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. 2021 Jul 15;239(6):1318–1335. doi: 10.1111/joa.13510

Osteological development of a small and fast metamorphic frog, Microhyla fissipes (Anura, Neobatrachia, Microhylidae)

Meihua Zhang 1, Wei Zhu 1, Bin Wang 1, Shouhong Wang 1, Liming Chang 1, Tian Zhao 1,✉, Jianping Jiang 1,✉
PMCID: PMC8602016  PMID: 34268788

Abstract

Describing osteological development is of great importance for understanding vertebrate phenotypic variations, form‐functional transitions and ecological adaptations. Anurans exhibit dramatic changes in their morphology, habitat preferences, diet and behaviour between the tadpole and frog stages. However, the anatomical details of their cranial and postcranial development have not been extensively studied, especially in Microhylidae. In this work, we studied the microhylid Microhyla fissipes, commonly known as the ornamented pygmy frog, a small‐sized frog with fast metamorphosis. Its osteological development was comprehensively described based on 120 cleared and stained specimens, including six tadpoles for each stage between 28 and 45, six juveniles and six adults. Additionally, 22 osteological traits of these specimens involved in food acquisition, respiration, audition and locomotion were selected and measured to reflect the changes in tadpole ecological functions during metamorphosis. Our study provides the first detailed qualitative and quantitative developmental information about these structures. Our results have confirmed that skeletal elements (viz., neopalatines, omosternum, clavicles and procoracoids) absent in adults are not detected during development. Our data reveal that morphologically, radical transformations of the cranial structures related to feeding and breathing are completed within stages 42–45 (72 h), but the relative length and width of these skeletons have changed in earlier stages. The postcranial skeletons correlated with locomotion are well developed before stage 42 and approach the adult morphology at stage 45. Indeed, the relative length of the pectoral girdle and forelimb reaches the adult level at stage 42 and stage 45, respectively, whereas that of the vertebral column, pelvic girdle and hind limbs increases from their appearance until reaching adulthood. Based on published accounts of 19 species from Neobatrachia, Mesobatrachia and Archaeobatrachia, cranial elements are among the first ossified skeletons in most studied species, whereas sphenethmoids, neopalatines, quadratojugals, mentomeckelians, carpals and tarsals tend to ossify after metamorphosis. These results will help us to better understand the ecomorphological transformations of anurans from aquatic to terrestrial life. Meanwhile, detailed morphological and quantitative accounts of the osteological development of Microhyla fissipes will provide a foundation for further study.

Keywords: functional transition, larval development, metamorphosis, Microhyla fissipes, osteological modification, tadpoles

Microhyla fissipes (Anura, Neobatrachia, Microhylidae) is known as a small‐sized and fast metamorphic frog. We provide the anatomical details of its cranial and postcranial development and quantify 22 skeletal traits mainly related to food acquisition, respiration and locomotion based on 120 cleared and stained specimens. The results will help us to better understand the ecomorphological transformations of anurans from aquatic to terrestrial life.

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1. INTRODUCTION

Amphibia consists of three extant orders (i.e., Anura, Caudata and Gymnophiona) and plays an essential role in understanding the water‐to‐land transition during the evolutionary history of vertebrates (Liu & Zheng, 2009; Vitt & Caldwell, 2013). Amphibians are typically characterized by their biphasic life cycle, with their larvae dwelling in water and the post‐metamorphosed adults living on land, and therefore, they are able to exploit resources from both aquatic and terrestrial ecosystems (Wassersug, 1975). Among the three extant groups of amphibians, anurans are outstanding in achieving metamorphosis in a relatively short time period and undergoing dramatic changes in their morphology, habitat preferences, diet and behaviour (e.g., respiration and locomotion) between the larval stage and adult stage (Handrigan & Wassersug, 2007; Harrington et al., 2013; Wassersug, 1975). Accordingly, anuran metamorphosis is a complex and spectacular event involving a transformation from entire suites of larval specializations to adult modes (Wassersug, 1975).

Developmental analyses of anuran osteology have proven helpful in understanding phenotypic variations, form‐functional transitions and ecological adaptations (Fabrezi et al., 2016). Osteological features during development have been recognized as taxonomically informative (Alcalde & Barg, 2006; Trueb et al., 2011) and as useful in cladistic analyses (Haas, 2003; Harrington et al., 2013; Pugener et al., 2003). For skeletons that are absent in adults, their development provides a dynamic view of resolving whether these skeletal elements fail to form, fuse to other skeletons, are absorbed during ontogeny or lost during evolution (de Sá & Trueb, 1991). The skeletal elements provide attachments for the musculature and nerves, and play a key role in body structure, movement and organ protection (Yang et al., 2008). For a species of the biphasic anurans, the osteological changes during development may reflect their changes in the modes of predation, respiration and locomotion before and after metamorphosis (Larson, 2005; Rose et al., 2015). Understanding these details helps us to better understand the anuran ecomorphological transformations from aquatic to terrestrial life.

Although osteological development of anurans has been studied since the 19th century (Parker, 1875), only a few studies have documented a relatively complete development of both the cranial and postcranial skeletons (Banbury & Maglia, 2006; Barrionuevo, 2013; Fabrezi et al., 2012; Hoyos et al., 2012; Maglia & Púgener, 1998; Púgener & Maglia, 1997; de Sá, 1988; de Sá & Trueb, 1991; Shearman & Maglia, 2015; Trueb & Hanken, 1992; Trueb et al., 2000; Vera & Ponssa, 2014; Wiens, 1989; Wild, 1997). Moreover, the quantitative data on cranial and postcranial changes over the course of larval development in anurans remain rare (Larson, 2005). Developmental studies that employ both qualitative and quantitative approaches are ideal because qualitative observation allows for a more detailed description and identification of differences, and quantitative analysis highlights subtle changes that might not be immediately apparent upon qualitative observation (Barrionuevo, 2018; Larson, 2008; Rose et al., 2015). These findings will facilitate a better understanding of the changes in functional roles during ontogeny (Larson, 2005).

Microhylidae, the third largest family of anurans, have 706 species in 12 subfamilies that are highly diversified in morphology, ecological preferences and life history (Duellman & Trueb, 1994; Frost, 2021; Wells, 2010). A thorough understanding of the osteological development of microhylids is valuable and needs to be studied (Trueb et al., 2011). First, tadpoles in this family are uniquely classified as morphotype II due to the presence of a single midventral spiracle and unkeratinized mouthparts (Orton, 1953). However, only two species in this family have been comprehensively studied for their osteological development to date (Fabrezi et al., 2012; de Sá & Trueb, 1991). Second, they only need a short time to change from a tadpole into a frog through metamorphosis (Fei et al., 2009). Third, some skeletal elements (i.e., neopalatines, omosternum, clavicles and procoracoids) are absent or reduced in some microhylid adults, and further investigation of their osteological development is necessary to interpret the skeletal absences in this family (Fabrezi et al., 2012; Trueb et al., 2011).

