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Research Article
Morphological diversity of tadpoles of fork-tongued frogs (Anura: Dicroglossidae) with different trophic specializations
expand article infoAnna B. Vassilieva‡§, Trung Duc Nguyen§, Pavel A. Sorokin‡
‡ A. N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, Russia
§ Southern Branch of Joint Vietnam–Russia Tropical Science and Technology Research Center, Ho Chi Minh City, Vietnam

Corresponding author: Anna B. Vassilieva ( vassil.anna@gmail.com )

Academic editor: Deepak Veerappan
© 2025 Anna B. Vassilieva, Trung Duc Nguyen, Pavel A. Sorokin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Vassilieva AB, Nguyen TD, Sorokin PA (2025) Morphological diversity of tadpoles of fork-tongued frogs (Anura: Dicroglossidae) with different trophic specializations. Vertebrate Zoology 75: 31-57. https://doi.org/10.3897/vz.75.e139103
ZooBank: urn:lsid:zoobank.org:pub:C2D9DEE5-7A46-4A24-88A3-F926CFDBA27C
Open Access

Abstract

Dicroglossid tadpoles are an important element of pond and stream communities in Southeast Asia. They are often found in syntopy but belong to different trophic guilds, from generalized detritophages to specialized predators. In the present research, the external and internal larval morphologies of seven anuran species of the family Dicroglossidae distributed in Vietnam (Fejervarya limnocharis, Fejervarya moodiei, Hoplobatrachus rugulosus, Limnonectes dabanus, Occidozyga lima, Occidozyga martensii, and Quasipaa verrucospinosa) are described and compared. An integrated approach is followed in the study by describing external and mouthparts morphologies, gross structure of the digestive tract, and the structure of chondrocranium and hyobranchium, which revealed the main trophic adaptations of tadpoles. Different trends in the evolution of larval macrophagy in Dicroglossidae led to the formation of fundamentally different larval forms. In the first case, the tadpoles are opportunistic predators that retain the ability to use a wide range of food resources (Hoplobatrachus); they have extremely specialized mouthparts but moderately transformed digestive tract and larval skeleton. In the other case, tadpoles are highly specialized obligate carnivores (Occidozyga) that have completely lost omnivory, with a radically transformed digestive tract, chondrocranium and hyobranchium. A comparison with generalized tadpoles revealed that in dicroglossids, the transformations of the larval skeleton due to carnivory mainly affect the labial cartilages and the palatoquadrate cartilage, as well as the hyobranchium. These transformations are common to macrophagous tadpoles of different taxonomic groups and result in greater larval morphological diversity.

Keywords

Adaptation, carnivory, chondrocranium, digestive tract, hyobranchium, larval feeding, macrophagy, Vietnam

Introduction

Among all terrestrial vertebrates, amphibians have a unique biphasic life cycle in which an aquatic larva transforms into a terrestrial adult through a complex process of reorganization, the metamorphosis. In the history of amphibians, the evolution of a biphasic ontogeny followed the path of increasing complexity of metamorphosis, which entailed a progressive divergence of the larval and adult stages (Alberch 1989; Reiss 2002; Herrel et al. 2019; Vassilieva and Smirnov 2021). This decoupling of ontogenetic phases culminates in anurans: their ­larva, the tadpole, differs strikingly from the adult form in morphology, physiology, and ecology. Thus, one species can occupy different ecological niches during its lifetime, and larvae and adults are embedded in different ecolo­gical communities. This process leads to the independent adaptive evolution of tadpoles and adult anurans; it is not uncommon for larvae of related species to differ more drastically than adults due to the diversity of their living conditions (e.g., Baldo et al. 2014; Vassilieva and Nguyen 2023).

Since feeding and growth are among the main characteristics of the larval stage (Wassersug 1975), the structures related to trophic function are the key morphological features of tadpoles. Body shape, eye and mouth position, the oral disc structure and armament, as well as the buccal cavity and digestive tract structure, are highly adaptive traits associated with food intake and digestion (e.g., Altig and Kelly 1974; Diaz-Paniagua 1985; Pfennig 1990; Vera Candioti 2007; Levis et al. 2015) and provide the basis for distinguishing tadpole ecomorphological guilds (Morin 1983; Altig and Johnston 1989; Marques et al. 2019). The larval skeleton, particularly the chondrocranium and hyobranchial apparatus, is also variable depending on the trophic specialization of the tadpoles and reflects the adaptive evolution of species (Haas 2003; Vera Candioti 2007; Fabrezi and Quinzio 2008).

Anurans of the family Dicroglossidae, which are widespread in Africa and Asia, can serve as good models for a comparative morphological analysis of tadpoles. This group is interesting in terms of the diversity of reproduction forms, including terrestrial clutches and viviparity (Brown and Iskandar 2000; Kusrini et al. 2015; Frederick et al. 2023), and the variety of trophic specializations of tadpoles, from nonfeeding endotrophic forms to pond and stream detritivores, facultative suspension feeders, and predators (Altig and Johnston 1989; Rowley and Altig 2012; Lalremsanga et al. 2013). Due to their very distinctive mouthparts, carnivorous tadpoles, especially members of the genus Hoplobatrachus, have attracted particular attention from researchers (Khan 1996; Grosjean et al. 2004; Altig et al. 2009). These tadpoles have also become known as dangerous invasive species due to their predation ability, which causes great damage to the larvae of native species (Mohanty and Measey 2019). Tadpoles of the genus Occidozyga are known for their unique mouthpart morphology, which is very different from that of other dicroglossids (Inthara et al. 2005; Haas et al. 2014). In addition, the highly specialized structure of the larval skeleton of O. baluensis prompted the authors who described it to call this tadpole “extreme” (Haas et al. 2014). However, the larval cranial morphology has been studied very fragmentarily in dicroglossids; apart from O. baluensis, some descriptive data on the structure of the chondrocranium and hyobranchium of single species of Limnonectes, Quasipaa and Nanorana exist (Haas 2003; Huang et al. 2014); some details of the chondrocranium of Hoplobatrachus tigerinus were provided by Ramaswami (1940); and short notes on the larval hyobranchium of some common Indian dicroglossids were published by Khan (2003). Even less attention has been given to the gross morphology of the larval digestive tract of dicroglossids; the most detailed description was reported by Haas et al. (2014).

Being almost omnipresent in Southeast Asia, various species of Dicroglossidae often live in syntopy in forest and agrarian ecosystems, and their tadpoles coexist in the same waterbodies (Inger 1985; Inthara et al. 2005). In particular, representatives of the genera Fejervarya, Hoplobatrachus and Occidozyga, which often occur in lowland agrarian landscapes, as well as Limnonectes and Quasipaa, which inhabit forest streams, are common in the southern and central regions of Vietnam (Nguyen et al. 2009; Vassilieva et al. 2016). Specifically, the Asian rice frog Fejervarya limnocharis (Gravenhorst, 1829), the Chinese tiger frog Hoplobatrachus rugulosus (Wiegmann, 1834), the Annam fanged frog Limnonectes dabanus (Smith, 1922), the green paddle frog Occidozyga lima (Gravenhorst, 1829) and the Marten’s puddle frog Occidozyga martensii (Peters, 1867) occur and breed syntopically in lowland forested and disturbed areas of Cat Tien National Park located at the foot of the southern end of the Truong Son ridge (Annamite Mountains) in southern Vietnam; the brackish frog Fejervarya moodiei (Taylor, 1920) inhabits mangroves on southern coasts of Vietnam and co-occurs with H. rugulosus; and the granular spiny Quasipaa verrucospinosa (Bourret, 1937) lives in association with streams in forested mountain areas of central Vietnam, often in co-occurrence with Limnonectes spp. These frogs (Fig. 1) differ in appearance, varying from small (nearly 30 mm for O. martensii) to rather large (up to 130 mm for H. rugulosus), but they are all similarly semiaquatic (or mostly aquatic as O. lima), and all are opportunistic predators that feed on a wide range of invertebrates: insects (beetles, orthopterans, ants, etc.) and their larvae, spiders, crabs, myriapods, snails, and, occasionally, froglets and other small vertebrates (Vassilieva et al. 2016; Mohanty et al. 2022). The aim of our study was to perform a comparative morphological analysis of tadpoles of several common dicroglossid species living in similar environments. This analysis reveals the extent to which they differ in their trophic specializations and how the adaptive evolution of trophic structures such as the oral apparatus, digestive tract and cartilaginous skeleton could have shaped the diversity of larvae of this group of amphibians.

Figure 1. 

Dicroglossid frogs inhabiting the lowland and mountain forests in southern and central Vietnam. A Fejervarya limnocharis (Cat Tien NP, female, SVL 50.2 mm), B Fejervarya moodiei (Can Gio, male, SVL 38.4 mm), C Limnonectes dabanus (Cat Tien NP, female, SVL 44.1 mm), D Hoplobatrachus rugulosus (Cat Tien NP, male, SVL 68.3 mm), E Quasipaa verrucospinosa (Kon Ka Kinh NP, female, SVL 70.0 mm), F Occidozyga lima (Cat Tien NP, male, SVL 27.6 mm), G Occidozyga martensii (Cat Tien NP, male, SVL 22.6 mm). Photos C, F, G courtesy of E. Galoyan.

Material and Methods

Specimen collection and sampling

Field observations and specimen collection occurred during the rainy seasons (April–July) of 2016–2023 in various nature conservation areas in central and southern Vietnam. Tadpoles and adult specimens of Fejervarya limnocharis, Hoplobatrachus rugulosus, Limnonectes dabanus, and Occidozyga spp. were collected in Cat Tien National Park (Dong Nai Province, Tan Phu District; approximate coordinates 11°26.45’N, 107°24.77’E, elevation: 140 m a. s. l.); Fejervarya moodiei in the Can Gio Applied Research and Testing Station of the Joint Vietnam-Russia Tropical Science and Technology Research Center (Ho Chí Minh City, Can Gio District; approximate coordinates 10°27.35’N, 106°53.61’E, elevation: –3 m a. s. l.); and Quasipaa verrucospinosa in Kon Ka Kinh National Park (Gia Lai Province, Mang Yang District; approximate coordinates 14°13.01’N, 108°18.89’E, elevation: 1020 m a. s. l.).

Tadpoles of midlarval stages were photographed alive, euthanized with 10% ethanol and fixed whole with 10% buffered formalin for laboratory morphological examination. Samples of the tail muscle were fixed with 96% ethanol for subsequent molecular analysis. Advanced tadpoles were staged according to Gosner’s (1960) staging system for larval anurans.

Taxonomy followed the constantly updated database “Amphibian Species of the World” by Frost (2024).

