Phylogenomics reveals rapid, simultaneous PNAS PLUS diversification of three major clades of Gondwanan at the Cretaceous–Paleogene boundary

Yan-Jie Fenga, David C. Blackburnb, Dan Lianga, David M. Hillisc, David B. Waked,1, David C. Cannatellac,1, and Peng Zhanga,1 aState Key Laboratory of Biocontrol, College of Ecology and Evolution, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, China; bDepartment of Natural History, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611; cDepartment of Integrative Biology and Collections, University of Texas, Austin, TX 78712; and dMuseum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720

Contributed by David B. Wake, June 2, 2017 (sent for review March 22, 2017; reviewed by S. Blair Hedges and Jonathan B. Losos) Frogs (Anura) are one of the most diverse groups of vertebrates The poor resolution for many nodes in anuran phylogeny is and comprise nearly 90% of living . Their world- likely a result of the small number of molecular markers tra- wide distribution and diverse biology make them well-suited for ditionally used for these analyses. Previous large-scale studies assessing fundamental questions in evolution, ecology, and conser- used 6 genes (∼4,700 nt) (4), 5 genes (∼3,800 nt) (5), 12 genes vation. However, despite their scientific importance, the evolutionary (6) with ∼12,000 nt of GenBank data (but with ∼80% missing history and tempo of diversification remain poorly understood. data), and whole mitochondrial genomes (∼11,000 nt) (7). In By using a molecular dataset of unprecedented size, including 88-kb the larger datasets (e.g., ref. 6), most data (>50%) are from the characters from 95 nuclear genes of 156 frog species, in conjunc- 12S and 16S mitochondrial ribosomal genes. The limited tion with 20 fossil-based calibrations, our analyses result in the amount of data also causes a wide range of estimates of di- most strongly supported phylogeny of all major frog lineages and vergence times for many nodes in the tree. For example, age

provide a timescale of frog evolution that suggests much younger estimates for the last common ancestor of extant , EVOLUTION divergence times than suggested by earlier studies. Unexpectedly, oftenreferredtoas“modern frogs” and containing 95% of our divergence-time analyses show that three species-rich clades extant anuran species, span ∼100 Mya (5, 7–11). Furthermore, (, , and Natatanura), which together com- divergences time estimates among the earliest neobatrachian prise ∼88% of extant anuran species, simultaneously underwent clades, such as the Heleophrynidae, , Calyp- rapid diversification at the Cretaceous–Paleogene (K–Pg) bound- tocephalellidae, Nasikabatrachidae, and , range ary (KPB). Moreover, anuran families and subfamilies containing from the Late Jurassic to early Cretaceous (∼150–100 Mya) arboreal species originated near or after the KPB. These results and have wide CIs (5, 7–11). In addition to these species-poor – suggest that the K Pg mass may have triggered explo- groups of neobatrachians, there are two species-rich clades: sive radiations of frogs by creating new ecological opportunities. (39% of extant anuran species, mostly Old World) This phylogeny also reveals relationships such as Microhylidae and Hyloidea (54%; mostly New World). The estimated ages being sister to all other ranoid frogs and African continental lineages of Natatanura forming a clade that is sister to a clade Significance of Eurasian, Indian, Melanesian, and Malagasy lineages. Biogeo- graphical analyses suggest that the ancestral area of modern frogs was Africa, and their current distribution is largely associ- Frogs are the dominant component of semiaquatic vertebrate ated with the breakup of Pangaea and subsequent Gondwanan faunas. How frogs originated and diversified has long attrac- fragmentation. ted the attention of evolutionary biologists. Here, we recover their evolutionary history by extensive sampling of genes and amphibia | Anura | nuclear genes | phylogeny | divergence time species and present a hypothesis for frog evolution. In contrast to prior conclusions that the major frog clades were estab- lished in the Mesozoic, we find that ∼88% of living frogs robust, reliable phylogeny is essential to understand the role originated from three principal lineages that arose at the end Aof macroevolutionary processes in generating biodiversity. of the Mesozoic, coincident with the Cretaceous–Paleogene However, resolution of evolutionary relationships among certain (K–Pg) mass extinction event that decimated nonavian dino- groups has been persistently difficult because of sparse genotypic saurs 66 Mya. The K–Pg extinction events played a pivotal role and phenotypic data. Frogs (Anura) are one such example; they in shaping the current diversity and geographic distribution of are one of the most diverse groups of , and currently modern frogs. comprise 6,775 described species, 446 genera, and 55 families (1) that are well represented on all continents. They exhibit great Author contributions: D.C.B., D.M.H., D.B.W., D.C.C., and P.Z. designed research; D.C.B., adaptive diversity within a highly constrained phenotype esti- D.M.H., D.B.W., D.C.C., and P.Z. designed and carried out taxon sampling; D.C.B. and mated to be 200 My old. Evolutionary convergence in body form, D.C.C. selected and vetted calibration points; Y.-J.F. and D.L. performed laboratory research; Y.-J.F., D.L., and P.Z. analyzed data; and Y.-J.F., D.C.B., D.L., D.M.H., D.B.W., life history, and behavioral traits is widespread in frogs, including D.C.C., and P.Z. wrote the paper. forms reflecting different microhabitat use by arboreal, aquatic, Reviewers: S.B.H., Temple University; and J.B.L., Harvard University. and fossorial species. These features make frogs a challenging but The authors declare no conflict of interest. fascinating model for addressing fundamental questions of mor- Freely available online through the PNAS open access option. phological, developmental, and biogeographical evolution. How- – Data deposition: The sequences reported in this paper have been deposited in the Gen- ever, despite intensive molecular phylogenetic studies (2 7), areas Bank database. For a list of accession numbers, see Dataset S1. of uncertainty and disagreement persist among clades that are 1 To whom correspondence may be addressed. Email: [email protected], catfish@ crucial for interpreting broad-scale macroevolutionary patterns. utexas.edu, or [email protected]. In addition, a general consensus on divergence times of the major This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. anuran lineages is also lacking (7, 8). 1073/pnas.1704632114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1704632114 PNAS Early Edition | 1of7 of each clade range from the Late Jurassic to the end of the Relationships within the Afrobatrachia mirror those found in Cretaceous, spanning ∼100 My, and relationships of family-level other studies (5–7, 14). taxa within each clade remain poorly resolved. Natatanura is a large clade of extant anurans (24% of species) In this study, we increased gene sampling by using a recently de- and mainly found in the Old World. Our ML and Bayesian to- veloped nuclear marker toolkit (12). Our new data include ∼88,000 nt pologies of Natatanura are identical. All nodes in the Bayesian of aligned sequences from 95 nuclear protein-coding genes covering tree have a BPP of 1.0, and only three nodes in the ML tree have 164 species (156 anuran species and 8 outgroups) from 44 of 55 frog BSs <90%. The 309-species topology is identical, but with low families; to our knowledge, this is the largest source of new data for support among the deeper branches, likely because of missing anuran phylogenetics. In addition, we enlarged this dataset to a total data. Notably, we found that endemic African continental lineages of 301 anuran species by incorporating previously published (Conrauidae, Odontobatrachidae, , Phrynoba- RAG1 and CXCR4 sequences so that all 55 extant frog families were trachidae, Ptychadenidae, ) form a clade that is included. Our goal was to propose a robust hypothesis of phyloge- the sister group to the clade of the remaining North American, netic relationships and divergence times of the major lineages. Our Eurasian, Melanesian, and Malagasy lineages (Ceratobatrachidae, results resolve previously intractable relationships, generate diver- Dicroglossidae, , , and Ranidae; Fig. gence times with narrow CIs, and provide perspectives on the evo- 1A and Figs. S1–S4). This African clade has low bootstrap sup- lutionary history and historical biogeography of frogs. port (56%) but high Bayesian support (1.0). The clade of the remaining non-African families is strongly supported (BS = Results and Discussion 100%), and the internal branches are strongly supported (BS = Data Characteristics. We assembled a de novo 164-species dataset 100%, BPP = 1.0), although they are short. In other studies, this by using 95 nuclear genes (Table S1) and 88,302 nt from 156 frog group of African lineages is not monophyletic (6, 7, 10, 14, 15). species and 8 outgroups; this matrix is 89.6% complete. To increase The phylogenetic position of the continental African lineages has coverage of anuran families, we added sequences of RAG1 and important biogeographic significance (as detailed later). CXCR4 from GenBank of 145 additional anuran species. This Relationships among the subfamilies of Microhylidae (one of 309-species dataset contains 88,386 nt and is 48.2% complete. the largest anuran families, including 8.8% of all species), which The 164-species and 309-species matrices are available from the have significant radiations on most continents and the large is- Dryad Digital Repository (doi:10.5061/dryad.12546). lands Madagascar and New Guinea, have proven difficult to resolve (4, 6, 7, 15–19). In contrast, our Bayesian tree has strong Higher-Level Phylogenetic Relationships of Frogs. Maximum-likelihood support; all 10 of the deepest nodes have a BPP of 1.0 (Fig. S2). (ML) and Bayesian analyses of concatenated genes in the 164- The ML topology is identical, but 3 of 10 nodes have a BS <90% species dataset produced identical trees except for two nodes (Fig. S1). ML analysis of our 309-species dataset (Fig. S4) re- with low support (Figs. S1 and S2). The ASTRAL species tree covered the same topology, but support is weaker. Nonetheless, differed from the ML tree at eight poorly supported nodes (Fig. S3). our tree is more strongly supported than others except for a Overall support is high: 94% of 155 nodes within frogs have a phylogenomic analysis (19) of 66 anchored loci for 48 taxa and 7 bootstrap value (BS) ≥70% (Fig. S1), and 97% have Bayesian Sanger-sequenced loci for 142 taxa. posterior probabilities (BPPs) ≥0.95 (Fig. S2). The ASTRAL Significantly, our study resolves relationships among one of species-tree method produced BSs ≥70% at 84% of the nodes the most diverse clades in the anuran phylogeny: Hyloidea, which (Fig. S3). Although ML analysis of the 309-species dataset has contains 54% of extant anuran species. ML and Bayesian anal- weaker support (78% of nodes have BSs ≥70%; Fig. S4), the yses strongly support 53 of 55 of nodes in Hyloidea (BS > 80% basic topology of the ML tree is similar. Therefore, we used the and PP = 1.0; Figs. S1 and S2). This is particularly unexpected ML tree as our primary hypothesis (Fig. 1A) for estimating because even the most species- and character-rich studies of the chronogram. hyloids (5–7) have recovered poorly resolved topologies. Our Archaeobatrachian relationships are identical to those of most expanded 309-species topology (Fig. S4) is similar to our primary recent studies (refs. 2, 5, 6, 13; but see refs. 4, 10), including an tree, even though many nodes have lower support, which is con- analysis of mitogenomes of 90 anuran species (7). Relationships sistent with the higher degree of missing data for this dataset of among the early branches of Neobatrachia are identical to the more species. mitogenomic phylogeny (7). We corroborate the placement of Two arrangements within hyloids are noteworthy. The deepest Heleophrynidae, which is from extreme southern Africa, as the divergences in Hyloidea are among southern South American sister taxon to all other neobatrachian frogs (Fig. 1A), as found taxa: in the temperate beech forests of Chile, by some authors (4–7, 9), but not by others (10, 11). We find that and and in Patagonia (Figs. S1–S4). Sooglossidae (known only from the Seychelles Islands) is the Similar relationships were previously reported (5, 20), albeit with sister taxon of Ranoidea (BS = 100%, BPP = 1.0; Figs. S1–S3)in weak support and a smaller sample of species. These relation- the 164-species analyses, and that Sooglossidae + Nasikabatrachidae ships support a southern South American origin of Hyloidea. (known only from the of peninsular India) form the Moreover, most previous studies (4, 6, 7, 21) supported Terrarana, Sooglossoidea, which is the sister group of Ranoidea (BS = 100%; a large New World tropical clade (15% of extant anuran species), Fig. S4) in the 309-species analyses. Sooglossoidea is placed as rather than southern South America groups, as the sister group the sister taxon of all other neobatrachians (11), all neobatrachian of all other hyloids. This difference in the placement of Terrarana frogs to the exclusion of Heleophrynidae (6), Hyloidea + may result from long-branch attraction of mtDNA sequences, be- Myobatrachidae + Calyptocephalellidae (4), and Ranoidea (3, 5, 7), cause Terrarana species apparently have higher rates of mtDNA but no previous studies recovered the placement of Sooglossoidea evolution than other hyloid lineages (22). with strong statistical support, including a mitogenome phylogeny (7). In summary, our analyses corroborate many of the deeper Taxa within the superfamily Ranoidea have been consistently neobatrachian nodes inferred by other studies, but with greater grouped into three clades (4–7): Microhylidae, Afrobatrachia support (all BS values = 100%). Furthermore, we find strong (i.e., epifamily Brevicipitoidae, which includes , support for many shallower nodes that are weakly supported in Hemisotidae, , and ), and Natata- other studies; only 4 of 155 nodes in our Bayesian tree have a nura (Fig. 1A). We found that Microhylidae is the sister group of posterior probability <1.0 and only 9 of 155 nodes have boot- Afrobatrachia + Natatanura (BS = 72%, PP = 1.0; Figs. S1 and strap support <75%. Relationships among the deepest branches S2); in contrast, many previous studies placed Microhylidae as in the Hyloidea, Microhylidae, and Natatanura, for which pre- the sister group of Afrobatrachia (4–6, 14) or of Natatanura (7). vious studies have conflicting results, are now strongly supported

