Zoosyst. Evol. 97 (1) 2021, 21-54 | DOI 10.3897/zse.97.57968 eee ee BERLIN Consequences of parallel miniaturisation in Microhylinae (Anura, Microhylidae), with the description of a new genus of diminutive South East Asian frogs Vladislav A. Gorin’, Mark D. Scherz*, Dmitriy V. Korost*, Nikolay A. Poyarkov!” Faculty of Biology, Department of Vertebrate Zoology, Lomonosov Moscow State University, Leninskiye Gory 1/12, Moscow 119234, Russia Sektion Herpetologie, Zoologische Staatssammlung Miinchen (ZSM-SNSB), Miinchhausenstr. 21, 81247, Miinchen, Germany Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany Geological Faculty, Petroleum Geology Department, Moscow State University, Leninskiye Gory 1, Moscow 119234, Russia oF WN PF Joint Russian-Vietnamese Tropical Research and Technological Center, Nghia Do, Cau Giay, Hanoi, Vietnam http://zoobank.org/C6E9DCB9-E56D-48E 1-A042-452A 1D2043EE Corresponding author: Nikolay A. Poyarkov (n.poyarkov@gmail.com) Academic editor: Rafe Brown # Received 25 August 2020 Accepted 14 December 2020 @ Published 12 January 2021 Abstract The genus Microhyla Tschudi, 1838 includes 52 species and is one of the most diverse genera of the family Microhylidae, being the most species-rich taxon of the Asian subfamily Microhylinae. The recent, rapid description of numerous new species of Microhyla with complex phylogenetic relationships has made the taxonomy of the group especially challenging. Several recent phylogenetic studies suggested paraphyly of Microhyla with respect to Glyphoglossus Giinther, 1869, and revealed three major phylogenetic lin- eages of mid-Eocene origin within this assemblage. However, comprehensive works assessing morphological variation among and within these lineages are absent. In the present study we investigate the generic taxonomy of Microhyla—Glyphoglossus assemblage based on a new phylogeny including 57 species, comparative morphological analysis of skeletons from cleared-and-stained speci- mens for 23 species, and detailed descriptions of generalized osteology based on volume-rendered micro-CT scans for five species— altogether representing all major lineages within the group. The results confirm three highly divergent and well-supported clades that correspond with external and osteological morphological characteristics, as well as respective geographic distribution. Accordingly, acknowledging ancient divergence between these lineages and their significant morphological differentiation, we propose to consider these three lineages as distinct genera: Microhyla sensu stricto, Glyphoglossus, and a newly described genus, Nanohyla gen. nov. Key Words Amphibians, integrative taxonomy, narrow-mouthed frogs, micro-computed tomography, Nanohyla gen. nov, osteology, sexual dimorphism, taxonomic revision Introduction Anuran amphibians of the family Microhylidae (nar- row-mouthed frogs) are globally distributed and diverse. This group currently comprises 12 subfamilies, 57 gen- era and over 700 recognized species, thus representing 10.3% of extant anuran diversity, making it the third largest anuran family after Hylidae and Strabomantidae (Streicher et al. 2020; Frost 2020). Microhylid frogs are morphologically and ecologically diverse including ter- restrial, arboreal and fossorial (burrowing) morphotypes (Wells 2010; Moen et al. 2015). Microhylids display ex- tensive variation in adult external morphology, osteology, and musculature; and in many cases, parallel speciali- zations associated with a burrowing lifestyle may have led to remarkable morphological convergence (Emerson 1971; Wu 1994; Trueb et al. 2011; Moen et al. 2015). The extensive homoplasy observed in Microhylidae hinders Copyright Viadislav A. Gorin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs Sa Nae ene Tip ate a oS Tiare a Figure 1. Distribution ranges of the three clades of the Microhyla—Glyphoglossus assemblage. Distribution area of Microhyla | is shown in yellow, of Microhyla II in red, and of Glyphoglossus in blue. Distributional data from Gorin et al. (2020). Question mark denotes the unconfirmed record of “Microhyla annamensis” from Khao Sebab in eastern Thailand by Taylor (1962). phylogenetic interpretations from morphological char- acters (Wu 1994): significant progress in understanding evolutionary relationships of microhylids was achieved only through molecular phylogenetic studies (de Sa et al. 2012, 2019a, 2019b; Tu et al. 2018; Streicher et al. 2020). Skeletal composition varies substantially among micro- hylids, showing features that are rare or absent in oth- er anuran clades (Noble and Parker 1926; Parker 1934; Zweifel 1972; 1986; de Sa and Trueb 1991). Although osteology and morphological evolution of microhylid subfamilies from the Americas (de Sa and Trueb 1991; Lehr and Trueb 2007; Trueb et al. 2011; Zweifel 1986, and references therein), Australasia (Zweifel 1972), and Madagascar (Scherz et al. 2015, 2016, 2017, 2019) re- ceived a decent amount of researchers’ attention, studies of the Asian microhylid subfamilies, to date, have pri- marily focused on description of long-neglected species diversity (Matsui et al. 2011; Hasan et al. 2014; Poyarkov et al. 2014, 2018a, 2018b, 2019, 2020; Vassilieva et al. 2014; Yuan et al. 2016; Suwannapoom et al. 2018, 2020; Nguyen et al. 2019, and citations therein). The first and only monographic revision of the fami- ly Microhylidae published over 85 years ago was largely based on osteological data (Parker 1934). In his review of Asian microhylid taxa, Parker only focused on the most zse.pensoft.net variable parts of the skeleton (such as the palatine region and pectoral girdle), but description of generalized osteolo- gy generally was not included (Parker 1934). In recent years skeletal morphology of only a few species in Microhylinae has been described in substantial detail, including the ge- nus Uperodon (Chandramouli and Dutta 2015; Garg et al. 2018), Kaloula borealis (Boring and Liu 1937; Zhang et al. 2020), and Glyphoglossus guttulatus (McPartlin 2010). The genus Microhyla Tschudi, 1838 currently com- prises 52 nominal species (Hoang et al. 2020; Poyarkov et al. 2020a, 2020b; Frost 2020) and several undescribed candidate species (Gorin et al. 2020). It is the second larg- est microhylid genus after Oreophryne (Frost 2020) and the most species species-rich taxon of the Asian subfam- ily Microhylinae. Over half of Microhyla species diver- sity was described within the last 15 years (29 species, see Frost 2020), but despite substantial progress in their taxonomy, this genus remains one of the most taxonom- ically challenging groups of Asian frogs. The small or medium-sized terrestrial frogs of the genus Microhyla are distributed all over the Oriental biogeographic region (Fig. 1) and exhibit significant variation in body size (adult body size varies from 10-46 mm) and ecomorphology (e.g. body shape, finger and toe disc expansion, and limb lengths) tied to their natural history (terrestrial, semi-ar- Zoosyst. Evol. 97 (1) 2021, 27-54 boreal, semi-fossorial). The smallest Microhyla species are amongst the smallest frogs in the world, approaching the lower body-size limit for the vertebrate bauplan (Das and Haas 2010; Kraus 2011). Phylogenetic analyses based on molecular data (Matsui et al. 2011; Garg et al. 2019; Gorin et al. 2020), provided novel insights into phylogeny of the genus and revealed significant inconsistencies with the traditional, morphology-based classifications (Park- er 1934; Dubois 1987; Fei et al. 2009). The preliminary mitochondrial DNA (mtDNA) based genealogies unex- pectedly suggested paraphyly of Microhyla with respect to the large-sized fossorial genus Glyphoglossus (Matsui et al. 2011; de Sa et al. 2012; Biju et al. 2019; Nguyen et al. 2019; Poyarkov et al. 2019). Additional multilocus phylogenetic (Garg and Biju 2019; Gorin et al. 2020) and phylogenomic (Tu et al. 2018; Peloso et al. 2016) studies supported monophyly of Microhyla, and agreed with one another in recovering the three main highly-divergent lin- eages within this group: the Glyphoglossus clade and two Microhyla clades (Microhyla 1 and Microhyla II hereafter, following Gorin et al. 2020). The three major clades of the Microhyla—Glyphoglossus assemblage were shown to have diversified in the middle Eocene (Garg and Biju 2019; Gorin et al. 2020), which makes the genus Micro- hyla, sensu lato (hereafter s. Jat.) older than other micro- hyline genera (Feng et al. 2017; Garg and Byu 2019). The lack of information on morphological variation among and within the lineages of the Microhyla—Glyphoglossus assemblage hinders further taxonomic assessment of di- versity within this group. Herein, we assess the status of the three lineages of the Microhyla—Glyphoglossus assemblage using an integra- tive taxonomic approach. We provide an updated mtD- NA-based genealogy including 57 species of the group. Based on traditional (cleared-and-stained specimens and external morphology) and digital (micro-Computed To- mography, or micro-CT) methods of comparative mor- phology we further report on osteological variation for 23 species of the genus Microhyla and three species of the genus Glyphoglossus, thus covering all major lineages for the first time. Based on analysis of morphological, oste- ological, molecular, and distribution data we recognize Glyphoglossus and Microhyla | sensu stricto (hereafter as s. str.) as valid genera. Additionally, we erect a new genus for Microhyla I, helping to stabilize the taxonomy of this clade. We further analyze miniaturization patterns, body size, and the evolution of sexual dimorphism in the Microhyla—Glyphoglossus assemblage. Material and methods Taxon sampling and examined specimens To assess the phylogenetic relationships within the Mi- crohyla—Glyphoglossus assemblage we used the mtD- NA and nuclear DNA (nuDNA) datasets from Gorin et al. (2020) with the addition of sequences of the recently 23 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 phyloge- ny (232 sequences, including 200 sequences of Microhy- la). 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, equita- bly selected (from preliminary analysis of mtDNA; not shown) to represent all major lineages within Microhyla, was used to estimate a robust, multilocus, time-calibrat- ed 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, spec- imen-associated locality data, museum voucher catalog numbers, and genetic data included in our study are pre- sented in Suppl. material 1: Table S1. Our osteological study was based on specimens housed in herpetological collections of the Zoological Muse- um 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 Cali- fornia Academy of Sciences (CAS, San Francisco, Cal- ifornia, USA). Altogether, for hand-preparation and his- tological 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 excep- tion of the MZ. 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\ 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. yunnan- ensis (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 Mor- phoSource database (www.morphosource.org) with per- mission. 