The microhylid Microhyla fissipes, commonly known as the ornamented pygmy frog, is widely distributed in both eastern and south‐eastern Asia (Fei et al., 2009; Frost, 2021). Microhyla fissipes is among the smallest extant anurans (Frost, 2021). Several skeletal elements of its sternum (i.e., omosternum) and pectoral girdle (i.e., clavicles and procoracoids) are completely absent in adults. This species has been selected for osteological development analyses in Microhylidae for several reasons. For instance, it is one of the dominant species in paddy fields and spawns multiple times every year. Its clutch size is relatively large (200–300) and, thus, we can easily acquire sufficient numbers of individuals from each stage. Importantly, for Microhyla fissipes tadpoles that are suspension feeders, the fast larval development period (roughly 30 days) allows this study to be finished in a short time (Wang et al., 2017). Here, in the present study, we (1) provided thorough descriptions and illustrations of the development of both the cranial and postcranial skeletons to record the fast metamorphic process, which is the first report of the developmental information in Microhylinae; (2) presented the relative dimensional changes in some skeletons involved in feeding, respiration, hearing and locomotion to better understand the fast transition from the aquatic to terrestrial life and (3) compared the relative ossification sequences of the cranial and postcranial skeletons among 19 species from Neobatrachia, Mesobatrachia and Archaeobatrachia.

2. MATERIALS AND METHODS

2.1. Animals: sampling and breeding

Adults of Microhyla fissipes were collected from Shangen Town (18.94667° N, 110.47833° E), Wanning City, Hainan Province, China, on 16 August 2016. They were then transferred to the laboratory at Chengdu Institute of Biology (CIB), Chinese Academy of Sciences. A pair of sexually mature individuals (male: snout‐vent length [SVL] = 18.39 mm, body weight = 1.005 g; female: SVL = 20.67 mm, body weight = 1.412 g) were selected and induced to breed. Following the protocol of Wang et al. (2017), both the male and female parent frogs were injected intraperitoneally with a diluted solution of luteinizing hormone‐releasing hormone (3 μg/ml in sterile 0.65% NaCl) at a ratio of 0.1 ml per gram of body weight at 18:00 on 15 May 2017 (Kouba et al., 2009). Then, the parent frogs were released into a breeding container (320 × 220 × 150 mm) to mate freely at 28°C. A total of 370 eggs were collected at 7:00 the next morning and transferred into three transparent plastic containers (420 × 320 × 110 mm) filled with dechlorinated tap water at a depth of 5 cm, with 100 eggs hatching in each container.

Taking species‐specific development into account (Gosner, 1960; Hall et al., 1997), the tadpoles were staged according to the microhylid developmental table (Table S1; Shimizu & Ota, 2003; Wang et al., 2017). From stages 28 to 45, a sufficient solution of Spirulina powder (Salt Research Institute of China National Salt Industry Corporation, Tianjin) was provided to the tadpoles once a day at 9:00 AM, and faeces were removed 12 h after feeding. Tadpoles were maintained at 28°C under natural light following the protocol in Wang et al. (2018). Half of the water in each container was renewed every 3 days to ensure a suitable environment for tadpole development. These processes continued for 30 days until the end of the experiment.

Specimen sampling began at stage 28, which is the last stage before fertilized eggs hatch for microhylid species (Shimizu & Ota, 2003). Six specimens at each stage (i.e., tadpoles from stages 28 to 45, froglets that completed the metamorphosis 1 week later and adults) were randomly selected, sacrificed by concentrated tricaine methanesulfonate (MS‐222) and transferred to 70% alcohol for preservation. In total, 120 individuals were prepared for observation and osteological data acquisition. We followed the skeletal terminology in de Sá and Trueb (1991) for the description of tadpoles and Lehr and Trueb (2007) for the description of adults. All animal procedures were approved by the Animal Care and Use Committee of CIB.

2.2. Osteological data acquisition

All preserved specimens were cleared and double stained using the methods in Hanken and Wassersug (1981) and were repositioned at CIB. The osteology of each specimen was observed, photographed and measured to the nearest 0.01 mm using a stereozoom (Leica S8 Apo, Germany) with an attached digital camera (Leica DFC450 C, Germany). Skeletal ossification sequences were determined based on more than three individuals of each stage. The photographs were scaled and edited in Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA).

2.3. Osteological measurements

Functional traits are morphological, structural, biochemical, physiological, phonological or behavioural characteristics of organisms that influence performance and fitness (Nock et al., 2016; Violle et al., 2007). It has been well established that the ratios between morphological traits are typically used to estimate their vital functions in ecosystems (Altig & Johnston, 1989; Zhao et al., 2017). Based on the ease of measurement in a large number of individuals (Dumay et al., 2004) and on previous studies showing that they can reflect tadpoles' ecological functions (i.e., food acquisition, respiration, audition and locomotion) during metamorphosis (Emerson, 1983; Gomes et al., 2009; Gradwell, 1972; Khan, 2003; Larson, 2005; Manjarrez et al., 2017; Manzano et al., 2008; Soliz & Ponssa, 2016; Xia et al., 2012), we measured 22 osteological traits for each specimen. Specifically, the SVL of each specimen was first measured and used as a correction to remove the effect of tadpole size. The relative dimensions (meaning one that has been divided by the SVL) of the skull length (SL), skull anterior width (SAW), skull medial width (SMW), skull posterior width (SPW), lower jaw length (LJL) and lower jaw width (LJW) are related to food acquisition. The relative sizes of the ceratohyale length (CHL), ceratohyale width (CHW), hypobranchial length (HBL) and hypobranchial width are related to both food acquisition and respiration. The relative auditory capsule length (ACL) and auditory capsule width (ACW) are correlated with audition. The relative vertebral column length (VCL), coracoid length (CL), scapula length, pelvic girdle length (PGL), humerus length (HL), radioulna length, hand length, femur length, tibiofibula length and tarsal and foot length are relevant to locomotion. The osteological traits mentioned above were measured as indicated in Figure 1.

FIGURE 1.