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Leiopelmatoidea hochstetteri Leiopelmatidae

Ascaphus truei Ascaphidae Discoglossus pictus Discoglossoidea Alytes obstetricans busuangensis Anura Bombina orientalis Bombina fortinuptialis Rhinophrynus dorsalis Rhinophrynidae Pipoidea Pipa pipa Pipa parva Pseudhymenochirus merlini Hymenochirus boettgeri Xenopus kobeli Xenopus epitropicalis Scaphiopus couchii Pelobatoidea Spea multiplicata Scaphiopodidae Spea intermontana Pelodytes ibericus Pelodytidae Pelobates syriacus Pelobatidae Brachytarsophrys feae Xenophrys omeimontis Ophryophryne microstoma Leptolalax alpinus Scutiger gongshanensis Leptobrachium chapaense Oreolalax jingdongensis Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae Mixophyes coggeri Crinia signifera Myobatrachidae Limnodynastes salmini acarpicus Rhinodermatidae darwinii gargola Alsodidae calcaratus Hyloidea Atelognathus reverberii taeniata Batrachylidae Batrachyla leptopus Batrachophrynus macrostomus Telmatobiidae vellardi Lepidobatrachus sp. Ceratophrys cornuta Cryptobatrachus boulengeri Gastrotheca pseustes Gastrotheca weinlandii Nyctimystes kubori Phyllomedusa tomopterna Neobatrachia Agalychnis callidryas Agalychnis lemur Hyloscirtus lindae Aplastodiscus perviridis Hypsiboas fasciatus Scinax ruber Osteocephalus taurinus Hyla chinensis Acris crepitans Dendropsophus parviceps Pseudis paradoxa Allobates femoralis Hyloxalus jacobuspetersi Dendrobatidae Ranitomeya imitator Eleutherodactylus planirostris Craugastor augusti Craugastor fitzingeri Pristimantis thymelensis pulcher Strabomantidae sulcatus Hypodactylus brunneus boiei occidentalis