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. Phylogenetic inference Nucleotide sequences were initially aligned in MAFFT v.6 (Katoh et al. 2002) with default parameters, and were zse.pensoft.net 24 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs 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 partition- ing schemes for our alignment were identified with Par- titionFinder 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; Tri- finopoulos et al. 2016). Clade stability was assessed by 1000 bootstrap (BS) replications and expected likelihood weights (ELW). One-thousand bootstrap pseudorepli- cates (ML BS) were employed, and nodes having ML BS values of 90 and above were considered strongly sup- ported, while nodes with values of 75-90 were regard- ed as significantly supported, lower values were consid- ered 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). Metropo- lis-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 MC- MCMC 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 support- ed, 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 ra- tio tests in PAML v4.7 (Yang 2007), which suggested the use of uncorrelated lognormal relaxed clock for our data- set. 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 dis- carded as burn-in. TreeAnnotator v1.8.0 (@¢n 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). Osteological preparation and double staining In order to observe both ossified and cartilaginous struc- tures, specimens were cleared and double stained with alcian blue for cartilage and alizarin red for bone. We zse.pensoft.net used the most delicate methodology of acid-free staining (following Walker and Kimmel 2006) to preserve min- ute skeletal elements of the smallest species. The proto- col included: (1) staining for about 24 hours in a solution of 0.05% alizarin red, 0.02% alcian blue, 45mM MgCl, 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% H,O, 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. Micro-CT scanning 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 Po- yarkov 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, Bel- gium) equipped with a Hamamatsu 10 Mp digital camera. Both specimens were mounted on a polystyrene baseplate and placed inside a hermetically sealed polyethylene ves- sel. Scans were conducted with a resolution of 3.7 um at 40 kV voltage and a current of 250 mA, with a rota- tion step of 0.3°. We used oversize mode, in which three blocks of sub-scan data were connected vertically, to ob- tain 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). Morphological descriptions and analyses Osteological terminology followed Trueb (1968, 1973), Scherz et al. (2017), Suwannapoom et al. (2018), and Poyarkov et al. (2014, 2018a). Terminologies used to de- scribe the shape of terminal phalanges (simple, knobbed, T-shaped, and Y-shaped) followed Parker (1927) and Garg et al. (2019). Comparative morphological and oste- ological 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 Poyar- kov et al. (2014, 2019); mensural data were taken with a Mitutoyo dial caliper (Mitutoyo Corporation, Kawasaki, Zoosyst. Evol. 97 (1) 2021, 27-54 Japan) to the nearest 0.1 mm. We recorded the follow- ing 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 FIT 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 com- piled data on maximum snout-vent length (SVL) sepa- rately for both sexes, for each species reported in literature and/or from our own measurements of voucher speci- mens following Gorin et al. (2020). Size (SVL) data for all Microhyla and Glyphoglossus species are summarized in Suppl. material 3: Table S3. Comparative morpholog- ical 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-bi- ased 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 gei- ger (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 dimor- phism was carried out using caper package (Orme et al. 2018) and plotted with ggplot2 (Wickham 2016). Species were binned into four size categories (terminology fol- lows Scherz et al. 2019) as follows: < 13 mm (state 1: “extremely miniaturized”); (2: 13-16 mm, “highly minia- turized’’); (3: 16—20 “miniaturized”’); (4: 20-24 “small’”). Results Phylogenetic relationships 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. gadjahma- dai, M. taraiensis, M. mixtura, M. fanjingshanensis, and 25 M. beilunensis, from clade II: M. perparva). We here re- port 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 rela- tionships for the Microhyla—Glyphoglossus assemblage (Fig. 2). All analyses concordantly resolved three strong- ly supported major clades within the group: Microhyla I, Microhyla I1, and Glyphoglossus, as indicated by Bayes- ian posterior probabilities of 1.0 and ML bootstrap node support of 100% (node support values are hereafter pro- vided as PP/BS); the majority of ingroup nodes also re- ceived 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 | + Glyphoglossus received no nodal support (-/70) (Fig. 2; Suppl. material 6: Figure SIA and Suppl. material 6: Figure S2). Phy- logenetic analyses of the nuDNA BDNF gene suggested monophyly of Mirohyla 1 + Microhyla II grouping with moderate to strong node support (0.90/97; Suppl. materi- al 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 1 + Microhyla II clade (0.46/90; Fig. 3; detailed in Suppl. material 6: FigureS1C). 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 data- set 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. The observed topological patterns within the Microhy- la-Glyphoglossus assemblage were congruent with earli- er results of Gorin et al. (2020) in recovering eight ma- jor 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 de- scribed 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, 1s widely dis- tributed from mainland southern China, Hainan and Tai- wan, 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, zse.pensoft.net 26 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs M. borneensis Microhyla sp. 1 M. malang: ieee é /M. achatina. a M. gadjahm M. irrawaddy M. mantheyi | M. minuta- . UATE Ees in og So Corry M. mymensinghensis M. chakrapanii M. mixtura M. beilunensis M. fanjingshanensis M. zeylanica 3% M. laterite . Sholigari 3 M. karunaratnei M. darreli M. eos Tea, “""200,,, 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 out- groups and node support values see Suppl. material 7: Figure S2. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 27-54 27 5 M. nepenthicola 8 M. borneensis i" Microhyla sp. 1 0.75/93 146 M. orientalis | 148 WM. orientalis 33 M, mantheyi. 62 M. gadjahmadai * 79 M. irrawaddy 193 icrohyia sp. 3 £ 63 WM oinaticola , pineti 0.83/95) 0.8602 | heymons! 0.53/64 A 5 M. mukhlesuri . 3 M. mukhlesuri 6 M. mukhlesuri 7 M. mukhlesuri Savina 32 co == 3 = > o 7) < =. = rOGN N=== 2h 77 2 S @ 77) a6 i. ERS Ne , mymensinghensis ween M. nymensiaghensis Mi Cr . chakrapanil Mncrohvese.? $= (Microhylas. : 143 M. okinavensis re M, okinavensis o SsS=S S —) — t<*) wn So 0.74/89 ; A berdmorei M. Cc yam 24 il . a5 M. berdmorei 1 0.93/93 185 M. taraiensis E 137 M. i pata os 151 M. ornata. mihintalei 91 M. zeylanica 7 M. laterite 180 M. sholigari 3 M. karunaratnei eli 3 M. superciliaris M. superciliaris = S = < M. 0.46/90 4 F 4 0.81/71 G 7 M. aurantiventris 152 M. palmipes 105 M. marmorata Z b 100 M. marmorata M f 7 iM, eS Icro ‘ , annectens 13 M. arboricola 16 M. arboricola Nanohyla Gen 168 M. pulchella . 78 M., hongiaoensis 132 M. nanapollexa 131 M. nanapollexa 204 Glyphoglossus guttulatus 23 Glypho lossus minutus . a ll 06 Glyphoglossus molossus G 207 Glyphoglossus yunnanensis 304 Giyp oglossus capsus d OUTGROUPS 0.2 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. zse.pensoft.net 28 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs Sumatra, Java and Bali in the south (Fig. 1). The Micro- hyla 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; 4 heymonsi Vogt, 1911; M. irrawaddy Po- yarkov, Gorin, Zaw, Kretova, Gogoleva, Pawangkhanant & Che, 2019; M. kodial Vineeth, Radhakrishna, Godwin, Anwesha, Rajashekhar & Aravind, 2018; M. malang Mat- sul, 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 can- didate 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. mukhlesu- ri Hasan, Islam, Kuramoto, Kurabayashi & Sumida, 2014; M. mymensinghensis Hasan, Islam, Kuramoto, Kuraba- yashi & 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, Am- arasinghe, Alhadi, Vineeth, Aravind, Senevirathne, Mee- gaskumbura & Biju, 2019; M. eos Byu, Garg, Kamei & Maheswaran, 2019; M. karunaratnei Fernando & Siriward- hane, 1996; M. Jaterite Seshadri, Singal, Priti, Ravikanth, Vidisha, Saurabh, Pratik & Gururaja, 2016; M. sholigari Dutta & Ray, 2000; M. superciliaris Parker, 1928: M. tetrix Suwannapoom, Pawangkhanant, Gorin, Juthong & Poyar- kov, 2020; and M. zeylanica Parker & Osman-Hill, 1949. The Microhyla ornata group (clade E), including M. mi- hintalei Wijayathilaka, Garg, Senevirathne, Karunarathna, Biu & Meegaskumbura, 2016; M. nilphamariensis How- lader, Nair, Gopalan & Merila, 2015; MZ ornata (Dumeril & Bibron, 1841); M. rubra (Jerdon, 1854); and M. taraien- sis 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 M- crohyla II clade is restricted to the montane forest areas in the Annamite (Truong Son) Mountains in East Indochina (Vietnam, eastern Laos, northeastern Cambodia), Malay- an Peninsula (Tittwangsa Mountain Range), mountains of Borneo (Sarawak, Sabah of Malaysia, Brunei and north- ern Kalimantan, Indonesia), and the southwestern-most islands of the Sulu Archipelago of the Philippines (Fig. 1). zse.pensoft.net 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; . 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) cov- ers the whole Thai-Malaysian Peninsula, parts of Indochi- na, 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, Yaa- kob & Lim, 2004); G. molossus Gunther, 1869; and G. yunnanensis (Boulenger, 1919). Divergence times 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. mate- rial 8: Figure S3. The results of the divergence time es- timation fully agree with Gorin et al. (2020), suggesting that the most recent common ancestor (MRCA) of Mi- crohyla 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 $3). This es- timate 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 1, Microhyla I, and Glyphoglossus clades initiated much later in the early to middle Oligocene (ca. 35—25 Ma, Gorin et al. 2020). Comparative osteology 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 Mi- crohyla—Glyphoglossus assemblage. Detailed informa- tion 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 mor- phology for cleared and stained representatives of these three clades is provided in Suppl. material 8, 9. Below, we provide comparative osteological descrip- tions for the three clades of the Microhyla—Glyphoglossus assemblage: Microhyla1, Microhyla II, and Glyphoglossus. Zoosyst. Evol. 97 (1) 2021, 27-54 29 as Figure 4. General osteology of the Microhyla—Glyphoglossus assemblage representatives. The full skeletons are shown for Glyphoglos- sus molossus (A — dorsal, B— ventral views), G/yphoglossus yunnanensis (C — dorsal, D— ventral views), Microhyla achatina (E — dor- sal, F — ventral views), Microhyla nepenthicola (G — dorsal, H — ventral views), and Nanohyla arboricola (1— 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. (A) Microhyla I clade This clade includes M. achatina, the type species of the genus Microhyla, and is the most widely distributed, species rich, and ecologically and morphologically di- verse 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 (MZ. 