Morphological examination

The description of the external morphology was based on 8–10 tadpoles of each species (6 for Quasipaa verrucospinosa) at stages 36–38. Tadpole morphometric parameters mostly followed Altig (2007) and included the following measurements: TL, total length (distance from tip of snout to tip of tail); BL, body length (distance from tip of snout to the end of belly on the level of lateral midline of tail myotomes); TaL, tail length (distance from the lateral end of belly to tip of tail); BW, maximal body width; BH, maximal body height; TH, maximal tail height (including fins); TBW, tail base width (the dorsal width of the tail right behind the end of body); SVL, snout-vent length (distance from tip of snout to vent tube opening); SSp, snout-spiracle length (distance from tip of snout to spiracle opening); DF, maximal dorsal fin height; VF, maximal ventral fin height; IN, internarial distance; IP, interpupilar distance; RP, rostro-pupilar distance (distance from tip of snout to the transversal line connecting the pupils); ED, horizontal eye diameter; ODW, horizontal oral disc width; LTRF, labial tooth (or keratodont) rows formula. All measurements were taken to the nearest 0.1 mm with digital calipers (Thingoo, China). For each species, the morphology of the larval chondrocranium and hyobranchium was examined in three to four tadpoles (stages 37–39) stained as whole mounts for cartilaginous tissue with Alcian blue and cleared with 1% KOH following Depew (2008). The skeletal elements were designated according to Haas (2003) and Haas et al. (2014). The digestive tract length (DTL) was measured by removing the entire digestive tract from three to four formalin-fixed tadpoles (stages 36–38) and extending it on a piece of filter paper. The gut contents were diluted with water and examined visually under a light microscope. Mouthpart and skeletal morphology were studied with a Leica EZ4 dissecting stereo zoom microscope (Germany) with a digital camera. Keratodont series were examined with a Keyence BZ-9000 digital microscope (Japan).

The voucher series of tadpoles were deposited in the herpetological collection of the Zoological Museum of the Lomonosov Moscow State University (ZMMU): Fejervarya moodiei, A-8127; Fejervarya limnocharis, A-8128; Hoplobatrachus rugulosus, A-8131; Limnonectes dabanus, A-8132; Occidozyga lima, A-8129; Occidozyga martensii, A-8130; and Quasipaa verrucospinosa, A-8126.

Larval identification and barcoding

Species identification of tadpoles was confirmed by DNA barcoding for all species except Quasipaa verrucospinosa. Tadpoles of Q. verrucospinosa were collected in a montane stream where adult frogs were observed; late larvae were kept in a plastic container for several days until the completion of metamorphosis, and the young metamorphs displayed characteristic features allowing an unequivocal attribution to the species (see Figs 1E, 4C, D).

The molecular genetic analysis was performed at the Center for the Collective Use of “Instrumental methods in ecology” (IPEE RAS). DNA was extracted from tissues with a QIAamp Blood and Tissue Kit (Qiagen, Germany).

A fragment of approximately 550 base pairs (bp) of the 16S rRNA mitochondrial gene from each sample was amplified using polymerase chain reaction (PCR) with the forward primer 16Sar: CGC CTG TTT ATC AAA AAC AT and reverse primer 16Sbr: CCG GTC TGA ACT CAG ATC ACG T (Palumbi et al. 2002). Each PCR mixture had a final volume of 15 μl, comprising a Master Mix 5x kit (Dialat, Russia), 1.5 μl each of the forward and reverse primers at a concentration of 5 picomoles, and 2 μl of DNA (20–100 ng/μl). The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 minutes and 35 cycles of denaturation at 95°C for 35 seconds, annealing at 55°C for 35 seconds and extension at 72°C for 45 seconds. The quality of the PCR products was controlled by electrophoresis on a 1.5% agarose gel. The PCR products were purified by enzymatic purification using the Exo/SAP Go Kit (Grisp, Portugal). Nucleotide sequences were determined using ABI PRISM 3130 and 3500 automated genetic analyzers (Applied Biosystems, USA) and a BigDye Terminator v 3.1 kit (Applied Biosystems, USA). Sequencing was performed with the primers used for amplification. Sequence alignment and editing were performed manually using the BioEdit 7.05 program (Hall 1999).

Basic Local Alignment Search Tool (BLAST) analyses on the GenBank online platform were performed to find regions of local similarity between our samples and the sequence database and calculate the statistical significance of the matches. All the distinct sequences were deposited in GenBank (sequence sets PP854639–PP854641, PP854646, PP854649–PP854651, PP855720, and PP855730–PP854732; see Table 1). The most similar sequences in the database (98–100% similarity) were used to validate the identification of our samples.

Results

Tadpole identification

A DNA barcoding approach based on the obtained sequences of the mitochondrial 16S rRNA gene (the sequence length was 521–538 bp for different taxa) supported the matching of tadpoles to the adult specimens of each taxon and the close genetic similarity of all frog species with conspecifics registered in GenBank (Table 1).

Table 1.
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Molecular identification of tadpole and adult specimens of dicroglossid species from Vietnam used in the study, based on 16S rRNA sequencing; sequences from GenBank with the highest total similarity and percent identity are listed.

Species Locality (province, district) Specimen GenBank accession number BLAST percent identity (GenBank accession number)
Fejervarya limnocharis (Gravenhorst, 1829) Dong Nai, Tan Phu Tadpoles: ABV-02087, ABV-02142, ABV-02143 (ZMMU A-8128); adults: ABV-02059 ♂, ABV-02060 ♀ PP854646 100 (MK621399)
Fejervarya moodiei (Taylor, 1920) Ho Chí Minh City, Can Gio Tadpoles: ABV-02012, ABV-02018 (ZMMU A-8127); adults: ABV-02017 ♀ PP854639 99.81 (MN453518)
ABV-02211 ♀ PP854640
ABV-02212 ♀ PP854641
Limnonectes dabanus (Smith, 1922) Dong Nai, Tan Phu Tadpoles: ABV-02451 (ZMMU A-8132); adults: ABV-02093 ♀, ABV-02094 ♀, ABV-02116 ♀ PP855650 99.63 (GU934329)
ABV-02138 ♂ PP855651 99.81 (MK688610)
Hoplobatrachus rugulosus (Wiegmann, 1834) Dong Nai, Tan Phu Tadpoles: ABV-02031, ABV-02089 (ZMMU A-8131) PP854649 99.81 (DQ283141)
Occidozyga lima (Gravenhorst, 1829) Dong Nai, Tan Phu Tadpoles: ABV-02083, ABV-02103, ABV-02126 (ZMMU A-8129); adults: ABV-02095 ♂, ABV-02096 ♀, ABV-02097 ♂, ABV-02098 ♂ PP855720 99.81 (MW007322)
Occidozyga martensii (Peters, 1867) Dong Nai, Tan Phu Tadpoles: ABV-02127 (ZMMU A-8130); adults: ABV-02049 ♂, ABV-02050 ♂, ABV-02051 ♂, ABV-02053 ♀ PP855731 98.35 (AF206467)
ABV-02048 ♂ PP855730 98.16 (AF206467)
ABV-02052 ♀ PP855732 97.98 (AF206467)

Natural history and habitats

Tadpoles of Fejervarya limnocharis were observed in lowland agricultural and disturbed habitats in shallow rain pools, often in road ruts and ditches, usually with silty bottoms and often without any aquatic or flooded vegetation. Tadpoles displayed little mobility, mostly lying on the bottom, occasionally changing position with short movements and, apparently, feeding on bottom detritus. In aquaria, tadpoles willingly ate fish flakes and scalded leaves of local green vegetables. In most cases, F. limnocharis larvae were the only tadpoles in the waterbody, rarely coexisting with larval Microhyla heymonsi and M. mukhlesuri.

Tadpoles of F. moodiei were observed in diverse types of natural pools, flooded areas and artificial ditches in disturbed open habitats along embankments on the edges of mangroves, often with abundant flooded vegetation (herbs and bushes). Larvae of Kaloula pulchra, Microhyla heymonsi and M. picta were found among coexistent tadpoles.

Tadpoles of Limnonectes dabanus were observed in lowland forests, mostly in calm backwaters of small rivulets or in shallow, slow-flowing waters of flooded forest areas in coexistence with larval Kalophrynus interlineatus, Occidozyga martensii and Microhyla minuta. They were epibenthic and displayed low mobility, usually hiding under leaf litter on the bottom.

Tadpoles of Quasipaa verrucospinosa were observed in montane forest fast-flowing rivulets with large rocks and sandy bottoms, coexisting with the larval megophryids Leptobrachella spp., Ophryophryne spp., and Brachytarsophrys intermedia. They were mostly epibenthic and rather active, hiding under rocks when disturbed.

Tadpoles of Hoplobatrachus rugulosus were commonly observed in lowland agricultural and disturbed habitats in diverse small and rather large temporary pools and ponds, both artificial (e.g., decorative or fire ponds and abandoned swimming pools) and natural. Tadpoles mostly stayed close to the bottom, moving in short, quick jerks, occasionally quickly surfacing vertically and returning down. They were observed to forage on microhylid egg clutches, which were formed as a one-layered patch of eggs floating on the surface. Tadpoles of H. rugulosus swam up to the clutches from below and bit the embryos out from the surrounding mucus. Tadpoles at advanced stages were observed to hunt other tadpoles by making quick lunges. In the laboratory, H. rugulosus tadpoles were successfully fed with scalded leaves of local green vegetables until metamorphosis. A diversity of tadpoles were observed in coexistence with H. rugulosus: Glyphoglossus guttulatus, Hylarana lateralis, Kaloula indochinensis, K. pulchra, Microhyla butleri, M. heymonsi, M. mukhlesuri, M. pulchra, Occidozyga lima, and Polypedates megacephalus. However, in some waterbodies, numerous H. rugulosus were the only tadpole species.

Tadpoles of Occidozyga martensii were common but usually not numerous in diverse shallow temporary forest ponds with soil or littered bottoms or in flooded areas of the lowland forest. They were epibenthic and rather secretive, hiding among the leaf litter. In contrast, tadpoles of O. lima were found mostly in open and agricultural habitats and in relatively large and deep ponds; they were often observed among water vegetation, sitting or crawling among algae and submerged macrophytes. In laboratory aquaria, feeding tadpoles of Occidozyga spp. with scalded green leaves and any type of fish food (flakes, powder or granules) was impossible, as these foods were completely ignored by even starving larvae; tadpoles were observed to make suction movements that propelled particles suspended in the water through the rhythmic protrusion and retraction of the lower lip.

External larval morphology

The main morphometric parameters and relevant body proportions for all the species studied are summarized in Table 2.

Table 2.
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Main morphometric characters (average ± SD, in mm) of the tadpoles (stages 36–38) of seven species of Dicroglossidae. For abbreviations, see Material and Methods.

