Morphological description is based on 9–10 larvae of medium larval stages. The external appearance of the tadpoles is shown in Fig. 4. The main morphometric parameters are summarized in Table 2.

Figure 4. 

External morphology and coloration in life of the tadpoles of three Kalophrynus species at stages 36–37: K. interlineatus (ZMMU A-7891), A dorsal view, B lateral view; K. honbaensis (ZMMU A-7892), C dorsal view, D lateral view; K. cryptophonus (ZMMU A-7893), E dorsal view, F lateral view. Scale bar: 5 mm.

Kalophrynus interlineatus. In dorsal view (Fig. 4A), body broadly elliptical, with maximum width at the gill level; snout bluntly rounded. In lateral view (Fig. 4B), body ovoid and slightly compressed dorsoventrally, with a flat dorsum and convex belly. Tail moderately long, broadly lanceolate, and slightly more than one and half times longer than the body; tail musculature rather weakly developed (tail base less than a third of the maximal body width); tail ending with terminal filament (approximately one-fourth of the tail length). Tail fins rather tall, reaching their maximum height at the middle third of the tail length; dorsal fin not extending on the trunk and ventral and dorsal fins nearly equal in height. Spiracle medial, transversal, narrow, and opening at the rear of the body; covering membrane rather long, with a serrated free margin. Vent tube medial and oblique. Eyes strictly lateral and moderately large. Nostrils sealed at larval stages. Nasolacrimal grooves and lateral line not visible. Mouth terminal, transversal, and relatively narrow. Lower labium not visible in dorsal view; upper labium short and rounded, with two lateral flaps hanging over narrow spoon-shaped lower labium. External papillae and keratinized mouthparts absent.

In life, at all larval stages, dorsal coloration dull brown with pink or reddish marbled pattern; belly paler, with a pinkish tint; tail fins densely pigmented, grayish-brown with irregular pink or orange spots and yellow or orange speckling; terminal, tapering parts of tail fins unpigmented, stem of the terminal filament gray; spiracle flap unpigmented. Iris black with golden speckling and wide golden ring around the pupil. In preservative, brown and reddish tints fading to pale gray, tail with a gray marbled pattern.

Kalophrynus honbaensis. In dorsal view (Fig. 4C), body subrectangular and slightly elongated, with a broad, concave snout. In lateral view (Fig. 4D), body subtriangular and compressed dorsoventrally, especially in the rostral part of the head. Tail rather long, nearly two and half times longer than the body or more in some specimens. Tail musculature rather well developed (tail base more than a third of the maximal body width); tail tip broadly acuminate. Tail fins moderately tall, reaching their maximum height at the distal third of the tail length; dorsal fin not extending on the trunk, ventral fin slightly higher than the dorsal fin. Spiracle medial, narrow, and opening at the middle of the belly; covering membrane rather short, with a finely serrated free margin. Vent tube medial and oblique. Eyes lateral and moderately large. Nostrils sealed at larval stages, narial openings developing only at premetamorphic stages 41–42. Nasolacrimal grooves faintly visible before stage 38, at more advanced stages visible from narial protuberances to the anteroventral margin of the orbit. Mouth terminal, transversal, and relatively wide (approximately half of the body width or even slightly more during earlier midlarval stages 35–36). In dorsal view, lower labium slightly visible, semicircular. Upper labium short and concave, with an even edge. Lateral lobes and keratinized mouthparts absent.

In life, at stages 35–41, body and tail uniformly dark brown with scarce irregular pinkish or orange speckles, belly and tail underside paler; tail fins pigmented, grayish with blurred orange spots; spiracle flap unpigmented. Iris black with goldish-silver speckling and narrow metallic ring around the pupil. In preservative, brown color fading to dull gray.