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. ma- terial 5: Table S5). Due to marked morphological varia- tion, providing a comprehensive morphological diagnosis of this speciose group remains a challenging task; below we summarize available information on skeletal traits. Skull Skull longer than wide, wider than long, or almost in equal proportions among species of Microhyla | (Fig. 5; Suppl. material 5: Table S5). Widest part of skull locat- ed posteriorly and giving head a triangular or trapezoid shape (Fig. 5). Frontoparietals longer than broad, nar- rowing anteriorly, in contact along medial border but not fused, lacking any dorsal crests; partially fused with or separated from exoccipital posteriorly and prootic pos- terolaterally. Exoccipitals always separate, in contact me- dially. Nasals large and widely separated, chondrified pe- ripherally; processus paraorbitalis broad, in some species blunt, posterior edge concave, anterior edge convex (Fig. 5). Sphenethmoid well-ossified, always clearly separat- ed from parasphenoid, with a concave posterior edge. Prootics ossified anteromedially; crista parotica carti- laginous 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 an- nulus 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. Quadrato- jugal 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 medi- al ramus. Vomers small, widely separated, triangular in shape. Neopalatines present or absent. Nasal capsules mineralized posteriorly or entirely cartilaginous. Men- tomeckelians ossified, connected to dentaries and to each other through Meckel’s cartilage. Dentary fused with an- gulosplenial. 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) pro- cesses of hyoid plate present, recurved, posterolateral pro- cesses slender, posteromedial processes strongly ossified, zse.pensoft.net 30 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs elongated, straight, wider at proximal ends, chondrified at distal ends, separated by a chondrified parahyoid (Suppl. material 10: Figure SSM). Vertebral column Vertebral column is diplasiocoelus, typically comprising eight presacral vertebrae (PSV) (Fig. 6C), with the ex- ception of extremely miniaturized species M. nepenthic- ola, which has PSV I and II fused (Fig. 6D). PSV H-VII procoelous and VIII amphicoelous. Transverse processes of PSV I-IV longer and wider than V—VII, transverse processes of PSV VI-—VIII oriented anterolaterally; ori- entation of transverse processes to other vertebrae varies (Suppl. material 5: Table S5). Transverse processes of sa- crum moderately expanded, with distal end about twice as wide as proximal end. Urostyle shorter than trunk ver- tebrae, bearing a weak dorsal crest that tapers posteriorly and vanishes at two-thirds of urostyle length (Fig. 6); its articulation with sacrum is bicondylar. Appendicular skeleton Pectoral girdle with a firmisternal arrangement. Cora- coids, scapulae, and suprascapulae present; first two ful- ly ossified; suprascapula largely chondrified. Coracoids robust with wide proximal end. Omosternum generally absent, except for MZ puchra, where a tiny cartilaginous omosternum is present (Suppl. material 5: Table S5). Pro- coracoids indistinct. Clavicles absent. Cartilaginous ster- num large, partially mineralized, fan-shaped or bifurcate (Suppl. material 10: Figure SSD, E); xiphisternum com- pletely cartilaginous. Hand skeleton including seven largely calcified carpal elements: carpale distale II, carpale distale IN—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 for- mula: 2-2-3-3; all phalanges ossified; distal phalanx of finger HI simple, conical-, bobbin- or T-shaped. Foot skel- eton with four tarsal elements, including ossified tarsale distale II-III, centrale and a prehallux; prehallux miner- alized 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 If T-shaped or simple. (B) Microhyla II clade 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 terrestri- al 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 zse.pensoft.net 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 anterior- ly, in contact along medial border and fused posteriorly, lacking any dorsal crests; posteriorly fused with exoccip- itals. Exoccipitals completely fused with each other (Fig. 5), except M. pulchella, (partial; Suppl. material 5: Ta- ble S5). Nasals large, broadly separated, chondrified pe- ripherally, processus paraorbitalis narrow and cultriform, posterior edge concave, anterior edge oblique (Fig. 5). Spenethmoids ossified, completely fused with parasphe- noid (in M. pulchella an indistinct suture remains later- ally, so the fusion is partial), extending posteroventrally nearly to level of prootics along parasphenoid. Prootics ossified anteromedially, crista parotica entirely cartila- gionous. Squamosal ossified, with well-developed long ventral and otic rami and poorly developed zygomat- ic ramus (Fig. 50; Suppl. material 5: Table S5). Oper- culum mineralized. Columella largely mineralized with only pars externa plectra cartilaginous, tympanic annulus completely chondrified (Suppl. material 10: Figure SSG). Premaxilla well-ossified, alary process oriented slightly anteriorly, distal part bending laterally. Maxilla well-ossi- fied; anteriorly in contact with labial portion of premaxil- la; edentate; pars facialis moderately high in lateral view. Quadratojugal reduced further than Microhyla | 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 | clade), broad posterior ramus, and short medial ramus. Vomers small, widely separated, triangular in shape. Neopalatines pres- ent as very thin elements. Mentomeckelians ossified, con- nected to dentaries and to each other through Meckel’s cartilage. Dentary fused with angulosplenial. Parasphe- noid 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 ossi- fied, 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 VHI amphicoelous. Transverse processes of PSV II-IV longer and wider than in PSV V—VIII; transverse processes of PSV II, VII and Zoosyst. Evol. 97 (1) 2021, 27-54 ol Figure 5. Cranial osteology of the Microhyla—Glyphoglossus assemblage representatives. The skulls are shown in dorsal / ventral / lateral views for G/yphoglossus 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, respec- tively). Note: figures display only calcified structures; cartilages are omitted due to limitations of micro-CT scanning. Scale bar equals 3 mm. zse.pensoft.net a2 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs sacrum sacrum sacrum sacrum sacrum urostyle urostyle urostyle urostyle urostyle 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 nepen- thicola (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. Table 1. Summary of osteological differences between Glyphoglossus, Microhyla s. str. and Nanohyla gen. nov. Diagnostic features of the new genus that are subjectively considered by us to be most reliable are highlighted in bold. Asterisk (*) denotes states ob- served in G. molossus exclusively. For species-specific data, see Suppl. material 5: Table SS. Character Glyphoglossus Nanohyla gen. nov. Skull shape Wider than long Subequal, longer than wide, or Subequal or longer than wide wider than long Frontoparietal—exoccipital junction Separated Separated Fused Exoccipitals Separated Separated Fused or incompletely fused Nasal capsules Ossified Ossified, partly mineralized, or Ossified, partly mineralized, or cartilaginous cartilaginous Neopalatines Obscured Present or absent Present Maxillary teeth Present or absent* Absent Absent Vomers Large Small Vomerine teeth Present or absent* Absent Absent Anterior ramus of pterygoid Thin and blunt or massive and Thin, blunt Thin, tapered blunt* Sphenethmoid and parasphenoid Separated Separated Fused or incompletely fused Otic ramus of squamosal Long Short Long Tympanic annulus Present Present or reduced Present Columella Fully ossified Poorly mineralised Poorly mineralised Crista parotica Fully ossified Posteriorly ossified Cartilaginous Clavicles Present or absent* Absent Absent Omosternum Absent Present Prehallux Ossified Mineralized Cartilaginous Terminal phalanges of finger III Simple T-shaped, knobbed, or simple T-shaped Distance between vomers Narrow Wide Wide VII oriented anterolaterally, [V and V posterolaterally, III and VI perpendicular to vertebral column axis, with ex- ception 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 posteri- orly; vanishes completely at 2/3 urostyle length (Fig. 6E). zse.pensoft.net Appendicular skeleton Pectoral girdle firmisternal. Coracoids, scapulae, and su- prascapulae present; coracoid and scapula fully ossified; suprascapula largely chondrified. Coracoids robust with wide proximal end. Cartilaginous omosternum present (Suppl. material 10: Figure SSF). Procoracoids indistinct. Clavicles absent. Sternum large, cartilaginous, partially Zoosyst. Evol. 97 (1) 2021, 27-54 33 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 scan- ning. Scale bar equals 1 mm. mineralized, bifurcate or fan-shaped; xiphisternum com- pletely cartilaginous. Manus skeleton with seven largely calcified carpal el- ements, including carpale distale I, carpale distale HI—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 fin- ger 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 ossi- fied, elongated and much more massive than metacarpals; phalangeal formula: 2-2-3-4-3; all phalanges ossified. Terminal phalanges of toe HI T-shaped. (C) Glyphoglossus clade In our analysis, three species of G/yphoglossus (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 G/yphoglossus inhabit lowland ar- eas of southern mainland China, Indochina, and Sunda- land (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 Calluel- la Stoliczka, 1872, now considered a junior synonym of Glyphoglossus based on its phylogenetic placement; Peloso et al. 2016). Owing to the morphological unique- ness of G. molossus, morphological features of this spe- cies 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 §anteri- orly, connecting medially with a suture along whole length, or anteriorly, separated or fused* (Fig. 5A) me- dially, lacking dorsal crests, separated or fused* with exoccipitals (separate) posteriorly. Nasals large, sepa- rated, chondrified peripherally; processus paraorbitalis well-developed, pointed laterally or anteriorly* (Fig. 5). Spenethmoid separate, well ossified, restricted to ante- rior 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 por- tion of premaxilla; teeth present or absent*; pars facialis moderately to notably high, and oriented towards proces- sus paraorbitalis of nasal*. Quadratojugal robust, with rounded cartilaginous articulation with angulosplenial, anteriorly articulating with or fused* to maxilla. Ptery- goid massive, with a long anterior ramus, broad posterior ramus, and short medial ramus. Vomers large, shape ei- ther complex or U-shaped*, defining lower floor of nasal capsule. Neopalatines obscured by postchoanal vomerine zse.pensoft.net 34 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs processes, fused or replaced completely*. Nasal capsules mineralized posteriorly or obscured by postchoanal vom- erine processes*. Mentomeckelians ossified, connected to dentaries, and to each other through Meckel’s carti- lage. 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 an- gulosplenial. 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 antero- lateral processes well-developed, recurved, posterolateral processes slender, posteromedial processes strongly ossi- fied, 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 ver- tebrae. 