Species TL BL TaL BW BH TH TBW SVL
Fejervarya limnocharis 24.4±2.1 9.1±0.9 15.3±1.4 5.3±0.7 3.9±0.5 4.2±0.4 1.8±0.1 11.3±0.9
Fejervarya moodiei 32.1±1.2 12.8±0.5 19.3±1.0 7.2±0.3 5.5±0.2 5.9±0.6 2.4±0.2 14.7±0.5
Limnonectes dabanus 25.0±1.7 8.8±0.4 16.2±1.3 5.3±0.4 3.8±0.3 3.8±0.4 1.9±0.2 10.7±0.7
Quasipaa verrucospinosa 51.8±8.1 17.4±2.2 34.4±6.0 11.1±1.3 9.3±1.4 12.3±1.5 5.5±1.0 19.8±2.7
Hoplobatrachus rugulosus 44.0±1.2 15.1±0.6 28.9±0.9 9.4±0.8 7.7±0.8 7.3±0.3 3.6±0.2 17.5±0.8
Occidozyga lima 27.0±4.6 7.1±1.0 19.9±3.7 4.1±0.6 3.1±0.4 5.8±1.2 2.3±0.4 8.3±1.4
Occidozyga martensii 23.0±3.6 6.7±0.8 16.2±2.9 4.0±0.5 2.8±0.5 3.0±0.5 0.4±1.8 7.7±1.7
SSp DF VF IN IP RP ED ODW
Fejervarya limnocharis 5.8±0.8 1.7±0.2 1.0±0.1 0.9±0.1 2.9±0.2 2.5±0.3 1.3±0.1 1.3±0.1
Fejervarya moodiei 8.4±0.4 2.4±0.3 1.7±0.2 1.4±0.1 3.9±0.2 4.0±0.2 1.6±0.1 3.2±0.1
Limnonectes dabanus 5.2±0.4 1.3±0.2 1.0±0.1 1.2±0.1 2.4±0.2 2.3±0.2 0.9±0.1 1.7±0.1
Quasipaa verrucospinosa 10.5±1.6 4.3±0.4 3.9±0.2 3.6±0.3 6.2±0.9 5.3±0.8 1.8±0.5 4.6±0.7
Hoplobatrachus rugulosus 11.2±0.5 2.8±0.2 2.1±0.1 2.0±0.1 4.3±0.2 4.1±0.4 2.1±0.2 3.8±0.2
Occidozyga lima 6.0±0.9 2.7±0.8 0.8±0.2 0.9±0.1 2.4±0.3 2.7±0.4 1.2±0.2 0.8±0.2
Occidozyga martensii 6.0±1.2 1.0±0.5 0.6±0.4 1.0±0.3 2.5±0.4 2.5±0.5 1.2±0.3 0.8±0.2

Fejervarya spp.

The tadpoles of both species are rather similar in their external appearance, but larval F. moodiei are markedly larger than F. limnocharis (at stages 37–38, TL 29.4–33.4 mm and 21.2–26.7 mm, respectively).

External view: In the dorsal view (Fig. 2A, C), the body is elliptical and oblong (BW/BL 0.58 ± 0.04 in F. limnocharis, 0.56 ± 0.01 in F. moodiei), with a maximum width in its middle part; the snout is broadly rounded in F. moodiei and bluntly narrowed in F. limnocharis. The tail is moderately long, approximately one and a half body lengths in F. limnocharis (TaL/BL 1.51 ± 0.07) and slightly longer in F. moodiei (TaL/BL 1.69 ± 0.13), with a moderately developed musculature (TBW/BW 0.34 ± 0.05 in F. limnocharis, 0.33 ± 0.02 in F. moodiei). Eyes are dorsolateral, moderately large, and slightly larger in F. limnocharis (ED/BL 0.14 ± 0.02) than in F. moodiei (ED/BL 0.12 ± 0.01). Nostrils are small and ovoid, without any rim or ornamentation. Nasolacrimal grooves and the lateral line system are faintly visible on the dorsal head surface in F. moodiei and almost inconspicuous in F. limnocharis. In the lateral view (Fig. 2B, D), in both species, the body is ovoid with a convex belly (BH/BL 0.42 ± 0.02 in F. limnocharis, 0.43 ± 0.01 in F. moodiei) and a slightly depressed rostral part of the head. The tail is narrowly lanceolate, almost equal in height or slightly taller than the body (TH/BH 1.10 ± 0.08 in F. limnocharis, 1.06 ± 0.11 in F. moodiei), with a tapering acuminate tip. Tail fins are moderately tall, with a maximum height at the middle of the tail length; the dorsal fin is markedly taller than the ventral fin and this difference is markedly more pronounced in F. limnocharis (DF/VF 1.75 ± 0.26 in F. limnocharis, 1.39 ± 0.11 in F. moodiei); the dorsal fin base does not extend on the trunk. The spiracle is sinistral, located mid-laterally in the posterior part of the body (SSp/BL 0.63 ± 0.03 in F. limnocharis, 0.66 ± 0.02 in F. moodiei) and represents a short, rather wide tube directed posterodorsally and entirely attached to the body wall. The vent tube is dextral, rather long, oblique, and fused to the ventral fin. Coloration: The pigmentation of the dorsum in F. limnocharis larvae is yellowish-gray, often with a greenish tint, without any conspicuous pattern. The distal part of the tail (approximately half the length of the tail) is dark gray or blackish; the belly is greenish-white. This coloration camouflages the tadpoles well against the muddy bottom; usually, only the dark tails make them visible. The pigmentation of the dorsum in F. moodiei larvae is dark brownish-gray, sometimes with a greenish tint and irregular black marbled speckling; the tail coloration is rather uniform. The belly is pale greenish or grayish, with a pearly gloss. In both species, the lateral tail vein is marked in the proximal part of the tail by a thin dark line. The iris is golden or copper. Mouthparts: In both species, the morphology of the external mouthparts is rather similar, differing in few details. The oral disc (Fig. 2E) is anteroventral, emarginated, rather wide (ODW/BW 0.35 ± 0.05 in F. limnocharis, 0.45 ± 0.03 in F. moodiei), and framed by short, finger-shaped, unpigmented marginal papillae, except for a wide gap in the upper labium and a narrow gap in the middle of the lower labium. In F. moodiei, on each side of the disc, a short double row of marginal papillae on the lateral part of the upper labium converges into a single row of thick bud-like papillae bordering the oral disc commissures and then into a crowded triple row of thinner marginal papillae framing the lateral and lower parts of the lower labium. One to three irregular rows of short submarginal papillae are present on the lateral part of the disc. In F. limnocharis, marginal papillae are less numerous and form a single or partially double row on the lower labium; submarginal papillae are absent, or only a few are present on the lower labium lateral to the tooth rows. In both species, the jaw sheaths are moderately strong (appearing more robust in F. moodiei), with fine, uniform serrations; the upper sheath is widely arched; and the lower sheath is V-shaped. In both species, the LTRF is 2(2)/3(1); keratodonts with narrowly spoon-shaped multicuspid (4–5 cusps) apices (Fig. 2D) are arranged in dense uniserial rows on flat, horizontal, unpigmented ridges. Row A2 is divided by a wide gap above the upper sheath; row P1 is subdivided medially by a very narrow, barely noticeable gap.

Figure 2. 

External morphology and mouthparts of Fejervarya spp. tadpoles. F. limnocharis (ZMMU A-8128) A dorsal view, B lateral view. F. moodiei (ZMMU A-8127), C dorsal view, D lateral view, E oral disc of F. moodiei, F keratodonts of F. limnocharis. Scale bars: 5 mm (A–D); 1 mm (E); 0.1 mm (F).

Limnonectes dabanus

External view: In the dorsal view (Fig. 3A), the body is rather oblong (BW/BL 0.60 ± 0.03), with a maximum width in its middle part; the snout is broadly rounded. The tail is moderately long, up to almost twice the length of the body (TaL/BL 1.84 ± 0.08); the tail musculature is moderately developed (TBW/BW 0.36 ± 0.01). The eyes are dorsolateral and moderately large (ED/BL 0.11 ± 0.01). Nostrils are ovoid, without any ornamentation. Nasolacrimal grooves are absent, and the lateral line system is not visible. In the lateral view (Fig. 3B), the body is narrowly elliptical and rather depressed (BH/BL 0.43 ± 0.03). The tail is lanceolate, with a tapering acuminate tip that is relatively narrow and more or less equal in height to the body (TH/BH 1.00 ± 0.10). Tail fins are relatively low, with a maximum height at the middle of the tail length; the dorsal fin is moderately taller than the ventral fin (DF/VF 1.33 ± 0.14); and the dorsal fin arises slightly posterior to the tail base. The spiracle is sinistral, located below the lateral midline of the flank in the posterior part of the body (SSp/BL 0.60 ± 0.02), and represents a short, rather wide tube directed posterodorsally and entirely attached to the body wall. The vent tube is dextral, remarkably long and wide, oblique, and fused to the ventral fin. Coloration: Coloration is camouflaged, mostly brownish; darker diffuse marbling is observed on the dorsal surface of the head and body; a more or less conspicuous dark lateral line runs from the snout through the eye to the posterior part of the head. On the proximal part of the tail side, a diffuse dark line runs along the midline marking the lateral tail vein, and diffuse dark blotches are observed on the dorsal surface; the rest of the tail, including the mostly transparent fins, displays a diffuse dark-brown marbled pattern. The belly is semitransparent, and the iris is golden with four radial black streaks. Mouthparts: The oral disc (Fig. 3C) is anteroventral, emarginated, rather small (ODW/BW 0.32 ± 0.02), and bordered by short, finger-shaped, unpigmented marginal papillae, except for a wide gap on the upper labium and a very narrow gap in the middle of the lower labium. The single row of papillae on each lateral part of the upper labium and oral disc commissure converges to a crowded double or triple row of slightly larger papillae on the lower labium; submarginal papillae are absent. The jaw sheaths are moderately strong, with fine, uniform serrations; the upper sheath is widely U shaped; the lower sheath is V shaped. Keratodonts have narrowly spoon-shaped apices with 4–6 marginal cusps (Fig. 3D) arranged on flat, unpigmented ridges, LTRF 2(2)/3(1), with row A2 divided by the upper sheath and row P1 divided by a narrow medial gap.

Figure 3. 

External morphology and mouthparts of Limnonectes dabanus tadpole (ZMMU A-8132). A dorsal view, B lateral view, C oral disc, D keratodonts. Scale bars: 5 mm (A, B); 1 mm (C); 0.1 mm (D). Abbreviations: mpu medial process of the upper sheath, ks keratinized spur, kpt keratinized tubercle.