FIGURE 1

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Measurements of Microhyla fissipes in a cleared and stained tadpole at stage 31 and a metamorphosed froglet at stage 45. (a) From left and right, the tadpole is in dorsal, ventral and left lateral views. (b) From left to right, the froglet is in dorsal and ventral views. (c) List of 16 functional traits associated with food acquisition, respiration, audition and locomotion. Scale bar = 1 mm

2.4. Statistical analyses

To remove the effects of the tadpole size variation, the ratios of these skeletal measurements to their SVL of 120 individuals were used for statistical analyses. Principal component analysis (PCA) was first conducted to create a functional space based on these corrected skeletal dimensions (i.e., ratios) of all individuals. Then, K‐means cluster analysis was used to put close individuals into the same functional group based on the PC1 and PC2 values. Each functional group is considered a distinct functional entity, which is typically used to incorporate intraspecific variability in functional ecological studies (e.g., Rudolf, 2020). Scatterplots were utilized to reflect the relationships of the relative skeletal dimensions versus stage to present the subtle changes during development. The fitting lines were used to show the general tendency of the relative skeletal size changes. All statistical analyses were conducted in R 3.2.2 (R development Core Team 2011) and SigmaPlot 12.5 (Systat Software Inc., USA).

2.5. Relative ossification sequence comparisons

Based on the literature (Banbury & Maglia, 2006; Barrionuevo, 2018; Dunlap & Sanchiz, 1996; Fabrezi et al., 2012; Gaudin, 1973; Haas, 1999; Maglia & Púgener, 1998; Púgener & Maglia, 1997; de Sá, 1988; de Sá & Trueb, 1991; Shearman & Maglia, 2015; Trueb et al., 2011; Trueb & Hanken, 1992; Wiens, 1989; Wild, 1997), the relative osteological ossification sequences of Microhyla fissipes and 18 species from Neobatrachia, Mesobatrachia and Archaeobatrachia were compared (see Table S2). To identify ossification heterochronic patterns within the phylogenetic context, a heatmap was utilized to show the relative cranial and postcranial ossification sequences of these species using SigmaPlot 12.5. The phylogenetic relationships of these species were reconstructed on the basis of their 2634 bp concatenated 12S rRNA and 16S rRNA mtDNA sequences. The gene sequences of every species were selected as far as possible from the specimens collected in closest geographic proximity to the specimens used for the skeleton descriptions and downloaded from GenBank (see Table S3). Then, the sequences were assembled and aligned using the ClustalW module in BioEdit v7.0.9.0 (Hall, 1999) with default settings. The best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITIONFINDER v1.1.1 (Lanfear et al., 2012). Gene trees (Figure S1) were reconstructed using maximum likelihood (ML) and Bayesian inference methods, implemented in PhyML v. 3.0 (Guindon et al., 2010) and MrBayes v. 3.2 (Ronquist & Huelsenbeck, 2003), respectively.

3. RESULTS

The chondrocraniums of the tadpoles were well developed at the end of early embryonic development (stage 28; Shimizu & Ota, 2003). Elements of the vertebral column, pectoral girdle, forelimb, pelvic girdle and hind limb appeared at stages 31, 33, 34, 32 and 33, respectively. Based on the osteological measurements at all stages, the first three PCA axes were retained, as their eigenvalue values were >1.0, which explained 87.65% of the total variation (PC1: 63.02%; PC2: 15.04%; PC3: 9.59%; Table 1). Specifically, PC1 was strongly correlated with the SVL‐corrected SAW, SL, SMW, CHW, ceratobranchial length (CBL), ceratobranchial width (CBW), LJW and CHL, which involve feeding and respiration; PGL, hind limb length, VCL, forelimb length (FLL) and pectoral girdle length, which involve locomotion; and ACW and ACL, which are related to audition. PC2 was strongly correlated with the SVL‐corrected SPW and ACL, which are related to food acquisition and audition, respectively. PC3 was strongly correlated with the SVL‐corrected LJL and SPW, which are related to feeding. In addition, three distinct functional groups were obtained based on K‐means cluster analyses (Figure 2). These groups were specimens that possessed only a chondrocranium (stages 28–30), specimens that were undergoing postcranial appearance and skeletal modification (stages 31–45), and specimens that had completed osteological metamorphosis (froglets and adults).

TABLE 1.

Variable loadings for principal components with eigenvalue greater than 1 from corrected morphometric characters

Functional traits PC1 (63.02%) PC2 (15.04%) PC3 (9.59%)
Skull anterior width (SAW/SVL) 0.96 −0.05 0.09
Skull length (SL/SVL) 0.92 0.19 0.25
Pelvis girdle length (PGL/SVL) −0.89 0.37 −0.02
Skull medial width (SMW/SVL) 0.88 0.16 0.09
Hind limb length (HLL/SVL) −0.87 0.43 0.00
Vertebral column length (VCL/SVL) −0.87 −0.07 0.11
Forelimb length (FLL/SVL) −0.86 0.43 0.08
Pectoral girdle length (PGL/SVL) −0.86 0.41 0.13
Ceratohyale width (CHW/SVL) 0.86 0.08 0.04
Ceratobranchial length (CBL/SVL) 0.84 −0.34 0.29
Ceratobranchial width (CBW/SVL) 0.82 −0.30 0.29
Auditory capsule width (ACW/SVL) 0.79 0.48 0.12
Lower jaw width (LJW/SVL) 0.71 0.38 −0.50
Ceratohyale length (CHL/SVL) 0.70 0.43 −0.33
Skull posterior width (SPW/SVL) 0.02 0.69 0.64
Auditory capsule length (ACL/SVL) 0.64 0.64 0.25
Lower jaw length (LJL/SVL) 0.49 0.38 −0.70
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All measurements are given in millimetre (mm).

FIGURE 2.

FIGURE 2

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K‐means cluster based on the PC1 and PC2. Coloured circles represent different functional groups. The black circles represent functional group 1 containing specimens at stages 28–30, the green circles represent functional group 2 including specimens at stages 31–45 and the red circles represent functional group 3 comprising specimens at stages 46 (froglets that completed the metamorphosis 1 week later) and 47 (adults)

3.1. Development of cranial skeletons related to feeding

Morphologically, the chondrocraniums of the tadpoles changed only in proportion before stage 39, except that the tectum synoticum appeared at stage 34. Metamorphosis started between stages 41 and 42 as radical structural reorganizations were seen in the chondrocranium and this process terminated at stage 45 with the chondrocranium approaching the adult form (Figures 3, 4, 5, 6).

FIGURE 3.

FIGURE 3

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Chondrocranium of Microhyla fissipes tadpole at stage 31 in dorsal (a), ventral (b) and anteroventral (c) views, the cranium and mandible in dorsal (d) and ventral (e) views and the hyobranchial skeleton in dorsal view (f). (d and e): ac., auditory capsule; ce., connecting element between suprarostrals; cqa., commissura quadratocranialis; ct., cornua trabecula; ep., ethmoid plate; ic., infrarostral cartilage; Mc., Meckel's cartilage; occ.con., occipital condyle; op., larval otic process; paq., pars articularis quadrati; pas., processus ascendens; pm., processus muscularis quadrati; pq., palatoquadrate; qe., processus quadrato‐ethmoidalis; sc., suprarostral cartilage. (f): ba.I–IV, branchial arches I–IV; cop., copula; cot., commissura terminalis; hp., hypobranchial plate; pa., processus articularis; pac., processus anterior of ceratohyale; plc., processus lateralis of ceratohyale; pre., pars reuniens; ppc., processus posterior of ceratohyale; sp., spiculum; ttm., taenia tecti marginalis. Scale bars = 1.0 mm

FIGURE 4.

