To assess the phylogenetic relationships within the Microhyla–Glyphoglossus assemblage we used the mtDNA and nuclear DNA (nuDNA) datasets from Gorin et al. (2020) with the addition of sequences of the recently described Mysticellus franki (Garg and Biju 2019) and Microhyla hongiaoensis (Hoang et al. 2020). We used the mtDNA dataset, consisting of 12S rRNA and 16S rRNA for all examined samples, for estimation of the phylogeny (232 sequences, including 200 sequences of Microhyla). A combined mtDNA + nuDNA dataset, joining the long 12S rRNA–16S rRNA mtDNA fragment and BDNF gene sequences for a reduced set of 120 samples, equitably selected (from preliminary analysis of mtDNA; not shown) to represent all major lineages within Microhyla, was used to estimate a robust, multilocus, time-calibrated phylogeny. In total, we analyzed GenBank sequences from 200 specimens of 49 nominal and three candidate Microhyla species, five species of Glyphoglossus, and 32 other microhylids, including representatives of all currently recognized microhyline genera. All taxa, specimen-associated locality data, museum voucher catalog numbers, and genetic data included in our study are presented in Suppl. material 1: Table S1.

Our osteological study was based on specimens housed in herpetological collections of the Zoological Museum of Lomonosov Moscow State University (ZMMU, Moscow, Russia), the Herpetology Lab of the Vertebrate Zoology department, Faculty of Biology, Lomonosov Moscow State University (HLMU; Moscow, Russia), the Museum of Comparative Zoology, Harvard University (MCZ, Cambridge, Massachusetts, USA), and the California Academy of Sciences (CAS, San Francisco, California, USA). Altogether, for hand-preparation and histological clearing-and-staining, we used 23 specimens, which included 17 nominal species of Microhyla I clade, and 4 species of Microhyla II clade, and representing all of the currently recognized species groups, with exception of the M. palmipes species group, and two species of Glyphoglossus. All specimens were adults, fixed in either 75% ethanol or in 4% buffered formalin with subsequent storage in 70% ethanol. Additionally, for micro-computed tomography (micro-CT) study we examined the smallest representatives of Microhyla I and Microhyla II clades (M. nepenthicola and M. arboricola, respectively). We also used micro-CT scans for one Microhyla (M. achatina, the type species of the genus; MCZ-A2683, ark:/87602/m4/M79961) and two species of Glyphoglossus: G. yunnanensis (CAS-H-242243, ark:/87602/m4/M49927) and G. molossus (the type species of the genus; CAS-H-243121, ark:/87602/m4/M49928), downloaded from the MorphoSource database (www.morphosource.org) with permission. Altogether, our morphological dataset included detailed information for 23 species of Microhyla and 3 species of Glyphoglossus. Detailed information on the species and specimens included in morphological study is presented in Suppl. material 2: Table S2.

Nucleotide sequences were initially aligned in MAFFT v.6 (Katoh et al. 2002) with default parameters, and were subsequently manually optimized in BioEdit 7.0.5.2 (Hall 1999). Genetic distances were calculated using MEGA 6.1 (Tamura et al. 2013). The optimal partitioning schemes for our alignment were identified with PartitionFinder 2.1.1 (Lanfear et al. 2012) using the greedy search algorithm under AICc criterion. Phylogenetic trees were reconstructed under maximum likelihood (ML) and Bayesian inference (BI). A ML analysis was implemented using the IQ-TREE webserver (Nguyen et al. 2015; Trifinopoulos et al. 2016). Clade stability was assessed by 1000 bootstrap (BS) replications and expected likelihood weights (ELW). One-thousand bootstrap pseudoreplicates (ML BS) were employed, and nodes having ML BS values of 90 and above were considered strongly supported, while nodes with values of 75–90 were regarded as significantly supported, lower values were considered to indicate lack of nodal support (Felsenstein 1985; Huelsenbeck and Hillis 1993).

Bayesian inference (BI) was performed in MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for one million generations, with sampling every 100 generations. We performed five independent MCMCMC runs and the initial 10% of trees were discarded as burn-in. We checked that the effective sample sizes (ESS) were all above 200 by exploring the likelihood plots using TRACER v1.6 (Rambaut et al. 2014). We assessed the clade support with posterior probabilities (PP) (Huelsenbeck and Ronquist 2001). Nodes with PP of 0.95 and above were considered strongly supported, nodes with values of 0.90–0.94 as significantly supported, while lower values were considered as no support (Huelsenbeck and Ronquist 2001; Wilcox et al. 2002). Molecular divergence time estimation was performed in BEAST v1.8.4 (Drummond et al. 2012). Molecular clock assumptions were tested using hierarchical likelihood ratio tests in PAML v4.7 (Yang 2007), which suggested the use of uncorrelated lognormal relaxed clock for our dataset. The models and partitioning scheme from our ML analysis were also incorporated into these subsequent divergence date estimations; we set the Yule model as the tree prior, assumed a constant population size, and used default priors for all other parameters. In BEAST, we conducted two runs of 200 million generations each, sampled every 4000 steps, parameter convergence was estimated in Tracer, and the first 10% of generations discarded as burn-in. TreeAnnotator v1.8.0 (in BEAST) was used to create our maximum clade credibility tree from the remaining samples. Calibration priors and all other details of this analysis followed Gorin et al. (2020).

In order to observe both ossified and cartilaginous structures, specimens were cleared and double stained with alcian blue for cartilage and alizarin red for bone. We used the most delicate methodology of acid-free staining (following Walker and Kimmel 2006) to preserve minute skeletal elements of the smallest species. The protocol included: (1) staining for about 24 hours in a solution of 0.05% alizarin red, 0.02% alcian blue, 45mM MgCl₂ and 70% ethanol; (2) maceration for about 24 hours at 37 °C in a saturated solution of sodium tetraborate with 1% trypsin; (3) bleaching for several hours in a solution of 1.5% H₂O₂ and 1% KOH; (4) clearing with successive changes of solutions of 25/50/75% glycerol with 0.25% KOH, for 1/3/5 days for each solution respectively; and final (5) storage in a 99% glycerol. Obtained skeletons were examined and photographed using a LEICA EZ4 dissecting stereo microscope (Leica Camera AG, Wetzlar, Germany) with a binocular-implemented ES-ESPERTS Digital camera BR-5101LC-UF.