Quasipaa verrucospinosa

External view: In the dorsal view (Fig. 4A), the body is widely elliptical (BW/BL 0.64 ± 0.01), with a maximum width at the gut level; the snout is broadly rounded. The tail is rather long, approximately twice the length of the body (TaL/BL 1.98 ± 0.15), with a well-developed musculature (TBW/BW 0.49 ± 0.04). Eyes are dorsolateral and rather small (ED/BL 0.10 ± 0.01). Nostrils are small and ovoid, without any ornamentation. Nasolacrimal grooves are absent; the lateral line system is clearly visible on the dorsal and lateral surfaces of the head, body and tail. In the lateral view (Fig. 4B), the body is depressed with a slightly convex belly (BH/BL 0.53 ± 0.01). The tail is broadly lanceolate and markedly taller than the body (TH/BH 1.32 ± 0.07), with a tapering, blunt tip. Tail fins are rather tall, with a maximum height in the middle part of the tail. The tail fins are approximately equal in height, or the dorsal fin is slightly taller than the ventral fin (DF/VF 1.09 ± 0.03); the dorsal fin base slightly extends on the dorsum. The spiracle is sinistral, located midlaterally in the middle part of the body (SSp/BL 0.60 ± 0.03) and represents a short, rather wide tube directed posterodorsally and entirely attached to the body wall. The vent tube is dextral, wide, oblique, and fused to the ventral fin. Coloration: The coloration is brown or yellowish-brown; the dorsal surface of the head and body lacks any distinct pattern. The tail is slightly paler than the body, with irregular, more or less distinct blackish spots on the flanks of the muscular portion and fewer, more diffuse round dark spots on the fins; the lateral tail vein is not marked by pigmentation. The belly is colored uniformly with the body flanks. The iris is golden with four radial black streaks. Mouthparts: The oral disc (Fig. 4E) is almost ventral, not emarginated, rather wide (ODW/BW 0.41 ± 0.02), and framed by small, thin, finger-shaped or conical, unpigmented marginal papillae, except for a wide gap in the upper labium. On each side of the disc, a triple row of papillae converges to a double row bordering the oral disc commissures and then to a crowded triple row of slightly longer marginal papillae that continuously frame the lower labium. A continuous, mostly single row of submarginal papillae (slightly larger than marginal papillae) extends below the lowest tooth row bordering the lower labium parallel to the marginal papillae. The oral disc commissures are deeply plicate, usually bearing submarginal papillae; their number varies from single, rather irregularly scattered papillae to double or triple rows. The jaw sheaths are moderately strong, with fine, uniform serrations; the upper sheath is widely arched; and the lower sheath is V shaped. Keratodonts with narrowly spoon-shaped, oblong, multicuspid (10–16 cusps) apices (Fig. 4F) are arranged on flat, unpigmented ridges; the LTRF is 6(2–6)/3(1). Row A2 is divided by a very narrow gap, row A3 has a somewhat wider gap, rows A4–6 are widely divided by the upper sheath, and row P1 is divided by a narrow but distinct medial gap.

Figure 4. 

External morphology and mouthparts of Quasipaa verrucospinosa tadpole (ZMMU A-8126). A dorsal view, B lateral view, scale bar equals to 5 mm; C premetamorphic tadpole, stage 43; D young metamorph (SVL 20.1 mm); E oral disc, scale bar equals to 1 mm; F keratodonts. Scale bars: 5 mm (A, B); 1 mm (E); 0.1 mm (F).

Hoplobatrachus rugulosus

External view: In the dorsal view (Fig. 5A), the body is elliptical and oblong (BW/BL 0.62 ± 0.05), with a maximum width in its middle part; the rostral part of the head is narrowed, with a rounded snout. The tail is rather long, up to twice the length of the body (TaL/BL 1.92 ± 0.09), with a well-developed musculature (TBW/BW 0.39 ± 0.04). The eyes are large (ED/BL 0.14 ± 0.01) and located dorsolaterally, with pupils oriented rostrolaterally. Nostrils are ovoid, without any ornamentation, and are located close to the eyes. Nasolacrimal grooves and the lateral line system are not visible. In the lateral view (Fig. 5B), the body is narrowly elliptical with a slightly convex belly (BH/BL 0.51 ± 0.06) and a depressed rostral part of the head. The tail is lanceolate, more or less equal in height or slightly lower than the body (TH/BH 0.96 ± 0.11), with a tapering acuminate tip. Tail fins are moderately tall, with a maximum height at the middle of the tail length, and the dorsal fin is slightly taller than the ventral fin (DF/VF 1.29 ± 0.08); the dorsal fin base does not extend on the trunk. The spiracle is sinistral, located below the lateral midline of the flank in the posterior part of the body (SSp/BL 0.74 ± 0.02), and represents a short, rather wide tube directed posterodorsally and entirely attached to the body wall. The vent tube is dextral, rather long, oblique, and fused to the ventral fin. Coloration: The pigmentation of the dorsum is brownish- or yellowish-gray, often with a greenish tint on the dorsum and flanks. The head and body do not display any distinct pattern, apart from darker marks around the nostrils. The dorsal surface of the muscular part of the tail bears darker irregular blotches, which are paired or alternate on both sides of the dorsal fin. The sides of the tail and fins are irregularly dark-speckled, and the distal part of the tail is often darker than the proximal part. A thin dark line marks the lateral tail vein in the proximal third or quarter of the tail. The belly is pale, grayish or cream, with a pearly gloss. The iris is golden with four black radial streaks. Mouthparts: The oral disc (Fig. 5C) is terminal, slightly emarginated, and moderately wide (ODW/BW 0.40 ± 0.04); the lower labium has a protruding semicircular medial part and smaller lateral lobes. The disc margins are bordered by a single continuous row of low, blunt, sometimes barely distinct unpigmented marginal papillae; submarginal papillae are absent. Jaw sheaths are large and extremely robust, forming a strong “predatory” beak with fine, uniform serrations. The upper sheath is widely arched, with a large, pointed, triangular medial projection; the lower sheath is very massive, bearing two pointed, triangular projections framing the medial projection on the upper sheath when the beak is closed. Inside the oral cavity, the upper beak bears an elongated medial process on the buccal roof; a low, oval, keratinized tubercle is present on the buccal roof between the lateral processes of the upper sheath (Fig. 5E). Two pointed, fang-like spurs are present on the buccal floor laterally to the mouth opening (Fig. 5F). Keratodonts have pointed, unicusped apices and are arranged on the low, flat, unpigmented ridges on the labia in uni- or biserial rows (Fig. 5D); LTRF 5(3–5)/6(3–5). On the upper labium, the keratodonts in the medial part of row A1 are larger than those of the other rows; row A2 is divided by a narrow gap in some specimens; and rows A3–6 are divided by the upper sheath and are usually single. On the lower labium, row P1 is biserial, much shorter than row P2 and formed by slightly larger teeth; row P2 is biserial at least in its medial part; rows P3–P6 are divided by the lower sheath; and rows P4–P6 are uniserial in most specimens.

Figure 5. 

External morphology and mouthparts of Hoplobatrachus rugulosus tadpole (ZMMU A-8131). A dorsal view, B lateral view, C oral disc, D keratodonts, E upper labium and mouth roof, F lower labium and lower sheath. Scale bars: 5 mm (A, B); 1 mm (C, E, F); 0.1 mm (D).

Occidozyga spp.

External view: The tadpoles of both species are similar in some features of their external appearance but also have a set of noticeable morphological differences.

In the dorsal view (Fig. 6A, C), the body is elliptical or pyriform (depending on gut fullness) and rather oblong (BW/BL 0.57 ± 0.02 in O. lima and 0.59 ± 0.02 in O. martensii), with a maximum width at the gut level; the rostral part of the head is narrowed. The tail is long, approximately two and a half times the length of the body in O. martensii and up to three times longer than the body in O. lima (TaL/BL 2.41 ± 0.11 and 2.78 ± 0.20, respectively), with a massive, strongly developed musculature (TBW/BW 0.55 ± 0.02 in O. lima and 0.45 ± 0.03 in O. martensii). The eyes are large (ED/BL 0.17 ± 0.02 in O. lima and 0.18 ± 0.01 in O. martensii) and dorsolateral, with pupils oriented rostrolaterally. The nostrils are very small, rounded, and located close to the medial line of the head. Nasolacrimal grooves and the lateral line system are not visible. In the lateral view (Fig. 6B, D), the body is subtriangular, with a depressed head narrowing rostrally and a flat belly (BH/BL 0.44 ± 0.02 in O. lima and 0.42 ± 0.03 in O. martensii). In O. lima, the tail is very characteristic in shape, much taller than the body (TH/BH 1.85 ± 0.20), with an extremely tall dorsal fin and tapering acuminate tip; the dorsal fin originates anteriorly to the tail base, has its maximum height at its proximal third, and is up to four times taller than the ventral fin (DF/VF 3.32 ± 0.66). In O. martensii, the tail is narrowly lanceolate and slightly taller than the body (TH/BH 1.07 ± 0.03), with a tapering acuminate tip; the dorsal fin originates posteriorly to the tail base, has its maximum height at its medial third, and is markedly taller than the ventral fin (DF/VF 1.63 ± 0.32). In both species, the spiracle is sinistral, located below the lateral midline of the flank in the posterior part of the body closer to the vent (SSp/BL 0.84 ± 0.06 in O. lima and 0.89 ± 0.04 in O. martensii) and represents a long, narrow tube directed posterodorsally and free in its distal part. In both species, the vent tube is dextral and rather short, disappearing rather precociously (at stages 38–39) along with the accelerated development of the hind limbs. Coloration: In both species, the pigmentation of the dorsum is brownish; the sides of the head and body below eye level are cream with darker marbling. Areas around the nostrils are brown or blackish. In O. martensii, a thin black stripe runs from the nostril through the eye to the level of the spiracle base; this line is less conspicuous in O. lima, but a clear oblique black streak below the eye is characteristic of this species, along with a dark patch on the shout tip. Both species have a thin black midlateral line on the sides of the tail running from the tail base to the proximal third of its length, along the lateral tail vein. In both species, the dorsal fin has a pinkish, reddish or orange tint, which is especially bright in O. lima. Diffuse black patches are more prominent on the dorsal surface of the tail and dorsal fin in O. lima and less distinct in O. martensii; the ventral fin bears a series of irregularly shaped black spots that are darker toward the end of the tail and more prominent and large in O. lima. The iris is golden-brown with radial black streaks. Mouthparts: In both species, the morphology of the mouthparts is very similar (Fig. 6E, F). The mouth is terminal, without a broadened oral disc, and narrow (ODW/BW 0.20 ± 0.03 in both species). The lower labium is protrusive, representing a narrow tube disrupted in its upper part that is horseshoe-shaped in cross section. The upper labium is small and semicircular, forming a flap overhanging the tubular lower lip. The jaw sheaths are small and rather delicate, with fine, uniform serrations; the upper sheath is slightly curved forward, with serrations only in its medial part; and the lower sheath is widely U-shaped. Kerato­donts are absent.

Figure 6. 