FIGURE 4

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Skull of Microhyla fissipes completed metamorphosis (stage 45). Skull in dorsal (a), ventral (b), anterior (c) and lateral (d) views, the cranium in dorsal (e), ventral (f) and left lateral (g) views, the mandible, hyoid apparatus and laryngeal apparatus in dorsal view (h). (e–g): exoc., exoccipital; fpar., frontoparietal; max., maxilla; nas., nasal; neopal., neopalatine; om., operculum; pmax., premaxilla; pro., prootic; prsph., parasphenoid; pter., pterygoid; spheth., sphenethmoid; sq., squamosal; vom., vomer. (h): angspl., angulosplenial; antlat p., anterolateral process; ar c., arytenoid cartilage; br p., bronchial process; den., dentary; Meck c., Meckel's cartilage; mmk., mentomeckelian; plat p., posterolateral process; pmed proc., posteromedial process. Scale bar = 1.0 mm

FIGURE 5.

FIGURE 5

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Developmental patterns of the skull of Microhyla fissipes. (a) Cleared and stained skulls of tadpoles from stages 28 to 45 and a froglet in dorsal (upper) and ventral (lower) views, which are related to feeding. Scale bar = 1.0 mm. Scatterplots of the relative skull length (SL/SVL, b); the relative skull anterior width (SAW/SVL), relative skull medial width (SMW/SVL) and relative skull posterior width (SPW/SVL, c); the relative auditory capsule length (ACL/SVL) and relative auditory capsule width (ACW/SVL, d) versus developmental stage

FIGURE 6.

FIGURE 6

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Developmental patterns of the viscerocranium of Microhyla fissipes. The cleared and stained lower jaw, hyobranchial skeletons and laryngeal apparatus in dorsal view (a), which are correlated with food acquisition and respiration. Scale bar = 1.0 mm. Scatterplots of the relative length and width of lower jaw (b), ceratohyale (c) and hypobranchial (d) versus developmental stage

3.1.1. Neurocranium

Stage 39

The paired anterior nasal walls grew laterally and remained independent from the trabecular plate. Posteriorly, the frontoparietals appeared as a slender bone along the orbit cartilage, which later grew in lateral‐medial and posterior‐anterior directions (Figure 5a).

Stage 40

Differentiation of the nasal walls and nasal capsule cartilages was underway. The cultriform process and the alae of the parasphenoid appeared, serving as the cranial floor to support part of the braincase. Endochondral ossification of the prootic appeared on the inner wall of the auditory capsules. Meanwhile, endochondral ossification of each exoccipital appeared around the jugular foramen (Figure 5a).

Stage 41

The cartilages of the nasal capsule rapidly developed into a more complex structure than in the last stage, and simultaneously, both the suprarostral and cornua trabeculae were resorbed (Figure 5a).

Stage 42

The climax of metamorphosis started at this stage because the chondrocranium underwent the most dramatic changes compared with earlier stages. Anteriorly, the paired cornua trabeculae completely disappeared, and therefore, the snout region of the tadpole was truncated in the external profile. The nasal capsules formed abruptly. In addition, the superior and inferior prenasal cartilages were well developed. The septum nasi projected forward as a short median prenasal process. The oblique cartilages, together with the lamina superior and lamina inferior and crista intermedia, were vertical in position. Posteriorly, both the processus ascendens and the otic process of palatoquadrate eroded, and the planum triangularis bore a processus maxillary anterior (Figure 5a).

Stage 43

Each of the nasals formed from a single ossification centre located dorsally between the posterior ends of the nasal capsules and the anterior ends of the planum antorbitale. The septomaxillae were ossified and attached to both the developing oblique and alary cartilages (Figure 5a).

Stage 44

The anterior and medial cartilages of the nasal capsule have developed into a well‐developed complex. The palatoquadrate was further reshaped; specifically, the processus pterygoideus was well differentiated, and the pars articularis migrated posterolaterally. The left and right frontoparietals were connected with one another. The parasphenoid was morphologically similar to that in adult specimens. The squamosal started to ossify at its ventral ramus as a tiny slender bone that was arranged posterolaterally (Figure 5a).

Stage 45

The chondrocranium had been reshaped into an adult form, with most cartilage that characterized the larvae resorbed and with augmenting ossification for the bony components as mentioned above. The pars articularis of the palatoquadrate was reduced and attached to the auditory capsule by ligaments. The anterior ramus of the pterygoid was partially ossified as a small slender bone. Bony components of both the suspensorium and jaw connections had moved towards the vertical orientation adjacent the auditory capsules (Figure 5a).

From stage 28 to adulthood (Table S4), (1) the relative SL decreased (Figure 5b), (2) both the relative SAW and the relative SMW decreased, while the relative SPW increased (Figure 5c), (3) the skull was medially wider than anteriorly from stage 43 (Figure S2A) and (4) both the relative length and width of the auditory capsule (ACL and ACW) decreased (Figure 5d).

3.1.2. Upper jaw

The suprarostral cartilages, the median connecting element and the cornua trabeculae were all resorbed between stages 41 and 42, resulting in the tadpole snout truncated from the external profile. The premaxillae and maxillae were detected at stage 44. The quadratojugals formed after metamorphosis (Figure 5a).

3.1.3. Lower jaw

Morphologically, transformations of the lower jaw began from stage 42 (Figure 6a). Specifically, the infrarostral cartilages were reshaped from a flattened inverted U‐shaped element into transverse bars with a distinct medial union, and Meckel's cartilages were distinctly elongated at stage 43. At stage 44, the dentaries and angulosplenials formed laterally and medially on Meckel's cartilages, respectively. Elongation of the Meckel's cartilages continued and reached their final form at stage 45. Both the relative LJL and the relative LJW exhibited inverted hump shapes; specifically, they decreased from stages 28 to 38, remained nearly stable during stages 39–41 and increased from stage 42 to adulthood (Figure 6b).