Kalophrynus cryptophonus. In dorsal view (Fig. 4E), body pyriform or subrectangular (depending on gut fullness), rather elongated, with a broad, blunt, and even or slightly concave snout. In lateral view (Fig. 4F), body subtriangular and markedly compressed dorsoventrally, especially in the rostral part of the head; rostral part rather long, nearly one third of the body length. Tail very long, nearly three and half times longer than the body or even more in some specimens. Tail musculature well developed (tail base more than a third of the maximal body width); tail tip tapering and acuminate. Tail fins low, reaching their maximum height at the distal half of the tail length; dorsal fin not extending on the trunk, ventral fin higher than the dorsal fin. Spiracle medial, transversal, narrow, and opening at the middle of the belly; covering membrane short, with an even free margin. Vent tube medial and oblique. Eyes dorsolateral and moderately large. Nostrils sealed at larval stages. Nasolacrimal grooves and lateral line not visible. Mouth terminal, transversal, slit-like, and relatively wide (more than a third of the body width at stage 38 and almost half of the body width at earlier stages). In dorsal view, lower labium not visible; upper labium short, straight or slightly concave, with an even edge. Lateral lobes and keratinized mouthparts absent.

In life, at early stages (26–31), larvae almost unpigmented, pinkish-white; at midlarval and late larval stages (35–39), skin semitransparent, body and tail uniformly grayish-brown with scarce irregular orange speckles; belly and tail paler than the dorsum; tail fins slightly pigmented, spiracle flap unpigmented. Iris black with scarce golden speckling and narrow, dotted ring around the pupil. In preservative, color fading to pale yellowish-gray.

The newly metamorphosed froglets of all three species were extremely small and fragile and rather similar externally (Fig. 5). All had a stocky appearance, with bluntly triangular snout, lateral bulging eyes, slender limbs with thin, short, and tapering digits, and smooth skin. Brownish or yellowish dorsal coloration contrasted with darker coloration on flanks. Unlike adults, all froglets displayed whitish or bluish dots on the dorsum and hind limb uppersides. In K. interlineatus (Fig. 5A), a darker triangular spot marked the dorsum from the nape of the head to the groins.

Figure 5. 

Young metamorphs of three species of Kalophrynus: A K. interlineatus (SVL 4.9 mm); B K. honbaensis (SVL 6.7 mm); C K. cryptophonus (SVL 5.5 mm).

Kalophrynus interlineatus. Tadpole digestive tract of roughly equal thickness, without distinct larval stomach; intestine arranged in a dense round six-looped spiral (Fig. 6A). Length of the digestive tract varying in fixed specimens (BL 4.2–4.7 mm) from 21.5 to 23.5 mm, depending on the tadpole size and gut fullness; DTL/BL 5.01 ± 0.08 (n = 4). In all tadpoles, the intestine evenly filled with a thin, brown or dark brownish-green detritus-like mass with fine particles of inorganic material.

Figure 6. 

Larval digestive tract of three Kalophrynus species, stages 37–38 (in preservative). A K. interlineatus with gut filled with fine detritus; B K. honbaensis with extended larval stomach and gut filled with ingested yolk; C K. cryptophonus with a whole partially digested egg in the larval stomach. Scale bar: 1 mm.

Kalophrynus honbaensis. Digestive tract including a well-defined large larval stomach with thin, extensible walls. Fully extended, filled stomach in satiated larvae occupying the entire ventral cavity; intestine arranged in three loops (Fig. 6B). Length of the digestive tract varying in fixed specimens (BL 5.3–6.3 mm) from 17.6 to 22.9 mm, depending on the tadpole size and stomach fullness; DTL/BL 3.39 ± 0.25 (n = 4). In most advanced tadpoles (stages 35–41), stomachs containing various amounts of amorphous whitish mass resembling the yolk of frog eggs; intestine filled with loose, amorphous brownish mass.

Kalophrynus cryptophonus. Tadpole digestive tract including a well-defined large larval stomach with thin, extensible walls. Fully extended, filled stomach in satiated larvae occupying the entire ventral cavity; intestine arranged in two loops (Fig. 6C). Length of the digestive tract varying in fixed specimens (BL 4.9–5.2 mm) from 12.3 to 14.9 mm, depending on the tadpole size and stomach fullness; DTL/BL 2.70 ± 0.11 (n = 4). In most advanced (stages 35–38) tadpoles from two bamboo stems, stomachs containing various amounts of amorphous whitish mass resembling the yolk of frog eggs and, in some cases, whole eggs of the same size as K. cryptophonus intact eggs; intestine filled with a loose, amorphous grayish mass.

