Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.
Studies have shown that some characters, such as morphological, biochemical and behavioural characteristics were shared among con-specifics of closely related frog species. It is hypothesized that closely related frogs would have similar vocal apparatus structure to produce similar call characteristics, in order to be recognized by con-specifics. The vocal apparatus structures of the frogs from Ranidae were, thus, investigated and calls were recorded from various locations in Sarawak, Borneo. The vocal apparatus was sliced and stained using haematoxylin and eosin staining method. The prepared slides were scanned using Dotslide System Olympus BX51. Captured images were further measured using Microscope Olympus BX51. The results showed that the vocal apparatus structure of ranids in Sarawak differed in length between pulvinar vocale and posterior margin (pvpm) or known as vocal cord stricter. This character was deemed to be the most significant character to discriminate the Sarawak ranids. It is suggested that histological analysis is also a good method for study of phenotypic variation of frog species, as shown by the vocal apparatus structure of the anurans. This study provides an alternative method to differentiate taxonomic identity among frog species.
The sound produced by the anurans including toads can be a survival strategy for the selection of fittest males by their conspecific females and a form of effective prezygotic mechanism. Most of the published works on anuran sound production were on true frogs or ranids, yet little is known about sound characteristics of toads. Here we describe the properties of mating calls from six selected toad species from six study sites in Sarawak. Males' mating calls were recorded and analyzed using Sound Ruler Acoustic Analysis ver 0.9.6.0. Call characteristic were subsequently compared for toad species differentiation. The results showed that each species differ in call characteristics, with pulsating note, rate of note repetition and pitch being the most apparent characters. The highest number of pulse notes belongs to Duttaphrynus melanostictus while Ansonia spinulifer exhibited highest pitch and rate of note repetition. These species' differences in mating call characteristics provide an additional method for anuran species classification besides morphological and molecular DNA data. It is also a very useful guide during field survey as the sound is audible and identification can be made without the need of capturing the toads.
Rakaman panggilan katak Borneo daripada genus Hylarana telah dijalankan di tujuh lokasi kajian di Sarawak, Malaysia. Sebanyak 12 individu daripada lima spesies katak Borneo telah berjaya dirakam dan dianalisis. Daripada 38 pembolehubah panggilan, hanya 27 daripadanya dapat menerangkan ciri-ciri panggilan daripada katak Hylarana Borneo. Saiz katak jantan mempengaruhi secara signifikan tenaga panggilan antara 90:50% amplitud puncak, tempoh nadi di antara 50%, nisbah tempoh panggilan untuk mencapai setengah modulasi frekuensi dan kadar ulangan not. Saiz katak Hylarana Borneo sedikit mempengaruhi frekuensi asas dan dominan panggilan. Di samping itu, taburan pembolehubah panggilan katak dan dendogram Euclid menyokong identiti taksonomi bagi setiap spesies katak Borneo Hylarana.
Recordings of frog calls from the genus Hylarana were conducted at seven study sites in Sarawak, Malaysia. The results showed that each species differs in terms of call characteristics, in which a high number of pulsed note belongs to H. baramica, while high repetition note belongs to the sibling species, H. glandulosa. Higher pitched and number of amplitude spectrum, however, belong to H. signata. Pulsed note and note repetition differed significantly among species, except for spectrum amplitude. Advertisement calls for each species were described in detail. This study provide a baseline data on advertisement call characteristics of frogs from the genus Hylarana for future studies of these frogs particularly and the Oriental frogs generally.