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 H-— IV longer and wider than V—VIII, transverse processes of PSV II, VII and VIII oriented anterolaterally, IV and V posterolaterally, HI and VI at right angle to vertebral column axis. In G. molossus transverse processes of PSV greatly shortened, I] 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 su- prascapulae present; first two fully ossified; suprascapu- lae largely chondrified. Coracoids robust, with proximal end, or both ends widened*. Omosternum absent. Pro- coracoids present or absent*. Clavicles present or ab- sent*. Cartilaginous sternum large, partially mineralized, fan-shaped; xiphisternum completely cartilaginous. Hand skeleton with six largely calcified carpal el- ements: carpale distale II, carpale distale HI—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 HI simple. Foot with four tarsal elements, including ossified tarsale distale H— II, centrale, and prehallux; prehallux enlarged and ossi- fied (Suppl. material 10: Figure SSC). Metatarsals fully zse.pensoft.net ossified, long, more massive than metacarpals; phalan- geal 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 1n 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 4 = 1.00). Sexual size dimorphism exhibits no phylogenetic sig- nal (Pagel’s 1 = 7.2 x 10°), changing sporadically across the tree, and 1s weakly positively correlated with log(male SVL) (PGLS, F, ,, = 5.478, adjusted R* = 0.07928, P = 0.02321; Fig. 9B). Most species of Microhyla J and II ex- hibit 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. Discussion A fully resolved taxonomic framework should approxi- mate the phylogenetic relationships of its members, allow- ing the user to roughly infer basic information from the framework itself (Wake 2013). This information includes monophyly of the recognized taxonomic groupings, and their differences in sets of biologically significant traits. A taxonomic framework that allows such information to be accurately inferred maximizes its utility. Additionally, the taxonomic framework should, ideally, be optimized for stability, reducing the need for additional taxonom- ic changes in future (Vences et al. 2013). All recent Zoosyst. Evol. 97 (1) 2021, 27-54 log(male SVL) SSS 2.5 4.6 (Microhyla l) ay Microhyla s. str. Dames J, 3D dimorphism (male SVL/female SVL) 0.7 1.2 Microhyla borneensis Microhyla sp. 1 41) Microhyla nepenthicola 2) Microhyla malang N Microhyla mantheyi ) Microhyla orientalis Microhyla minuta Microhyla sp. 3 Microhyla koadial Microhyla gadjahmadai Ancestral male SVL O <20mm Microhyla achatina (= ) Microhyla pineticola Microhyla heymonsi Microhyla fodiens Microhyla chakrapanii © O <16mm Microhyla mymensinghensis Microhyla mukhlesuri Microhyla fissipes Microhyla mixtura Microhyla sp. 2 Microhyla beilunensis Microhyla okinavensis Microhyla picta Microhyla pulchra Microhyla berdmorei @) Microhyla nilphamariensis Microhyla taraiensis Microhyla ornata Microhyla mihintalei Microhyla rubra Microhyla zeylanica = Microhyla laterite ay Microhyla sholigari “) Microhyla karunaratnei Microhyla superciliaris Microhyla tetrix Microhyla butleri Microhyla aurantiventris =) Microhyla palmipes (Microhyla ll) 4 = Nanohyla petrigena ) Nanohyla perparva Nanohyla 4 » Gen. nov. a Nanohyla annectens Nanohyla annamensis Nanohyla marmorata i) 4 Nanohyla pulchella ay M Nanohyla hongiaoensis ay Nanohyla arboricola Nanohyla nanapollexa Glyphoglossus guttulatus Glyphoglossus minutus Glyphoglossus molossus Glyphoglossus yunnanensis Glyphoglossus capsus Kaloula baleata 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. phylogenetic studies of the subfamily Microhylinae agree that (1) Glyphoglossus and Microhyla s. lat. are closely related, and (11) Microhyla s. lat. consists of two deep- ly-divergent lineages (Microhyla I and I of Gorin et al. [2020]). The relationship between these two Microhyla clades and Glyphoglossus evidently cannot be resolved with mitochondrial DNA alone (e.g., Matsui et al. 2011; Poyarkov et al. 2018b, 2019; Nguyen et al. 2019; Li et al. 2019; Gorin et al. 2020), likely due to a combination of the considerable age of these splits (>40 Ma), resulting in saturation and loss of phylogenetic signal, and the moder- ately rapid succession in which they apparently occurred. Nuclear data, especially multilocus datasets, do, however, support the monophyly of Microhyla s. lat. (Peloso et al. 2016; Tu et al. 2018; Garg and Biju 2019; Gorin et al. 2020; and the present paper). However, as will become evident in the following, we find there to be substantial evidence supporting the treatment of the two major clades within Microhyla s. lat. as separate genera. Although present evidence indicates that we can be moderately confident in the respective monophyly of Mi- crohyla s. lat. and Glyphoglossus, it is also worth noting that the two lineages within Microhyla s. lat. are very old. The Microhyla—Glyphoglossus assemblage radiated within a narrow period in the middle Eocene, with the origin of Glyphoglossus dating to 44.1 Ma (38.5-49.6), while the basal split within Microhyla s. lat. is estimat- ed at 43.9 Ma (37.8-48.2) (Suppl. material 4: Table S4, Suppl. material 8: Figure S3). These two estimates are very close and their 95% credibility intervals overlap, suggesting near-simultaneous origin of Glyphoglossus, Microhyla I, and Microhyla II. These estimates are no- tably older than the ages of all other microhyline genera (except Chaperina), which may have diverged in the late Eocene (the split between Micryletta and Mysticellus [40.9 Ma, 33.3—47.7]) or Oligocene (the split between Kaloula and Uperodon 27.4 Ma [19.4—34.9], and the split between Phrynella and Metaphrynella is estimat- ed at 23.0 Ma [16.2—29.1]; Suppl. material 4: Table S4, Suppl. material 8: Figure S3). Similar results were also reported by Garg and Biju (2019), who provided even older estimates for all microhyline genera. Thus, the two clades within Microhyla s. lat. are of equal or greater age than other genera in this subfamily. While age has not historically been taken into account in most higher taxonomy, it is nonetheless desirable for taxa of equal rank to be of generally comparable age (Hennig 1966; Vences et al. 2013). In addition to their substantial age, we have identi- fied a number of important osteological and external zse.pensoft.net 36 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs 4.5 A 4.0 O ®@ ey > 7) 2 = 3.5 Lo & 7) — ; hod A A A a” 3.0 “e AD @ Glyphoglossus A Eo A A\ Microhyla s. str. oN || i Nanohyla Gen. nov. Oo 2.5 A A 2.5 3.0 3.5 4.0 4.5 log(male SVL) 1.2 o = = © oO = dimorphism (male SVL/female SVL) ° ro) 0.7 2.5 3.0 @ Glyphoglossus A Microhyla s. str. i Nanohyla Gen. nov. -- — PGLS: y ~ 0.17x + 0.36, R? = 0.08, P = 0.02321 — GLS: y ~0.10x + 0.59, R? = 0.10, P = 0.01243 3.5 4.0 4.5 log(male SVL) Figure 9. Relationships between body size among sexes (A), and between male body size and sexual size dimorphism (B) in Mi- crohyla s. str., Nanohyla gen. nov., and Glyphoglossus. The line in (A) represents x=y. morphological differences that distinguish the three clades within this assemblage, including the two clades within Microhyla s. lat. These include body size and shape, num- ber and shape of metatarsal tubercles, adaptation to bur- rowing lifestyle, extent of toe webbing, relative size of the first finger (FI) (Fig. 10), and the presence of an external tympanum (Fig. 11). The absence of an externally visible tympanum traditionally was regarded as one of the key diagnostic characters of the genus Microhyla (Boulenger, 1882; Parker 1934; Garg et al. 2019). In all species of Mi- crohyla I, the tympanum is hidden under the skin of the supratympanic fold. However, a closer examination of all species of Microhyla II demonstrates that six (of nine) taxa actually have an external tympanum that is discernable in breeding males (Fig. 11). The presence of an externally vis- ible tympanum in the majority of the Microhyla II species zse.pensoft.net suggests it may be an important character for diagnosing this clade from Microhyla I (Suppl. material 5: Table S5). Furthermore, there are pronounced differences in the pat- terns of geographical distribution among the three clades of the Microhyla—Glyphoglossus assemblage (Fig. 1) which, along with their ecological differences, suggest that they may warrant recognition as separate genera of Micro- hylinae. The available hypothesis of the biogeographic his- tory of this assemblage (Gorin et al. 2020) demonstrated that the group originated in Southeast Asia. The smaller members of Microhyla II clade are closely associated with perhumid montane forests, and their distribution is limited by mountain ridges among Borneo, the Thai-Malay Penin- sula and Indochina (Fig. 1). At the same time, large-sized burrowing species of Giyphoglossus can aestivate during the dry season, and have become more widely distributed Zoosyst. Evol. 97 (1) 2021, 27-54 37 Figure 10. Palmar views of hands (above) and thenar views of feet (below) of the representative Microhyla s. str. and Nanohyla gen. nov. species: NV. annamensis (A, B), N. arboricola (C, D), M. minuta (KE, F), and M. tetrix (G, H). Arrow indicates outer metatarsal tubercle. Not to scale. Line drawings by Valentina D. Kretova. across Southeast Asia and seasonally dry plains of Central Indochina and Myanmar (Fig. 1; Gorin et al. 2020). Micro- hyla J is the most diverse clade in terms of morphological and ecological adaptations, and species of this group have colonized almost the entire Asian Realm, including south- ern and eastern China (Fig. 1). The cumulative evidence suggests to us that continuing to recognize the superficially similar Microhyla I and II clades as members of a single genus would conceal infor- mation on the ancient divergence between these lineages, as well as the differences between them in a number of bi- ologically relevant organismal traits. Put another way, rec- ognizing the two clades as separate genera would enhance the diagnosability of the respective genera, make them more comparable units to other genera, and fully stabilize the taxonomy of the Microhyla—Glyphoglossus assemblage (if coalescent phylogenomic reconstructions were to reveal the clades to be paraphyletic with respect to Glyphoglossus, no taxonomic changes would be necessary). Splitting them would therefore be in accordance with all three of the Prior- ity Taxon Naming Criteria (TNCs) of Vences et al. (2013): Monophyly, Clade Stability, and Diagnosability, as well as the secondary TNCs Time Banding and Biogeography. We also contend that this solution is superior to the obvious al- ternatives, which are (1) sinking all three clades into a single genus, or (11) recognizing the two clades within Microhyla Ss. lat. as subgenera. The former would maximize mono- phyly and clade stability, but would seriously compromise the diagnosability of the genus, whereas the latter would continue to satisfy the three priority TNCs but would not optimize under the Time Banding TNC. In the following, we therefore divide Microhyla s. lat. (hitherto containing 52 species), into two genera consisting of 43 (Microhyla 1) and nine (Microhyla II) species each. As there are no available zse.pensoft.net 38 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs names for Microhyla II, we formally describe it as a new ge- nus, and provide revised taxonomic accounts of Microhyla s. lat. and Glyphoglossus. Taxonomic accounts Nanohyla Poyarkov, Gorin & Scherz, gen. nov. http://zoobank.org/0624CCB0-DC63-40F9-A7B7-7F8627B491 BB Figs 10, 11; Suppl. material 5: Table S5 Chresonymy. Microhyla (partim)—Boulenger 1900; Smith 1923; Inger and Frogner 1979; Inger 1989; Bain and Nguyen 2004; Poyarkov et al. 2014; Hoang et al. 2020. Microhyla (Microhyla) (partim)—Dubois 1987 (as a part of the subgenus Microhyla). Type species. Microhyla annectens