Leptodactylus albilabris EVOLUTION Lithodytes lineatus Physalaemus pustulosus Physalaemus cuvieri Pleurodema somuncurensis Pleurodema thaul Melanophryniscus stelzneri Amazophrynella minuta peltocephala Schismaderma carens Rentapia hosii Neobatrachia gargarizans Bufonidae Duttaphrynus melanostictus Rhinella marina nebulifer Anaxyrus canorus Anaxyrus punctatus Sooglossus thomasseti Sooglossidae Phrynomantis microps pyburni Microhylidae Chiasmocleis ventrimaculata Stereocyclops incrassatus Elachistocleis ovalis Gastrophryne olivacea Stumpffia pygmaea Platypelis tuberifera Anodonthyla boulengerii Paradoxophyla palmata Scaphiophryne boribory Scaphiophryne marmorata Microhylidae Kalophrynus pleurostigma Dyscophus antongilii heymonsi conjuncta Kaloula pulchra Cophixalus cheesmanae Oreophryne sp. Liophryne schlaginhaufeni Xenorhina sp. Ranoidea Callulops wilhelmanus Mantophryne lateralis Cophixalus sp. Hemisus marmoratus Hemisotidae Breviceps macrops Afrobatrachia Brevicipitidae Callulina kreffti Phlyctimantis boulengeri Hyperolius bolifambae Hyperoliidae Leptopelis parkeri Leptodactylodon ovatus Arthroleptis poecilonotus Nyctibates corrugatus Arthroleptidae Scotobleps gabonicus Trichobatrachus robustus Astylosternus diadematus Odontobatrachus natator Odontobatrachidae B Ptychadena oxyrhynchus Ptychadenidae 0.2 natalensis Phrynobatrachidae K-Pg boundary euskircheni Petropedetidae crassipes Conrauidae subsigillata Strongylopus grayii Pyxicephalidae Amietia lubrica Liurana xizangensis Ceratobatrachidae Natatanura Cornufer pelewensis Quasipaa spinosa 0.1 Limnonectes fujianensis Hoplobatrachus tigerinus Dicroglossidae Fejervarya limnocharis Boophis madagascariensis Aglyptodactylus madagascariensis Mantellidae Buergeria oxycephala odontotarsus Polypedates megacephalus Rhacophoridae 0.0 Rhacophorus dennysi nigromaculatus Amolops ricketti Net diversification rate Net diversification Amolops loloensis guentheri sp. Papurana latouchii Ranidae Odorrana schmackeri 200 150 100 50 0 berlandieri Rana virgatipes Rana draytonii Time before present (Mya) Rana amurensis Rana chensinensis

200 150100 50 0

Fig. 1. Time-calibrated phylogenetic tree of frogs and the pattern of net diversification rate across time. (A) Evolutionary chronogram based on 95% nuclear genes and 20 fossil age constraints. Gray bars represent the 95% credibility interval of divergence time estimates. Divergence time estimates and corre- sponding 95% credibility intervals for all nodes are provided in Table S2. Note that the initial diversification of the three major frog clades: Hyloidea (blue), Microhylidae (purple), and Natatanura (green) took place simultaneously near the KPB (dashed red line). (B) Rate-through-time plot of extant frogs indicates an increase in diversification rate at the end of the Cretaceous. in Hyloidea and Natatanura, and most nodes are well-supported include the position of southern South American taxa as the in Microhylidae. Some clades that were not well-supported in the earliest branches of hyloids, and the identification of a clade of mitogenomic phylogeny (7) are rejected in this study. Our findings endemic African taxa as the sister group of all other Natatanura.

Feng et al. PNAS Early Edition | 3of7 A New Timescale for Extinction and Diversification. Our extensive Rate-through-time analysis indicates that a surge in net diver- sample of nuclear loci produced not only a strongly supported sification rates of frogs occurred immediately following the KPB phylogeny for frogs but also younger divergence time estimates (Fig. 1B), which suggests a clear impact of the KPB extinction on with smaller CIs than previous studies. The estimated divergence frog diversification. The rapidity of the diversification is reflected times in the 164-species and the 309-species data sets are similar in the short branches of the deepest nodes in each clade. In (Figs. S5 and S6); the average time deviation among comparable Hyloidea, all but one branch connecting the 10 deepest nodes nodes between the two analyses is ∼5%. Following a thorough are <4.6 My in duration. In Microhylidae, the branches con- literature review, we selected 20 fossil calibration points, of necting the four deepest nodes are <4.0 My long. In Natatanura, which 13 are within crown Anura (Fig. S7). Jackknife removal of the branches connecting the six deepest nodes are <2.1 My. Even each fossil resulted in highly congruent sets of date estimates. though these branches are short, they are strongly supported. The overall average time differences were less than 1% except Another line of evidence demonstrates three mass for the two outlier fossils (the salamander Chunerpeton and the preceding explosive speciation. The branches subtending the salientian Triadobatrachus), and even the two outliers only led to Hyloidea, Natatanura, and Microhylidae are long: 47.3, 32.1, and overall average time differences of 3–5.4% (Fig. S8). 33.8 My, respectively (Fig. 1A and Table S2). The lack of other For this discussion, we focus on the 164-species tree (Fig. 1A). extant lineages originating from these three stem lineages cor- Overall, our divergence times are notably younger than those roborates our suggestion that an extinction event simultaneously found by most other studies (5, 10, 23, 24) (Fig. 1A and Table S2), decimated these lineages near the KPB. and especially much younger than those in the mitogenome phy- Even though the fossil record indicates mass extinction at the logeny (7). For example, we estimate the last common ancestor of KPB for several taxa, including birds, squamates, and mammals, crown-group Anura to be during the Upper Triassic at 210.0 Mya and subsequent radiation post-KPB (29–31), molecular data of- (95% CI, 199.1–220.4 Mya), and the age of crown Neobatrachia in ten indicate that many clades had diversified, at least in the sense the Lower Cretaceous at 142.1 Mya (95% CI, 132.8–149.8 Mya). In of lineage splitting, before the KPB event (32–36). Unlike nona- contrast, many other estimates place the age of crown Anura be- vian dinosaurs, whose demise is well documented in the rocks, the tween the Permian and Middle Triassic and the origin of Neo- fossil record of frogs is so far largely uninformative about survival/ batrachia between Upper Triassic and Upper Jurassic (Fig. S9). extinction across the KPB (37–40). In the present study, the three A striking pattern is the synchronous origin of three species-rich parallel combinations of precise node ages overlapping the KPB, neobatrachian clades—Hyloidea, Microhylidae, and Natatanura— preceded by a long branch and followed by very short but strongly at the Cretaceous–Paleogene (K–Pg) boundary (KPB;Fig. 1A, supported branches, indicate three extinctions followed by rapid dashed red line). The diversification synchronicity of the three divergence. These three radiations, which are coincident with the frog clades still existed when all calibration constraints in frogs late Cretaceous extinction, account for 88% of extant frog species. were excluded (Fig. S10), suggesting that this pattern is unlikely This molecular phylogenetic perspective strongly suggests that the result of the choice of calibration points. The K–Pg boundary frogs experienced a major extinction at the KPB. (KPB), dated precisely at 66 Mya (25), marks one of Earth’s great extinctions, largely attributed to the impact of the Chicxulub Historical Biogeography of Frogs. We performed a biogeographic bolide. However, other factors such as climate warming and the analysis on the 309-species data set using BioGeoBEARS (41). Indian Deccan volcanism, possibly related to the bolide impact, The DEC+J model performs significantly better than the DEC likely contributed to this extinction event (26). Although a near- model (Akaike information criterion; Table S3), indicating the KPB origin was separately reported for Hyloidea (5, 8, 10, 20, 27) importance of the J parameter, which models long-distance or and Microhylidae (10, 16) (Fig. S9), our study found that these “jump” dispersal. Our interpretation is that dispersal into a new three major clades originated at the same time, very near the KPB. area is accompanied by near-instantaneous speciation. The prac- The contemporaneous origin of the three large clades is highlighted tical effect of adding the J parameter to the model is that ancestral in our results by the narrow 95% CIs for each and their overlap with ranges often comprise one area rather than several. the KPB. Nine of the 10 deepest nodes of Hyloidea have relatively The most recent common ancestor (MRCA) of extant frogs narrow CIs that overlap the KPB (9.6–14.0 My; Fig. 1A and Table was distributed in Eurasia + North America + Australia or North S2). Similarly, within Microhylidae, the CIs of the four deepest America + Australia (Fig. 2 and Fig. S11), a Pangaean origin. In nodes encompass the KPB, and the CIs are similarly narrow (11.1– contrast, neobatrachians (i.e., modern frogs) originated in Gond- 11.9 My). Finally, in Natatanura, the CIs of the six deepest nodes all wanaland, most likely in Africa (relative probability, 93.6%; Fig. 2). overlap the KPB, also with similarly narrow CIs (10.4–10.7 My). The major neobatrachian lineages are also Gondwanan in origin: The use of internal calibration points for these clades is unlikely to Hyloidea in South America and Ranoidea, Afrobatrachia, and be driving this pattern because there was only one internal cali- Natatanura in Africa (relative probability, >90%; Fig. 2). bration point (within the Natatanura) used within these three clades. Breakup of land masses may be associated with three diver- There are several reasons why our study may have produced gence events in the evolution of frogs. The first of these was the younger divergence times compared with previous studies. First, split of neobatrachians from ancestral groups (Fig. 2A), dated at we included diverse outgroups, including lungfish, coelacanth, 172–181 Mya, which agrees with the breakup of Pangaea into salamanders, caecilians, and amniotes. Second, we used a new Laurasia and Gondwana in the Early Jurassic (∼180 Mya) (42). set of calibration points rather than uncritically recycling cali- This breakup event, which isolated the MRCA of Neobatrachia bration points from other studies; these calibration points have in Gondwana, is associated with the initial diversification of the been carefully reexamined based on the fossil record. The choice clade. The second breakup event caused the split between the of calibration points has significant impact on divergence time two major lineages of neobatrachians, Procoela (Hyloidea and estimation for frogs and may be an important reason that older Myobatrachoidea, mostly South American) and Diplasiocoela divergence times were obtained in previous studies (27). Third, (Ranoidea + Sooglossoidea, mostly African), as well as the split our phylogenetic estimate is based on a much larger dataset between the African and South American pipids (Fig. 2B). (88 kb; 62% of sites are variable) derived solely from 95 nuclear Spreading of the South Atlantic Ocean sea floor started in the loci (rather than primarily from mitochondrial loci) that exceeds Early Cretaceous (135 Mya), but the final physical separation other studies by at least sevenfold. We attribute the greater pre- between Africa and South America took place approximately cision (i.e., smaller CIs) to the large amount of information within 105 Mya (42). Our estimates for timing of these divergences, our dataset. A similar increase in precision was found for pletho- 125–130 Mya and ∼120 Mya, respectively, are highly congruent dontid salamanders by using the same nuclear marker toolkit (28). with this rifting process. The third breakup event took place