3.2. Development of cranial skeletons related to food acquisition and respiration

3.2.1. Hyobranchial skeleton

The hyobranchial skeletons began to be modified from stage 42, when erosions of the ceratobranchials commenced (Figure 6a). At stage 43, the processus articularis and processus posterior of the ceratohyale and the speculum and commissurae terminals of ceratobranchials IV‐I were first resorbed, followed by the branchial arches. As resorption proceeded, the hypobranchial plate was expanded into the hyoid plate by stage 44, the ceratobranchial I was modified into the anterolateral processes and the posterior processes of the hypobranchial plate were changed into posteromedial processes. At stage 45, the ceratohyale was changed into the adult form, which was thin, long, curved and distally articulated with the anteroventral wall of the auditory capsule. In froglets that have completed metamorphosis 1 week later, the cartilaginous hyoid plate and the ossified posteromedial processes were separated by a distinct narrow transverse gap. In adults, the median part of the hyoid plate was partly calcified and the posteromedial processes were more robust. The posterolateral processes were new outgrowths from the lateral sides of the hyoid plate. The relative CHL was decreased from stages 28 to 39, remained stable at stages 40 and 41 and increased from stage 42 to the adult stage, while the relative CHW was decreased from stage 28 to the adult stage (Table S4; Figure 6c). Both the relative CBL and the relative CBW were increased from stages 28 to 34, then decreased from stages 35 to 44 and were completely resorbed by stage 45 (Table S4; Figure 6d).

3.2.2. Laryngeal apparatus

The arytenoids appeared at stage 32. The cricoid appeared at stage 44, and the bronchial processes appeared at stage 45. The oesophageal process is absent in this species (Figure 6a).

3.3. Development of the postcranial skeletons correlated with locomotion

3.3.1. Vertebral column

Both chondrification and ossification of the vertebral column appeared following an anteroposterior sequence (Figure 7a). The neural arches of Presacrals I–IV chondrified as distinct paired arcs at stage 31, those of Presacrals V–VIII appeared at stage 32 and those of the sacrum emerged at stage 34. The transverse processes of Presacrals II–IV appeared at stage 36, and those of Presacrals V–VIII and the sacrum appeared at stage 38. At stage 39, both the prezygapophyses and postzygapophyses had developed. At stage 40, the neural arches started to meet at the midline, and the centrums of Presacrals I–III ossified. The centra rings had closed at stage 41. The transverse processes of the sacrum attached to the ilial shaft of the pelvic girdle at stage 42. The rod‐like hypochord, which lies ventral to the notochord, chondrified at stage 34. The first and second coccygeal vertebrae chondrified at stages 35 and 40, respectively. These elements fused and formed the urostyle in specimens that completed metamorphosis a week later. By stage 44, the vertebral column was almost ossified. At the end of metamorphosis (stage 45), the vertebral column had approached the adult morphology except for the transverse processes of Presacrals V–VIII, ossification of the sacrum and fusion of the urostyle. The relative VCL increased from its appearance (stage 31) to the adult stage (Table S4; Figure 7b; Figure 8).

FIGURE 7.

FIGURE 7

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Developmental patterns of postcranial skeletons of Microhyla fissipes, which are related to locomotion. The development and the relative length of vertebral column (a, b), pectoral girdle and forelimb (c, d), pelvic girdle and hind limb (e, f) versus developmental stage. The arrows in C2 indicate the sternum. Scale bar = 1.0 mm

FIGURE 8.

FIGURE 8

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The mean relative length of the skull and postcranial structures versus developmental stage from their appearance to the adult in Microhyla fissipes

3.3.2. Sternum

The sternum extended from the coracoids at stage 45, it presented an inverted V‐shaped and enlarged after metamorphosis (Figure 7c2). The omosternum, which is absent in adults, was not observed in any investigated developmental stages.

3.3.3. Pectoral girdle

The pectoral girdle, formed only by coracoids and scapulae, appeared as two halves at stage 33 (Figure 7c1). The suprascapulae chondrified at stage 36. The dermal cleithrums appeared along the anterior dorsal edges of the suprascapulae at stage 41 (Figure 7c2). Ossification of coracoids and scapulae occurred at stages 41 and 42, respectively. The two halves of the pectoral girdle medially connected at stage 41 and fused at stage 42. Clavicles and procoracoids were not detected throughout development. The relative length of the pectoral girdle increased from its emergence (stage 34) and reached the adult level at stage 42 (Table S4; Figure 7d; Figure 8).

3.3.4. Forelimb

The primordia of the humeri were detected at stage 34 and attached to the glenoid bridges formed by scapulae and coracoids (Figure 7c1). The crista ventralis of the humerus was well developed and resembled the adult form at stage 41 (Figure 7c3). The radius and ulna appeared at stage 34, fused at the proximal and distal ends at stage 39 and completely fused and left a shallow groove between them at stage 41 (Figure 7c4). The diaphyses of the humeri and radioulnas were ossified well at stage 41. The appearances of the five carpals appeared as follows: ulnare at stage 34, radiale at stage 35, element Y and distal carpals 2 and 3–5 at stage 37. Metacarpal V appeared at stage 34, metacarpals IV and III appeared at stage 35 and metacarpal II appeared at stage 37. The digital phalanges for II–V were 0‐0‐1‐1 at stage 36, 0‐1‐3‐2 at stage 38 and 2‐2‐3‐3 at stage 39, which were the same condition as the adult phalanges. Interestingly, the terminal phalanges were distally Y‐shaped in larvae but T‐shaped in adults. The prepollex appeared by stage 37, comprising two elements. The relative length of the forelimb increased from appearance (stage 34) and approached the adult level at stage 45 (Table S4; Figure 7d; Figure 8).

3.3.5. Pelvic girdle

The left and right sides of the pelvic girdle appeared as two independent structures at stage 32, approached each other posteromedially at stage 38 (Figure 7e1) and fused medially at stage 39 (Figure 7e2). Ossifications occurred in the iliac diaphysis at stage 40. A triangular protuberance appeared on the dorsal base of the ilium at stage 42, and approached the adult form. At stage 44, both the ilia and ischia were well ossified. The relative length of the pelvic girdle increased from its appearance (stage 32) to the adult stage (Table S4; Figure 7f; Figure 8).

3.3.6. Hind limbs

The femora were observed at stage 33, and were proximally connected with the acetabulums of the pelvic girdle (Figure 7e1). The tibia and fibula chondrified as two independent elements at stage 34, united with each other at their proximal and distal ends by stage 37 and completely fused with a shallow groove between them at stage 39 (Figure 7e4). Ossifications started on the diaphyses of the femora and tibiofibulae at stage 41. The tibia and fibula appeared at stage 34 (Figure 7e1) and were synchondrotically fused at both ends by stage 37. Distal tarsal 1, distal tarsal 2–3 and Element Y appeared by stage 38. Metatarsals III–V appeared at stage 35, and metatarsals II and I appeared at stage 37. The appearance of toe phalanges I–V was 0‐0‐0‐1‐0 at stage 36, 0‐1‐2‐3‐2 at stage 37 and 2‐2‐3‐4‐3 at stage 39, which were in the same condition as the adult phalanges (Figure 7e5). The prehallux appeared at stage 38, comprising two elements. The relative length of the hind limb increased from its appearance (stage 33) to the adult (Table S4; Figure 7f; Figure 8). The tibiofibulae had been longer than the femora since stage 38 (Figure S2B).