External morphology and mouthparts of Occidozyga spp. tadpoles. O. lima (ZMMU A-8129): A dorsal view, B lateral view. O. martensii (ZMMU A-8130): C dorsal view, D lateral view, E mouth of O. lima, F keratinized beak of O. lima. Scale bars: 5 mm (A–D); 1 mm (E, F).

Gross digestive tract morphology

Among the tadpoles of the species studied, the morphology of the digestive tract differs mainly in the length of the intestine (and, accordingly, the number of intestinal loops) and the presence or absence of a well-defined, muscularized stomach-like dilatation (“stomach”) in the anterior portion of the tract, assumingly homologous to the larval stomach (manicotto glandulare) characteristic of generalized tadpoles. A comparison of the characteristics is provided in Table 3.

Table 3.
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Features of the larval digestive tract morphology in the seven studied dicroglossid species. For abbreviations, see Material and Methods.

Species DTL/BL Number of intestinal loops “Stomach”
Fejervarya limnocharis 14.46 ± 1.40 18 not defined
Fejervarya moodiei 16.32 ± 1.13 18 not defined
Limnonectes dabanus 12.60 ± 1.05 12 not defined
Quasipaa verrucospinosa 12.22 ± 0.85 13 not defined
Hoplobatrachus rugulosus 7.37 ± 0.65 9–11 present
Occidozyga lima 3.53 ± 0.41 5 present
Occidozyga martensii 2.83 ± 0.54 5 present

In both species of Fejervarya, the digestive tract is very long (DTL/BL 12.84–15.35 in F. limnocharis and 15.56–17.61 in F. moodiei), of roughly equal thickness, arranged in a densely packed spiral (Fig. 7A) and filled with a uniform detritus-like mass, with abundant mineral particles (clay, sludge); in F. moodiei, this mass obviously includes small plant particles.

Figure 7. 

Digestive tracts of dicroglossid tadpoles at stages 36–38 (liver removed). A Fejervarya limnocharis, B Limnonectes dabanus, C Quasipaa verrucospinosa, D Hoplobatrachus rugulosus, E Occidozyga martensii, F Occidozyga lima. Abbreviation: ls stomach-like dilatation (“stomach”).

In Limnonectes dabanus and Quasipaa verrucospinosa, the digestive tract is roughly similar to that of Fejervarya spp. (Fig. 7B, C): it is long (DTL/BL 11.88–13.80 in L. dabanus and 11.27–12.92 in Q. verrucospinosa, respectively), of roughly equal thickness, arranged in a densely packed spiral and filled mostly with a uniform detritus-like mass, with abundant mineral particles. In L. dabanus, the gut contents include small plant particles and rare chitinous fragments (presumably, remnants of small water crustaceans or their exuviae).

In Hoplobatrachus rugulosus, the digestive tract is somewhat shortened in comparison with the species mentioned above (DTL/BL 6.62–7.83) and shows signs of functional differentiation. Its anterior portion forms a well discernible stomach-like dilatation: a large loop in the right part of the abdominal cavity, which is markedly widened and most likely serves for massive food storage, but is thin-walled, semitransparent and weakly muscularized; the rest of the intestine forms a densely packed spiral (Fig. 7D). The gut contents included, in addition to rough detritus, plant fragments and mineral particles, wholly ingested frog eggs, remains of small crustaceans and intact or damaged microhylid tadpoles.

In both species of Occidozyga, the digestive tract is very short (DTL/BL 2.32–3.39 in O. martensii and 3.27–4.00 in O. lima, respectively), forms a few loops, and its anterior part forms a well-defined expanded “stomach” with firm, opaque muscularized walls, which is easily distinguishable from the intestine even when empty (Fig. 7E, F) and separated from it by a clearly visible sphincter. The “stomach” and gut contents of both species included, in addition to small quantities of detritus and mineral particles, wholly ingested larvae and pupae of mosquitoes (Diptera) and partially digested coverings of small worms or worm-like insect larvae.

Larval skeletal morphology

As a basis for a comparative study of the larval skeletons of all studied species of Dicroglossidae, the most detailed description of these structures is given for Fejervarya species, which display the most complete set of features without signs of reduction.

Fejervarya spp.

In both species, the larval skeleton is very similar, differing only in some fine details of the morphology of the chondrocranium.

The chondrocranium (Fig. 8A, B) is broadly elliptical, with the maximum width at the level of the subocular bars. Cartilaginous roofing of the cranial cavity includes a well-developed transversal bridge (taenia tecti transversalis, ttt) and a relatively wide medial cartilaginous bridge (taenia tecti medialis, ttm) connecting a rather broad tectum synoticum (ts) with ttt and delimiting paired, ovoid parietal fenestrae (fp). Cranial base is totally chondrified, fenestra basicranialis is absent. The maximal length of the otic capsules (oc) is nearly 27–28% of the length of the chondrocranium; capsules are well chondrified, roughly elliptic with a longer axis oriented rostrolatero-occipitally; cristae paroticae (crp) are faintly developed. The trabecular horns (th) are relatively long, moderately broad (slenderer in F. limnocharis), weakly divergent, with slightly broadened rostral ends. Palato­quadrate cartilage (more robust in F. moodiei) is connected to the braincase with a moderately wide quadratocranial commissure (qcc) anteriorly (at the angle nearly 60° in F. limnocharis and 70° in F. moodiei) and a narrowing ascending process (appq) posteriorly (at the angle nearly 75° in F. limnocharis and 80° in F. moodiei). The rostral part of the palatoquadrate, the pars articularis quadrati (paq), is moderately solid and bears a slightly widened articular process (arp, forming a joint with Meckel’s cartilage) and a short quadratoethmoid process (qep). Anteriorly, the lateral margin of the palatoquadrate forms a well-developed, rather tall (and markedly broader in F. moodiei), subtriangular muscular process (mp) protruding dorsally and slightly curving medially. The subocular bars (sb) are moderately wide (broader in F. moodiei), delimiting large, elliptic subocular fenestrae (fs). A thin, spike-shaped pseudopterygoid process (ppp) is present. The posterior palatoquadrate curvature (ppqc) is slightly concave on F. limnocharis and strongly concave with a bulging posterior margin in F. moodiei. A rather weak larval otic process (lop), especially thinning rostrally, connects the posterior curvature of the palatoquadrate with the anterolateral part of the crista parotica on the outer side of the otic capsule. Suprarostral cartilage (src) (Fig. 8C) is entire, with fused alae and corpora; the corporal parts (cor p) are connected ventrally and divided dorsally by a deep medial slit; a relatively small oval aperture is present between each corporal and alar part; and alar parts (ala p) are articulated with the rostrolateral end of the trabecular horns by expanded posterior processes (pp). The lower jaw (Fig. 8D) is rather gracile and is composed laterally of paired Meckel’s cartilage (Mc) and medially of paired infrarostral cartilage (irc) connected by a medial symphysis. Meckel’s cartilage is rather slender, bearing a relatively robust, hooked retroarticular process (rap). The infrarostral cartilage is flattened vertically and arranged in a wide U-shaped arc.

Figure 8. 

Skeleton of Fejervarya spp. larvae, stages 37–38. Dorsal view of the chondrocranium: A F. limnocharis, B F. moodiei, C supralabial cartilage of F. moodiei; D lower jaw of F. moodiei. Ventral view of the hyobranchium: E F. limnocharis, F F. moodiei. Abbreviations: ala p alar part of the suprarostral, alp anterolateral process, ap anterior process, appq ascending process of the palatoquadrate, arp articular process of the palatoquadrate, bb basibranchial, bh basihyal, cor p corporal part of the suprarostral, cbr ceratobranchial, chy ceratohyal, crp crista parotica, fp parietal fenestra, fs subocular fenestra, hbp hypobranchial plate, irc infrarostral cartilage, lop larval otic process, Mc Meckel’s cartilage, mp muscular process of the palatoquadrate, oc otic capsule, pp posterior process, ppch posterior process of the ceratohyal, ppp pseudopterygoid process, ppqc posterior palatoquadrate curvature, pru pars reuniens, qcc quadratocranial commissure, qep quadratoethmoid process, rap retroarticular process, sb subocular bar, sp spicula, src suprarostral cartilage, th trabecular horn, ts tectum synoticum, ttm taenia tecti medialis, ttt taenia tecti transversalis, ubp urobranchial process. Scale bar: 1 mm.

The hyobranchial skeleton (Fig. 8E, F) is composed of basihyal (bh), ceratohyals (chy), basibranchial (bb), pars reuniens (pru), hypobranchial plates (hpb), and a moderately voluminous (approximately 68–70% of the total length of the hyobranchium) reticulated branchial basket formed by four ceratobranchials (cbr). The basihyal is rather small, delicate, narrowly fusiform. Ceratohyals are moderately robust, with a massive lateral portion, prominent triangular anterior process (ap) oriented rostrally and a well-developed, rather narrow, triangular anterolateral process (alp) slightly curving medially (at the angle 70° in both species); the posterior process (ppch) is large and triangular. The pars reuniens is short, wider than long; the basibranchial is short, subquadrate in shape, bears a rather short and blunt urobranchial process (ubp), and articulates posteriorly to the moderately large hypobranchial plates. Ceratobranchials are moderately long, rather slender, and flattened, bearing numerous delicate, branching lateral projections. Four thin, rather short (approximately one-third of the length of the ceratohyals) spike-like spiculae (sp), often with slightly branching free tips, are fused to the proximal end of each ceratobranchial I–IV dorsally (see also Fig. 14A).

Limnonectes dabanus

The skeletal morphology of this species basically resembles that of Fejervarya spp. but differs in a set of features.

The chondrocranium (Fig. 9A) is broadly elliptical, with a maximum width at the level of the subocular bar, but more gracile; the trabecular horns and palatoquadrate cartilage are slenderer; the quadratocranial commissure connecting to the braincase at the angle nearly 55° and ascending process of the palatoquadrate connecting to the braincase at nearly a right angle are slightly longer and narrower; the taenia tecti transversalis and medialis are narrower; the fenestrae parietales are relatively larger, making the larval skull of L. dabanus look lighter and more fenestrated, somewhat broader than in Fejervarya. The elongate, elliptic or subrectangular basicranial fenestra occupies the central part of the cranial base and is covered ventrally by a thin parasphenoid bone. The length of the otic capsules is nearly 28–30% of the length of the chondrocranium. Pseudopterygoid and quadratoethmoid processes are present. The posterior palatoquadrate curvature is concave with a bulging posterior margin. The major difference from Fejervarya is the absence of the larval otic process; only a small knob-like tubercle, the anterior process of the crista parotica (apcp), is observed on the anterolateral side of the otic capsule. Cristae paroticae are underdeveloped. Unlike in Fejervarya, the distal end of each trabecular horn bears a prominent pointed outgrowths directed posterolaterally. The suprarostral cartilage (Fig. 9B), lower jaw (Fig. 9C) and hyobranchium (Fig. 9D) are similar in shape to those of Fejervarya; the basihyal is absent (at least in the stages examined), and the branchial basket is nearly the same size (approximately 70% of the whole length of the hyobranchium). The triangular, pointed anterior process of the ceratohyal is oriented rostrally and a well-developed, rather narrow, triangular anterolateral process (alp) is curving medially (at the angle about 60°). Ceratobranchials are slender, with numerous branching lateral projections, and are fused together distally. Spiculae I–III are thin, spike-like; spicula IV is short and somewhat flattened.