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Permian Triassic Jurassic Cretaceous Paleogene Neogene E Hynobiidae E Cryptobranchidae 87.4% N Ascaphidae O Leiopelmatidae 78.9% E Alytidae Anura E Bombinatoridae 42.4% N Rhinophrynidae S Pipa 68.8% Pipidae A Xenopus 50.0% 44.4% A Hymenochirus A Pseudhymenochirus N Scaphiopodidae E 64.6% Pelodytidae 57.9% E Pelobatidae E Megophryidae A Heleophrynidae 93.1% O Myobatrachidae 52.4% S Calyptocephalellidae S Rhinodermatidae S Alsodidae Hyloidea S S Batrachylidae S Telmatobiidae 93.6% S Ceratophryidae S Hemiphractidae Neobatrachia SNE Hylinae Hylidae O Pelodryadinae ': Middle Jurassic 94.2% (~180 Mya) S Phyllomedusinae S Dendrobatidae S Ceuthomantidae S S N Eleutherodactylidae SN Craugastoridae 63.7% 27.3% S Strabomantidae SNEA I Bufonidae S Allophrynidae S Centrolenidae S Leptodactylidae S (: Early Cretaceous S Odontophrynidae (~125 Mya) I Nasikabatrachidae I Sooglossidae A Hoplophryninae Microhylidae A Phrynomerinae EVOLUTION Seychelles 79.5% S India Otophryninae S N Gastrophryninae M Cophylinae 95.5% 48.0% M E Kalophryninae 51.0% 94.3% I Melanobatrachinae ): Late Cretaceous 94.6% O E 91.8% (~66 Mya) 93.6% M Dyscophinae Ranoidea 93.1% E I A Hemisotidae A Brevicipitidae Afrobatrachia A Hyperoliidae Africa (A) A Arthroleptidae Eurasia (E) A Odontobatrachidae South America (S) A Phrynobatrachidae A Ptychadenidae North America (N) A Pyxicephalidae Madagascar (M) 92.5% A Conrauidae A Petropedetidae India-Seychelles (I) Natatanura E Ceratobatrachidae Australia (O) E I Dicroglossidae I Nyctibatrachidae E + N + O 88.9% E NS Ranidae N + O 93.5% I Micrixalidae Other I 88.6% Ranixalidae M Mantellidae E I Rhacophoridae 250 200 150 10016M 50 0

Fig. 2. Ancestral-area estimates for 69 terminal taxa (families, subfamilies, and genera) of extant frogs using the DEC+J model in BioGeoBEARS. Circles on nodes represent the set of possible ancestral areas, and the color is associated with the area legends. The probabilities are given next to circles for the most probable ancestral area. Circles without values indicate that the probability of the ancestral area is >99%. Three important landmass breakup events are indicated: (A) the break-up of Pangea with division into Laurasia and Gondwana in the Middle Jurassic coincident with the origin of neobatrachian frogs; (B) the separation of Africa and South America in the Early Cretaceous coincident with the divergence of Ranoidea and Hyloidea as well as between the African and New World pipids; and (C) the separation of the Seychelles and India in the Late Cretaceous coincident with the divergence between the Sooglossidae and Nasikabatrachidae. Anuran taxa that contain at least some arboreal species are indicated in green and a tree icon (we do not imply that the last common ancestor of each of these families was arboreal). Note that all clades containing arboreal frogs originated after the KPB. when the Sooglossidae (Seychelles) and the Nasikabatrachidae but connected intermittently via Antarctica (42). From the late (India) diverged (Fig. 2C). The Seychelles/India land mass con- Cretaceous to the early Tertiary, the Earth experienced a period tinued to exist until 66 Mya, when new rifting severed the Sey- of global warming (44). Climate data from plant fossils, sediments, chelles from India (43). We estimate Sooglossidae split from and geochemical indicators show that the mean annual tempera- Nasikabatrachidae at ∼66 Mya, which is considerably younger ture of the Antarctic Peninsula region was 10–20 °C from 100 to than the previous estimates (77–130 Mya) (10, 11, 23), in con- 50 Mya (45), which is sufficiently warm to allow dispersal of frogs gruence with the geological event. through this region. Thus, the origin of the Australian myoba- The MRCA of Hyloidea and of Myobatrachidae + Calyp- trachids and pelodryadine hylids is most likely explained by dis- tocephalellidae occurred in South America (Fig. 2). The divergence persal from South America to Australia through Antarctica, and between Myobatrachidae (Australia) and Calyptocephalellidae later extinction in Antarctica because of the formation of ice sheets. (South America) and the split between Phyllomedusinae (South The role of Antarctica in dispersal of frogs among Gondwanan America) and Pelodryadinae (Australia and New Guinea) took land masses has received little attention (but see ref. 46), but place ∼100 Mya and ∼50 Mya, respectively (Fig. 2). During this our time-calibrated phylogeny predicts that paleontological period, South America and Australia were distantly separated research in the Cretaceous and early Cenozoic of Antarctica