We followed Micro-CT scanning of M. nepenthicola (ZMMU A-6028-1) and M. arboricola (ZMMU A-5051), using protocols of Suwannapoom et al. (2018) and Poyarkov et al. (2018). Scanning was conducted at the Petroleum Geology Department, Faculty of Geology, Lomonosov Moscow State University, using a SkyScan 1 172 desktop scanner (Bruker micro-CT, Kontich, Belgium) equipped with a Hamamatsu 10 Mp digital camera. Both specimens were mounted on a polystyrene baseplate and placed inside a hermetically sealed polyethylene vessel. Scans were conducted with a resolution of 3.7 μm at 40 kV voltage and a current of 250 mA, with a rotation step of 0.3°. We used oversize mode, in which three blocks of sub-scan data were connected vertically, to obtain a general tomogram. We used 3D Slicer (Kikinis et al. 2014) for construction and processing of 3D-models. Scans were deposited in MorphoSource (http://www.morphosource.org/Detail/ProjectDetail/Show/project_id/1183).

Osteological terminology followed Trueb (1968, 1973), Scherz et al. (2017), Suwannapoom et al. (2018), and Poyarkov et al. (2014, 2018a). Terminologies used to describe the shape of terminal phalanges (simple, knobbed, T-shaped, and Y-shaped) followed Parker (1927) and Garg et al. (2019). Comparative morphological and osteological data for other genera were taken from a number of revisions of Microhylinae (Parker 1934; Boring and Liu 1937; Duellman and Trueb 1986; Dubois 1987; Fei et al. 2009; McPartlin 2010; Chandramouli and Dutta 2015; Garg et al. 2019; Garg and Biju 2019; Poyarkov et al. 2018b; Zhang et al. 2020; Suwannapoom et al. 2020). External morphology was described following Poyarkov et al. (2014, 2019); mensural data were taken with a Mitutoyo dial caliper (Mitutoyo Corporation, Kawasaki, Japan) to the nearest 0.1 mm. We recorded the following external morphology characters: snout-vent length (SVL, measured as distance from tip of snout to cloaca), body shape (slender, stocky, or stout, following Bain and Nguyen 2004), snout profile (in lateral and dorsal view), dorsal skin texture (smooth, shagreened, feebly granular or tuberculate), relative length of first finger (FI length: ≤ 1/2 of FII length, ≥ 1/2 of FII length, or reduced to a nub), widths of discs on fingers and toes, number and shape of metatarsal tubercles, the presence (vs absence) of dorsomedial grooves on fingers and toes, of a distinct dorsomedial (vertebral) line, of superciliary tubercles, and of externally visible tympanum, the level to which the tibiotarsal articulation of an adpressed leg reaches (not reaching the eye, to the eye, to the snout, far beyond the snout), and the development of toe webbing (rudimentary, basal, well-developed, developed to discs; webbing and subarticular tubercle formulas follow Savage 1975).

To assess body size and sexual dimorphism evolution in the Microhyla–Glyphoglossus assemblage, we compiled data on maximum snout-vent length (SVL) separately for both sexes, for each species reported in literature and/or from our own measurements of voucher specimens following Gorin et al. (2020). Size (SVL) data for all Microhyla and Glyphoglossus species are summarized in Suppl. material 3: Table S3. Comparative morphological analyses were conducted in R 3.6.3 (R Core Team 2014). Analyses of SVL measurements were carried out using their natural logarithms. Sexual dimorphism was expressed as a ratio of male to female SVL (female-biased species have > 1, male-biased species have < 1). The tree and morphological dataset were pruned to reflect taxa represented in both, using the treedata() function in geiger (Harmon et al. 2008). Continuous trait evolution was mapped to the phylogeny using the contMap() function of phytools (Revell 2012). Phylogenetic Least Squares (PGLS) analysis of the log of male SVL against dimorphism was carried out using caper package (Orme et al. 2018) and plotted with ggplot2 (Wickham 2016). Species were binned into four size categories (terminology follows Scherz et al. 2019) as follows: ≤ 13 mm (state 1: “extremely miniaturized”); (2: 13–16 mm, “highly miniaturized”); (3: 16–20 “miniaturized”); (4: 20–24 “small”).

Our final aligned matrix of mtDNA data contained 232 sequences (length 2478 bp), representing 49 of the 52 currently recognized species of the genus Microhyla s. lat., three undescribed candidate species of Microhyla, and five species of Glyphoglossus. Our final alignment of the nuDNA BDNF gene was 720 bp long, and included all of the taxa sampled for the mitochondrial matrix but for six Microhyla s. lat. species (from clade I: M. gadjahmadai, M. taraiensis, M. mixtura, M. fanjingshanensis, and M. beilunensis; from clade II: M. perparva). We here report on mitochondrial-only and nuclear-only phylogenies first, and concatenated phylogenies afterwards.

Both BI and ML phylogenetic methods resulted in identical topology of mtDNA-based genealogical relationships for the Microhyla–Glyphoglossus assemblage (Fig. 2). All analyses concordantly resolved three strongly supported major clades within the group: Microhyla I, Microhyla II, and Glyphoglossus, as indicated by Bayesian posterior probabilities of 1.0 and ML bootstrap node support of 100% (node support values are hereafter provided as PP/BS); the majority of ingroup nodes also received strong support (PP/BS ≥ 0.95/95%). Although the Microhyla–Glyphoglossus assemblage was recovered to be a monophyletic group with strong support (1.0/100), the relationships among the three main clades within it remained essentially unresolved according to the mtDNA dataset, and the grouping of Microhyla I + Glyphoglossus received no nodal support (-/70) (Fig. 2; Suppl. material 6: Figure S1A and Suppl. material 6: Figure S2). Phylogenetic analyses of the nuDNA BDNF gene suggested monophyly of Mirohyla I + Microhyla II grouping with moderate to strong node support (0.90/97; Suppl. material 6: Figure S1B), despite the short length of this marker. Relationships at shallower nodes within the respective clades were less strongly resolved than in the mtDNA phylogeny. The combined mtDNA + nuDNA analyses (3207 bp) yielded a topology largely congruent with that of the nuDNA alone, but with lower node support values for the Mirohyla I + Microhyla II clade (0.46/90; Fig. 3; detailed in Suppl. material 6: Figure S1C). Thus, while the three major clades in the Microhyla–Glyphoglossus assemblage are strongly and consistently recovered as monophyletic, the monophyly of Microhyla s. lat. remains tentative, with practically no signal in the mitochondrial dataset but some signal in BDNF. As the combined dataset yielded a better resolved phylogeny that is also more consistent with previous work (e.g. Tu et al. 2018), we use that tree for further analyses (time tree, ancestral state reconstruction) and discussion below.