Boulenger, 1900. Etymology. The genus name is derived from the Greek vavoc (nanos), meaning “dwarf”, “pygmy”, and the myth- ological figure, Hylas (Ancient Greek: "YAac), which 1s probably derived from the Ancient Greek verb “diaw” meaning “to bark” (Bourret 1942). In classical mythology, Hylas, son of King Theiodamas, was a youth who served as Heracles’ companion, lover, and servant. Heracles took Hylas with him on the Argonauts’ expedition, during which Hylas was kidnapped by nymphs of the spring in Pegae, Mysia, and turned into an echo. Heracles left the ship and was searching for Hylas for a great length of time, calling his name: “His adjunxit Hylan nautae quo fonte relictum / Clamassent ut littus Hyla! Hyla! omne sonaret’ (“The mariners cried on Hylas till the shore / Then Re-echoed Hylas! Hylas! soothed...”; Virgil 1916, Ecl. 6, 43). The ge- nus name refers to the small body size (< 25 mm) of all known Nanohyla species, while maintaining resemblance to its sister genus Microhyla, from which it is separated herein. The new genus name is feminine in gender. Suggested common name. Pygmy Narrow-mouthed Frogs. Taxonomic content. Nine species, including: Nanohyla annamensis comb. nov. (Smith, 1923); Nanohyla annect- ens comb. nov. (Boulenger, 1900); Nanohyla arboricola comb. nov. (Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014); Nanohyla hongiaoensis comb. nov. (Hoang, Nguyen, Luong, Nguyen, Orlov, Chen, Wang & Jiang, 2020); Nanohyla marmorata comb. nov. (Bain & Nguyen, 2004); Nanohyla nanapollexa comb. nov. (Bain & Nguyen, 2004); Nanohyla petrigena comb. nov. (Inger & Frogner, 1979); Nanohyla perparva comb. nov. (Inger & Frogner, 1979); and Nanohyla pul- chella comb. nov. (Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014). Photos of Nanohyla gen. nov. members are presented in Fig. 11. Diagnosis. The new genus 1s assigned to the subfamily Microhylinae on the basis of phylogenetic affinities and zse.pensoft.net the following combination of morphological character states: vomers small, confined to the anterior and medial margins of choanae; clavicles and, in most cases, procora- coids absent, maxillary arcade edentate (Parker 1934). Nanohyla gen. nov. differs from other Microhylinae gen- era by the following combination of osteological char- acter states: (1) frontoparietals fused with exoccipitals; (2) exoccipitals fused with each other (incomplete fusion in N. pulchella), (3) neopalatines present; (4) spheneth- moids completely fused with parasphenoid (incomplete fusion in N. pulchella); (5) crista parotica entirely carti- laginous; (6) otic ramus of squamosal well-developed; (7) tympanic annulus well-developed; (8) transverse process- es of presacral vertebrae with the following orientation: IV and V posterolaterally, II, VI and VII anterolaterally, III and VI at right angle to body axis; (9) clavicles ab- sent; (10) omosternum present, cartilaginous; (11) pre- hallux cartilaginous; (12) terminal phalanges of longest fingers and toes T-shaped. The combination of diagnostic external morphological characters includes: (13) small to extremely small frogs (adult SVL 11.8—25.8 mm); (14) snout rounded or pointed in profile; (15) supratympanic fold present; (16) ridge on posterior sides of choanae ab- sent; (17) first finger (FI) length less than 2 FI or reduced to a nub; (18) finger discs present, at least on FI-—FIV; (19) dorsal median longitudinal grooves on finger discs generally present (with the exception of N. perparva); (20) toes dorsolaterally flattened, prominent discs pres- ent; (21) dorsal median longitudinal grooves on toe discs present; (22) metatarsal tubercle single (inner metatarsal tubercle present, outer absent); (23) dorsomedial line ab- sent; (24) superciliary tubercles absent; (25) tibiotarsal articulation of adpressed hindlimb reaching well beyond snout; (26) toe webbing well-developed (at least one-half webbed); (27) skin on dorsum feebly granular to tuber- cular; (28) tympanum externally distinct at least in males (N. annamensis, N. annectens, N. arboricola, N. marmo- rata, N. nanapollexa, N. pulchella) or barely distinct (N. hongiaoensis, N. perparva, N. petrigena), (29) terrestrial or scansorial semi-arboreal microhabitat preference. Phylogenetic definition. The genus Nanohyla gen. nov. includes all species sharing a more recent common ances- tor with Nanohyla annectens than with Microhyla achati- na and Glyphoglossus molossus. Distribution. The distribution area of Nanohyla gen. nov. covers montane forests of the Annamite (Truong Son) Mountains in Vietnam, eastern Laos, and north-eastern Cambodia, the Titiwangsa Mountain Range in the south- ernmost Thailand and peninsular Malaysia, mountains of Borneo (including Sabah and Sarawak of Malaysia, Brunei, and Kalimantan of Indonesia) and the Sulu Ar- chipelago of the Philippines (see Fig. 1). The occurrence of Nanohyla gen. nov. in Cardamom Mountains in east- ern Thailand (the record of “M. annamensis” from Khao Sebab by Taylor [1962], see Fig. 1) 1s questionable (see Poyarkov et al. 2014, 2020a). Zoosyst. Evol. 97 (1) 2021, 27-54 é \ leis, N. petrigena | N.-.pulchelle Figure 11. Members of the new genus Nanohyla gen. nov. in life (males): N. annectens from Genting Highlands, Pahang, Malaysia (A), NV. annamensis from Bidoup — Nui Ba N.P., Lam Dong, Vietnam (B), NV. arboricola from Chu Yang Sin N.P., Dak Lak, Viet- nam (C), N. hongiaoensis from Bidoup — Nui Ba N.P., Lam Dong, Vietnam (D), NV. marmorata from Kon Chu Rang N.R., Gia Lai, Vietnam (E), N. nanapollexa from Kon Plong, Kon Tum, Vietnam (F), NV. perparva from Gunung Mulu, Sarawak, Malaysia (G), N. petrigena from Gunung Mulu, Sarawak, Malaysia (H), and N. pulchella from Bidoup — Nui Ba N.P., Lam Dong, Vietnam (1). Insets show tympanic area of the each species; white arrow points at the tympanic rim of the external tympanum. Photos by Nikolay A. Poyarkov (A-C, D-F, I), Vu Dang Hoang Nguyen (D), and Indraneil Das (G, H). Morphological comparison. The new genus Nanohyla gen. nov. differs from its sister genus Microhyla Tschudi, 1838 s. str. by the well-developed (vs poorly-developed) otic ramus of the squamosal, frontoparietals and exoccip- itals fused (vs separated or slightly fused), exoccipitals fused with each other (vs always separated), omosternum present (vs usually absent), sphenethmoid and parasphe- noid fused completely or partially (vs separated), cartilag- inous crista parotica (vs mineralized posteriorly), cartilag- inous prehallux (vs mineralized), tympanum externally visible or barely visible (vs concealed beneath skin), inner metatarsal tubercle well-developed, outer generally absent (vs two metatarsal tubercles well-developed), and in having digits dorso ventrally flattened, FI often reduced to a nub or shortened (vs variably longer). The new genus differs from the closely related genus G/yphoglossus Gunther, 1869 by its smaller adult size with SVL < 25mm (vs SVL > 25mm), skull longer than wide or almost equal (vs wider than long), alary process of premaxilla oriented slightly anteriorly (vs posteriorly), neopalatines present (vs obscured by vomers), vomers small, indistinct (vs large, well-developed), omo- sternum present (vs absent), terminal phalanges T-shaped (vs simple), tibio-tarsal articulation reaching well beyond snout (vs to the anterior border of the eye, or less), by body habitus short, triangular-shaped (vs stout, balloon-shaped), and by inner metatarsal tubercle not enlarged (vs enlarged, shovel-shaped). Nanohyla gen. nov. differs from Kaloula Gray, 1831 by its much smaller adult body size SVL < 25 mm (vs SVL > 38 mm), procoracoids absent (vs present), postchoanal portion of vomer absent (vs present), neopal- atines present (vs obscured), prehallux formed by two el- ements (vs one), tibio-tarsal articulation reaching well be- yond snout (vs to shoulder), absence (vs presence) of ridge on posterior margin of choanae, inner metatarsal tubercle not enlarged (vs enlarged and spatulate), and by body habi- tus short, triangular-shaped (vs robust). The new genus can be distinguished from Uperodon Dumeril & Bibron, 1841 by its smaller adult size, SVL < 25 mm (vs SVL > 34 mm), postchoanal portion of vomer absent (vs present), neopal- atines present (vs obscured), tibio-tarsal articulation reach- ing well beyond snout (vs posterior border of eye, or less), absence (vs presence) of ridge on posterior margins of cho- anae, inner metatarsal tubercle not enlarged (vs enlarged or spatulate), and by body habitus short, triangular-shaped (vs robust and globular). Nanohyla gen. nov. differs from Phrynella Boulenger, 1887 by its smaller adult size, SVL < zse.pensoft.net 40 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs 25 mm (vs SVL > 30 mm), medial process of the precho- anal part of vomer absent (vs present), neopalatines present (vs absent), procoracoids absent (vs present), vertebral col- umn diplasiocoelus (vs procoelus), metatarsal tubercules separate (vs united), by tibio-tarsal articulation reaching well beyond snout (vs to tympanic region), by body habitus short, triangular-shaped (vs robust and flattened), and by generally dull brownish coloration of inguinal and dorsal surfaces (vs greenish coloration of dorsum and bright-red coloration of inguinal area, and ventral surfaces of limbs). The new genus further differs from Metaphrynella Parker, 1934 by its smaller adult size, SVL < 25 mm (vs SVL > 25 mm), skull longer than wide or almost equal (vs wider than long), neopalatines present (vs absent), omosternum pres- ent (vs absent), vertebral column diplasiocoelus (vs pro- coelus), tibio-tarsal articulation reaching well beyond snout (vs to tympanic region), absence (vs presence) of a ridge on posterior margins of choanae, metatarsal tubercules sepa- rate (vs united and enlarged), and by finger webbing absent (vs present). The new genus differs from Mysticellus Garg & Biju, 2019 by its short triangular-shaped body habitus (vs slender), supratympanic fold present (vs absent), fin- ger and toe tips enlarged with prominent discs (vs slight- ly enlarged), toe webbing well-developed (vs rudimenta- ry), supernumerary carpal tubercles absent (vs prominent subarticular tubercles alternating with additional smaller tubercles), and the two prominent blackish-brown ‘false- eye’ inguinal spots absent (vs present). Nanohyla gen. nov. differs from Micryletta Dubois, 1987 by its snout longer than eye diameter, and having eye less (vs more) prominent in lateral and dorsal aspects, finger and toe tips enlarged with prominent discs (vs slightly enlarged), toe webbing well-developed (vs rudimentary or absent), supernumerary carpal tubercles absent (vs present), omosternum present (vs absent), neopalatines present (vs absent), tibio-tarsal ar- ticulation reaching well beyond snout (vs to anterior border eye, or less), supratympanic fold present (vs absent), and body habitus short, triangular-shaped (vs slender). Finally, the new genus is distinguished from Chaperina Mocquard, 1892 by clavicles and procoracoids absent (vs present), postchoanal portion of vomer absent (vs present), omoster- num present (vs absent), terminal phalanges T-shaped (vs simple), tibiotarsal articulation reaching well beyond snout (vs anterior border of eye), belly dull-colored (vs bright saffron-yellow belly with dark pattern), and by absence of spine-like projections on limbs (vs a long, narrow dermal spine projecting from calcaneus). Larval morphology. Description of the larval stages of the Nanohyla gen. nov. members are sparse and often not detailed. Poyarkov