Feng et al. PNAS Early Edition | 5of7 could shed light on the early evolution and dispersal, espe- sequenced on a Illumina HiSEq.2500 sequencer. Approximately 3 GB of Illu- cially of hyloids. mina HiSeq paired-end 90-bp reads were obtained. These reads were bio- The nearly worldwide distribution of Microhylidae presents a informatically sorted by barcode sequence and assembled into consensus longstanding and challenging biogeographic puzzle. Vicariant sequences. NGS protocol of library construction and bioinformatic analyses have been described previously (51). All sequences were compared by using origin and long-distance oceanic dispersal have been proposed to BLAST against GenBank to ensure that the target sequences were amplified. explain the current distribution of this family (10, 15, 16, 18, 23). The remaining sequences were further checked for frame shift or stop codons. Which scenario dominates interpretation depends largely on the GenBank accession numbers for the new sequences are given in Dataset S1. time and location of origin for the Microhylidae. Our analyses place the initial divergence of the Microhylidae at ∼66 Mya, with GenBank Data. To provide a comprehensive phylogeny at the family level, a relative probability of 79.5% for an African origin (Fig. 2). By we retrieved RAG1 and CXCR4 sequences from GenBank for 145 additional species this time, Gondwana was already highly fragmented and Africa of frogs (Dataset S1). These data plus our new sequences comprise a combined was separated from South America and Madagascar by ocean. dataset that contains 301 frog species from all recognized anuran families. Accordingly, overseas dispersal of the major microhylid lineages on Gondwanan landmasses is required. Phylogenetic Analyses. The 95 nuclear protein coding genes were aligned by using the ClustalW algorithm as implemented in MEGA v6 (52). Ambiguously The origin and diversification of the second large clade, aligned regions were culled using GBlocks v.0.91b (53) with the “codon” Natatanura, is controversial. Two hypotheses have been pro- model (−t = c), smaller block (−b4 = 3), and all gaps allowed (−b5 = a). All posed. The Out-of-Africa hypothesis (47), argues that the ori- refined alignments were then concatenated into a concatenated supermatrix. gin of the Natatanura lies in Africa, with subsequent dispersal Two supermatrices were built: 164-species dataset (156 frogs and 8 outgroups) to other continents. The Out-of-India hypothesis (48) postulates and 309-species dataset (301 frogs and 8 outgroups). PartitionFinder v.1.1.1 that the ancestor of Natatanura originated on the Indian plate (54) was used to select models and partitioning schemes for the two and was confined there until the plate collided with Asia ap- supermatrices according to the Bayesian information criterion. A total of proximately 55 Mya. We find that Natatanura originated in 285 data partitions were selected as the best partitioning scheme that cor- responded to the three separate codon positions for each of the 95 genes Africa (relative probability, 92.5%; Fig. 2). Considering that (Table S4). The ML tree was estimated by using RAxML version 8.0 (55) with Natatanura is the sister group of Afrobatrachia (apparently of the GTR + Γ + I model assigned to each partition. Support for nodes in the African origin) and the basal split within the Natatanura phylog- ML tree was assessed with a rapid bootstrap analysis (option –fa)with eny separates all African continental lineages from other Asian, 1,000 replicates. The Bayesian tree was inferred using MrBayes 3.2 (56) using Indian, and Madagascar lineages (Fig. 2), an African origin seems the models and partitions identified by PartitionFinder. Two Markov chain more likely. The endemic Indian natatanurans (Nyctibatrachidae, Monte Carlo (MCMC) runs were performed with one cold chain and three Micrixalidae, and Ranixalidae) are nested within Asian lineages, heated chains (temperature set to 0.1) for 50 million generations and sampled and the divergences between them and their closest Asian rela- every 1,000 generations. Chain stationarity was visualized by plotting likeli- hoods against the generation number by using TRACER v1.6 (beast.bio.ed.ac. tives occurred between 55 and 60 Mya (Fig. 2), which is consistent – ∼ uk/Tracer). The effective sample sizes were greater than 200 for all parameters with the timing of the India Asia collision ( 55 Mya) (49). In- after the first 10% of generations were discarded. Species tree analysis with- triguingly, the divergence between the Asian tree frogs (Rhaco- out gene concatenation was performed by using the Accurate Species TRee phoridae) and the Malagasy mantellids also happened during this ALgorithm (ASTRAL) (57) under the coalescent model. The individual input period (Fig. 2), implying that the Indian plate served as a stepping gene trees were inferred from partitioned ML analyses by using RAxML with stone for the long-distance dispersal from Asia to Madagascar. the same GTR + Γ + I model assigned to each codon position of each gene. The The three long branches of similar duration leading to Hyloidea, species tree analysis was conducted by using ASTRAL under the multilocus − = Microhylidae, and Natatanura suggest that the K–Pg mass ex- bootstrapping option with 200 replicates ( r 200). tinction event may have triggered explosive radiations by emptying ecological space. Notably, all terminal taxa (family and subfamily Divergence Time Analyses. Divergence time estimation was conducted by – using the program MCMCTREE in the PAML package (58). The ML topology ranks) with arboreal species originate after the K Pg boundary was used as the reference tree. Twenty calibration points were used to (Fig. 2). The rebounding of forests after the massive loss of veg- calibrate the clock (Fig. S7). The ML estimates of branch lengths for each of etation (50) at the KPB may have provided new ecological op- the 95 nuclear genes were obtained by using BASEML (in PAML) programs portunities for the subsequent radiation of largely arboreal groups under the GTR + Γ model. Based on the mean estimate from the 95 genes such as Hylidae, Centrolenidae, and Rhacophoridae. The obser- using a strict molecular clock with a 450-Mya root age (the divergence be- vation that no lineages of frogs originating before the KPB tween Latimeria and Protopterus; ref. 59), the prior for the overall substitution (archaeobatrachians, Heleophrynidae, Myobatrachoidea, and rate (rgene gamma) was set at G (1, 11.96, 1). The prior for the rate-drift Sooglossoidea) have truly arboreal species, and that all origins parameter (sigma2 gamma) was set at G (1, 4.5, 1). The 20 calibration points were specified with soft boundaries by using 2.5% tail probabilities above of arboreality (e.g., within hyloids or natatanurans) follow the – and below their limits; this is a built-in function of MCMCTREE. The inde- KPB, supports the hypothesis that the K Pg mass extinction pendent rate model (clock = 2 in MCMCTREE) was used to specify the rate shaped the current diversity of frogs. priors for internal nodes. The MCMC run was first executed for 10,000,000 generations as burn-in, then sampled every 1,000 generations until a total of Materials and Methods 10,000 samples was collected. Two MCMC runs using random seeds were Taxon Sampling and Data Collection. Our sampling included 156 frog species compared for convergence, and similar results were found. from 44 of the 55 recognized families (1). Eight outgroup species, including two salamanders, one caecilian, one bird, one crocodile, one mammal, and Rate-Through-Time Analysis. We investigated the diversification tempo of two lobe-finned fishes, were used in all phylogenetic analyses. Total DNA frogs using the program Bayesian Analysis of Macroevolutionary Mixtures was isolated from frozen or ethanol-preserved tissues (liver or muscle) by (BAMM), v2.5 (60). The 309-species chronogram was used as the input tree. proteinase K digestion followed by standard salt extraction protocol. Pre- To account for incomplete taxon sampling, the time tree was pruned to viously published PCR primers and PCR protocols (12) were used to amplify family level, and the sampling fraction of each family was calculated based 95 unlinked nuclear protein-coding genes (including RAG1 and CXCR4) from on the number of species following AmphibiaWeb. The BAMM analysis was the DNA extracts in 96-well plates. The amplification products were se- run for 100 million generations at a temperature increment parameter of quenced using a next-generation sequencing (NGS) strategy as described by 0.01 and sampled event data every 1,000 generations. The first 20% samples Feng et al. (51). Briefly, all amplification products from a single species were were removed as burn-in. The rate-through-time plot was summarized and vi- pooled together and purified. The amplification product pool of a sample was sualized by using BAMMtools (61) from the remaining 80% event data samples. then randomly sheared to small fragments (200–500 bp), ends were repaired, and a species-specific barcode linker was added. All indexed amplification Biogeographic Analyses. Based on the current distribution pattern of frogs, we product pools were mixed together. A sequencing library was constructed defined seven biogeographic areas: Africa, Eurasia (Europe and Asia with with the pooled DNA by using the TruSeq DNA Sample Preparation kit and exception of Indian plate), India (including Sri Lanka and the Seychelles),