Kalophrynus interlineatus. Larval chondrocranium (Fig. 7A) widely elliptic, compressed dorsoventrally, delicate, and widely fenestrated. Cranial floor mostly cartilaginous with membranous central part covered ventrally with a thin, laced bony plate of the parasphenoid. Braincase opened dorsally; tectum synoticum narrow, weak, and smoothly arched forward, without a medial process. Otic capsules rounded and rather small (approximately 27% of chondrocranium length). Palatoquadrate cartilage mostly narrow, with a broadened reinforced rostral part, connected to the braincase anteriorly by rather narrow quadratocranial commissures and posteriorly by short, thin ascending processes. Articular process of the palatoquadrate rather narrow. Defined muscular process absent, lateral margins of palatoquadrate with low elevated marginal ridges protruding dorsally. Subocular bar thin, narrow, and fragile. Posterior part of palatoquadrate connected to the otic capsule by the larval otic process. Subotic process on the ventral surface of the palatoquadrate well defined. Lateral posterior process long, narrow, and markedly extending beyond the lateral limits of the otic capsule. Crista parotica forming a bump-like outgrowth. Subocular fenestrae large and elliptically elongated. Choanal openings wide and elliptical. Ethmoid plate short, subquadrate. Trabecular horns rather elongate, with rostral ends fused to the suprarostral cartilage with faintly visible suture. Suprarostral cartilage entire, forming a rather wide thin plate convex rostrally. Mandible (Fig. 7B) delicate, formed by short Meckel’s cartilages tapering medially, with prominent dorsal processes. Infrarostral cartilage extremely thin, subtle, and filiform. Hyobranchium (Fig. 7C) length approximately 85% of the total head length. Ceratohyals narrow, with faintly formed rounded anterior processes and very prominent, thin, spike-like anterolateral processes, slender posterior processes and lateral processes formed with thin sheet of cartilage. Basihyal (anterior copula) absent. Basibranchial (posterior copula) slender and fused to narrow hypobranchial plates. Ceratobranchials I–IV long, thin, widely expanded, joined with numerous branchial commissures (up to 21–22 between ceratobranchials I and II) and forming a voluminous branchial basket (approximately 75% of the total length of the hyobranchium). Three well-defined, flattened spiculae fused dorsally to each hypobranchial plate.

Figure 7. 

Larval skeleton of three Kalophrynus species, stages 37–38: K. interlineatus, A chondrocranium, B mandible, C hyobranchium; K. honbaensis, D chondrocranium, E mandible, F hyobranchium; K. cryptophonus, G chondrocranium, H mandible, I hyobranchium. Scale bar: 1 mm. Abbreviations: alp – anterolateral process, ap – anterior process, appq – articular process of the palatoquadrate, asp – ascending process, bb – basibranchial, bh – basihyal, cbr – ceratobranchials, chy – ceratohyal, cp – crista parotica, hbp – hypobranchial plate, irc – infrarostral cartilage, lop – larval otic process, lp – lateral process, lpp – lateral posterior process of the palatoquadrate, Mc – Meckel’s cartilage, oc – otic capsule, pq – palatoquadrate, qcc – quadratocranial commissure, sb – subocular bar, sf – subocular fenestra, src – suprarostral cartilage, th – trabecular horns, ts – tectum synoticum.