et al. (2014) provided descrip- tions, photos and illustrations of tadpole morphology for N. annamensis, N. arboricola and N. pulchella. Vassilieva et al. (2017) provided a detailed description of develop- ment, larval morphology and anatomy for N. arboricola. Le et al. (2016) provided a brief description of tadpole morphology of N. marmorata. Leong (2004) provided a short description and photographs of larval and meta- zse.pensoft.net morph morphology for N. annectens. Brief descriptions and figures depicting larvae of N. petrigena and N. per- parva are found in the original description of these spe- cies by Inger and Frogner (1979), as well as in Inger and Steubing (2005) and Haas et al. (2020). Larval stages of N. hongiaoensis and N. nanapollexa remain unknown. As with almost all larvae in Microhylidae, labial teeth and mandibles are absent from the oral discs of Nanohyla tadpoles. Most species of Nanohyla have larval morphol- ogy resembling that of many pond-breeding Microhyla species (Poyarkov et al. 2014) with rather short-tailed transparent or semi-transparent Orton’s type II tadpoles (Orton 1953), that are mid-water column (neustonic) feeders with comparatively unexpanded lower labium and anteriorly directed terminal mouths, lateral orientation of eyes, spiraculum located in a medial position on the ven- ter, spiracular flap with crenulate margins, and tail lacking terminal filament (Altig and Johnston 1989; Donnelly et al. 1990; Leong 2004). In contrast, many species of Mi- crohyla s. str. are surface suspension feeders, and demon- strate greatly expanded lower labium and dorso-terminal mouth orientation; they may have terminal filament on tail and smooth margins of spiracular flap (e.g., Leong 2004; Hendrix et al. 2008; Poyarkov et al. 2014). A peculiar exception 1s the case of N. arboricola, which is an obligate phytotelm-breeding species that reproduces in water-filled tree hollows (Vassilieva et al. 2017). The oophagous tadpoles of this species differ from larvae of pond-dwelling Microhyla and Nanohyla species in many aspects, including external morphology (extremely long tails, dorsolateral position of the eyes, dark pigmenta- tion), morphology of digestive tract (large, extensible stomach with comparatively short intestine), and charac- teristic oral morphology (Vassilieva et al. 2017). Nano- hyla nanapollexa was suggested as phytotelm-breeder as a Single specimen of this species was recorded in a water-filled tree hollow (Gorin et al. 2020), although the details of reproductive biology and tadpole morphology of this species are still unknown. Taxonomic comment. Microhyla pulverata Bain & Nguyen, 2004 was considered a junior synonym of N. marmorata based on the phylogenetic results of Gorin et al. (2020); the same study also reported on three putative candidate species within N. arboricola, N. perparva, and N. petrigena, indicating that our knowledge on diversity of Nanohyla is still incomplete. Certain variation in diagnostically important charac- ters of Nanohyla gen. nov. requires further comments. Bain and Nguyen (2004) reported on significant varia- tion in size and shape of the outer metatarsal tubercle in N. marmorata which was reported to vary from almost indistinct to “conical.” We have examined a large series of N. marmorata (see Poyarkov et al. 2014; Nguyen et al. 2019) and found that in this species the outer meta- tarsal tubercle usually is not discernable or is indistinct; we assume that this discrepancy might be explained with the differences in preservation of specimens examined by Zoosyst. Evol. 97 (1) 2021, 27-54 us and by Bain and Nguyen (2004). Hoang et al. (2020) reported two metatarsal tubercles in their diagnosis of N. hongiaoensis, however in the holotype description they refer to the outer metatarsal tubercle as “indistinct;” it is also not discernable in their photo of holotype’s foot (Ho- ang et al. 2020:fig. 3F). In all the remaining species of Nanohyla gen. nov. it is absent, and we therefore consid- er this state to be diagnostic for the genus (in comparison to Microhyla s. str., which has two metatarsal tubercles in all species but M. maculifera, see comment below). It 1s not clear why Bain and Nguyen (2004), or Poyarkov et al. (2014; and other preceding studies) did not recognize the presence of externally visible tympanum in most of spe- cies of the genus (Fig. 11). In species of Nanohyla gen. nov., smaller tubercles and other dermal structures of the skin become flattened and less distinct after fixation and preservation; this has also been reported in other anurans (Poyarkov et al. 2015, 2017, 2019; Nguyen et al. 2018, 2019, 2020). It is likely that the presence of the tympa- num was artifactually concealed from Bain and Nguyen (2004), since their description was based exclusively on museum specimens. In some species of Nanohyla gen. nov., we were not able to detect an externally visible tym- panum (N. hongiaoensis, N. perparva, N. petrigena). It is not clear whether this reflects an actual character state in these species, or if this apparent state relates to the small sample size of specimens and photographs available to us. Further studies are needed to clarify variation of the external tympanum in Nanohyla gen. nov. Microhyla Tschudi, 1838 Synonymy (fide Frost 2020). Microhyla Tschudi, 1838. Type species. “Hy/aplesia achatina Boie, 1827” (nomen nudum) (= Microhyla achatina Tschudi, 1838), by monotypy. Micrhyla Duméril & Bibron, 1841. Ex errore. Siphneus Fitzinger, 1843. Type species: Engystoma or- natum Dumeril & Bibron, 1841. Dendromanes Gistel, 1848. Nomen substitutum for Mi- crohyla Tschudi, 1838. Diplopelma Gunther, 1859. Nomen substitutum for Siph- neus Fitzinger, 1843. Scaptophryne Fitzinger, 1861 “1860.” Type species: Scaptophryne labyrinthica Fitzinger, 1861 “1860” (nomen nudum). Copea Steindachner, 1864. Type species: Copea fulva Steindachner, 1864. Ranina David, 1872 “1871”. Type species: Ranina sym- etrica David, 1871, by monotypy. Junior homonym of Ranina Lamarck, 1801. Etymology. The genus name is derived from the Greek Luukpos (mikros), meaning “small,” and “Hylas” (for ori- gin of this name see above). Common name. Narrow-mouthed Frogs. 41 Taxonomic content. 42 species: M. achatina Tschudi, 1838; M. aurantiventris Nguyen, Poyarkov, Nguyen, Nguyen, Tran, Gorin, Murphy & Nguyen, 2019; M. bei- lunensis Zhang, Fei, Ye, Wang, Wang & Jiang, 2018; M. berdmorei (Blyth, 1856); M. borneensis Parker, 1928: M. butleri Boulenger, 1900; M. chakrapanii Pillai, 1977; M. darevskii Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; M. darreli Garg, Suyesh, Das, Jiang, Wijayathilaka, Amarasinghe, Alhadi, Vineeth, Aravind, Senevirathne, Meegaskumbura & Biju, 2019; M. eos Biju, Garg, Kamei & Maheswaran, 2019; M. fan- Jingshanensis Li, Zhang, Xu, Lv & Jiang, 2019; M. fis- sipes Boulenger, 1884; M. fodiens Poyarkov, Gorin, Zaw, Kretova, Gogoleva, Pawangkhanant & Che, 2019; M. gad- Jahmadai Atmaja, Hamidy, Arisuryanti, Matsui & Smith, 2018; M. heymonsi Vogt, 1911; M. irrawaddy Poyarkov, Gorin, Zaw, Kretova, Gogoleva, Pawangkhanant & Che, 2019; M. karunaratnei Fernando & Siriwardhane, 1996; M. kodial Vineeth, Radhakrishna, Godwin, Anwesha, Ra- jashekhar & Aravind, 2018; M. laterite Seshadri, Singal, Priti, Ravikanth, Vidisha, Saurabh, Pratik & Gururaja, 2016; M. malang Matsui, 2011; VM. mantheyi Das, Yaakob & Sukumaran, 2007; M. mihintalei Wiyjayathilaka, Garg, Senevirathne, Karunarathna, Biju & Meegaskumbura, 2016; M. minuta Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; M. mixtura Liu & Hu in Hu et al. 1966; M. mukhlesuri Hasan, Islam, Kuramo- to, Kurabayashi & Sumida, 2014; M. mymensinghensis Hasan, Islam, Kuramoto, Kurabayashi & Sumida, 2014; M. nepenthicola Das & Haas, 2010; M. nilphamariensis Howlader, Nair, Gopalan & Merila, 2015; M. okinaven- sis Stejneger, 1901; M. orientalis Matsui, Hamidy & Eto, 2013; M. ornata (Duméril & Bibron, 1841); M palmi- pes Boulenger, 1897; M. picta Schenkel, 1901; M. pin- eticola Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014; M. pulchra (Hallowell, 1861); M. rubra (Jerdon, 1854); M. sholigari Dutta & Ray, 2000; M. superciliaris Parker, 1928; M. taraiensis Khatiwada, Shu, Wang, Thapa, Wang & Jiang, 2017; M. tetrix Su- wannapoom, Pawangkhanant, Gorin, Juthong & Poyar- kov, 2020; M. zeylanica Parker & Osman-Hill, 1949; and, tentatively, M. maculifera Inger, 1989. Revised diagnosis. Microhyla s. str. differs from all other Microhylinae genera by the following combination of os- teological characters: (1) frontoparietals generally sepa- rated from exoccipitals (partially fused in M. mukhlesuri, M. picta and Microhyla sp. 2); (2) exoccipitals separate; (3) neopalatines present (in M. berdmorei, M. butleri, M. minuta, M. orientalis, M. pineticola, M. superciliaris and M. tetrix) or absent (in M. achatina, M. heymonsi, M. fis- sipes, M. malang, M. mukhlesuri, M. nepenthicola, M. nilphamariensis, M. okinavensis, M. picta, M. pulchra and Microhyla sp. 2); (4) sphenethmoids not fused to parasphenoid; (5) crista parotica ossified posteriorly; (6) otic ramus of squamosal poorly developed; (7) tympanic annulus well-developed (reduced in MZ. heymonsi, M. ne- penthicola, M. nilphamariensis, M. orientalis, M. pinet- zse.pensoft.net 42 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs icola, M. superciliaris and M. tetrix); (8) orientation of transverse processes of presacral vertebrae VI-—VIII an- terolateral, other vertebrae with inconsistent orientation; (9) clavicles absent; (10) omosternum absent (cartilagi- nous omosternum present only in M. pulchra); (11) prehal- lux cartilaginous; (12) terminal phalanges of the longest fingers T-shaped (in M. achatina, M. berdmorei, M. but- leri, M. fissipes, M. heymonsi, M. malang, M. minuta, M. nepenthicola, M. nilphamariensis and M. pineticola), knobbed (in M. minuta, M. mukhlesuri, M. nilphamarien- sis, M. superciliaris and M. tetrix), or simple (in M. okina- vensis, M. orientalis, M. picta and M. pulchra), terminal phalanges of the longest toe T-shaped (in M. achatina, M. berdmorei, M. butleri, M. heymonsi, M. malang, M. nepenthicola and M. pineticola), knobbed (in M. minuta, M. mukhlesuri, M. nepenthicola, M. superciliaris and M. tetrix), or simple (in M. fissipes, M. okinavensis, M. orientalis, M. picta, M. pulchra and Microhyla sp. 2). The combination of diagnostic external morphological characters includes: (13) body size medium to extreme- ly miniaturized (adult SVL 12.8-45.8 mm); (14) snout rounded or pointed in profile; (15) supratympanic fold present; (16) ridge on posterior margins of choanae ab- sent; (17) FI length greater than % FII; (18) discs present on every finger, only FII—FIV, or absent; (19) dorsomedial grooves on fingers present or absent; (20) toe discs pres- ent or absent; (21) dorsomedial grooves on toes present or absent; (22) two metatarsal tubercles (except M. macu- lifera with a single metatarsal tubercle); (23) dorsomedial line present or absent; (24) superciliary tubercles present (M. palmipes and M. superciliaris) or absent (all remain- ing species); (25) tibiotarsal articulation reaching well beyond snout (in M. berdmorei, M. darevskii, M. man- theyi and M. tetrix) or less; (26) toe webbing from basal to developed to discs; (27) skin on dorsum from smooth to tubercular; (28) tympanum externally indistinct; (29) ter- restrial or subfossorial microhabitat preference. Phylogenetic definition. The genus Microhyla s. str. in- cludes all species that share a more recent common an- cestor with Microhyla