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1704632114 Feng et al. Madagascar, North America (northern Mexico, United States, and Canada), South number of ancestral areas allowed at each node was set to four. We com- PNAS PLUS America, and Australia (Australia, New Zealand, and New Guinea; Fig. 2). pared the DEC and DEC+J models to determine the influence of founder- Connectivity of these biogeographic areas was modeled with three dispersal event dispersal on biogeographic patterns. The AIC criterion selected the probability categories: 0.01 for well-separated areas, 0.5 for moderately sep- DEC+J as the best-fitting model (Table S3), and this was subsequently used arate areas, and 1.0 for well-connected areas. Area connectivity and dispersal to infer the most likely biogeographic history of anurans. probability were modeled in seven time slices: 0–30 Mya, 30–66 Mya, 66–90 Mya, – – – – 90 120 Mya, 120 160 Mya, 160 200 Mya, and 200 270 Mya (Table S5). ACKNOWLEDGMENTS. This work was supported by National Natural Science Biogeographic analyses were performed by using BioGeoBEARS (41). We Foundation of China Grants 31672266 and 31372172 (to P.Z.), National Youth used the 309-species chronogram generated by our divergence time analyses Talent Support Program Grant W02070133 (to P.Z.), National Science Fund for as the input phylogeny. The current distribution of each frog species was Excellent Young Scholars of China Grant 31322049 (to P.Z.), and National Science assigned based on data from AmphibiaWeb (Table S6). The maximum Foundation Grant DEB-1202609 (to D.C.B.) and DEB-1441652 (to D.B.W.).

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Feng et al. PNAS Early Edition | 7of7 Supporting Information

Feng et al. 10.1073/pnas.1704632114

Table S1. Descriptive statistics for the 95 loci used in this study Parsimony Relative GC of GC of third Gene Variable informative composition total position Substitution No. name Length, bp sites sites variability mean, % mean, % model (BIC)

1 ADNP 822 86 507 0.0445 47.6 45.1 GTR+I+G 2 ANKRD50 996 61 484 0.0261 42.1 32.2 GTR+I+G 3 ARID2 873 83 587 0.0342 44.0 32.8 HKY+I+G 4 ARSI 852 44 485 0.0431 44.2 41.9 GTR+I+G 5 B3GALT1 780 49 332 0.0402 40.1 41.9 HKY+I+G 6 BPTF 546 29 258 0.0980 54.4 70.1 SYM+I+G 7 CAND1 1,176 50 514 0.0238 42.6 35.3 GTR+I+G 8 CASR 750 39 346 0.0515 46.5 64.0 GTR+I+G 9 CELSR3 1,281 91 693 0.0682 48.5 52.6 GTR+I+G 10 CHST1 693 47 391 0.0417 46.5 47.6 GTR+I+G 11 CILP 1,158 92 661 0.0325 41.6 32.7 GTR+I+G 12 CPT2 792 63 546 0.0588 47.5 52.6 SYM+I+G 13 CXCR4 651 51 379 0.0347 47.3 60.2 HKY+I+G 14 DBC1 810 32 383 0.0713 45.9 50.4 GTR+I+G 15 DCHS1 363 41 173 0.0566 49.6 46.5 GTR+I+G 16 DET1 711 39 329 0.0397 42.9 42.7 SYM+I+G 17 DISP1 1,062 53 510 0.0344 44.0 51.1 GTR+I+G 18 DISP2 981 98 653 0.0494 42.8 43.3 GTR+I+G 19 DMXL1 1,002 80 653 0.0316 43.1 37.4 GTR+I+G 20 DNAH3 957 73 487 0.0293 41.1 37.5 HKY+I+G 21 DOLK 741 66 489 0.0504 49.0 57.8 GTR+I+G 22 DOPEY1 600 49 303 0.0343 42.1 49.6 GTR+I+G 23 DSEL 1,305 77 763 0.0211 39.2 31.3 GTR+I+G 24 ENC1 1,098 53 604 0.0308 45.7 44.1 GTR+I+G 25 EVPL 1,014 111 664 0.0404 42.0 43.1 GTR+I+G 26 EXOC8 1,143 67 658 0.0463 40.9 39.3 GTR+I+G 27 EXTL3 1,269 114 622 0.0386 46.8 49.9 GTR+I+G 28 FAT1 1,545 137 1071 0.0235 38.7 30.0 GTR+I+G 29 FAT4 756 41 467 0.0290 41.7 35.6 HKY+I+G 30 FEM1B 996 60 552 0.0553 49.2 51.4 GTR+I+G 31 FICD 525 25 271 0.0364 45.0 43.5 GTR+I+G 32 FILIP1 810 61 492 0.0328 37.1 31.7 GTR+I+G 33 FLRT3 1,059 148 559 0.0252 42.5 42.0 GTR+I+G 34 FREM2 1,152 76 688 0.0356 40.7 36.1 GTR+I+G 35 FUT9 771 58 357 0.0250 40.5 42.8 HKY+I+G 36 FZD4 762 37 329 0.0382 45.9 53.3 GTR+I+G 37 GGPS1 414 19 212 0.0366 36.4 31.1 GTR+I+G 38 GLCE 438 26 220 0.0433 48.8 50.3 HKY+I+G 39 GPER 519 30 260 0.0439 44.5 58.2 GTR+I+G 40 GRIN3A 651 33 309 0.0337 41.1 33.4 GTR+I+G 41 GRM2 687 46 374 0.0831 54.6 68.3 GTR+I+G 42 HYP 1,299 370 653 0.0414 47.4 52.2 SYM+I+G 43 IRS1 1,020 48 496 0.0391 50.9 48.9 SYM+I+G 44 KBTBD2 1,125 94 638 0.0204 44.4 42.8 GTR+I+G 45 KCNF1 765 38 374 0.0396 46.4 58.2 SYM+I+G 46 KIAA2013 537 46 300 0.0820 53.8 69.3 HKY+I+G 47 LCT 666 62 450 0.0453 45.3 44.3 GTR+I+G 48 LIG4 1,056 87 618 0.0262 38.8 37.3 GTR+I+G 49 LINGO1 1,137 65 594 0.0335 46.0 45.6 HKY+I+G 50 LINGO2 1,332 108 805 0.0266 40.4 39.9 GTR+I+G 51 LPHN2 582 38 252 0.0361 48.3 50.9 SYM+I+G 52 LRRC8D 1,155 62 554 0.0325 40.8 41.4 HKY+I+G 53 LRRN1 837 48 424 0.0563 51.0 56.5 HKY+I+G 54 LRRN3 1,113 100 607 0.0288 37.8 38.4 HKY+I+G

Feng et al. www.pnas.org/cgi/content/short/1704632114 1of14 Table S1. Cont. Parsimony Relative GC of GC of third Gene Variable informative composition total position Substitution No. name Length, bp sites sites variability mean, % mean, % model (BIC)