Figure 2. 

Diversity of the Microhyla–Glyphoglossus assemblage based on an updated mtDNA-genealogy derived from the analysis of 2478 bp of alignment including 12S rRNA, tRNAVal, 16S rRNA gene fragments. Black circles correspond to well-supported (PP ≥ 0.95; BS ≥ 90) and white circles to moderately supported (0.95 > PP ≥ 0.90; 90 > BS ≥ 75) nodes; no circles indicate unsupported nodes. Letters A–I denote the species groups of Gorin et al. (2020). Photos by Nikolay A. Poyarkov, Indraneil Das, Vladislav A. Gorin, Parinya Pawangkhanant, Luan Thanh Nguyen, and Evgeniya N. Solovyeva. For full version of this tree showing the outgroups and node support values see Suppl. material 7: Figure S2.

Figure 3. 

Bayesian inference tree of the Microhyla–Glyphoglossus assemblage derived from the combined mtDNA + nuDNA analysis of 3207 bp of alignment including 12S rRNA, tRNAVal, 16S rRNA and BDNF gene fragments. Black circles correspond to well-supported (PP ≥ 0.95; BS ≥ 90) and white circles to moderately supported (0.95 > PP ≥ 0.90; 90 > BS ≥ 75) nodes; no circles indicate unsupported nodes. Letters A–I denote the species groups of Gorin et al. (2020). For voucher specimen information and GenBank accession numbers see Suppl. material 1: Table S1. Yellow, red, and blue color denotes Microhyla I, Microhyla II, and Glyphoglossus, respectively. Numbers at tree nodes correspond to PP/BS support values, respectively (shown only for moderately supported nodes). For full version of this tree showing the outgroups and node support values see Suppl. material 6: Figure S1.

The observed topological patterns within the Microhyla–Glyphoglossus assemblage were congruent with earlier results of Gorin et al. (2020) in recovering eight major species groups within Microhyla s. lat. (clades A–H, see Figs 2–3), and the genus Glyphoglossus (clade I, see Figs 2–3). The only difference with results of Gorin et al. (2020) is the phylogenetic placement of the recently described M. hongiaoensis as sister species to M. pulchella (Figs 2–3). Since a detailed description of phylogenetic relationships within the genus Microhyla was provided by Gorin et al. (2020), we only focus here on a general description of the most important basal nodes, crucial for discussion in the present study.

The most species-rich clade, Microhyla I, is widely distributed from mainland southern China, Hainan and Taiwan, and the Ryukyu Archipelago of Japan in the north, through the Indochina Peninsula, to India, and Sri Lanka in the west, and through the Malayan Peninsula to Borneo, Sumatra, Java and Bali in the south (Fig. 1). The Microhyla I clade contained the following species, clustered in seven species groups (Fig. 2): The Microhyla achatina group (clade A), including M. achatina Tschudi, 1838; M. borneensis Parker, 1928; M. fodiens Poyarkov, Gorin, Zaw, Kretova, Gogoleva, Pawangkhanant & Che, 2019; M. gadjahmadai Atmaja, Hamidy, Arisuryanti, Matsui & Smith, 2018; M. heymonsi Vogt, 1911; M. irrawaddy Poyarkov, Gorin, Zaw, Kretova, Gogoleva, Pawangkhanant & Che, 2019; M. kodial Vineeth, Radhakrishna, Godwin, Anwesha, Rajashekhar & Aravind, 2018; M. malang Matsui, 2011; M. mantheyi Das, Yaakob & Sukumaran, 2007; M. minuta Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; M. nepenthicola Das & Haas, 2010; M. orientalis Matsui, Hamidy & Eto, 2013; M. pineticola Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; and two undescribed candidate species, Microhyla sp. 1 and Microhyla sp. 3.

The Microhyla fissipes group (clade B), including M. beilunensis Zhang, Fei, Ye, Wang, Wang & Jiang, 2018; M. chakrapanii Pillai, 1977; M. fanjingshanensis Li, Zhang, Xu, Lv & Jiang, 2019; M. fissipes Boulenger, 1884; M. mixtura Liu & Hu, 1966 in Hu et al. (1966); M. mukhlesuri Hasan, Islam, Kuramoto, Kurabayashi & Sumida, 2014; M. mymensinghensis Hasan, Islam, Kuramoto, Kurabayashi & Sumida, 2014; M. okinavensis Stejneger, 1901; and an undescribed candidate species, Microhyla sp. 2.

The Microhyla berdmorei group (clade C), including M. berdmorei (Blyth, 1856); M. picta Schenkel, 1901; and M. pulchra (Hallowell, 1861).

The Microhyla superciliaris group (clade D), including M. darreli Garg, Suyesh, Das, Jiang, Wijayathilaka, Amarasinghe, Alhadi, Vineeth, Aravind, Senevirathne, Meegaskumbura & Biju, 2019; M. eos Biju, Garg, Kamei & Maheswaran, 2019; M. karunaratnei Fernando & Siriwardhane, 1996; M. laterite Seshadri, Singal, Priti, Ravikanth, Vidisha, Saurabh, Pratik & Gururaja, 2016; M. sholigari Dutta & Ray, 2000; M. superciliaris Parker, 1928; M. tetrix Suwannapoom, Pawangkhanant, Gorin, Juthong & Poyarkov, 2020; and M. zeylanica Parker & Osman-Hill, 1949.

