Gracixalus gracilipes, G. quangi, and G. supercornutus are small, morphologically and molecularly similar treefrogs with green blood from Vietnam, Laos and southern China. The breeding biologies, eggs, embryos and larvae of these three species are poorly known, and the male advertisement call of only G. quangi is known; this species has a hyperextended vocal repertoire. We provide new information on the breeding habitats, eggs, embryos and tadpoles of these three species and describe the calls of G. gracilipes and G. supercornutus. All three species deposit egg clumps on leaves overhanging shallow pools and puddles in forests. Like G. quangi, the calls of G. gracilipes and G. supercornutus are non-stereotypical, with individual calls highly variable in structure, duration, amplitude and frequency. Both calls are frequency modulated and have a dominant frequency of 4.1-4.7 kHz and many harmonics. The functional significance of these variations is unknown, and it is not known how common hyperextended call repertoires are within the genus.
Forested habitats of central and northern Vietnam and parts of Laos and southern China are home to a group of three, relatively small (male SVL < 25 mm) frogs with pointed snouts, green blood and turquoise bones (Rowley et al., 2011). These three species, Gracixalus quangi, G. gracilipes and G. supercornutus, are morphologically and molecularly most similar to each other (Rowley et al., 2011). G. quangi, the only species for which vocalisations are known, has a non-stereotypical, hyperextended vocal repertoire (Rowley et al., 2011). Calls of this species are highly variable in structure, duration, amplitude and frequency (Rowley et al., 2011).
Gracixalus quangi, G. gracilipes and G. supercornutus deposit eggs in clear jelly on leaves and branches above nonflowing water (Bain and Nguyen, 2004; Delorme et al., 2005; Rowley et al., 2011), but none of the eggs, embryos and larvae are well known. We provide new data on the eggs and early embryos of the three species and a small tadpole of G. quangi and describe the vocalisations of G. gracilipes and G. supercornutus for the first time.
Materials and methods
Eggs, embryos and tadpoles were collected in Vietnam as follows: Gracixalus gracilipes (1 clutch, AMS R 177672; Hoang Lien National Park, Lao Cai Province; 22.3483 N, 103.7302 E, 1887 m; June 2012), G. quangi (5 clutches, AMS R 173421, 173424-173427; Pu Hoat Nature Reserve, Nghe An Province; 19.700 N, 104.739 E, 1083 m; June 2010) and G. supercornutus (3 clutches, AMS R 176267, 176273, 176287; Kon Ka Kinh National Park, Gia Lai Province; 14.224 N, 108.335 E, 1468 m, August 2011; and one clutch AMS R 173899; Ngoc Linh Nature Reserve, Kon Tum Province; 15.08 N, 107.96 E, ∼1900 m, July 2009). Eggs, embryos and tadpoles were euthanised with Benzocaine, and prior to fixation in 5% formalin, tissue samples from each were preserved in 95% ethanol or buffered salt solution. Voucher specimens were examined, measured, and photographed with a dissecting microscope. Photographs from the field were also examined. Staging followed the Gosner (1960) table, egg terminology is that of Altig and McDiarmid (2007), and tadpole terminology is that of Altig and McDiarmid (1999). Specific identification of eggs, embryos and tadpoles from each site (using samples from G. gracilipes, AMS R 177672, G. quangi, AMS R 173426, and G. supercornutus, AMS R 176273, 176287) was confirmed by matching mitochondrial 16S RNA sequences with that of adults of each species (G. gracilipes: AMS R 177667; G. quangi AMS R 173410, 173411, 173417, 173423; G. supercornutus AMS R 173395-173396, 173428, 173887; ∼530 bp; GenBank accession numbers KT374013-KT374016, JN862537-862545).
The calls of five male G. gracilipes and four G. supercornutus were recorded in situ with an Edirol R-09HR WAVE/MP3 Recorder (96 kHz sampling rate and 24-bit encoding) and a Røde NTG-2 condenser shotgun microphone. Calls were recorded at a distance of about 0.2-0.5 m and ambient temperatures were taken immediately after recordings with a Kestrel 3500 hand-held weather meter. Calls were analysed with Raven Pro 1.3© software (http://www.birds.cornell.edu/raven). Audiospectrograms in the figures were calculated with a fast-Fourier transform (FFT) of 512 points, 50% overlap and 188 Hz grid-spacing with Hanning windows. Temporal and spectral parameters of calls were measured from oscillograms (waveforms) and audiospectrograms by the definitions of Cocroft and Ryan (1995). We broadly categorized calls as “clicks”, relatively short notes (∼6-20 ms) of high amplitude and “whistles”, relatively long notes (∼150-400 ms) of lower amplitude and a more narrow frequency band. Narrower categorization of call types was not possible because the calls varied from one extreme to the other and often combined in variable ways.