55 LRRTM4 1,155 60 590 0.0248 45.1 51.5 GTR+I+G 56 MB21D2 1,029 42 422 0.0260 44.0 43.8 HKY+I+G 57 MED1 627 45 329 0.0434 46.5 40.7 GTR+I+G 58 MED13 525 39 277 0.0278 43.5 42.5 GTR+I+G 59 MGAT4C 792 51 400 0.0297 36.6 33.9 HKY+I+G 60 MIOS 972 53 464 0.0865 53.0 63.2 GTR+I+G 61 MSH6 1,350 101 821 0.0426 44.2 44.1 GTR+I+G 62 MYCBP2 1,068 80 551 0.0216 42.1 34.8 GTR+I+G 63 NHS 1,044 88 639 0.0248 42.1 32.8 HKY+I+G 64 NTN1 585 22 238 0.0904 52.2 66.8 HKY+I+G 65 P2RY1 744 41 342 0.0311 39.7 48.9 GTR+I+G 66 PANX2 735 49 362 0.0429 37.8 37.2 GTR+I+G 67 PCDH1 1,479 117 774 0.0277 45.5 42.7 GTR+I+G 68 PCDH10 849 53 487 0.0582 60.3 82.8 GTR+I+G 69 PCLO 906 43 480 0.0256 39.1 24.4 GTR+I+G 70 PDP1 1,071 60 491 0.0391 46.1 44.2 GTR+I+G 71 PIK3CG 978 90 572 0.0258 38.6 35.6 GTR+I+G 72 PPL 1,389 141 998 0.0418 44.9 51.3 GTR+I+G 73 RAG1 1425 84 763 0.0331 44.3 43.9 HKY+I+G 74 RAG2 906 62 653 0.0363 43.0 44.6 GTR+I+G 75 RERE 456 28 216 0.0336 52.0 43.7 SYM+I+G 76 REV3L 675 52 512 0.0327 39.9 28.2 GTR+I+G 77 ROR2 972 78 489 0.0274 43.5 40.3 GTR+I+G 78 RP2 510 41 272 0.0393 45.6 51.7 HKY+I+G 79 SACS 1,146 56 579 0.0252 40.2 34.3 GTR+I+G 80 SALL1 1,497 96 862 0.0223 43.1 35.0 GTR+I+G 81 SETBP1 795 64 443 0.0353 43.7 42.3 GTR+I+G 82 SH3BP4 1,158 79 717 0.0403 44.9 43.8 GTR+I+G 83 SLITRK1 1,185 103 636 0.0714 46.9 55.4 GTR+I+G 84 SOCS5 999 55 504 0.0460 49.0 47.2 GTR+I+G 85 SPEN 948 80 577 0.0503 41.4 39.8 GTR+I+G 86 STON2 921 74 602 0.0506 47.6 55.1 SYM+I+G 87 SVEP1 840 81 566 0.0294 41.3 27.2 HKY+I+G 88 TTN 999 250 575 0.0232 41.1 28.0 GTR+G 89 VCPIP1 1,125 77 578 0.0592 46.8 48.7 GTR+I+G 90 VPS18 975 81 586 0.0651 42.8 49.4 GTR+I+G 91 WFIKKN2 1,155 82 699 0.0617 47.0 53.6 GTR+I+G 92 ZBED4 1077 92 518 0.0352 40.2 37.3 GTR+I+G 93 ZEB1 819 55 484 0.0270 42.5 33.4 HKY+I+G 94 ZFPM2 1,251 119 652 0.0304 45.2 41.4 GTR+I+G 95 ZHX2 993 78 649 0.0422 44.5 45.2 GTR+I+G

Feng et al. www.pnas.org/cgi/content/short/1704632114 2of14 Table S2. Detailed node information of the concatenated analyses and divergence analyses Node BS PP Age, Mya 95% CI

1 N/A 1.0 433.4 416.3–455.2 2 N/A 1.0 422 410.8–427.5 3 100 1.0 348.4 343.0–351.5 4 100 1.0 319 316.9–322.7 5 100 1.0 252.2 246.9–259.7 6 100 1.0 297.9 287.7–309.1 7 100 1.0 271.7 266.8–274.7 8 100 1.0 153 130.5–166.3 9 100 1.0 210 199.1–220.4 10 100 1.0 193.8 178.6–207.0 11 100 1.0 194.1 183.2–204.1 12 100 1.0 138.5 122.9–158.6 13 100 1.0 102.2 74.8–125.5 14 100 1.0 42.8 33.0–54.7 15 100 1.0 10 7.4–13.3 16 100 1.0 182.5 172.0–192.7 17 100 1.0 159.4 146.7–171.4 18 100 1.0 117.6 104.2–130.9 19 100 1.0 11.6 8.4–15.5 20 100 1.0 104.1 91.7–117.7 21 100 1.0 57.9 45.3–69.9 22 100 1.0 45.3 34.2–57.9 23 100 1.0 172.6 162.5–182.6 24 100 1.0 128 115.6–141.5 25 100 1.0 41.9 32.1–54.1 26 100 1.0 9.3 6.8–12.3 27 100 1.0 112.3 99.9–125.6 28 100 1.0 74.4 66.2–86.2 29 100 1.0 50.6 44.3–57.9 30 100 1.0 24.8 20.5–29.8 31 100 1.0 17 13.1–21.6 32 100 1.0 32.4 27.1–38.0 33 100 1.0 18.7 15.2–22.9 34 100 1.0 15.5 12.1–19.6 35 100 1.0 142.1 132.8–149.8 36 100 1.0 130 121.1–137.8 37 100 1.0 119.9 110.7–129.1 38 100 1.0 99.5 88.6–111.3 39 100 1.0 72.6 63.4–84.8 40 100 1.0 65.7 56.2–77.6 41 100 1.0 72.6 67.4–78.5 42 100 1.0 61.5 54.3–68.3 43 100 1.0 68 63.2–73.4 44 100 1.0 61.8 56.5–67.9 45 100 1.0 20.1 14.7–27.0 46 100 1.0 26.7 21.2–33.5 47 100 1.0 13.4 9.8–17.7 48 100 1.0 66.4 61.8–71.7 49 100 1.0 4.9 3.5–6.7 50 100 1.0 64.8 60.3–70.0 51 98 1.0 63.9 59.5–69.1 52 100 1.0 18.2 13.1–24.6 53 94 1.0 62.6 58.2–67.9 54 100 1.0 43.9 35.5–51.9 55 100 1.0 19.2 14.2–25.2 56 100 1.0 58.9 54.4–64.2 57 100 1.0 47.5 42.0–53.4 58 100 1.0 24.5 20.0–29.7 59 100 1.0 16.7 12.8–21.3 60 100 1.0 49.5 44.9–54.4 61 100 1.0 32.3 26.6–38.3 62 100 1.0 25.2 19.6–31.0

Feng et al. www.pnas.org/cgi/content/short/1704632114 3of14 Table S2. Cont. Node BS PP Age, Mya 95% CI

63 100 1.0 46.2 41.6–50.9 64 99 1.0 43.9 39.4–48.6 65 100 1.0 41.1 36.8–45.8 66 100 1.0 24.7 20.0–29.9 67 100 1.0 35.3 30.6–40.2 68 100 1.0 63.6 59.2–68.9 69 100 1.0 34.6 28.7–40.8 70 100 1.0 28.9 23.1–35.4 71 67 1.0 62.1 57.6–67.4 72 100 1.0 48.5 43.2–53.7 73 100 1.0 45.1 40.0–50.4 74 100 1.0 30.2 24.5–36.0 75 100 1.0 43.2 38.2–48.4 76 91 0.84 39.2 34.0–44.6 77 81 0.84 40.7 35.6–45.9 78 81 1.0 59.9 55.5–64.9 79 36 1.0 58.1 53.8–63.1 80 100 1.0 32 24.8–40.0 81 100 1.0 52.8 48.2–58.0 82 100 1.0 36.2 29.6–42.5 83 100 1.0 39 34.0–44.4 84 100 1.0 25.8 21.1–30.8 85 100 1.0 6.2 4.5–8.4 86 100 1.0 48 43.1–53.2 87 100 1.0 35.4 30.8–40.5 88 100 1.0 25.4 22.4–28.9 89 100 1.0 21 18.5–24.0 90 100 1.0 18 15.4–20.9 91 100 1.0 17.1 14.6–20.0 92 87 0.99 16.4 13.9–19.4 93 100 1.0 19.4 16.9–22.5 94 100 1.0 16.5 14.0–19.4 95 100 1.0 12.3 9.7–15.2 96 100 1.0 122.9 114.1–131.0 97 100 1.0 100.9 92.8–108.6 98 100 1.0 67.1 61.0–72.9 99 39 1.0 63.6 57.7–69.2 100 100 1.0 59.6 53.9–65.3 101 100 1.0 48.1 42.1–53.8 102 100 1.0 27.9 23.0–32.8 103 100 1.0 21 16.6–25.9 104 40 1.0 62 56.1–67.4 105 100 1.0 56.8 50.8–62.6 106 100 1.0 32.3 25.2–39.9 107 57 NR 30.3 23.3–38.1 108 100 1.0 47.7 41.3–54.3 109 100 1.0 7.2 5.2–9.8 110 35 1.0 60.9 55.1–66.2 111 100 1.0 54.8 49.2–60.4 112 99 1.0 52.7 47.2–58.2 113 100 1.0 45.9 40.1–51.5 114 100 1.0 15 11.1–20.1 115 100 1.0 20.4 17.6–23.5 116 100 1.0 16.9 14.1–20.0 117 42 NR 19.7 16.9–22.8 118 100 1.0 16.8 14.3–19.8 119 100 1.0 14.2 11.9–16.9 120 100 1.0 12.5 10.1–15.2 121 72 1.0 96.6 88.6–104.1 122 100 1.0 82 73.7–89.7 123 100 1.0 53.8 45.2–62.8 124 100 1.0 34.3 26.7–42.7