The Microhyla ornata group (clade E), including M. mihintalei Wijayathilaka, Garg, Senevirathne, Karunarathna, Biju & Meegaskumbura, 2016; M. nilphamariensis Howlader, Nair, Gopalan & Merilä, 2015; M. ornata (Duméril & Bibron, 1841); M. rubra (Jerdon, 1854); and M. taraiensis Khatiwada, Shu, Wang, Thapa, Wang & Jiang, 2017.

The Microhyla butleri group (clade F), including M. aurantiventris Nguyen, Poyarkov, Nguyen, Nguyen, Tran, Gorin, Murphy & Nguyen, 2019; and M. butleri Boulenger, 1900.

The Microhyla palmipes group (clade G), including M. palmipes Boulenger, 1897. The distribution area of the Microhyla II clade is restricted to the montane forest areas in the Annamite (Truong Son) Mountains in East Indochina (Vietnam, eastern Laos, northeastern Cambodia), Malayan Peninsula (Titiwangsa Mountain Range), mountains of Borneo (Sarawak, Sabah of Malaysia, Brunei and northern Kalimantan, Indonesia), and the southwestern-most islands of the Sulu Archipelago of the Philippines (Fig. 1). It contains the following nine species (clade H, Fig. 2) of the M. annectens group: M. annamensis Smith, 1923; M. annectens Boulenger, 1900; M. arboricola Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; M. hongiaoensis Hoang, Nguyen, Luong, Nguyen, Orlov, Chen, Wang & Jiang, 2020; M. marmorata Bain & Nguyen, 2004; M. nanapollexa Bain & Nguyen, 2004; M. perparva Inger & Frogner, 1979; M. petrigena Inger & Frogner, 1979; and M. pulchella Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014.

Finally, the Glyphoglossus clade (clade I, Fig. 2) covers the whole Thai-Malaysian Peninsula, parts of Indochina, including Myanmar, and also penetrates northward, as far as southern mainland China; and southward as far as Sumatra and Borneo (Fig. 1). It contains the following five species: G. capsus (Das, Min, Hsu, Hertwig & Haas, 2014); G. guttulatus (Blyth, 1856); G. minutus (Das, Yaakob & Lim, 2004); G. molossus Günther, 1869; and G. yunnanensis (Boulenger, 1919).

Estimated node-ages (mean age estimate ± 95% highest posterior density interval [95% HPD]) for main nodes are detailed in Suppl. material 4: Table S4 and Suppl. material 8: Figure S3. The results of the divergence time estimation fully agree with Gorin et al. (2020), suggesting that the most recent common ancestor (MRCA) of Microhyla and Glyphoglossus originated around the early Eocene ca. 50.9 million years ago (hereafter Ma, 95% HPD 44.2–58.7) (Suppl. material 8: Figure S3). This estimate coincides with some previous estimates (48.8 Ma; 45.9–53.2, Feng et al. 2017), but is significantly younger than other reports (61.5, 56.6–66.5, Garg and Biju 2019). The Microhyla–Glyphoglossus assemblage radiated into the three major clades in the middle Eocene (44.1, 38.5–49.6), notably later than other estimates (48.7, 44.1–53.2, Garg and Biju 2019). Subsequent diversification of each genus-level radiations of Microhyla I, Microhyla II, and Glyphoglossus clades initiated much later in the early to middle Oligocene (ca. 35–25 Ma, Gorin et al. 2020).

A total of 26 species examined for osteological variation allows us to clarify similarities and variation in skeletal morphology among and within the three clades of the Microhyla–Glyphoglossus assemblage. Detailed information on species’ characters’ states is presented in Suppl. material 5: Table S5. Overall skeletal morphology and the main osteological features for representatives of each clade are illustrated in Figures 4–7. Skull and hand morphology for cleared and stained representatives of these three clades is provided in Suppl. material 8, 9.

Below, we provide comparative osteological descriptions for the three clades of the Microhyla–Glyphoglossus assemblage: Microhyla I, Microhyla II, and Glyphoglossus.

This clade includes M. achatina, the type species of the genus Microhyla, and is the most widely distributed, species rich, and ecologically and morphologically diverse group of the Microhyla–Glyphoglossus assemblage Clade I includes most small- to medium-sized terrestrial species, along with several large species; they are adapted to fossorial (M. picta), or semi-fossorial (M. fodiens, M. rubra, M. mihintalei) lifestyles (Fig. 2). This diversity is also reflected in osteological features, which demonstrate conspicuous variation among species (Fig. 4; Suppl. material 5: Table S5). Due to marked morphological variation, providing a comprehensive morphological diagnosis of this speciose group remains a challenging task; below we summarize available information on skeletal traits.

Figure 4. 

General osteology of the Microhyla–Glyphoglossus assemblage representatives. The full skeletons are shown for Glyphoglossus molossus (A – dorsal, B – ventral views), Glyphoglossus yunnanensis (C – dorsal, D – ventral views), Microhyla achatina (E – dorsal, F – ventral views), Microhyla nepenthicola (G – dorsal, H – ventral views), and Nanohyla arboricola (I – dorsal, J – ventral views). Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Scale bar equals 5 mm.

