4H ). In Gosner stage 42 ( Fig. 5A ) (age 37-38, total body length 48.7 ± 0.22 mm), the fore limbs were emerged. At this stage the larva attained maximum total body length. The mouth was slightly shifted from anterior to the nostril.

Mouth restructuring and tail reabsorption (Gosner stage 43 to 44)

The atrophying of tail began in Gosner stage 43 ( Fig. 5B ) (age 39-41 days, total body length 27.13 ± 0.43 mm). The angle of mouth was widened and reached between nostril and eye. Both dorsal and ventral tail fins started to shrink. Truncate digital discs on both limbs were distinct. In Gosner stage 44 ( Fig. 5C ) (age 42 days, total body length 23.01 ± 0.42 mm), widening of mouth continued up to beneath the eye. Dorsal and ventral tailfins disappeared. Tail was greatly reduced to 12.38± 0.43 mm. The metamorphosing tadpoles were observed in the boundary of water and land and started to come out from water. Ventral parts of head, trunk and limbs turned to pale yellow colour. Dorsum of head, trunk and limbs were blue coloured with dark sport. The patches in the limb were jointed as small darkened striations.

Metamorphosis (Gosner stage 45)

In Gosner stage 45 (age 43 days), the mouth was extended up to the posterior margin of the eye. The dark blue-green rounded tail stump was visible at the base of cloaca. The snout vent length was reduced as 19.31 ± 0.41 mm.

Metamorphosed froglet (Gosner stage 46)

The tail completely disappeared. Hind limbs and fore limbs were well developed and metamorphosis was completed in 44 days and the juvenile froglet emerged ( Fig. 5D ).

MORPHOMETRIC VARIATION

The morphometric measurements of different developmental stages are shown in Table 2 . The total length of larva in Gosner stage 26 was 17.27± 0.09 mm, gradually increased to 48.85 ± 0.01mm in Gosner stage 42. Tail length followed a similar trend up to Gosner stage 42, later declined drastically and disappeared at Gosner stage 46 ( Fig. 7 ). Thus total length was reduced as 17.17 ± 0.06 mm. The upper and lower tail fin height was more or less the same in the beginning stages, the length of upper tail fin was slightly increased in later stages. In all graphs, the total body length and tail length showed a proportionate increase in different developmental stages ( Fig. 2 ). Similar observations were found in the graph plotted for snout-vent length and body height as a function of the total body length. The measurement of the body height, body length and diameter of eyes were highly correlated with developmental stages ( Table 3 ). The correlation between total body length and all parameters was significant at the level of 0.05 ( Fig. 8 ).

ORAL MORPHOLOGY

The following description of oral morphology is based on tadpoles at Gosner stage 32. The mouth was slightly protruding ventrally. The oral disc was elliptical. Two rows of marginal and submarginal soft unpigmented moderate sized, roundended papillae were present on the margin of lower labium. The upper labium margin with moderate sized rounded papilla that shows a wide dorsal gap (55-65% of the width of oral disc) ( Fig. 9 ). The upper mouth sheath was nearly shaped as an inverted ‘U’, with keratinised, moderately sharp and equal sized serrations. Lower mouth sheath was ‘V’ shaped with well keratinised sharp, equal sized serrations. The number of anterior and posterior keratodont rows were 7 (A-1-2-3-4-5-6-7) and 3 (P-1-2-3) respectively. A-1 and A-2 were entire, A-3 was divided medially by a conspicuous gap. A-4-5-6-7 were completely separated by upper mouth sheath. Three undivided keratodont rows (P-1-2-3) were present on the lower labium ( Fig. 10 ). All keratodont rows were biserial except A6 and A7 (uniserial). The order of relative lengths of anterior and posterior tooth rows were A2>A1>A3>A4>A5>A6>A7 and P3>P2>P1 respectively. Labial Tooth Row Formula (LTRF) 7(3-7)/3, Keratodont Row Formula (KRF), 2:5+5/3. Keratodonts (17-20 µm long) with spatulate apex bearing 8-10 sharp marginal denticles ( Fig. 11 ).

Figures

FIG. 12. — Bayesian inference tree of selected species of Rhacophoridae Hoffman, 1932 (1858). For Rhacophorus malabaricus Jerdon, 1870, the sequences are newly generated (A, B), or from GenBank (C, D).

FIG. 2. — Leafs hiding a foam nest of Rhacophorus malabaricus Jerdon, 1870.

FIG. 3. — Developmental stages of Rhacophorus malabaricus Jerdon, 1870: A, Gosner stage 1; B, Gosner stage 13; C, Gosner stage 15; D, Gosner stage 22; E, Gosner stage 26; F, Gosner stage 27; G, Gosner stage 29; H, Gosner stage 31. Scale bars: 1 mm.

FIG. 4. — Developmental stages of Rhacophorus malabaricus Jerdon, 1870: A, Gosner stage 34; B, Gosner stage 35; C, Gosner stage 36; D, Gosner stage 37; E, Gosner stage 38; F, Gosner stage 39; G, Gosner stage 40; H, Gosner stage 41. Scale bars: A-E, 3 mm; F-H, 4 mm.

FIG. 6. — Habitus of the tadpole of Rhacophorus malabaricus Jerdon, 1870 stage 36: A, lateral view; B, dorsal view. Scale bar: 10 mm.

FIG. 5. — Developmental stages of Rhacophorus malabaricus Jerdon, 1870: A, Gosner stage 42; B, Gosner stage 43; C, Gosner stage 44; D, Gosner stage 46.

FIG. 7. — Comparison of length-age ratio of tadpole of Rhacophorus malabaricus Jerdon, 1870.

FIG. 8. — Correlation between morphometric parameters (total length and tail length) and stages A, and B, correlation between morphometric parameters (tail length and snout-vent length) and stages C, and D.

FIG. 9. — Oral disc morphology of Rhacophorus malabaricus Jerdon,1870 (Gosner stage 32). Abbreviations:A3G, third anterior tooth gap;A1 to A7 anterior tooth rows 1 to 7; P1 to P3 posterior tooth rows 1 to 3. Scale bar: 100 µm.

FIG. 10. — SEM image of oral disc morphology of Rhacophorus malabaricus Jerdon, 1870 (Gosner stage 32). Abbreviations: A3G, third anterior tooth gap; A1 to A7, anterior tooth rows 1 to 7; LJSS, lower jaw sheath serration; LMP, lower marginal papillae; P1 to P3, posterior tooth rows 1 to 3; UJSS, upper jaw sheath serration; UMP, upper marginal papillae. Scale bar: 100 µm.

FIG. 11. — SEM image of keratodonts of second anterior tooth row (A2) of Rhacophorus malabaricus Jerdon, 1870 (Gosner stage 32). Abbreviations: KD, kera- todonts; MD, marginal denticles. Scale bar: 10 µm.