Figure 9. 

Skeleton of Limnonectes dabanus larvae, stage 38: A chondrocranium (dorsal view), B supralabial cartilage, C lower jaw, D hyobranchium (ventral view). Abbreviations: alp anterolateral process, ap anterior process, apcp anterior process of the crista parotica, appq ascending process of the palatoquadrate, cbr ceratobranchials, fp parietal fenestra, irc infrarostral cartilage, Mc Meckel’s cartilage, pp posterior process, ppp pseudopterygoid process, qep quadratoethmoid process. Scale bar: 1 mm.

Quasipaa verrucospinosa

This species is basically similar to Fejervarya spp. in terms of the morphology of its larval skeleton, with few features.

The chondrocranium (Fig. 10A) is broadly elliptical, with the maximum width at the level of the posterior curvature of the palatoquadrate. Fenestra basicranialis is almost totally chondrified and covered ventrally by a well ossified parasphenoid bone. Otic capsules are rather rounded, approximately of the same relative size as those in Fejervarya (28% of the length of the skull). Cristae paroticae are faintly developed. The palatoquadrate cartilage is moderately robust, the subocular bar is curved smoothly medially, and the quadratocranial commissure connecting to the braincase at the angle nearly 65° and ascending process connecting to the braincase at the right angle are rather narrow. On the anterior part of the palatoquadrate, the quadratoethmoid process is present, and the pseudopterygoid process is reduced. The posterior palatoquadrate curvature is concave with a bulging posterior margin. The larval otic process is absent, and a small tubercle-shaped anterior process of the crista parotica is observed on the anterolateral surface of the otic capsule. The supralabial cartilage (Fig. 10B) and the lower jaw (Fig. 10C) do not differ markedly from the same structures in Fejervarya. In the hyobranchium (Fig. 10D), the basihyal is absent or can be identified in a rudimentary form in the largest tadpoles. The urobranchial process on the basibranchial is short and blunt but well defined. The ceratohyal is more robust than in Fejervarya, and the branchial basket is approximately the same relative size (approximately 70% of the hyobranchial length). The anterolateral process of the ceratohyal is rather low and blunt, very slightly curving medially at the angle nearly 80°. The ceratobranchials are rather slender, with lateral projections. Ceratobranchials I–III are fused distally; ceratobranchial IV has a loose end, approaching the the distal part of ceratobranchial III but not fusing to it. Spiculae I–III are thin, spike-like and similar to the spiculae of Fejervarya spp., whereas spicula IV forms a pair of flat, rather large cartilaginous plates contacting each other medially.

Figure 10. 

Skeleton of Quasipaa verrucospinosa larvae, stage 36. A chondrocranium (dorsal view), B supralabial cartilage, C lower jaw, D hyobranchium (ventral view). Abbreviations: alp anterolateral process, ap anterior process, apcp anterior process of the crista parotica, appq ascending process of the palatoquadrate, cbr ceratobranchials, chy ceratohyal, irc infrarostral cartilage, Mc Meckel’s cartilage, pp posterior process, qep quadratoethmoid process, ubp urobranchial process. Scale bar: 1 mm.

Hoplobatrachus rugulosus

Compared with that of Fejervarya spp., the morphology of the larval skeleton of this species is markedly modified.

The chondrocranium (Fig. 11A) is rather broad, stout, and roughly as wide as long, with well-chondrified posterior skull roofing: the taeniae tecti are extremely widened, and the parietal fenestrae are strongly reduced. A narrow fenestra basicranialis occupies the central part of the partially chondrified cranial base. Otic capsules are solid, subrectangular, and approximately the same relative size as those in Fejervarya (28–29% of the length of the skull). The crista parotica represents a low tubercle on the lateral side of the otic capsule. The trabecular horns are rather broad, partially fused proximally at nearly 60% of their length; only their distal ends are divergent. The palatoquadrate cartilage is rather firm and massive. The pars articularis quadrati is widened, bearing a robust articular process; the quadratoethmoid process is wide and triangular; the pseudopterygoid process is absent. The muscular process is tall and massive, with bold and rigid dorsal margins, and is only slightly curved medially. The subocular bar is curved laterally, forming wide subocular fenestra. The quadratocranial commissure connecting to the braincase at the angle nearly 45° is broadened and solid; the ascending process connecting to the braincase at a slightly obtuse angle (nearly 100°) is solid and flattened vertically. Larval otic process is very short and solid. The supralabial cartilage (Fig. 11B) represents an entire, firm and resilient cartilaginous plate without defined posterior processes and curved rostrally, with a ventral protrusion corresponding to the pointed beak-like medial projection on the upper sheath. The lower jaw (Fig. 11C) is very robust, formed by extremely massive Meckelian cartilages and a large, solid infrarostral cartilage with two triangular protrusions on its dorsal edge corresponding to the pointed tooth-like projections on the lower sheath. In the hyobranchium (Fig. 11D), the branchial basket is rather reduced (nearly 55–56% of the total length of the hyobranchium) relative to the large and very robust ceratohyals; anterior processes of the ceratohyals are blunt and massive, whereas anterolateral processes are absent. The basihyal is small but thick; the pars reuniens is weakly chondrified, and the basibranchial bears a very short urobranchial process that forms a low, transversally oblong tubercle. Ceratobranchials are somewhat thickened, with numerous branching lateral projections. Spiculae have a modified structure, representing short (nearly 1/5–1/4 of the length of the ceratobranchials) flat structures; spicula I is the narrowest, with an acuminate apex; spicula II is broadly triangular; and spiculae III and IV are broadened cartilaginous plates with irregular margins (see also Fig. 14B).

Figure 11. 

Skeleton of Hoplobatrachus rugulosus larvae, Stage 37. A chondrocranium (dorsal view), B supralabial cartilage, C lower jaw, D hyobranchium (ventral view). Abbreviations: appq ascending process of the palatoquadrate, bb basibranchial, bh basihyal, cbr ceratobranchials, chy ceratohyal, crp crista parotica, fp parietal fenestra, irc infrarostral cartilage, lop larval otic process, Mc Meckel’s cartilage, mp muscular process of the palatoquadrate, paq pars articularis of the palatoquadrate, qep quadratoethmoid process, src suprarostral cartilage, th trabecular horns, ubp urobranchial process. Scale bar: 1 mm.

Occidozyga spp.

In these tadpoles, the skeletal morphology is highly modified compared with that of larval Fejervarya spp. and certain notable differences are observed between the two species of Occidozyga.

The chondrocranium (Fig. 12A, B) is oblong, roughly subrectangular in shape, more elongated in O. lima than in O. martensii, and rather solid. In the posterior part of the cranial roof, the tectum synoticum is well chondrified and solid, whereas the taenia tecti transversalis and taenia tecti medialis are rather thin in both species, forming wide parietal fenestrae. The cranial base is totally chondrified. Otic capsules are elliptic, relatively larger than in Fejervarya spp. (30–31% of the total length of the skull in O. lima and 36–37% in O. martensii); cristae paroticae are absent. The trabecular horns are very short, broadened, and well divergent in O. martensii and nearly parallel in O. lima. In both species, palatoquadrate cartilages are massive, robust, and connected to the braincase only anteriorly with extremely broad quadratocranial commissures; ascendant processes are absent. In both species, the anterior part of the palatoquadrate cartilage is narrowed, bearing a solid, rather tall muscular process (ridge-shaped in O. lima and subtriangular in O. martensii) protruding dorsally and markedly curving medially; the most rostral part of the palatoquadrate (pars articularis, paq) forms a long outgrowth protruding rostroventrally and bearing robust ventral and muscular processes (sensu Haas et al. 2014). The pseudopterygoid and quadratoethmoid processes are absent. In the posterior portion of the palatoquadrate, subocular bars are shifted rearward and severely shortened, and subocular fenestrae are small and open posteriorly. In O. martensii, the posterior end of the subocular bar is fused to a narrow cartilaginous bar oriented dorsally and then curving laterally, which presumably corresponds to the posterior palatoquadrate curvature; laterally, it is fused to the larval otic process connecting it with the otic capsule and forms a rather long outgrowth protruding rostrolaterally and resembling the lateral process of the palatoquadrate found in some other tadpoles. In O. lima, the posterior end of the subocular bar is free but bears a small tapering outgrowth directed dorsolaterally, toward a similar portion of cartilage pointing toward it; the latter is connected with the otic capsule by a thin cartilaginous bridge, apparently representing the larval otic process. Thus, the two laterally directed pointed outgrowths obviously resemble rudiments of a disrupted and partially reduced posterior palatoquadrate curvature.

Figure 12. 

Skeleton of Occidozyga spp. larvae, stages 37–38. Dorsal view of the chondrocranium: A O. lima, B O. martensii. Supralabial cartilage: C O. lima, D O. martensii. Lower jaw: E O. lima, F O. martensii. Hyobranchium (ventral view): G O. lima, H O. martensii. Abbreviations: bb basibranchial, cbr ceratobranchials, chy ceratohyal, crp crista parotica, fp parietal fenestra, hbp hypobranchial plate, irc infrarostral cartilage, lop larval otic process, Mc Meckel’s cartilage, mp muscular process of the palatoquadrate, paq pars articularis of the palatoquadrate, ppqc posterior palatoquadrate curvature, qcc quadratocranial commissure, rap retroarticular process, r ppqc rudiments of the posterior palatoquadrate curvature, sb subocular bar, src suprarostral cartilage, sp spicula, th trabecular horn, ttm taenia tecti medialis, ttt taenia tecti transversalis. Scale bar: 1 mm.

In both species, the suprarostral cartilage (Fig. 12C, D) represents an entire solid and resilient cartilaginous plate, arching forward and articulating dorsally with the trabecular horns. The morphology of the lower jaw (Fig. 12E, F) is similar in O. lima and O. martensii and differs from the generalized structure by unusually elongated Meckel’s cartilage and its almost vertical orientation; the infrarostral cartilage is rather narrow but dense and firm.