Skull

Skull longer than wide, wider than long, or almost in equal proportions among species of Microhyla I (Fig. 5; Suppl. material 5: Table S5). Widest part of skull located posteriorly and giving head a triangular or trapezoid shape (Fig. 5). Frontoparietals longer than broad, narrowing anteriorly, in contact along medial border but not fused, lacking any dorsal crests; partially fused with or separated from exoccipital posteriorly and prootic posterolaterally. Exoccipitals always separate, in contact medially. Nasals large and widely separated, chondrified peripherally; processus paraorbitalis broad, in some species blunt, posterior edge concave, anterior edge convex (Fig. 5). Sphenethmoid well-ossified, always clearly separated from parasphenoid, with a concave posterior edge. Prootics ossified anteromedially; crista parotica cartilaginous with posterior margin mineralized. Squamosal ossified, with a well-developed ventral ramus and poorly developed otic and zygomatic rami. Operculum slightly mineralized. Majority of columella (stapes) mineralized, with only pars externa plectra cartilaginous; tympanic annulus completely chondrified (Suppl. material 10: Figure S5H). Premaxilla well-ossified, its alary process oriented slightly anteriorly, distal part bending laterally. Maxilla well-ossified; anteriorly in contact with labial portion of premaxilla (eleutherognathine condition); edentate; pars facialis moderately high in lateral view. Quadratojugal reduced, with a chondrified posterior articulation with angulosplenial; not in anterior contact with maxilla. Support of upper jaw taken over by pterygoid, with long anterior ramus, broad posterior ramus, and short medial ramus. Vomers small, widely separated, triangular in shape. Neopalatines present or absent. Nasal capsules mineralized posteriorly or entirely cartilaginous. Mentomeckelians ossified, connected to dentaries and to each other through Meckel’s cartilage. Dentary fused with angulosplenial. Parasphenoid smooth; cultriform process of parasphenoid narrowing anteriorly, terminating at level of sphenethmoid with a chondrified notch (Fig. 5). Hyoid plate completely cartilaginous, anterolateral (alary) processes of hyoid plate present, recurved, posterolateral processes slender, posteromedial processes strongly ossified, elongated, straight, wider at proximal ends, chondrified at distal ends, separated by a chondrified parahyoid (Suppl. material 10: Figure S5M).

Figure 5. 

Cranial osteology of the Microhyla–Glyphoglossus assemblage representatives. The skulls are shown in dorsal / ventral / lateral views for Glyphoglossus molossus (A / B / C, respectively), Glyphoglossus yunnanensis (D / E / F, respectively), Microhyla achatina (G / H / I, respectively), Microhyla nepenthicola (J / K / L, respectively), and Nanohyla arboricola (M / N / O, respectively). Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Scale bar equals 3 mm.

Vertebral column

Vertebral column is diplasiocoelus, typically comprising eight presacral vertebrae (PSV) (Fig. 6C), with the exception of extremely miniaturized species M. nepenthicola, which has PSV I and II fused (Fig. 6D). PSV II–VII procoelous and VIII amphicoelous. Transverse processes of PSV II–IV longer and wider than V–VIII, transverse processes of PSV VI–VIII oriented anterolaterally; orientation of transverse processes to other vertebrae varies (Suppl. material 5: Table S5). Transverse processes of sacrum moderately expanded, with distal end about twice as wide as proximal end. Urostyle shorter than trunk vertebrae, bearing a weak dorsal crest that tapers posteriorly and vanishes at two‐thirds of urostyle length (Fig. 6); its articulation with sacrum is bicondylar.

Figure 6. 

Axial skeleton composition in the Microhyla–Glyphoglossus assemblage representatives. The vertebral columns are shown in dorsal view for Glyphoglossus molossus (A), Glyphoglossus yunnanensis (B), Microhyla achatina (C), Microhyla nepenthicola (D) and Nanohyla arboricola (E). Numerals (I–VIII) correspond to the numbers of presacral vertebrae (PSV); I+II denotes fusion of the two first PSV. Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Scale bar equals 3 mm.

Appendicular skeleton

Pectoral girdle with a firmisternal arrangement. Coracoids, scapulae, and suprascapulae present; first two fully ossified; suprascapula largely chondrified. Coracoids robust with wide proximal end. Omosternum generally absent, except for M. puchra, where a tiny cartilaginous omosternum is present (Suppl. material 5: Table S5). Procoracoids indistinct. Clavicles absent. Cartilaginous sternum large, partially mineralized, fan-shaped or bifurcate (Suppl. material 10: Figure S5D, E); xiphisternum completely cartilaginous.

Hand skeleton including seven largely calcified carpal elements: carpale distale II, carpale distale III–V fused into a single large element, prepollex (consisting of two elements), Element Y, radiale, and ulnare (Fig. 7C–D). Metacarpals long and fully ossified; hand phalangeal formula: 2-2-3-3; all phalanges ossified; distal phalanx of finger III simple, conical-, bobbin- or T-shaped. Foot skeleton with four tarsal elements, including ossified tarsale distale II–III, centrale and a prehallux; prehallux mineralized in all species examined (Suppl. material 10: Figure S5A). Metatarsals fully ossified, long and relatively more massive than metacarpals; foot phalangeal formula: 2-2-3-4-3; all phalanges ossified. Terminal phalanges of toe III T-shaped or simple.

This is a compact clade of nine species belonging to the M. annectens group previously recovered by Gorin et al. (2020), encompassing minute- or small-sized terrestrial or semi-arboreal species with short triangular-shaped body habiti, inhabiting montane forests in Indochina and Sundaland (Fig. 2). The clade Microhyla II is rather uniform in skeletal composition, and examined species share a set of osteological characters that clearly separate this group from the two other clades of the Microhyla–Glyphoglossus assemblage (Suppl. material 5: Table S5).

Skull

Skull longer than wide or almost equal (Figs 4, 5); widest portion posterior, giving head triangular shape (Fig. 5). Frontoparietals longer than broad, narrowing anteriorly, in contact along medial border and fused posteriorly, lacking any dorsal crests; posteriorly fused with exoccipitals. Exoccipitals completely fused with each other (Fig. 5), except M. pulchella, (partial; Suppl. material 5: Table S5). Nasals large, broadly separated, chondrified peripherally, processus paraorbitalis narrow and cultriform, posterior edge concave, anterior edge oblique (Fig. 5). Spenethmoids ossified, completely fused with parasphenoid (in M. pulchella an indistinct suture remains laterally, so the fusion is partial), extending posteroventrally nearly to level of prootics along parasphenoid. Prootics ossified anteromedially, crista parotica entirely cartilagionous. Squamosal ossified, with well-developed long ventral and otic rami and poorly developed zygomatic ramus (Fig. 5O; Suppl. material 5: Table S5). Operculum mineralized. Columella largely mineralized with only pars externa plectra cartilaginous, tympanic annulus completely chondrified (Suppl. material 10: Figure S5G). Premaxilla well-ossified, alary process oriented slightly anteriorly, distal part bending laterally. Maxilla well-ossified; anteriorly in contact with labial portion of premaxilla; edentate; pars facialis moderately high in lateral view. Quadratojugal reduced further than Microhyla I clade, not in anterior contact with maxilla; support of upper jaw taken over by pterygoid. Pterygoid with long anterior ramus (pronounced concavity along ramus that is much more laterally oriented than in Microhyla I clade), broad posterior ramus, and short medial ramus. Vomers small, widely separated, triangular in shape. Neopalatines present as very thin elements. Mentomeckelians ossified, connected to dentaries and to each other through Meckel’s cartilage. Dentary fused with angulosplenial. Parasphenoid smooth; cultriform process of parasphenoid broad, completely fused with sphenethmoid laterally (Fig. 5O), terminating at level of neopalatines with a chondrified notch. Hyoid plate completely cartilaginous, anterolateral processes of hyoid plate present, recurved, posterolateral processes slender, posteromedial processes strongly ossified, elongated, straight, chondrified at distal ends, wider at proximal ends, separated by a chondrified parahyoid.