The breeding sites for Gracixalus gracilipes in Hoang Lien National Park, Lao Cai Province included a large (>100 × 100 m) grassy swamp, small (<10 × 10 m) muddy pools (fig. 1A), flooded grassy ditches at the edge of a road (fig. 1B) and seeps at the edge of limestone cliffs. At all locations, 1-5 calling males were observed 10-30 cm above the water on blades of grass or leaves of herbs.
We observed a single breeding site for Gracixalus quangi (June 2010) at a seep adjacent to a stream (fig. 1C-D; Rowley et al., 2011) in Pu Hoat Nature Reserve, Nghe An Province. There was no standing water at the site, and it was densely vegetated with ferns and herbs less than 1.0 m tall. Most of the 15 males observed were calling, one female was observed on vegetation at the site (an area of <10 m2), and eggs and embryos were observed attached to leaf tips < 50 cm above the water.
Gracixalus supercornutus was observed after a thunderstorm in a large breeding aggregation in a high-elevation (∼1500 m asl) swamp in Kon Ka Kinh National Park, Gia Lai Province. The swamp was about 100 × 100 m in extent, water < 50 cm deep occurred throughout, the substrate was mud, and emergent vegetation was dense (fig. 1E-F). Hundreds of individuals were observed, and at least twenty males were heard calling at once. Calling males were at a density of at least 1/m2, and almost all were calling from emergent vegetation < 0.5 m above the water. A few calling males were observed on vegetation along the edges of the swamp, up to 1.5 m above the water level. Males appeared territorial, jumping towards any males in close proximity while calling and raising their bodies off the substrate in response to their reflections in a camera.
Eggs, embryos and tadpoles
The small clutches of eggs of Gracixalus with prominent, clear jelly layers are laid in small clumps on leaves above nonflowing water (fig. 2A-C). The relatively firm consistency of the jelly usually does not allow the clutch to droop. Eggs were typically placed on the front of leaves but were occasionally positioned on the backs of green leaves.
The single clutch of G. gracilipes examined contained 5 ova and was on the front of a fern leaf (fig. 2A). Egg diameters ranged from 5.1-5.3 mm, and ovum diameter was 2.0-2.1 mm. The clutch size of G. quangi ranged from 7-18 (mean = 10.2; ), and one clutch contained a dead embryo. Embryos at stage 19 (fig. 2D) have a large yolk and long, branched gills. A tadpole at stage 24 (AMS R 173427) removed from the egg jelly was 9.8 mm TL, the oral disc was fully formed, tooth ridges for LTRs A-1-2 and P-1-2 were present, a small amount of keratinization occurred on the upper jaw sheath and a hint of keratinization occurred on the lower jaw sheath. Slight remnants of the external gills persisted on the left side, two, round adhesive glands were present (fig. 2E), and the ventral marginal papillae were discernible. By stage 26 (10.6 mm TL), the oral disc was better defined, both jaw sheaths and labial teeth in all tooth rows except P-3 were visible, and the adhesive glands remained visible although probably no longer functional. Given the relatively young developmental stage of all tadpoles found, the definitive tooth row formula may differ.
Eggs of G. supercornutus (fig. 2C) were as above, but two clutches that were deposited on dead leaves had slid into a monolayer. Clutch size ranged from 5-16 (mean = 9.0, ), egg diameters ranged from 5.7-6.1 mm with one jelly layer, and ovum diameter ranged from 2.4-2.6 mm. The ova and early embryos were uniformly pale brown, and at stage 18, the gill anlage was large and the tail was wrapped around against the yolk to the left. Separate, round anlagen of adhesive glands were present.