3.4. Comparisons of ossification sequences among M. fissipes and other anuran species

The ossification timing of M. fissipes is provided in Figure 9. The relative cranial and postcranial ossification sequences of M. fissipes were compared with those of 18 anuran species from 12 families (Figure 10). The initial ossification occurred on cranial skeletons in seven species of Neobatrachia (Microhyla fissipes of Microhylidae, Boana lanciformis of Hylidae and Chacophrys pierottii of Ceratophryidae), Mesobatrachia (Xenopus laevis of Pipidae, Spea multiplicata and S. intermontana of Scaphiopodidae) and Archaeobatrachia (Discoglossus sardus of Alytidae). The initial ossification occurred at the same stage in the skeletons of the cranium and vertebral column in five species of Neobatrachia (Hamptophryne boliviana and Gastrophryne carolinensis of Microhylidae, Leptodactylus chaquensis of Pyxicephalidae, Pyxicephalus adspersus of Leptodactylidae) and Archaeobatrachia (Pelobates cultripes of Pelobatidae). Skeletons of the cranium, vertebral column, girdles and limbs presented ossification at the same stage in four species of Neobatrachia (Bufo bufo and Epidalea calamita of Bufonidae, Chacophrys pierottii of Ceratophryidae) and Mesobatrachia (Spea multiplicata of Scaphiopodidae). The vertebral column skeletons ossified first in Phyllomedusa vaillantii (Neobatrachia, Phyllomedusidae). The hind limb skeletons first ossified in Bombina orientalis (Archaeobatrachia, Bombinatoridae). The pelvic girdle and hind limbs presented ossification first in Ceratophrys cornuta (Neobatrachia, Ceratophryidae). Interestingly, the pectoral girdle was among the last ossified structures in the studied taxa, excluding two species of Neobatrachia (Boana lanciformis of Hylidae and Pyxicephalus adspersus of Pyxicephalidae).

FIGURE 9.

FIGURE 9

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Skeletal appearance and ossification of Microhyla fissipes from stages 28 to 45, a froglet (46) amd a adult (47). Dermal bones appear as red, while endochondral skeletons appear as blue and then ossify as red

FIGURE 10.

FIGURE 10

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The phylogenetic hypothesis and the relative ossification sequence comparisons of 19 species from 12 families. (a) The maximum likelihood (ML) and Bayesian inference (BI) tree of these species are reconstructed based on their 2634bp DNA sequences of the 12S rRNA and 16S rRNA mt DNA genes. The ML bootstrap support and Bayesian posterior probability were provided in Figure S1. See information on samples and GenBank Accession number in Table S3. (b) The different colours of the heatmap represent the relative ossification sequences. Specifically, the black grids mean that the skeletons are absent in the species, while the white grids mean that the endochondral skeletons ossified/dermal bones appear after metamorphosis. And then, the more red of the colour grid, the earlier stage skeletal ossification occurs

For cranial skeletons, most of the studied species shared the following ossification features: (1) the frontoparietals, parasphenoid, exoccipitals and prootics tended to be among the first ossified skeletons; (2) the sphenethmoids, vomers, neopalatines, quadratojugals and mentomeckelians were among the skeletons that ossified after metamorphosis; (3) the neopalatines, quadratojugals and mentomeckelians were lost in some species; (4) for skeletons related to respiration, the septomaxillae ossified earlier or at the same stage as nasals; (5) dermal bones of the maxillary arch appeared earlier or at the same stage as those of the mandible and (6) for the suspensory apparatus, the ossification of the squamosals occurred earlier or at the same stage as that of the pterygoids.

For M. fissipes, the frontoparietals initially ossified (stage 39), the neopalatines were absent, the squamosals ossified (stage 44) earlier than the pterygoids (stage 45), the septomaxillae ossified at the same stage as the nasals (stage 43) and the maxillary arch and mandible presented ossification at the same stage (stage 44).

4. DISCUSSION

4.1. Osteological transformation of Microhyla fissipes related to food acquisition

In anurans, the transition from aquatic to terrestrial living settings requires a dramatic reorganization of the morphological structures of tadpoles (e.g., those related to predation, respiration and locomotion) (Soliz et al., 2017). One of the crucial steps is the mouth configuration because the differences in feeding modes between premetamorphosed tadpoles and postmetamorphosed frogs are much more complex than those in either salamanders or caecilians (Barrionuevo, 2018). In tadpoles of the microhylid M. fissipes, the chondrocraniums are the widest anteriorly and bear jaw articulations at stages 28–41, which is similar to other filter feeding tapoles (Candioti, 2007; Trueb & Hanken, 1992). The presence of a wider mouth in tadpoles probably facilitates the consumption of more suspension food by providing a larger food collecting area (Duellman & Trueb, 1994). This is because suspension feeders acquire nutrients depending largely on how much water can pass through their pharynxes per unit time (Wassersug & Hoff, 1979).

Radical transformations of the chondrocranium occur between stages 41 and 42 (48 h), such as remodelling of the palatoquadrates, complete erosion of the suprarostral cartilages and the median connecting element, elongation of the Meckel's cartilages and posterior migration of the jaw joints. Then, from stages 42 to 45 (72 h), the jaw joints migrate posteriorly until they connect with the anterior margins of the auditory capsules, reaching the adult form. At stage 44, bones involved in frog feeding appear. Specifically, the premaxillae and maxillae appear and contribute to the formation of the maxillary arch, and angulosplenials and dentaries appear and protect the Meckel's cartilages medially and laterally, respectively. After metamorphosis, quadratojugals appear, and the cartilaginous infrarostrals ossify into bony mentomeckelians. These modifications and reinforcements of the mouth configuration work to prepare for tongue protrusion to capture prey on land (Trueb et al., 2011).

4.2. Osteological reorganization of Microhyla fissipes related to food acquisition and respiration

The hyobranchium plays vital roles in feeding and breathing behaviours in tadpoles (Rose, 2009). This is because the main force driving the flow of water through tadpoles is the cyclical pumping action of the hyobranchial apparatus (Gradwell, 1972; Larson & Reilly, 2003; Seale et al., 1982). Specifically, each ceratohyal acts as the piston for the buccal pump (Wassersug & Hoff, 1979). The ceratohyale of M. fissipes tadpole morphologically changes into the long and slender hyale from stages 43 to 45 (48 h). The relative CHL is decreased from stages 28 to 40 and then increased from stage 41 to the adult stage, while the relative CHW decreases from stage 28 to the adult stage. The skeletal surface area affects musculature attachment (Trueb, 1973), and the muscle cross‐sectional area is proportional to the force generated by a muscle (Ryerson, 2008). These accounts give insights into the phenomenon of the decreasing efficiency of filter feeding that occurs in later stages during larval ontogeny (Larson, 2005). In frogs, the long and slender hyales connect the hyoid plate to the prootic portions of the skull. And the hyales facilitate the hyoid plate acting as a spring, which is critical in tongue protrusion of frogs to capture prey on land (Emerson, 1977).