Kalophrynus honbaensis. Larval chondrocranium (Fig. 7D) subrectangular, slightly longer than wide (maximum length/maximum width 1.1), flattened dorsoventrally, rather robust, well chondrified, and moderately fenestrated. Cranial floor mostly cartilaginous with membranous central part covered ventrally with a thin, laced bony plate of the parasphenoid. Braincase opened dorsally; tectum synoticum very thin and narrow, weak, and smoothly arched forward, with faintly marked triangular medial process. Otic capsules ovoid, of moderate size (approximately 34% of the chondrocranium length). Palatoquadrate cartilage with massive anterior part forming a large, robust articular process and connected to the braincase by a wide quadratocranial commissure. Low dorsal ridges on lateral margins of the palatoquadrate instead of the muscular process. Subocular bar rather weak, narrowing posteriorly; ascending process absent. Posterior part of the palatoquadrate connected to the otic capsule by a very short larval otic process. Subotic process absent. Lateral posterior process thin, short, and almost not extending beyond otic capsule. Crista parotica not defined. Subocular fenestrae narrowed and elongated. Choanal openings wide and elliptical. Ethmoid plate short and wide, trapeziform. Trabecular horns short, broadened rostrally and widely divergent with rostral ends fused to the suprarostral cartilage with distinct suture. Suprarostral cartilage entire, forming a rather wide thin plate somewhat narrowed and concave medially. Mandible (Fig. 7E) formed by short, thick Meckel’s cartilages tapering medially, with prominent dorsal processes. Infrarostral cartilage thin, delicate, filiform, ω-shaped. Hyobranchium (Fig. 7F) approximately 70% of the total head length. Ceratohyals relatively robust, with prominent, sharp anterior processes and rather long triangular anterolateral processes connected to anterior processes by a thin membranous sheet. Basihyal thin, horizontal, and rod-like. Basibranchial narrow and fused with widened hypobranchial plates. Ceratobranchials I–IV moderately long and thin, joined with branchial commissures (up to 13–15 between ceratobranchials I and II) and forming a moderately large branchial basket (68% of the total length of the hyobranchium). Three well-defined, flattened spiculae fused to each hypobranchial plate dorsally, the lateralmost being the widest.

Kalophrynus cryptophonus. Larval chondrocranium (Fig. 7G) roughly elliptical, elongated (maximum length/maximum width 1.4), rather robust, well chondrified, and moderately fenestrated. Cranial floor mostly cartilaginous with membranous central part covered ventrally with thin, laced bony plate of the parasphenoid. Braincase opened dorsally; tectum synoticum reduced, only small condensation of chondrified tissue visible medially in the membranous chondrocranium roof between otic capsules. Otic capsules ovoid, rather small (approximately 29% of chondrocranium length). Palatoquadrate cartilage with robust anterior part and large articular process connected to the braincase by relatively wide quadratocranial commissures. Subocular bar reduced posteriorly, ascending and lateral posterior processes absent. Crista parotica not developed. Subocular fenestrae narrow, open-ended posteriorly. Choanal openings wide and elliptical. Ethmoid plate short and wide. Trabecular horns short, broad, and widely divergent with rostral ends fused to suprarostral cartilage without a visible suture. Suprarostral cartilage entire, forming a rather broad semilunar plate somewhat narrowed medially. Mandible (Fig. 7H) Ω-shaped, with bar-like Meckel’s cartilages tapering medially. Infrarostral cartilage thin but resilient, round in cross-section. Hyobranchium (Fig. 7I) approximately 65% of the total head length. Ceratohyals robust, with wide rounded anterior processes; anterolateral processes indistinct; posterior processes large, robust, and triangular. Basihyal thin and narrowly spindle-shaped. Basibranchial broad and fused with widened hypobranchial plates. Ceratobranchials I–IV rather shortened, slightly thickened, joined with branchial commissures (up to 9–10 between ceratobranchials I and II) and forming a rather reduced branchial basket (48% of the total length of the hyobranchium). Three well-defined, flattened and slightly widened spiculae fused to each hypobranchial plate dorsally, the lateralmost being the widest.

The existing knowledge on reproductive modes, development and larval morphology in Kalophrynus frogs is very fragmentary and moreover complicated by the taxonomic confusion in older publications. Some cases of erroneous species attributions to K. pleurostigma by Berry (1972), Inger (1985), Lim and Ng (1991), and Leong and Chou (1999) were later resolved by Matsui et al. (2012, 2017) and Zug (2015).

Apparently, many (if not most) Kalophrynus species tend to breed in more or less confined waterbodies. These waterbodies can be small, shallow pools as for K. sinensis (Maglangit et al. 2020), burrows in the ground as for K. calciphilus (Dehling 2011) or diverse kinds of phytotelmata, including pitcher plants Nepenthes, hollows in trees and stumps or bamboo stems as for K. limbooliati and K. yongi and possibly for K. eok, K. barioensis and K. puncak (Lim and Ng 1991; Leong and Chou 1999; Das and Haas 2003; Matsui 2009; Matsui and Nishikawa 2011; Fukuyama et al. 2021). K. meizon is rather opportunistic, using small pools, hollows in logs and burrows for reproduction (Inger 1985). In contrast, K. interlineatus and K. palmatissimus breed in various open waterbodies, including large temporary ponds and flooded areas of the forest (Kiew 1984; Manthey and Grossmann 1997; Vassilieva et al. 2016). Thus, the pond-breeding K. interlineatus, on the one hand, and K. cryptophonus breeding in very small water volumes inside bamboo internodes, on the other hand, represent the extremes of the known spectrum of reproductive habits within the genus, whereas K. honbaensis, which spawns in large tree hollows, displays a transitional state.