The call of G. gracilipes consists of a series of highly variable, non-stereotypical call types (fig. 3). Individual call components were highly variable. Although some calls were broadly similar in structure, no two calls were the same within or among the five individuals recorded. At an ambient temperature of 17.3-18.1°C, calls ranged from about 6-20 ms (“clicks”) to 150-250 ms (“whistles”). Frequency varied among calls, the dominant frequency ranging from 4.1-5.1 kHz, and harmonics were present at 8.1-9.6, 12.4-16.1, 17.6-18.8, and ∼22 kHz. A fundamental frequency was just visible at 0.2 kHz in some calls. Calls were generally frequency and amplitude modulated (generally a ∼0.3 kHz change in frequency within a call), but not in a constant or predictable manner (fig. 3). The temporal order of the call types did not appear fixed.
The call of G. supercornutus consists of a series of highly variable, nonstereotypical calls (fig. 4) including clicks and longer notes. Individual call components were highly variable but clicks were often temporarily grouped. Although some calls were broadly similar in structure, no two calls were the same within or among the four individuals recorded. At an ambient temperature of 22.6-23.7°C, calls ranged from about 6-9 ms (“clicks”) to ∼370 ms. Frequency varied among calls, the dominant frequency ranged from 3.6-4.1 kHz, and harmonics were present at 7.1-8.1, 10.5-12.2, 13.3-16.1, and 17.3-20.1 kHz. A fundamental frequency was just visible at 0.2 kHz. Calls were frequency and amplitude modulated, but not in a constant or predictable manner (fig. 4). The temporal order of the call types did not appear fixed.
Gracixalus gracilipes, G. quangi and G. supercornutus have similar breeding biologies. All species call from vegetation overhanging shallow forest pools or puddles or from emergent vegetation in larger flooded areas and deposit egg in clumps on leaves overhanging these waterbodies (Bain and Nguyen, 2004; Delorme et al., 2005; Rowley et al., 2011; this study).
The observation that eggs on dead leaves had slid into a monolayer indicates that the outer surfaces of the jelly layers are not strongly adherent with these substrates. Our inability to understand the significance of the substrates chosen for oviposition emphasizes our poor understanding of the biology of arboreal eggs in general. Clutch size, survival of embryos in arboreal clumps, the developmental effects of position of a given egg within a clump, and variations in yolk content have not been studied. The minimum number of eggs/clump of 4-5 as well as the maximum of 15-18 seems small relative to the sizes of the ova and the frogs, and a given pair may deposit the entire complement in multiple clumps. In some cases the area of the ovipositional surfaces may be a limiting factor.
Clutch structure is likely modified by whether the leaf is wet or dry at the time of oviposition and whether the leaf is alive or dead. Wetness reduces the effective tackiness of outer egg jelly layers, and variations in the hydrophobicity of the surfaces of live versus dead leaves and among leaves of different plants, between the front and backs of leaves, and different densities, lengths and shapes of trichromes on the leaves surely modify proper adhesion of the egg jelly to the leaf surface.
Clumps of Gracixalus were usually placed on the upper surface of a live leaf, near the leaf tip. This may guarantee that the eggs are properly wetted by dew or minimal rain but also quickly drained of excess water. The distal reduction in leaf width and the multiple points of a serrated leaf margin increases water drainage (Meng et al., 2014). Eggs that were laid on dead leaves had slid into a monolayer, presumably because the surfaces of dead leaves are less hydrophobic and more easily wetted. The margins of jelly layers on a wettable substrate have a less prominent meniscus which indicates a reduction of the hydrophobic interaction between the jelly and leaf. Because egg jellies in a monolayer have reduced surface area for gas exchange, these embryos likely have reduced viability, and the deteriorating processes of a dead leaf may reduce proper gas exchange through the enlarged contact surface with the egg jellies. The reduced height of the egg jellies may also impede proper cell divisions and the eventual growth of the embryos, and such eggs would suffer accordingly with any amount of dehydration.
We note that the eggs of G. gracilipes we examined were pigmented, and the ones in Bain and Nguyen (2004) were nonpigmented. The coloration of the embryos and the opaque, white jelly they illustrate differ from those we observed. The meniscus at the margin of the clump is reduced, and the ova appear to be depressed into a spheroid. We suggest that this clutch was somehow compromised, and that the ova may not have developed properly.