The hyobranchium (Fig. 12G, F) is similar in both Occidozyga species but differs strongly from the same skeletal element in the other studied tadpoles. The ceratohyal is extremely large and robust, with wide and massive anterior and posterior processes and a long and massive lateral process, whereas the branchial basket is severely reduced (approximately 37–40% of the total length of the hyobranchium). The basihyal is absent. The pars reuniens is shortened, and the basibranchial is narrow, strongly elongated, and almost rod-like, especially in O. lima; the urobranchial process is absent. The hypobranchial plate is broad and solid; laterally, it fuses with ceratobranchial I. In O. lima, the rather wide and solid, flattened proximal part of the ceratobranchial I is connected laterally to a thin, rod-like distal part by a very fragile, filiform strand of cartilage. In O. martensii, the connection of the proximal and distal parts of ceratobranchial I is disrupted. The distal portion of ceratobranchial I is connected to the distal end of ceratobranchial II by a thin terminal commissure; the commissure between ceratobranchials II and III is disrupted, whereas the short commissure between ceratobranchials III and IV is preserved, though extremely thin and fragile. Ceratobranchials are thinned and simple, without lateral transverse branchial processes. The structure of the spiculae differs in both species. In O. lima, spicula I is present as a short, flat outgrowth at the base of ceratobranchial I; it is directed posteriorly and reaches the base of ceratobranchial II. In O. martensii, spicula I represents a very thin cartilaginous rod extending from the base of ceratobranchial I (but not fused to it) and directed posteriorly across the base of ceratobranchial II (see Fig. 14C); in both species, other spiculae are absent.

Discussion

Dicroglossid larvae are a common element of tadpole communities in South and Southeast Asia, and many species have been described to date (e.g., Inger 1985; Chou and Lin 1997; Inthara et al. 2005; Haas et al. 2022; Raj et al. 2023, and many individual descriptions). The vast majority of them are omnivorous/detritophagous and have a more or less generalized morphology with a moderately long tail (approximately 1.5–2 times longer than body), moderately developed fins, dorsolaterally positioned eyes and an anteroventrally positioned mouth. Most of them belong to the guilds of lentic–benthic or nonspecialized lotic–benthic tadpoles (guild II.12) in the classification of Altig and Johnston (1989). In our study, the pool-dwelling, detritophagous tadpoles of Fejervarya limnocharis and F. moodiei with TaL/BL values of 1.5–1.7, rather low fins not extending beyond the tail–body junction, an anteroventral oral disc bordered with a gaping row of rather large marginal papillae and LTRF 2(2)/3(1), are very similar to the larvae of closely related species (Raj et al. 2023) and can be considered typical representatives of guild 12a. The tadpoles of Limnonectes dabanus, although they belong to stream communities, inhabit a wide range of slow-flowing lowland and montane waterbodies and possess typical features of unspecialized tadpoles of the same guild II.12; at the same time, the structures of their oral disc and LTRF 2(2)/3(1) are identical to those of other lotic–benthic Limnonectes larvae (e.g., McLeod 2008; Ampai et al. 2015; Flores et al. 2023).

Larval Quasipaa verrucospinosa, which are found only in cascade mountain streams, are rather similar to other congeneric tadpoles described to date (Inthara et al. 2009; Chuaynkern et al. 2018) and display some features characteristic of more specialized lotic tadpoles, i.e., longer tails (nearly twice the body length) with a well-developed musculature and taller fins, which can be considered adaptations to locomotion in flowing water. The oral disc in this species is bordered with smaller and more densely packed marginal papillae and bears more keratodont rows (six) on the upper labium than typical generalized larvae, which is also considered a feature of stream-adapted tadpoles (Altig and Johnston 1989).

In contrast, the studied predatory dicroglossid tadpoles, Hoplobatrachus rugulosus and Occidozyga spp., belong to guilds of carnivorous macrophages with a set of characteristic features, such as a strongly muscled tail (particularly long in Occidozyga spp., up to three times longer than the body), a terminal mouth, and relatively large eyes oriented rostrolaterally. However, the mouthparts of Hoplobatrachus and Occidozyga are specialized in two very different ways. The actively hunting larval H. rugulosus possess an oral disc, although it is more rigid than in detritophagous tadpoles, and its marginal papillae are severely reduced. The adaptation to predation in this species is based on the increased armament of the oral apparatus: the powerful, cuspate beak is adapted to heavy loads, which is also due to the additional support on the palate by a medial outgrowth. The keratinous “fangs” in the corners of the mouth, a unique feature of the genus, apparently contribute to grasping and holding large resisting prey; the keratinous tubercle on the roof of the oral cavity, which was not mentioned for Hoplobatrachus larvae in the studies by Grosjean et al. (2004) and Altig et al. (2009) but noted by Khan (1996) for H. tigerinus, probably serves the same purpose. Large, pointed, and arranged in biserial rows, the keratodonts of H. rugulosus larvae differ strikingly from the smaller, spoon-shaped, multicuspid keratodonts of the detritophagous tadpoles of Fejervarya spp., L. dabanus and Q. verrucospinosa; moreover, the reduction in the internal papilla on the oral roof and oral floor in Hoplobatrachus larvae, noted by Khan (1996), is a sign of adaptation to carnivory. According to these features, larval H. rugulosus, as well as other congeneric larvae, can be referred to as typical representatives of the carnivorous tadpoles of guild II.13a sensu Altig and Johnston (1989).

The oral apparatus of Occidozyga is fundamentally different. It is adapted not to grasp large victims but to suck relatively small live prey through a narrow mouth with a protruding, semitubular lower lip. The upper lip, which forms a type of valve covering the mouth opening, could conceivably prevent the backward movement of the captured food. The oral disc is practically absent, and the only armament of the oral apparatus is a miniature beak, which is rather capable of fixing the prey but not tearing it. Due to these features and a less mobile hunting mode close to the bottom, larval Occidozyga spp. belong to the guild of benthic macrophagous tadpoles, II.14a (Altig and Johnston 1989).

Different mouthpart structures determine the different ways of feeding of predatory tadpoles of H. rugulosus and Occidozyga spp. and their different food spectra. Our observations contradict the conclusion of Grosjean et al. (2004) that Hoplobatrachus tadpoles are obligate carnivores. The contents of the digestive tract show that, despite their highly specialized mouthparts, H. rugulosus are omnivorous opportunists rather than strict carnivores. Thus, their gut contains not only tadpoles and invertebrates but also amphibian eggs (which many generalized tadpoles feed on), ground plant matter, and microalgae. These findings confirm that H. rugulosus tadpoles are capable not only of hunting but also of filtering organic particles from water and scraping surfaces. This finding is supported by our laboratory observations in which H. rugulosus tadpoles developed normally while feeding on plant food, as well as previous field observations in their natural habitat and stable isotope data. The latter demonstrated that, in the same temporary waterbody in Cat Tien National Park in different years, H. rugulosus tadpoles either occupied the highest trophic level as top predators or were on the same trophic level with omnivorous coexisting tadpoles (Vassilieva et al. 2017a).

On the other hand, tadpoles of O. lima and O. martensii, with their highly specialized mouthparts devoid of oral discs and keratodonts, are unable to scrape or bite off plant material and are apparently unable to effectively filter small organisms and organic matter suspended in the water. Laboratory observations also confirmed that they are incapable of growing when only plant food or various types of dry fish food are offered. In other words, they are obligate predators, feeding exclusively on living prey, and the small amount of detritus found in their digestive tract is probably the remains of concomitantly ingested matter. Interestingly, O. baluensis larvae, generally very similar to the Occidozyga tadpoles studied here, have been observed to swallow whole relatively large prey (such as aquatic coleopteran larvae) and even to attack live tadpoles of other species (Haas et al. 2014).

The gross morphology of the digestive tract of dicroglossid tadpoles also confirms their segregation into different trophic guilds. Many examples of anuran larvae have shown that the length of their intestine, as well as the differentiation of a stomach-like dilatation, correlate well with the trophic specialization of the species (Altig and Kelly 1974; Wickramasinghe et al. 2007; Fabrezi and Cruz 2020). Among the species studied here, the generalized detritivorous tadpoles Fejervarya spp., L. dabanus and Q. verrucospinosa have a typical long (DTL/BL > 10) spiral intestine, forming dense loops, and lack a clearly differentiated muscular “stomach”. In contrast, the strictly macrophagous larvae of Occidozyga spp. have a much shorter digestive tract (DTL/BL < 4) and a well-defined, voluminous and muscularized “stomach” separated from the intestine by a sphincter (Haas et al. 2014; present study). In H. rugulosus, the larval digestive tract shows a somewhat intermediate state, as the intestine is spiraled but shorter than that in nonspecialized omnivores (DTL/BL nearly 7–8), with fewer loops; the enlarged portion, serving for ingested food storage and corresponding to the larval stomach, is long, weakly muscularized and lacks a sphincter in its distal part. Thus, on the one hand, the adaptation of the digestive tract of H. rugulosus to store a large volume of food also characterizes these tadpoles as voracious predators capable of grasping and tearing relatively large mobile prey. Not surprisingly, in large numbers, Hoplobatrachus tadpoles are capable of the total extermination of all other tadpoles in a waterbody. On the other hand, despite the obvious signs of specialization to macrophagy, the digestive tract of this species is still adapted for the digestion of a variety of food, both of animal and plant origin.

Trophic specialization of both generalized and predatory tadpoles is also clearly displayed in the morphology of their chondrocranium and hyobranchium.

The detritophagous Fejervarya spp., Limnonectes dabanus and Quasipaa verrucospinosa have a similar shape and structure of their rather generalized larval skull. The main difference between Fejervarya spp. and the other two species is the structure of the posterior part of the palatoquadrate. In both species of Fejervarya, the posterior palatoquadrate curvature is connected to the otic capsule by the larval otic process, whereas in L. dabanus and Q. verrucospinosa, only a small, knob-shaped anterolateral process of the crista parotica is present that does not reach the palatoquadrate. The cartilaginous elements of the jaw apparatus are also similar in all these species and seem to lack striking specializations.

In contrast, the predatory tadpoles of H. rugulosus and Occidozyga spp. display a set of morphological modifications related to macrophagy, although differences in their feeding strategies lead to markedly different adaptations. The resemblance between H. rugulosus and Occidozyga spp. is limited to very few characters, such as very short, stout trabecular horns and an entire supralabial cartilage, which shows no signs of subdivision into alar and corporal parts and has no defined posterior processes.

Due to the almost continuous cartilaginous roofing in the occipital part of the skull, the massive pars articularis of the palatoquadrate and powerful muscular process, the solid supralabial cartilage and robust lower jaw, the chondrocranium of H. rugulosus appears to be adapted to greater efforts than the skulls of the generalized tadpoles. However, its general structure is not fundamentally different from that of detritophagous dicroglossids.