Vertebral column

Vertebral column diplasiocoelus, including eight presacral vertebrae, with the exception of one of the smallest species of the group, M. arboricola, which has PSV I and II fused (Fig. 6E). PSV II–VII procoelous and VIII amphicoelous. Transverse processes of PSV II–IV longer and wider than in PSV V–VIII; transverse processes of PSV II, VII and VIII oriented anterolaterally, IV and V posterolaterally, III and VI perpendicular to vertebral column axis, with exception of M. marmorata, which has transverse processes of PSV VI oriented anterolaterally. Transverse processes of sacrum notably expanded, with distal end more than twice as wide as proximal. Urostyle shorter than trunk vertebrae, bearing a weak dorsal crest, tapering posteriorly; vanishes completely at 2/3 urostyle length (Fig. 6E).

Appendicular skeleton

Pectoral girdle firmisternal. Coracoids, scapulae, and suprascapulae present; coracoid and scapula fully ossified; suprascapula largely chondrified. Coracoids robust with wide proximal end. Cartilaginous omosternum present (Suppl. material 10: Figure S5F). Procoracoids indistinct. Clavicles absent. Sternum large, cartilaginous, partially mineralized, bifurcate or fan-shaped; xiphisternum completely cartilaginous.

Manus skeleton with seven largely calcified carpal elements, including carpale distale II, carpale distale III–V (fused into a single large element), prepollex (consisting of two separate elements), Element Y, radiale, and ulnare (Fig. 7E). Metacarpals long and fully ossified; phalangeal formula: 2-2-3-3; all phalanges ossified, with exception of M. arboricola, in which metacarpals and phalanges ossified only peripherally (Fig. 7E); distal phalanx of finger III T-shaped. Foot skeleton with four tarsal elements, including ossified tarsale distale II–III, centrale and a prehallux; prehallux cartilaginous in all species examined (Suppl. material 10: Figure S5B). Metatarsals fully ossified, elongated and much more massive than metacarpals; phalangeal formula: 2-2-3-4-3; all phalanges ossified. Terminal phalanges of toe III T-shaped.

Figure 7. 

Hand skeleton composition in the Microhyla–Glyphoglossus assemblage representatives. The hands are shown in ventral view for Glyphoglossus molossus (A), Glyphoglossus yunnanensis (B), Microhyla achatina (C), Microhyla nepenthicola (D) and Nanohyla arboricola (E). Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Scale bar equals 1 mm.

In our analysis, three species of Glyphoglossus (of nine recognized) were examined, so the variation of skeletal characters in this genus might be underestimated. All Glyphoglossus species are adapted to fossorial lifestyle, and are easily distinguished from all other members of the group by their large body size, stocky and globular habitus, and enlarged inner metacarpal tubercle used for burrowing. Species of Glyphoglossus inhabit lowland areas of southern mainland China, Indochina, and Sundaland (Fig. 2). A broad range of morphological variation has been documented: G. molossus is notably different from G. yunnanensis and G. guttulatus (until recently, both were classified as members of the genus Calluella Stoliczka, 1872, now considered a junior synonym of Glyphoglossus based on its phylogenetic placement; Peloso et al. 2016). Owing to the morphological uniqueness of G. molossus, morphological features of this species are marked with an asterisk (*).

Skul

Skull notably wider than long (Fig. 4). Skull widest at mid-length, giving head a widened, rounded shape. Frontoparietals longer than broad, narrowing anteriorly, connecting medially with a suture along whole length, or anteriorly, separated or fused* (Fig. 5A) medially, lacking dorsal crests, separated or fused* with exoccipitals (separate) posteriorly. Nasals large, separated, chondrified peripherally; processus paraorbitalis well-developed, pointed laterally or anteriorly* (Fig. 5). Spenethmoid separate, well ossified, restricted to anterior third of brain case or nearly closing lateral wall of brain case* (Fig. 5C). Prootics ossified anteromedially or completely*, crista parotica mineralized medially or completely*. Squamosal ossified, with well-developed ventral ramus and less developed, but distinct otic and zygomatic rami. Operculum cartilaginous or ossified*. Columella largely ossified, with only pars externa plectra cartilaginous; tympanic annulus completely chondrified. Premaxilla well-ossified, alary process oriented slightly posteriorly, distal portion straight or bending laterally*. Maxilla well-ossified, anteriorly contacting labial portion of premaxilla; teeth present or absent*; pars facialis moderately to notably high, and oriented towards processus paraorbitalis of nasal*. Quadratojugal robust, with rounded cartilaginous articulation with angulosplenial, anteriorly articulating with or fused* to maxilla. Pterygoid massive, with a long anterior ramus, broad posterior ramus, and short medial ramus. Vomers large, shape either complex or U-shaped*, defining lower floor of nasal capsule. Neopalatines obscured by postchoanal vomerine processes, fused or replaced completely*. Nasal capsules mineralized posteriorly or obscured by postchoanal vomerine processes*. Mentomeckelians ossified, connected to dentaries, and to each other through Meckel’s cartilage. In G. molossus, ventral portion of mentomeckelian cartilage protruding and greatly mineralized, forming a unique beard-like structure, shaping the characteristically flattened snout profile* (Fig. 2). Dentary fused with angulosplenial. Parasphenoid smooth, its cultriform process broad, tapering anteriorly or not*, terminating at level of sphenethmoid or nasal capsules*, with a chondrified notch. Hyoid plate completely cartilaginous, its anterolateral processes well-developed, recurved, posterolateral processes slender, posteromedial processes strongly ossified, elongated, straight, chondrified at distal ends, wider at proximal ends, separated by a chondrified parahyoid. Each posteromedial process bears two bony flanges; one oriented laterally, another medially.