Branchial baskets play key roles in screening out nonfood materials (Khan, 2003) and gas exchanges (Wassersug & Murphy, 1987). Lacking keratinized mouthparts, larval M. fissipes feed by their gills entrapping particles suspended in the water column. The gills also act as one of the important sites of aquatic O2 and CO2 exchange prior to the onset of metamorphosis (Burggren & Infantino, 1994). In M. fissipes, the absorption of the branchial baskets morphologically occurs from later stage 42 and completely finishes at stage 44. This process takes 48 h. However, both the relative HBL and width are decreased from stages 35 to 44. According to our observations, Microhyla fissipes tadpoles nearly do not feed from stage 42 to the end of metamorphosis (72 h). This is probably because jaw and hyobranchium reshaping results in the loss of their functions (Wassersug & Hoff, 1982). However, the hypobranchial plate is remarkably enlarged from stage 42 and modified into the hyoid plate at stage 45 (72 h). In most species of the frogs, the hyoid plate not only provides the attachment for muscles involved in feeding, respiration and vocalization but also moves during these behaviours (Emerson, 1977; Nishikawa, 2000). It has been reported that the lungs of M. fissipes tadpoles are enlarged and well developed before the branchial baskets are absorbed (Wang, 2019). Microhyla fissipes tadpoles at this stage could survive with little water, which indicates that respiration through their gills may have been partly substituted by their lungs (Burggren & Infantino, 1994). Fast radical transformations are required to reduce the danger of ephemeral breeding water evaporation (Fabrezi et al., 2012; Haas & Richards, 1998).

4.3. Postcranial development of Microhyla fissipes related to locomotion

Tadpoles have elongated fishlike body plans and move in water by undulating their muscular tail (Zhao et al., 2017). Locomotion changes from the axial‐driven mode in tadpoles to the limb‐driven mode in frogs occur through metamorphosis (Senevirathne et al., 2020). Frogs have a set of unique postcranial structures specialized for saltatory locomotion, such as the shortened trunk, rod‐like urostyle, mobile ilio‐sacaral, sacro‐urostylic joints and elongated hind limbs (Lutz & Rome, 1994; Shubin & Jenkins, 1995). The reduced number of presacral vertebrae and a single urostyle make saltation more efficient (Handrigan & Wassersug, 2007). In M. fissipes, vertebral skeletons are initially visible at stage 31, and ossification occurs from stage 40. The iliosacral contacts are acquired at stage 42, which allows distinct joint mobility and is related to diverse locomotor activities (Jenkins & Shubin, 1998). The urostyle plays a major role in transmitting thrust from the hind limbs to the axial column during limb‐driven locomotion (Jenkins & Shubin, 1998; Shubin & Jenkins, 1995). It is formed after metamorphosis in M. fissipes. The relative length of the vertebral column increases from its appearance to adulthood. Indeed, the postmetamorphic period is also required to complete structural maturation and to achieve full locomotor functionality (Soliz & Ponssa, 2016; Soliz et al., 2020).

The pectoral girdle functionally absorbs the impact loading stress and, then, transfers the propulsive thrust to the body during landing (Emerson, 1983). Unlike most anurans, the omosternum, clavicles and procoracoids are lost in M. fissipes and in some other microhylids (Parker, 1934; Zhang, 2019). At stage 42, the morphology and the relative length of the pectoral girdle approach the adult form, which indicates that the pectoral girdle has been well prepared for saltatory locomotion. The forelimbs play crucial roles in supporting the body (Manzano et al., 2008), absorbing impact forces (Essner et al., 2010) and controlling the take‐off angle during landing (Nauwelaerts & Aerts, 2006). Forelimb skeletons of M. fissipes were well developed before erupting through the outer body layer at stage 42. For instance, the complete fusion of the radius and ulna is related to posture and shock‐absorbing adaptation (Jenkins & Shubin, 1998; Liem et al., 2001). The ossification of the humeri and radioulnas strengthens load bearing. The well‐developed humeral ventral crests functionally enlarge the attachment area for musculature and make the forelimbs stronger (Trueb, 1973). In addition, the relative length of the forelimb increases rapidly from its appearance to stage 42.

The internal development and sudden eruption of anuran forelimbs at stage 42, which is common to all metamorphosing frogs, probably occurs to avoid affecting the tadpole locomotion performance and decrease their predation risk (Dudley et al., 1991; Wassersug, 1989; Wassersug & Sperry, 1977). The relative length of the forelimbs increases slowly from stage 43 and reaches the adult morphology when complete metamorphosis occurs (stage 45). It has been reported that shorter forelimbs could keep the frog's centre of mass backward and increase propulsion when jumping (Emerson, 1983; Nauwelaerts & Aerts, 2006).

The pelvic girdle connects the hind limbs to the axial structure and transmits the leaping forces to the movable iliosacral joint near the mid‐body (Reilly et al., 2016). In M. fissipes, the pelvic girdle reaches the adult morphology at stage 42. Its relative length significantly increases from its appearance to the adult, which ensures the iliosacral joint is in the middle of the body. Reilly et al. (2016) reported that the iliosacral joint near the mid‐body creates unique biomechanical consequences for controlling the posture of the trunk and head relative to the pelvis and legs during jumping locomotion. Anurans are unique in vertebrates whose hind limb development precedes forelimb development (Bininda‐Emonds et al., 2007). The femora are visible at stage 33. The tibiae and fibulae are observed at stage 34, are longer than the femora from stage 38 and completely fused at stage 39. The femora and tibiofibulae obviously ossify centrally at stage 41. With the tail being reabsorbed from stages 42 to 45 (72 h), the hind limbs start to function as the principal propulsive agent in locomotion (Enriquez‐Urzelai et al., 2015; Manzano et al., 2008). Similar to the pelvic girdle, the relative length of hind limb significantly increases after its appearance to the adult. According to our observations, the leaping ability of M. fissipes increases from stage 43 to adulthood. This is because structural maturation and relatively longer hind limbs enhance jumping performance (Enriquez‐Urzelai et al., 2015; Gómez & Lires, 2019; Manzano et al., 2008). Moreover, similar to the findings of Soliz et al. (2018), the development of the glenoid fossa and the acetabulum is intimately related to the development of the epiphyses of the humerus and femur. This result contradicts the ‘in‐out’ mechanism hypothesis in which the development of the forelimb precedes that of the pectoral girdle (Valasek et al., 2011).