It has previously been shown that in Asian microhylid genera Microhyla and closely related Nanohyla phytotelm-breeding species have larger eggs and smaller clutches than pond-breeding species (Vassilieva et al. 2017b). The same pattern is revealed in Kalophrynus: the pond-breeding K. interlineatus has smaller egg diameters and much larger clutches than the phytotelm-breeding K. honbaensis and K. cryptophonus. These findings are in agreement with the extremely scarce published data on egg size in other Kalophrynus species provided by Berry (1972) and Matsui (2009): 2.3 mm and 1.88–2.14 mm in phytotelm-breeding K. limbooliati and K. yongi, respectively. The duration of the embryonal period and degree of embryonization also progress from K. interlineatus to K. honbaensis and K. cryptophonus, leading to the hatching of the larvae at more advanced developmental stages, as also occurs in N. arboricola.

No trophic eggs were found in any of the tree hollows or bamboo stems with K. honbaensis and K. cryptophonus larvae during field observations. Presumably, these tadpoles do not rely on parental provisioning and feed on unfertilized eggs from the clutches from which they themselves hatched and/or eat fertilized eggs and embryos from other conspecific clutches laid in the same phytotelmata. Such feeding mode is characteristic of tadpoles of N. arboricola, which were observed to swallow conspecific fertilized eggs and developing embryos during experimental feeding in aquaria (Vassilieva et al. 2017b) or to extract eggs from clutches suspended on the walls of the hollows where they lived (Vassilieva, personal observations 2018). The large number of K. honbaensis and K. cryptophonus tadpoles of different development stages in some of the hollows or bamboo internodes allows us to assume that different mating pairs may share a common breeding site.

All studied Kalophrynus species develop with the larval stage. More or less detailed descriptions of the external larval morphology exist for very few species: K. palmatissimus (Kiew 1984; Manthey and Grossmann 1997), K. limbooliati (Berry 1972 – as “K. pleurostigma smaller form”; Lim and Ng 1991; Leong and Chou 1999 – as K. pleurostigma), K. kiewi (Berry 1972 – as “K. pleurostigma larger form”), K. meizon (Inger 1985 – as K. pleurostigma), K. yongi (Matsui 2009), and K. intermedius (Haas et al. 2022); the previous description of K. cryptophonus tadpole by Vassilieva et al. (2014) was based on a very early developmental stage and is revised in the present work. While sharing characteristic features common for most microhylid larvae of Orton’s type II (Orton 1953; Donnelly et al. 1990), such as simplified mouthparts without keratinized elements, a medial spiracle and a medial anal tube, as well as a set of features common for Kalophrynus larvae, such as small size and terminal mouth without papillae, the described tadpoles display a variety of body shapes and proportions. The endotrophic larvae of the nepenthiphilous species K. limbooliati and K. intermedius are small, scarcely pigmented, with a rounded body, gut filled with yolk and moderately long tail with blunt tip and medium-tall fins (Lim and Ng 1991; Haas et al. 2022). Among the species studied by us, the tadpole of K. interlineatus is externally most similar to another pond-dweller, larval K. palmatissimus: both have a rounded body, convex belly, strictly lateral eyes, a rather short and broadly lanceolate tail with a weakly developed muscular portion, tall fins and a thin, fragile terminal filament. This morphological plan is characteristic of many other microhylid tadpoles coexisting with K. interlineatus in the same temporary ponds, e.g., for some Microhyla or Micryletta species (Vassilieva et al. 2016). In contrast, the tadpole of K. cryptophonus closely resembles larval K. yongi by elongated body, dorsolateral eyes, and extremely long tail with well-developed musculature and low fins. The same features are also characteristic for the larval N. arboricola and many nonmicrohylid obligatorily oophagous tadpoles (Lannoo et al. 1987; Lehtinen et al. 2004; Vassilieva et al. 2017b); this allows us to suggest that the tadpole of K. yongi may also not be endotrophic, as has been assumed by Matsui (2009), but oophagous, and the yolk in its intestine is not embryonic but rather from eaten eggs. The external larval morphology of K. honbaensis displays an intermediate state, with lateral eyes and a moderately long tail with less reduced fins. Possibly, this set of features reflects the evolutionary transition from pond to phytotelm breeding through the use of large cavities for reproduction.