On the contrary, the unusually elongated, compact chondrocrania of Occidozyga spp. display a set of unique features not found in any other tadpoles known to date apart from the congeneric O. baluensis. The larval cranial morphology and head musculature of O. baluensis were thoroughly described in detail by Haas et al. (2014). In this study, the authors emphasized, among other cranial features of the species, the unique morphology of the palatoquadrate cartilage, including its highly modified pars articularis with long muscular and ventral processes directed anteroventrally, the extremely broadened quadratocranial commissure, the absence of the ascending process, the reduced subocular bars and subocular fenestrae and the presence of a small, rod-like, isolated cartilage positioned in front of the otic capsule. Haas et al. (2014) homologized this cartilage (described as cartilago praeotica) with the posterior palatoquadrate curvature.

Our study revealed that the most significant differences between the species of Occidozyga were mainly in the posterior portion of the palatoquadrate. In O. martensii, the posterior palatoquadrate curvature (ppqc) is complete and confluent to the larval otic process (lop), whereas in O. lima, the lop is preserved, but the ppqc is disrupted and represented by two rudimentary portions: the medial one is fused to the blunt end of the subocular bar and the lateral one is fused through the lop to the otic capsule. This condition is very unusual, including for dicroglossids, in which the lop is often absent, for example, in Limnonectes and Quasipaa (Haas 2003; present study), but the ppqc is always fully developed. In support of the homology proposed by Haas et al. (2014), we presume that the cartilago praeotica can represent a preserved fragment of the ppqc formed as a result of a gradual reduction in the rear portion of the palatoquadrate in the series O. martensii — O. lima — O. baluensis.

Among recently described tadpoles, similar chondrocranial transformations have been observed in other specialized macrophagous species, which are obligate phytotelm-dwelling oophages. Thus, in Vampyrius vampyrus (formerly Rhacophorus vampyrus, Rhacophoridae), the quadratocranial commissure is strongly broadened compared to other known rhacophorid chondrocrania, the ascending and larval otic processes disappear, the subocular bar is shortened and ends bluntly without any connection to the braincase or otic capsule, and the subocular fenestra are small and open posteriorly (Vera Candioti et al. 2021). The progression of specialization to obligate oophagy in the tadpoles of Kalophrynus (Microhylidae) allowed us to trace the accompanying gradual remodeling of the palatoquadrate, including the reduction of the posterior quadratocranial curvature and related structures (Vassilieva and Nguyen 2023); in fact, the rear part of the palatoquadrate in K. cryptophonus larvae closely resembles the conditions observed in O. lima and O. baluensis. In another obligatory oophagous microhylid tadpole, Nanohyla arboricola (formerly Microhyla arboricola), the unique morphology of the palatoquadrate also includes several similar features. In the original description, the presence of the ascending process in this species was erroneously stated (Vassilieva et al. 2017b). A subsequent examination of newly obtained specimens revealed that, in fact, in N. arboricola, the palatoquadrate is connected to the braincase only by a broad quadratocranial commissure, whereas the ascending and larval otic processes are absent; the subocular fenestra is unclosed and the subocular bar is continued posteriorly by a laterally directed, loosely ending cartilaginous process that is presumably homologous to the posterior palatoquadrate curvature (Fig. 13). Somewhat similar features (expanded palatoquadrate and reduced subocular bar) can be found in the extremely bizarre phytotelm-dwelling tadpole of Phyllodytes gyrinaethes (Hylidae), which is also presumed to be oophagous (Vera Candioti et al. 2017). The carnivorous tadpoles of Hymenochirus boettgeri and Pseudohymenochirus merlini (Pipidae), although very peculiar in their cranial morphology, are also characterized by certain similarities in the structure of the palatoquadrate: a massive quadratocranial commissure and the absence of ascending and larval otic processes (Klinger-Strobel et al. 2020; Lukas et al. 2024). These findings corroborate that macrophagous specialization drives similar transformations of the larval chondrocranium even in phylogenetically distant anuran groups.

Figure 13. 

Schematic depiction of the chondrocranium of Nanohyla arboricola larvae (ventral view). Abbreviations: oc otic capsule, ppqc posterior palatoquadrate curvature, qcc quadratocranial commissure, sb subocular bar.

The hyobranchial apparatus has a more or less uniform generalized morphology in all the studied detritophagous tadpoles (Fejervarya spp., L. dabanus, and Q. verrucospinosa), with few minor interspecific differences. In the carnivorous H. rugulosus, the hyobranchium follows, to some extent, the pattern of rearrangement characteristic of many macrophagous tadpoles, which implies the hypertrophy of the ceratohyals and the reduction of the ceratobranchials, as was shown, for example, for predatory larvae of Ceratophrys and Lepidobatrachus (Vera Candioti 2005; Fabrezi and Quinzio 2008) and oophagous tadpoles of V. vampyrus, N. arboricola and Kalophrynus spp. (Vassilieva et al. 2017b; Vera Candioti et al. 2021; Vassilieva and Nguyen 2023). Although the ceratohyal in H. rugulosus is markedly enlarged and robust, the branchial basket, which is somewhat reduced in relative size, still preserves the whole set of ceratobranchial elements without any signs of simplification. Hence, in this species, the hyobranchium maintains a functional balance: it is still capable of performing both filtering and respiratory functions in addition to effectively creating negative pressure in the oral cavity.

Compared with H. rugulosus, the hyobranchium of Occidozyga spp., which is very similar in all the species studied, including O. baluensis (Haas et al. 2014), is much more specialized. The enormous ceratohyal and rather broad hypobranchial plate, along with the substantially reduced in size and simplified ceratobranchials, indicate that in these tadpoles, the filtering and respiratory functions are heavily biased in favor of enhanced force of pumping. It can be assumed that the capacity for powerful suction made it possible for Occidozyga larvae to swallow even relatively large but soft prey, as reported, for example, by Haas et al. (2014). The great similarity of the hyobranchium of Occidozyga spp. with that of another specialized macrophagous tadpole with a suctorial mode of feeding, Dendropsophus nanus (formerly Hyla nana, Hylidae), supports their common adaptation to the intensive suction of prey with the water current (Vera Candioti et al. 2004; Haas et al. 2014). At the same time, most likely, owing to the poor respiratory function of the gill apparatus, the substantial elongation of the tail in Occidozyga tadpoles and the extremely hypertrophied, tall and richly vascularized dorsal fin in O. lima are related.

Apart from the ceratohyals and branchial baskets, the remodeling of the hyobranchium in the studied dicroglossid tadpoles also includes spiculae — cartilaginous outgrowths of ceratobranchials supporting the ventral velum of the branchial cavity. In all the detritophagous tadpole species, all the spiculae I–IV develop normally. Among them, spiculae I–III have a typical stick-like shape, but spicula IV can transform into a flattened plate as, for example, in Quasipaa; however, in carnivorous tadpoles, all spiculae are notably modified (Fig. 14). In H. rugulosus, all the spiculae are present but are shortened and markedly broadened, probably due to the need to withstand the greater stress of ingesting large and actively resisting prey. In Occidozyga spp., spiculae are mostly reduced; only spicula I is preserved, but its apparently rudimentary state and unusual orientation make its functional value unclear. In O. baluensis, the only spicula, spicula I, is a small, posteriorly directed outgrowth of the base of ceratobranchial I (Haas et al. 2014). A very similar condition is observed in O. lima, but in O. martensii, this cartilage is slightly longer, thinner and has a free proximal end. Vera Candioti et al. (2004) did not mention the presence of spiculae in D. nanus with the most similar hyobranchium morphology; most likely, in this species, the reduction in spiculae has proceeded even further to complete disappearance.

Figure 14. 

Transformation of the spiculae in the hyobranchia of carnivorous dicroglossid tadpoles. A Fejervarya limnocharis (generalized state), B Hoplobatrachus rugulosus, C Occidozyga martensii.

Conclusions

Our study of the external and internal larval morphology of several species contributes to the knowledge of the diversity of dicroglossid tadpoles. Most of the studied species belong to the generalized type of omnivorous or detritophagous tadpoles, in which the external appearance and mouthparts somewhat differ in lentic (Fejervarya) and lotic (Limnonectes, Quasipaa) forms. Other tadpoles (Hoplobatrachus and Occidozyga) deviate from the generalized type and have morphological characteristics of predators but also exhibit substantial differences between themselves, as their evolution followed two different trends.

Numerous morphological features of Hoplobatrachus tadpoles (strong tail, large, rostrally oriented eyes, extended forward jaw apparatus and especially its powerful armament, stomach-like dilatation of digestive tract, and robust chondrocranium) indicate their adaptation to macrophagy that can be advantageous if the prey is abundant. Nevertheless, in the absence of radical morphological rearrangements, tadpoles of H. rugulosus are rather omnivorous opportunists capable of utilizing a variety of food resources. In contrast, tadpoles of the genus Occidozyga are highly specialized predators whose morphology has undergone far more radical transformations compared to generalized tadpoles. The unique structure of their mouthparts, digestive tract, chondrocranium and hyobranchium suggests they are obligate carnivores ingesting their food by intense suction but no longer capable of rasping plants or tearing off large prey. Thus, during evolution along the path of extreme specialization, Occidozyga tadpoles “burn their ships”, giving up all flexibility in the use of food resources and losing the ability to return to omnivory in the absence of suitable prey.

These trends clearly demonstrate that, as a result of the independent evolution of the anuran larval stage, similar adult amphibians from related taxa can develop highly divergent tadpoles. Due to different food specializations, tadpoles of various dicroglossid species can coexist in the same temporary waterbodies but occupy very different trophic niches, from planktonophages and detritophages to predators of different types, thus avoiding direct competition for food resources.

Acknowledgments

The authors thank the Administration of Cat Tien National Park for the opportunity to perform long-term research in 2016–2023 in accordance with Agreement No. 37/HD on the scientific cooperation between Cat Tien National Park and the Joint Vietnam–Russia Tropical Science and Technology Research Center (JVRTSTRC). The fieldwork in Kon Ka Kinh National Park was conducted in 2017 with the permission of the Department of Forestry of the Ministry of Agriculture and Rural Development of Vietnam (permit No. 432/TCLN-BTTN) and the Forest Protection Departments of the Peoples’ Committee of Gia Lai Province (permit No. 142/SNgV-VP). We thank the Administration and the rangers of the Park for facilitating the research. We are deeply indebted to Nguyen Van Thinh and Vu Manh for organizing the fieldwork from part of the JVRTSTRC. We are very grateful to Vitaly Trounov for his extensive help with the field and photographic work and to Fedor Shkil for his kind assistance with microphotography. We also thank Eduard Galoyan for kindly providing photos of the frogs. The study was performed as part of the research project of the JVRTSTRC (E-1.2 “Conservation, Restoration and Sustainable Use of Tropical Forest Ecosystems”, Part 1.9 “Research on Amphibians and Reptiles”) and supported by the Russian International Scientific Collaboration Program Mega-Grant No. 075-15-2022-1134.

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