The composition of Asian rhacophorid genera remain in a state of flux, and knowing various facets of the breeding biologies sometimes focuses these decisions. For example, Philautus is now considered to have only direct development (Frost, 2014) even though tadpoles have been previously described with this name. The composition of Chiromantis with various patterns of oviposition is another debate. All species of Gracixalus for which information is available (these three species plus G. lumarius that oviposits above tree holes; Rowley et al., 2014) are known to lay eggs that have clear, reasonably firm jelly layers on leaves or tree trunks above nonflowing water and have exotrophic development.
Frog vocalisations have traditionally been described as highly stereotypic, particularly as they promote species recognition (Wells and Greer, 1981). However, the acoustic repertoires of an increasing number of frog species are not stereotyped. Rhacophorid frogs in particular are known to have extended vocal repertoires that include distinct call types, sometimes in no predictable order (Jehle and Arak, 1998; Narins et al., 2000; Rowley et al., 2011).
The vocal repertoire of a frog species is likely to reflect the selection pressure for premating reproductive isolation within the species, their ecological environment, and their social behavior (Capranica, 1976). The repertoire of all three species of Gracixalus described here indicates that they have a relatively sophisticated signalling system. In some species, female frogs may actually prefer the most complex calls (Rand and Ryan, 1981), perhaps because more complex calls may be correlated with larger body size (Rand and Ryan, 1981) or better body condition.
Different vocalisations in a species’ repertoire may have a distinct communicative significance; some components may attract mates (advertisement calls), while others may be territorial calls (Narins and Capranica, 1978). Indeed, much of the variation in the call structure in calling males is thought to be a result of competitive interactions between males (Wells, 1977, 1988; Jehe and Arak, 1998).
The calls of Gracixalus gracilipes, G. quangi (Rowley et al., 2011), and G. supercornutus are similar in terms of dominant frequency and duration. Calls of all three species are a mix of high-pitched whistling and sharp clicking that sounds somewhat similar to the human ear. Their calls are difficult to quantitatively compare because of their extreme variability within and among different individuals and under differing social and environmental conditions. In particular, G. supercornutus was the only species recorded in a large chorus, and other species, including rhacophorids, have been known to modify their calls depending on social context (Rand and Ryan, 1981; Wells and Greer, 1981; Arak, 1983; Ramer et al., 1983). In other species of frog with large vocal repertoires, isolated males may produce only a single note type. In this study, we heard no males calling in isolation. G. gracilipes was only heard calling in groups of <5 males relatively widely spaced (<1 m apart), yet all individuals displayed a diversity of call types.
It is not known why Gracixalus have developed such a range of calls. The functional significance of different call types is also unknown. We can only suspect that the clicks serve to defend territories, as clicks have been shown to be territorial in nature in other frog species (Narins and Capranica, 1978; Wells and Greer, 1981). Clicks were also most common in G. supercornutus, the only species observed in a large breeding aggregation. Playback experiments (e.g., Narins and Capranica, 1978) may help elucidate the functional significance of the hyperextended vocal repertoire of frogs in the genus Gracixalus.
The Vietnamese Ministry of Agriculture and Rural Development, Vinh University, the Ministry of public security (Nghe An Province), and Le Trong Nguyen and staff at the Forest Protection Department of Nghe An Province kindly facilitated surveys and issued permission to collect at Pu Hoat Nature Reserve (permit numbers 1124/DHV-QHQT, 1130/DHV-HTQT, and 15/2010/GP), and the Vietnamese Ministry of Agriculture and Rural Development and staff at Hoang Lien National Park kindly facilitated surveys and issued permissions (Permit numbers 19/BTTNVN and A13041165/A72-P2). Nguyen Dinh Cong and Luu Dam Cu (Vietnam National Museum of Nature, Hanoi) facilitated specimen loans. Dinh Quoc Thang and staff at Ngoc Linh Nature Reserve kindly facilitated surveys and issued permission to collect (Permit numbers 776/BNN-KL, 748/BNN-KL). The People’s Committee of Kon Tum Province issued permit number 548/UBND-DN for Rowley for work in the Province. Research was carried out by Rowley under Animal Care and Ethics approval #09-01 and #12-02 from the Australian Museum Animal Care and Ethics Committee. Research was supported by funding from ADM Capital Foundation, Ocean Park Conservation Foundation Hong Kong, and The John D. and Catherine T. MacArthur Foundation. For all this assistance we are most grateful.
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