Vertebral column

Vertebral column diplasiocoelus, with eight presacral vertebrae. PSV II–VII procoelous, PSV VIII amphicoelous. PSV I very unusual in shape in G. molossus, with highly extended condylar arms. Transverse processes of PSV II–IV longer and wider than V–VIII, transverse processes of PSV II, VII and VIII oriented anterolaterally, IV and V posterolaterally, III and VI at right angle to vertebral column axis. In G. molossus transverse processes of PSV greatly shortened, II and VI–VIII oriented anterolaterally, IV oriented posterolaterally, III and V at the right angle to the body axis* (Fig. 6A). Sacral transverse processes moderately expanded, with the distal end about twice as wide as the proximal end. The urostyle notably shorter than the trunk vertebrae (Fig. 6), bearing a dorsal crest that tapers posteriorly and vanishes at about one third of the urostyle length (Fig. 6B), or continues almost to the end of the urostyle* (Fig. 6A).

Appendicular skeleton

Pectoral girdle firmisternal. Coracoids, scapulae, and suprascapulae present; first two fully ossified; suprascapulae largely chondrified. Coracoids robust, with proximal end, or both ends widened*. Omosternum absent. Procoracoids present or absent*. Clavicles present or absent*. Cartilaginous sternum large, partially mineralized, fan-shaped; xiphisternum completely cartilaginous.

Hand skeleton with six largely calcified carpal elements: carpale distale II, carpale distale III–V fused into a single large element, prepollex (consisting of two elements), element Y, radiale and ulnare (Fig. 7A–B). Metacarpals long and fully ossified; phalangeal formula: 2-2-3-3; all phalanges ossified, notably shortened in G. molossus*; distal phalanx of finger III simple. Foot with four tarsal elements, including ossified tarsale distale II–III, centrale, and prehallux; prehallux enlarged and ossified (Suppl. material 10: Figure S5C). Metatarsals fully ossified, long, more massive than metacarpals; phalangeal formula: 2-2-3-4-3; all phalanges ossified. Terminal phalanx of toe III simple, conical.

Body size and sexual dimorphism evolution

Clades I and II of Microhyla are inferred to have independently reduced in body size from a moderately small common ancestor (males estimated at 25.3 mm, 95% CI 18.8–34.2; Fig. 8). Within Microhyla I, two clades arose from miniaturized common ancestors, the Microhyla superciliaris species group (common ancestor estimated at 17.7 mm), and the M. achatina species group (common ancestor estimated at 19.6 mm; a second clade, composed of Microhyla sp. 3 and M. kodial, likely independently reduced in size with a common ancestor of 18.3 mm). A few lineages have also reduced in body size below 20 mm independently (Fig. 8), giving a total of eight transitions to SVL < 20 mm. The common ancestor of all Microhyla II species was apparently miniaturized (male SVL estimated at 18.1 mm), and most lineages reduced further. Two lineages, M. annamensis + M. marmorata and M. pulchella, have increased in body size independently and repeatedly from miniaturized ancestors, to their modern body sizes. In Microhyla I, the M. berdmorei species group substantially increased in body size. Among Glyphoglossus, G. molossus is an extreme outlier in body size, and is substantially larger than other equivalent-level clades. Across the entire assemblage, male SVL exhibits substantial phylogenetic signal (Pagel’s λ = 1.00).

Figure 8. 

Continuous ancestral state reconstruction of male body size (left) and sexual size dimorphism (right) in the Microhyla–Glyphoglossus assemblage. Species names in purple have at least one sex with maximum SVL ≤ 20 mm, in fuchsia at least one sex with maximum SVL ≤ 16 mm. Circles at nodes are based on inferred ancestral male SVL.

Sexual size dimorphism exhibits no phylogenetic signal (Pagel’s λ = 7.2 × 10^-5), changing sporadically across the tree, and is weakly positively correlated with log(male SVL) (PGLS, F_1,51 = 5.478, adjusted R² = 0.07928, P = 0.02321; Fig. 9B). Most species of Microhyla I and II exhibit female-biased size dimorphism (above the y = x line in Fig. 9A), and among these, species with the smallest males exhibit the greatest degree of size dimorphism (Fig. 9B). Only six species are male-biased (Microhyla sp. 1, M. mantheyi, M. mukhlesuri, M. superciliaris, M. mihintalei, and G. molossus), including both the largest (G. molossus) and the smallest (Microhyla sp. 1) species in our dataset.

Figure 9. 

Relationships between body size among sexes (A), and between male body size and sexual size dimorphism (B) in Microhyla s. str., Nanohyla gen. nov., and Glyphoglossus. The line in (A) represents x=y.

Among vertebrates, numerous clades of fishes, frogs, and squamate reptiles compete for the title of the smallest absolute body size, with several converging around body lengths (defined vastly differently in the three clades) of 8–12 mm (Hanken and Wake 1993). This apparent size limit has invoked the idea of physiological constraints preventing the evolution of smaller body sizes (Alexander 1996; Hedges and Thomas 2001; Scherz et al. 2019). As such, species exhibiting miniaturization provide interesting opportunities to understand the lower size limits of vertebrate physiology and development, whereas clades exhibiting miniaturized body plans offer opportunities to understand the dynamics of size evolution. Moreover, miniaturization is often associated with major morphological rearrangements (Hanken 1985; Hanken and Wake 1993; Polilov 2015), and is thought to have played a significant role in generation of some key innovations, such as the mammalian inner ear (Lautenschlager et al. 2018). It is therefore of great interest to also understand the consequences of miniaturization from a broad array of cases.