achatina than with Nanohyla an- nectens and Glyphoglossus molossus. Distribution. Frogs of the genus Microhyla are widely distributed across the East (southern China, including Tai- wan and Hainan islands, and Ryukyu Archipelago of Ja- pan), Southeast (Myanmar and Indochina, Malayan Pen- insula, Sumatra, Java, Bali, and Borneo), and South Asia (Bangladesh, Nepal, Indian subcontinent to north-eastern Pakistan in the west and Sri Lanka in the south) (Fig.1). Taxonomic comment. In the last phylogenetic revision of Microhyla, Gorin et al. (2020) included all species of the genus in their analysis, except M. darevskii, M. fusca Andersson, 1942, and M. maculifera. Microhyla darevskii was described from five formalin-fixed specimens and morphologically appears to be very close to the members of M. berdmorei species complex (Poyarkov et al. 2014). zse.pensoft.net Although the phylogenetic position of M. darevskii 1s not known, this species can be confidently assigned to the genus Microhyla s. str. based on morphological data. Microhyla fusca was described from a single specimen collected from southern Vietnam (Andersson 1942), and was recently demonstrated to be a junior synonym of . butleri (Poyarkov et al. 2020a). Microhyla maculifera remains the most enigmatic species of the group due to the lack of molecular data and uncertainties regarding morphological characters. This species was described from only two specimens (Inger 1989), and no additional specimens have been reported since that time, despite numerous field survey efforts. This small-sized species 1s unique among its con- geners in having comparatively short hindlimbs, large and wide head, less triangular than in other Microhyla, comparatively stout body habitus (Fig. 12), and a single metatarsal tubercle (vs two). Microhyla maculifera is dif- ferent from the members of the genus Nanohyla gen. nov. by having FI longer than 2 of FII (vs FI shorter than 2 of FI or reduced to a nub), lack of discs on fingers and rudimentary discs on toes (vs digital discs well-devel- oped), absence (vs presence) of dorsal median grooves on tips of fingers and toes, having comparatively short hindlimbs with tibiotarsal articulation reaching to snout (vs to well beyond snout), and toe webbing being basal (vs well-developed; Inger 1989). Due to the lack of mo- lecular data, the phylogenetic position and generic place- ment of “Microhyla’ maculifera remains uncertain; we tentatively retain this species Microhyla s. str. pending data or future phylogenetic studies, which might suggest another arrangement. Glyphoglossus Giinther, 1869 Synonymy (fide Frost 2020). Glyphoglossus Gunther, 1869 “1868”. Type species: Glyphoglossus molossus Gunther, 1869 “1868,” by monotypy. Calluella Stoliczka, 1872. Type species: Megalophrys guttulata Blyth, 1856 “1855,” by original designation. Colpoglossus Boulenger, 1904. Type species: Colpoglos- sus brooksi Boulenger, 1904, by monotypy. Dyscophina Van Kampen, 1905. Type species: Dyscophi- na volzi Van Kampen, 1905, by monotypy. Calliglutus Barbour & Noble, 1916. Type species: Cal- liglutus smithi Barbour & Noble, 1916, by monotypy. Kalluella Gee & Boring, 1929. Ex errore. Etymology. The genus name ts derived from the Ancient Greek yAvon (gluphé), meaning “a carving,” and Greek yA@ooa (glossa), meaning “tongue.” Common name. Balloon Frogs. Taxonomic content. Nine species, including: G. brooksii (Boulenger, 1904); G. capsus (Das, Min, Hsu, Hertwig Zoosyst. Evol. 97 (1) 2021, 27-54 43 Figure 12. Holotype of Microhyla maculifera Inger, 1989 (FMNH 231271, adult male) in dorsal (A) and ventral (B) aspects. Scale bar denotes 5 mm. Field Museum of Natural History. FMNH 231271. Created by Field Museum of Natural History, Amphibian and Reptile Collection and licensed under CC-BY-SA 4.0. & Haas, 2014); G. flavus (Kiew, 1984); G. guttulatus Revised diagnosis. G/yphoglossus Gunther, 1869 dif- (Blyth, 1856); G. minutus (Das, Yaakob & Lim, 2004); fers from other Microhylinae genera by the combina- G. molossus Ginther, 1869; G. smithi (Barbour & Noble, — tion of the following osteological characters: (1) fronto- 1916); G. volzi (Van Kampen, 1905); and G. yunnanensis _ parietals separated from exoccipitals (fused to them in (Boulenger, 1919). G. molossus), (2) exoccipitals separated from each other; zse.pensoft.net 44 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs (3) neopalatines obscured by a postchoanal portion of vomers; (4) sphenethmoids separated from parasphenoid; (5) crista parotica ossified; (6) otic ramus of squamosal well-developed; (7) tympanic annulus well-developed; (8) orientation of transverse processes of presacral vertebrae as follows: IV and V posterolateral, I, VII and VIII an- terolateral, HI and VI at right angle to body axis (in G. mo- lossus IV posterolateral, II, VI- VIII anterolateral, III and V at right angle to body axis); (9) clavicles present (absent in G. molossus), (10) omosternum absent; (11) prehallux ossified; (12) terminal phalanges of the longest finger and toe simple. The combination of diagnostic external mor- phological characters includes: (13) large to medium-sized frogs (adult SVL 30.9-94.9 mm); (14) snout rounded or bluntly flattened; (15) supratympanic fold present; (16) ridge on posterior margins of choanae poorly developed or absent; (17) first finger (FI) length greater than % FII; (18) discs on digits absent; (19) two metatarsal tubercles; (20) dorsomedial line absent; (21) superciliary tubercles absent; (22) tibiotarsal articulation of the adpressed hind- limb reaching eye or shorter; (23) toe webbing moderate- ly developed (at least one-third webbed, in G. molossus three-quarters webbed); (24) skin on dorsum from feebly granular to tubercular; (25) external tympanum invisible; (26) fossorial microhabitat preference. Phylogenetic definition. The genus G/yphoglossus in- cludes all species sharing a more recent common ancestor with Glyphoglossus molossus than with Microhyla acha- tina and Nanohyla annectens. Distribution. From south-western China across Indochi- na to Myanmar, Thai-Malay Peninsula, islands of Suma- tra and Borneo (Fig. 1). Taxonomic comment. Until recently G/yphoglossus was considered to be a monotypic genus, until it was syn- onymized with Calluella based on phylogenetic data of Peloso et al. (2016). However, available phylogenetic studies (Tu et al. 2018; Garg and Biju 2019; Gorin et al. 2020) have not all included comprehensive sampling of Sundaland species (e.g., C. volzi, C. smithi, C. flavus, and C. brooksi). In our opinion, the variable taxonomic sam- pling included in previous analyses (Matsui et al. 2011; Peloso et al. 2016; Tu et al. 2018; Garg and Biju 2019; Gorin et al. 2020) creates uncertainty which, along with the significant morphological disparity among G. mo- lossus and the other species of Glyphoglossus examined (Parker et al. 1934), suggests that the generic taxonomy of the group may not be fully resolved. Body size evolution in the Microhyla— Glyphoglossus assemblage Among vertebrates, numerous clades of fishes, frogs, and squamate reptiles compete for the title of the smallest ab- solute body size, with several converging around body lengths (defined vastly differently in the three clades) of zse.pensoft.net 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 (Alexan- der 1996; Hedges and Thomas 2001; Scherz et al. 2019). As such, species exhibiting miniaturization provide inter- esting 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 1s often associated with major morpho- logical rearrangements (Hanken 1985; Hanken and Wake 1993; Polilov 2015), and is thought to have played a sig- nificant 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 con- sequences 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 Sa et al. 2012, 2019b). Here, we have demonstrated that the Microhylinae are a partic- ularly 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 parsi- mony 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 miniaturiza- tion events, with Nanohyla miniaturizing first and inde- pendently from all Microhyla species; their most recent common ancestor 1s 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 Nano- hyla appears to have been coupled with the loss of meta- tarsal tubercles, whereas these are retained in even the smallest Microhyla. Likewise, the first finger of Nanohy- /a 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 Zoosyst. Evol. 97 (1) 2021, 27-54 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 Stumpf- fia species. Also, they have not lost any phalanges, even when fingers are reduced in length, whereas other minia- turized 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 fu- sion 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 fea- tures 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 com- monalities and differences both within these clades, and in comparison between these clades and other miniatur- ized frogs, highlights the extent to which miniaturization occurs through a combination of determinism and con- tingency. Nanohyla and Microhyla apparently share the reduction of the quadratojugal and loss of its connection to the maxilla, and the resulting take-over of suspensori- um support by the pterygoid (Fig. 5). This arrangement is sometimes seen in other miniaturized microhylids (e.g., Anodonthyla eximia Scherz, Hutter, Rakotoarison, Riemann, Rodel, 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 ex- treme body size reduction that would be expected to re- sult in such a degree of change. Thus, caution 1s always recommended when interpreting features as consequenc- es 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 micro- hylid, with long, slender limb bones and a subtriangular skull. Glyphoglossus molossus, however, shows extreme 45 osteological modification associated with its more fosso- rial 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 1s 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). Sexual size dimorphism in the Microhyla— Glyphoglossus assemblage 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, apparent- ly rapidly and independently, to male-biased dimorphism. Remarkably, even species with diminutive males can be male-biased, exemplified by Microhyla sp. 1. In gener- al, male-biased dimorphism is thought to be associated with territoriality and physical combat among male frogs (Shine 1979). These transitions to male-biased dimor- phism may, therefore, be associated with changes in nat- ural 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 under- stand common drivers of these changes. Conclusions Miniaturized amphibians are characterized by a high proportion of cryptic species, along with numerous an- atomical homoplasies, muddying our estimates of their evolutionary relationships and diversity (e.g., Hanken and Wake 1993; Rovito et al. 2013; Parra-Olea et al. 2016; Rakotoarison et al. 2017; Scherz et al. 2019; Gorin et al. 2020). Integrative taxonomic approaches, optimally com- bining the results of molecular phylogenetic analyses with morphological, acoustic and behavioral data, represent the most promising approach for better understanding of spe- cies boundaries, diversity and evolutionary relationships in microhylid frogs, including the genus Microhyla (Hasan et al. 2014; Garg et al. 2019; Poyarkov et al. 2018a, 2019; Gorin et al. 2020). Many recent phylogenetic studies of miniaturized frogs demonstrate that the diversity of these zse.pensoft.net 46 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs groups is unexpectedly