4.4. Comparisons of osteological development among Microhyla fissipes and other anurans

Although the initial ossification timing is relatively plastic and susceptible to temperature (Barrionuevo, 2020), wild‐ or laboratory‐raised conditions (Sheil et al., 2014) and other external factors (Trueb & Hanken, 1992), the relative ossification sequences are almost invariable within a species or population (Wiens, 1989; Yeh, 2002). The relative ossification sequences describe the order in which the bones begin to ossify (Yeh, 2002). Based on comparisons of 19 anuran species from 12 families, the cranial skeletons are among the first ossified elements except for P. vaillantii (vertebral column), B. orientalis (hind limb) and C. cornuta (pelvic girdle and hind limb). This is probably because the vertebrate head is an important structure involving sensory functions, prey capture and defence mechanisms and is directly linked to evolutionary success (Paluh et al., 2020). In Microhylidae (Table S2; Table S5), the initial ossification of M. fissipes (Microhylinae) occurs in the cranium at stage 39, while that of H. boliviana (Gastrophryninae) and G. carolinensis (Gastrophryninae) occur simultaneously in the cranium and vertebral column at stages 36 and 33, respectively. The ossification process differences may be related to phylogenetic relationships, developmental conditions with the former specimens collected from the laboratory and the latter collected from the field or other factors (Sheil et al., 2014). More investigations of microhylid osteological development are required to solve this issue (Trueb et al., 2011).

For anurans, the ossifications of the skull were probably adapted to meet functional requirements based on comparisons among these 19 species. The parasphenoid, frontoparietals, exoccipitals and prootics that form the braincase and protect the brain are among the first ossified bones (Harrington et al., 2013). These are followed by the skeletons related to respiration and feeding (Yeh, 2002). Specifically, the septomaxillae that are closely related to breathing (West & Jones, 1975) appear earlier than the nasals that protect the nasal capsule dorsally in nine species and occur at the same stage in seven species, including M. fissipes. The dermal bones of the maxillary arch emerge earlier than those of the mandible in 17 species, excluding M. fissipes and X. laevis. For the maxillary arch, the anterior premaxillae emerge earlier or together with the medial maxillae in these species apart from X. laevis, while the posterior quadratojugals appear later than the two bones in 15 species and are absent in four species. For the mandible, the medial angulosplenials appear earlier or together with the lateral dentaries in all species, and the anterior mentomeckelians ossify later than them in 17 species, including M. fissipes, and are absent in X. laevis. The squamosals and pterygoids act to protect and strengthen the palatoquadrates (Bardua et al., 2020), with the squamosals emerging earlier than the pterygoids in 14 species, including M. fissipes, and at the same stage in four species. The vomers that prevent prey from escaping appear after metamorphosis in six species, including M. fissipes. Neopalatines appear after metamorphosis in eight species and are absent in eight species, including M. fissipes. Later‐developing elements (e.g., neopalatines, quadratojugals) are indeed more labile in terms of their presence or absence (Hanken, 1984). Unfortunately, it is not possible to correlate the ossification variation with either systematic relationships or ecological relationships because of insufficient data (Trueb et al., 2011). More investigations of anuran osteological development are required to better understand these issues.

5. CONCLUSION

Our study of the cranial and postcranial development of Microhyla fissipes comes to the following conclusions:

  • (1) The detailed developmental information of some skeletons is first described, such as, arytenoids related to frog respiration and sound production are unexpectedly observed at stage 32; the tibiofibula‐to‐femur ratio, as an indicator of high jumping ability, is greater than 1 from stage 38; the humeral crista ventralis develops from stage 40 and approaches the adult form at stage 41; the chondrocranium is wider medially than anteriorly from stage 43 and skeletal elements (i.e., neopalatines, omosternum, clavicles and procoracoids) absent in adults are not detected during development.

  • (2) Microhyla fissipes is a fast metamorphic anuran. For instance, the ceratohyales change into their adult morphology between stages 44 and 45 (24 h), the branchial baskets are completely absorbed during stages 42–44 (48 h) and the jaw articulations migrate from anteriorly to posteriorly from stages 42 to 45 (72 h).

  • (3) Cranial structures of Microhyla fissipes related to feeding and breathing undergo radical transformations within stages 42–45, but the relative length and width of these skeletons change in earlier stages. These skeletal reshapings probably result in the loss of their function, and thus, the tadpoles cannot feed until the process is completed. Radical transformations in a later and short time could increase the survivability during development.

  • (4) Postcranial skeletons correlated with locomotion have well developed before stage 42 and approached adult morphology at stage 45. The relative length of pectoral girdle and forelimb has reached adult level at stage 42 and stage 45, respectively, whereas that of vertebral column, pelvic girdle and hind limbs has increased from appearance to the adult. These accounts present direct evidence that postcranial skeletons are well prepared for landing at the end of metamorphosis, but the postmetamorphic period is also requisite for achieving full locomotory functionality.

  • (5) Based on published accounts of cranial and postcranial development of species from 12 anuran families, contrary to the pectoral girdle, the cranial skeletons are among the first ossified structures in most studied species.

CONFLICT OF INTEREST

All authors have no conflict of interests.

AUTHOR CONTRIBUTIONS

Meihua Zhang conceived and designed the experiments, acquired and analysed the data, drafted the manuscript; Wei Zhu contributed to data analysis and interpretation; Bin Wang, Shouhong Wang and Liming Chang prepared figures and tables, reviewed drafts of the paper; Tian Zhao and Jianping Jiang conceived and designed the experiments, analysed the data, authored or reviewed drafts of the paper. All authors critically revised the final version of the manuscript and approved it.

Supporting information

Fig S1

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Fig S2

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Table S1

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Table S2

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Table S3

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Table S4

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Table S5

Click here for additional data file. (74KB, doc)

ACKNOWLEDGEMENTS

This work is supported by Important Research Project of Chinese Academy of Sciences (KJZG‐EW‐L13), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA19050201), Construction of Basic Conditions Platform of Sichuan Science and Technology Department (2019JDPT0020) to J.P. Jiang and the National Natural Science Foundation of China (31700353) to T. Zhao.

Zhang, M. , Zhu, W. , Wang, B. , Wang, S. , Chang, L. , Zhao, T. et al. (2021) Osteological development of a small and fast metamorphic frog, Microhyla fissipes (Anura, Neobatrachia, Microhylidae). Journal of Anatomy, 239, 1318–1335. 10.1111/joa.13510

Contributor Information

Tian Zhao, Email: zhaotian@cib.ac.cn.

Jianping Jiang, Email: jiangjp@cib.ac.cn.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in the supplementary material of this article.

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Associated Data

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Table S1

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Table S2

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Table S3

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Table S4

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Table S5

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Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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