Differences in the morphology of the larval digestive tract in the three studied species also reflect the evolutionary transition from micro- to macrofeeding. Suspension-feeding tadpoles, such as K. interlineatus, have a relatively long, uniform, spiraled gut evenly filled with thin homogeneous food (Altig and Kelly 1974; Wassersug and Pyburn 1987). In contrast, oophagous tadpoles, such as K. honbaensis and K. cryptophonus, tend to have a shorter digestive tract and large larval stomach (manicotto glandulare) as an adaptation to food storage and digestion in intermittent macrophagy (Lannoo et al. 1987; Liang et al. 2002). The same differences are observed between other suspension-feeding and oophagous microhylids (Vassilieva et al. 2017b); and, again, K. honbaensis displays an intermediate state of gut shortening.

The morphology of the larval chondrocranium and brachial skeleton of the three Kalophrynus species also differs markedly. In K. interlineatus, these structures are in many aspects similar to those of other pond-dwelling microhylid tadpoles studied to date, in particular, the representatives of the neotropical genera Hamptophryne, Chiasmocleis, Gastrophryne, Dermatonotus, and Elachistocleis (de Sá and Trueb 1991; Vera Candioti 2007; Trueb et al. 2011; Fabrezi et al. 2012; Ferreira and Weber 2021), as well as the Asian genera Uperodon, Microhyla, and Kaloula (Ramaswami 1940; Haas 2003; Bowatte and Meegaskumbura 2011; Vassilieva 2021; Zhang et al. 2021). All of these tadpoles share features such as a generally broad and depressed shape of the skull, an underdeveloped muscular process of the palatoquadrate, well-developed lateral posterior process, and the presence of larval otic and subotic processes; the wide cristae paroticae are mostly characteristic of neotropical microhylids, but in Asian species they are rather reduced. Other typical features of the microhylid tadpoles are a small mandible with a very thin, delicate infrarostral cartilage and hyobranchium with narrow, widely separated ceratohyals and a voluminous branchial basket. These features are recognized as adaptations for pumping large volumes of water through the buccal cavity and thus for suspension feeding (Wassersug and Hoff 1979). In contrast, the larval chondrocranium and branchial skeleton of K. cryptophonus are greatly modified. The complete reduction of the posterior part of the palatoquadrate along with ascending, larval otic and lateral posterior processes is among its most striking features. A similar reduction is found in the rhacophorid oophagous tadpole Vampyrius vampyrus (former Rhacophorus vampyrus) and is thought to be related to macrophagy (Vera Candioti et al. 2021). The infrarostral cartilage in K. cryptophonus seems to be adapted to stronger mechanical stresses than in K. interlineatus, which may be related to the need to suck out viscous mucus and swallow larger food. The hyobranchium of K. cryptophonus also sharply differs from that of K. interlineatus in greatly enlarged, massive ceratohyals giving space for the attachment of potent depressor muscles and, on the contrary, significantly reduced and simplified branchial baskets. This can be attributed to the increasing pumping force during the ingestion of large food and the reduction in the filtering function during the transition to macrophagy (Wassersug and Hoff 1979). The larval skeleton of K. honbaensis displays a somewhat intermediate state between these “suspension-feeding” and “macrophagous” extremes: in this species, the reduction of the posterior part of the palatoquadrate is not complete, although it affects the ascending and lateral posterior processes; the mandible is reinforced whereas the infrarostral cartilage is still thin and weak; and the hyobranchium is more adapted to macrophagous feeding, although its branchial basket is less reduced than in K. cryptophonus.