high, at both the species and su- praspecific levels, due to a combination of overlooked di- versity (cryptic species) and microendemism (Oliver et al. 2017; Rakotoarison et al. 2017; Clemente-Carvalho et al. 2011; Poyarkov et al. 2018a; Zimkus et al. 2012; Black- burn et al. 2008; Louren¢go-de-Moraes et al. 2018; Rodri- guez et al. 2013; Kohler et al. 2008; Scherz et al. 2019). The present analysis of the Microhyla—Glyphoglossus as- semblage diversity represents a case in point: miniaturized taxa, that were previously assigned ad hoc to Microhyla s. lat., were demonstrated to belong to two deeply diver- gent clades, together closely related to the genus Glypho- glossus, which consists of species of a much larger body size and very different ecology. Upon closer examination of their phylogenetic relationships from molecular data, as well as morphology, ecology, and biogeography, we found that these deep clades were older than most other Microhylinae genera, and sufficiently different to justify recognition as distinct genera. This yielded the new genus Nanohyla gen. nov. described herein. This result further underlines the importance of genetic data, useful for inde- pendently elucidating diversity and evolutionary relation- ships within groups with extensive homoplasies (Mott and Vieites 2009; Heideman et al. 2011; Scherz et al. 2019). The Microhyla—Glyphoglossus assemblage (perhaps better now called the Microhyla—Nanohyla—Glyphoglos- sus assemblage) shows highly dynamic body size evolu- tion, and a propensity to miniaturize, with at least nine separate miniaturization events inferred across Microhyla and the new genus Nanohyla. Convergence in body size in these two genera has generated some homoplasies, but both have unique, apomorphic features. It is clear, how- ever, that, in order to gain a comprehensive understand- ing of the evolution of miniaturization in these frogs, much more extensive sampling of outgroups is need- ed. The Microhylidae, however, form an ideal group in which to study the evolution of miniaturization, which is one of several phylogenetically recurring frog ecomorphs (Moen et al. 2015). Acknowledgements We express our sincere gratitude to V.F. Orlova, R.A. Nazarov, and E.A. Galoyan (ZMMU, Moscow, Russia) for permission to study specimens under their care. We deeply appreciate the help of those colleagues who pro- vided various assistance, including specimens and mate- rials for examination: C. Suwannapoom, E.N. Solovye- va, M. Hasan, H. Okamiya, D.M.S.S. Karunarathna, P. Pawangkhanant, A. de Silva, W. Juthong, E.A. Galoyan, K.D. Milto, L.T. Nguyen, A. Haas, D.P. Bickford, and I. Das. We are grateful to N.A. Formozov, A.N. Kuznetsov, A.A. Polilov, L.P. Korzun, V.V. Shakhparonov, I.B. Sol- datova, and K.B. Gerasimov for many useful comments and suggestions on the discussion of our hypotheses and results. We thank A.A. Bannikova, I.V. Artyushin, A.B. Vassilieva, A.V. Diakova, S.E. Farisenkov, M.M. Perfilov, zse.pensoft.net T.V. Duong, A.S. Dubrovskaya, P.V. Yushchenko, and S.S. Idiatullina for valuable comments and help during our work on this project. We are deeply thankful to N.V. Kryukova who guided and helped us with making skele- tal preparations and histological staining. We are grateful to D.R. Gafurova and A.N. Khomyak for assistance with micro-CT scanning. We thank V.D. Kretova who prepared line drawings for this study, and I. Das, P. Pawangkhanant, E. N. Solovyeva, V. D. H. Nguyen, L. T. Nguyen, T. V. Nguyen and vnherps.com for providing us with photos of several species of Microhyla and Nanohyla. We thank J. Mata, A. Resetar and the Field Museum of Natural Histo- ry (Chicago, USA) for providing the photo of Microhyla maculifera. We also thank R. Brown and one anonymous reviewer for constructive feedback on this manuscript. Funding This work was supported by the Russian Foundation of Ba- sic Research to Nikolay A. Poyarkov (RFBR grant No. 19- 34-90167). Creation of dataset accessed on MorphoSource was made possible by the University of Florida (The 0 Vert (openVertebrate) Thematic Collection Network (TCN),; NSF DBI-1701714; NSF DBI-1702263). The funders had no role in study design, data collection and analysis, deci- sion to publish, or preparation of the manuscript. References Alberch P, Gale EA (1983) Size dependence during the development of the amphibian foot: colchicine-induced digital loss and reduction. Journal of Embryology and Experimental Morphology 76(1): 177-197. Alberch P, Gale EA (1985) A developmental analysis of an evolution- ary trend: digital reduction in amphibians. 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Zimkus BM, Lawson L, Loader SP, Hanken J (2012) Terrestrialization, miniaturization and rates of diversification in African puddle frogs (Anura: Phrynobatrachidae). PLoS ONE 7(4): e35118. https://doi. org/10.1371/journal.pone.0035118 Zweifel RG (1986) A new genus and species of microhylid frog from the Cerro de la Neblina Region of Venezuela and a discussion of relationships among New World microhylid genera. American Mu- seum Novitates 2863: 1—24. Zweifel RG (1972) Results of the Archbold Expeditions. No. 97. A revision of the frogs of the subfamily Asterophryinae, Family Microhylidae. Bulletin of the American Museum of Natural History 148: 411-546. Supplementary material | Table SI Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Microsoft Word Document (.docx) Explanation note: Museum voucher information, geo- graphic localities, and GenBank accession numbers of specimens and sequences used in this study. Asterisk (*) denotes sequences that were included in the align- ment for timetree calibration. Exact locality informa- tion unknown for specimens obtained via pet trade or those published in earlier works. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968 suppl 1 zse.pensoft.net Supplementary material 2 Table S2 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Microsoft Word Document (.docx) Explanation note: Museum voucher information and geo- graphic localities of osteological specimens examined. For abbreviations see Materials and methods; SVL given in mm. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.97.57968 .suppl2 Supplementary material 3 Table S3 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Microsoft Word Document (.docx) Explanation note: Body size data for the Microhyla— Glyphoglossus assemblage members (from Gorin et al. 2020, with modifications). For both sexes of each spe- cles maximal body size data is given. Question mark denotes “no data.” For voucher IDs of specimens see Gorin et al. (2020). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.97.57968.suppl3 Supplementary material 4 Table S4 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Microsoft Word Document (.docx) Explanation note: Results of divergence time estimates. Node No. — estimated tree node, for node names see Suppl. material 8: Figure S3; divergence time given in million years before present (Ma). Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- mons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended Zoosyst. Evol. 97 (1) 2021, 27-54 to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for oth- ers, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968.suppl4 Supplementary material 5 Table S5 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Microsoft Word Document (.docx) Explanation note: Osteological comparison of the Micro- hyla-Glyphoglossus assemblage members. For char- acter definitions and state descriptions see Materials and methods; Latin numerals (I-VIII) refer to numbers of presacral vertebrae. Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- mons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for oth- ers, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968.suppI5 Supplementary material 6 Figure S1 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Adobe PDF file Explanation note: Comparison of bayesian inference trees of the Microhyla—Glyphoglossus assemblage derived from the analysis of: (A) 2478 bp of mtD- NA fragment including 12S rRNA, tRNAVal and 16S rRNA genes; (B) 720 bp of BDNF nuDNA gene; (C) the combined mtDNA + nuDNA dataset of 3207 bp including 12S rRNA, tRNAVal, 16S rRNA and BDNF gene fragments. For voucher specimen infor- mation and GenBank accession numbers see Suppl. material 1: Table S1. Yellow, red, and blue color de- notes Microhyla I, Microhyla Il, and Glyphoglossus, respectively. Numbers at tree nodes correspond to PP/ BS support values, respectively. Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- mons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and au- thor(s) are credited. Link: https://do1.org/10.3897/zse.97.57968 .suppl6 53 Supplementary material 7 Figure S2 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Adobe PDF file Explanation note: Updated mtDNA-genealogy of the Microhyla—Glyphoglossus assemblage. For voucher specimen information and GenBank accession num- bers see Suppl. material 1: Table S1. Yellow, red, and blue color denotes Microhyla 1, Microhyla WJ, and Glyphoglossus, respectively. Numbers at tree nodes correspond to PP/BS support values, respectively. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968 .suppl7 Supplementary material 8 Figure S3 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Adobe PDF file Explanation note: Bayesian chronogram resulted from *BEAST analysis of the 3207 bp-long concatenated mtDNA + nuclear DNA dataset. Node values corre- spond to node numbers, for estimated divergence times (in Ma) see Suppl. material 4: Table S4. Red cir- cles correspond to calibration points used in molecular dating analysis, for details see Gorin et al. (2020). Blue bars correspond to 95%-confidence intervals. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968.suppl8 Supplementary material 9 Figure S4 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Adobe PDF file Explanation note: Hand preparations of the three represen- tatives of the Microhyla—Glyphoglossus assemblage. Pictures provided for G. guttulatus (A — dorsal view of zse.pensoft.net 54 Vladislav A. Gorin et al.: Parallel miniaturisation and a new genus of Microhylinae frogs the skull, B — lateral view of the skull, palmar view of the hand), M. fissipes (D — dorsal view of the skull, E — lateral view of the skull, F — palmar view of the hand) and N. marmorata (G — dorsal view of the skull, H — lateral view of the skull, I— palmar view of the hand). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968 suppl9 Supplementary material 10 Figure S5 Authors: Vladislav A. Gorin, Mark D. Scherz, Dmitriy V. Korost, Nikolay A. Poyarkov Data type: Adobe PDF file zse.pensoft.net Explanation note: Variable states of osteological char- acters in the Microhyla—Glyphoglossus assemblage. (A) mineralized prehallux of MZ. butleri; (B) cartilag- inous prehallux of N. annamensis; (C) ossified pre- hallux of G. guttulatus, (D) fan-shaped sternum of . picta; (E) bifurcate sternum of M. nilphamariensis, (F) pectoral girdle of M. annectens (omosternum shown by an arrow); (G, H, I) — mineralized stapes of N. pulverata, M. berdmorei and miniaturized M. minuta (shown with an arrow) respectively; (J, K, L) — verte- bral column of G. guttulatus, M. fissipes and N. mar- morata respectively; (M) — hyoid of M. okinavensis; (N, O) — palatine region of N. marmorata and M. fis- sipes respectively (neopalatine shown with an arrow). Copyright notice: This dataset 1s made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.97.57968.suppl10