Frogs, and especially microhylids, have a particular propensity to miniaturize, with several microhylids in a variety of subfamilies achieving adult body sizes of 12 mm or smaller (Clarke 1996; Lehr and Coloma 2008; Das and Haas 2010; Rittmeyer et al. 2012; Rakotoarison et al. 2017; Scherz et al. 2019; Oliver et al. 2017). Despite this diversity, there are surprisingly few studies that have looked at miniaturization in a comparative context within the Microhylidae (e.g., de Sá et al. 2012, 2019b). Here, we have demonstrated that the Microhylinae are a particularly interesting clade of microhylids in which to study miniaturization, because they have converged repeatedly on extremely small body sizes.

Body size evolution in the Microhyla–Glyphoglossus was discussed in a study based on the maximum parsimony analysis of trait evolution, categorizing SVL into a series of bins (Gorin et al. 2020). Our analysis, which instead uses ancestral state reconstruction of continuous traits (Revell 2012) on our dated phylogeny, is largely congruent with that of Gorin et al. (2020) but provides better estimation of ancestral states and the timing of transitions in body size. Our results show clearly that this assemblage has undergone repeated miniaturization events, with Nanohyla miniaturizing first and independently from all Microhyla species; their most recent common ancestor is inferred to have been only a small frog (ca 25.3 mm in males). Within Microhyla, two large clades converged further toward the minimum size range, but six other lineages independently also became miniaturized (crossing the threshold of SVL < 20 mm). These replicates provide an opportunity to understand the relationship of certain morphological features with extreme body size reduction. Miniaturization of Nanohyla appears to have been coupled with the loss of metatarsal tubercles, whereas these are retained in even the smallest Microhyla. Likewise, the first finger of Nanohyla is often reduced to a nub, whereas it is always at least half the length of the second finger in Microhyla. This is reminiscent of the patterns seen in Stumpffia Boettger, 1881 frogs from Madagascar, where digit reduction is a hallmark of each major clade, and where the first finger is always the first to reduce (Rakotoarison et al. 2017). Unlike Stumpffia, however, even the smallest Microhyla and Nanohyla do not show reduction of the second and fourth fingers, although Microhyla tetrix presents bizarre hand morphology with a particularly thick and long third finger (Poyarkov et al. 2020b) reminiscent of the third-finger-only phenotype seen in the smallest Stumpffia species. Also, they have not lost any phalanges, even when fingers are reduced in length, whereas other miniaturized frogs often show finger or toe formula reduction (Alberch and Gale 1983, 1985; Scherz et al. 2019). Still, there has been a tendency for the terminal phalanx of F1 to transition from T-shaped to knobbed to simple in miniaturization series, indicative of a strong reduction despite the lack of loss of this element.

In the vertebral column, Microhyla nepenthicola (Fig. 6D) and Nanohyla arboricola (Fig. 6E) exhibit fusion of the first two presacral vertebrae, potentially linked to their extremely small body size. In the skull, both Nanohyla and Microhyla show forward displacement of the jaw articulation in miniaturized species, but other features are unique to each group, including the long otic ramus of the squamosal of Nanohyla (vs the reduction of the otic ramus of Microhyla), or the expansion of the sphenethmoid of Nanohyla along the parasphenoid (vs lack of expansion in Microhyla). The wide array of commonalities and differences both within these clades, and in comparison between these clades and other miniaturized frogs, highlights the extent to which miniaturization occurs through a combination of determinism and contingency. Nanohyla and Microhyla apparently share the reduction of the quadratojugal and loss of its connection to the maxilla, and the resulting take-over of suspensorium support by the pterygoid (Fig. 5). This arrangement is sometimes seen in other miniaturized microhylids (e.g., Anodonthyla eximia Scherz, Hutter, Rakotoarison, Riemann, Rödel, Ndriantsoa, Glos, Roberts, Crottini, Vences & Glaw [Scherz et al. 2019]), but, surprisingly, in the present case the loss of quadratojugal connection to the maxilla does not appear to be related to body size; even the largest species of Microhyla in the M. berdmorei group show the pterygoidal suspensorium support, but are not inferred to have passed through a period of extreme body size reduction that would be expected to result in such a degree of change. Thus, caution is always recommended when interpreting features as consequences of miniaturization, when they may have arisen through other selective pressures. Interestingly, some species within Nanohyla and Microhyla increased again in body size from an ancestral body size that was <18 mm. These species would be worthy of future investigation, because cases of post-miniaturization body-size increases can leave behind hallmarks (e.g., potentially irrevocable loss of anatomical features such as fingers), which can lead to morphological innovation (Hanken and Wake 1993).

Finally, although they are not miniaturized, it is worth briefly remarking on the osteology of Glyphoglossus, and especially the bizarre G. molossus. The osteology of G. yunnanensis is rather typical of a large-bodied microhylid, with long, slender limb bones and a subtriangular skull. Glyphoglossus molossus, however, shows extreme osteological modification associated with its more fossorial lifestyle, from its thickened hind- and forelimb bones to its small, rounded skull, to its highly modified first presacral vertebra. The peculiar flattened snout in this species is formed by a large chondrified beard-looking structure, not co-ossified to rostral and mandibular bones. Its limb and skull modifications resemble other strong burrowers, e.g., Breviceps gibbosus (Linnaeus, 1758) and Barygenys maculata Menzies & Tyler (Menzies 2020; Van Dijk 2001).

As is typical for frogs (Shine 1979), most members of the Microhyla–Glyphoglossus assemblage exhibit slight female-biased size dimorphism. There is a weak, but significant, positive correlation between log(male SVL) and sexual size dimorphism, with those species with the smallest males having the strongest female-biased size dimorphism, and dimorphism decreasing with increasing male SVL. They thus conform to Rensch’s rule (Rensch 1950). This may reflect a greater constraint on female body size, associated with the cost of reproduction in these frogs, which, even in the smallest species, produce clutches of many dozens of eggs.

Only a handful of species have transitioned, apparently rapidly and independently, to male-biased dimorphism. Remarkably, even species with diminutive males can be male-biased, exemplified by Microhyla sp. 1. In general, male-biased dimorphism is thought to be associated with territoriality and physical combat among male frogs (Shine 1979). These transitions to male-biased dimorphism may, therefore, be associated with changes in natural history of these lineages. Yet, this condition does not appear to be evolutionarily stable, because in no cases are a pair of sister species both male-biased. At present, too little is known of the ecology of these species to understand common drivers of these changes.