Feng et al. www.pnas.org/cgi/content/short/1704632114 4of14 Table S2. Cont. Node BS PP Age, Mya 95% CI

125 100 1.0 63.9 56.7–70.9 126 100 1.0 39.9 31.9–48.0 127 100 1.0 48.9 43.3–55.1 128 92 1.0 46.2 40.7–51.9 129 94 1.0 42.5 36.9–48.3 130 100 1.0 36.9 31.4–42.6 131 100 1.0 34.8 29.2–40.4 132 100 1.0 20.9 16.1–26.5 133 100 1.0 64.5 59.1–69.8 134 56 1.0 63.1 57.7–68.4 135 64 1.0 61 55.5–66.2 136 100 1.0 54.5 48.3–60.1 137 97 1.0 61.3 55.8–66.5 138 80 1.0 58.3 52.7–63.9 139 100 1.0 47 40.1–53.6 140 100 1.0 7.2 5.3–9.6 141 100 1.0 62.7 57.3–67.8 142 100 1.0 39.4 31.2–47.1 143 100 1.0 61.2 56.0–66.4 144 100 1.0 45.9 39.6–52.2 145 91 1.0 42.8 36.8–49.0 146 100 1.0 26.6 21.2–32.8 147 100 1.0 59.1 54.1–64.3 148 100 1.0 53.7 48.6–59.0 149 100 1.0 42.7 35.9–48.9 150 100 1.0 42.5 37.3–48.1 151 100 1.0 26.2 22.0–31.1 152 100 1.0 22.5 18.1–27.2 153 100 1.0 30.6 27.1–34.3 154 100 1.0 26.9 24.0–30.2 155 100 1.0 18.8 14.8–22.9 156 100 1.0 24.4 21.6–27.5 157 100 1.0 17.7 14.9–20.7 158 100 1.0 14.4 11.5–17.4 159 100 1.0 22.2 19.5–25.3 160 100 1.0 16.4 14.2–18.9 161 100 1.0 11.4 8.7–14.1 162 100 1.0 12.6 10.6–14.7 163 100 1.0 6.5 5.1–8.3

The node number corresponds to Fig. S5. BS represents ML bootstrap support value in Fig. S1. PP represents Bayesian posterior probability in Fig. S2. N/A, not applicable.

Table S3. Models and parameters of ancestral range estimation of frogs Model LnL Parameters de jAIC w

DEC −257.30 2 0.13 0.02 — 518.6 5.0 × 10−22 DEC+J −207.26 3 0.02 1.0 × 10−12 0.04 420.5 1

Models include dispersal-extinction cladogenesis (DEC), and the same model allowing for founder event speciation (+J). AIC, Akaike Information Criterion; d, rate of range expansion by adding an area; e,rateof range reduction through extirpation in an area; j, relative per-event weight of jump dispersal at cladogenesis; ln L, log-likelihood; w, Akaike weights. The best-fit model is the DEC+J model.

Feng et al. www.pnas.org/cgi/content/short/1704632114 5of14 Table S4. Comparisons of partitioning schemes using AIC, AICc, and BIC Partitions Rank No. of parameters Ln L AIC AICc BIC

285p 1 2,989 −2,869,254.946 5,743,956.247 5,744,203.285 5,772,548.75 190p 2 2,124 −2,875,870.896 5,755,641.612 5,755,762.151 5,775,929.993 3p 3 357 −2,891,347.478 5,783,408.955 5,783,411.863 5,786,760.486 2p 4 346 −2,895,102.496 5,790,896.993 5,790,899.724 5,794,145.256 95p 5 1,277 −2,919,155.093 5,840,791.627 5,840,830.889 5,852,852.715 1p 6 335 −2,931,059.583 5,862,789.166 5,862,791.726 5,865,934.16

The Ln L and number of parameters are represented with results from BIC.

Feng et al. www.pnas.org/cgi/content/short/1704632114 6of14 Table S5. BioGeoBEARS dispersal multipliers for seven slices

Dispersal rates were assigned as 1 for neighboring areas connected by land bridge, 0.5 for neighboring but not connected areas, and 0.01 for nonneighboring areas or areas separated by large ocean barriers. A, Africa; E, Eurasia (Europe and Asia with exception of Indian plate); I, India-Seychelles; M, Madagascar; N, North America; O, Australia (Australia, New Zealand, and New Guinea); S, South America. The paleogeography maps are based on ref. 42 and from the website jan.ucc.nau.edu/rcb7/index.html.

Feng et al. www.pnas.org/cgi/content/short/1704632114 7of14 Table S6. Taxonomic information and geographical distribution used for biogeographical analysis No. Species Distribution

1 Allophryne ruthveni Allophrynidae South America 2 Alsodes gargola Alsodidae South America 3 Eupsophus calcaratus Alsodidae South America 4 Alytes obstetricans Alytidae Eurasia 5 Discoglossus pictus Alytidae Eurasia 6 Arthroleptis poecilonotus Arthroleptidae Africa 7 Arthroleptis variabilis Arthroleptidae Africa 8 Astylosternus diadematus Arthroleptidae Africa 9 Leptodactylodon ovatus Arthroleptidae Africa 10 Leptopelis kivuensis Arthroleptidae Africa 11 Leptopelis parkeri Arthroleptidae Africa 12 Nyctibates corrugatus Arthroleptidae Africa 13 Scotobleps gabonicus Arthroleptidae Africa 14 Trichobatrachus robustus Arthroleptidae Africa 15 Ascaphus truei Ascaphidae North America 16 Atelognathus reverberii Batrachylidae South America 17 Batrachyla leptopus Batrachylidae South America 18 Batrachyla taeniata Batrachylidae South America 19 Barbourula busuangensis Bombinatoridae Eurasia 20 Bombina fortinuptialis Bombinatoridae Eurasia 21 Bombina orientalis Bombinatoridae Eurasia 22 Brachycephalus ephippium Brachycephalidae South America 23 Ischnocnema guentheri Brachycephalidae South America 24 Breviceps macrops Brevicipitidae Africa 25 Breviceps mossambicus Brevicipitidae Africa 26 Callulina kreffti Brevicipitidae Africa 27 Callulina laphami Brevicipitidae Africa 28 Probreviceps durirostris Brevicipitidae Africa 29 Spelaeophryne methneri Brevicipitidae Africa 30 brauni Bufonidae Africa 31 Sclerophrys maculata Bufonidae Africa 32 Mertensophryne micranotis Bufonidae Africa 33 Nectophrynoides tornieri Bufonidae Africa 34 Schismaderma carens Bufonidae Africa 35 Bufo bufo Bufonidae Eurasia 36 Bufo gargarizans Bufonidae Eurasia 37 Bufo japonicus Bufonidae Eurasia 38 Bufotes viridis Bufonidae Eurasia 39 Epidalea calamita Bufonidae Eurasia 40 Ingerophrynus divergens Bufonidae Eurasia 41 Ingerophrynus galeatus Bufonidae Eurasia 42 Leptophryne borbonica Bufonidae Eurasia 43 Rentapia hosii Bufonidae Eurasia 44 Phrynoidis aspera Bufonidae Eurasia 45 Phrynoidis juxtaspera Bufonidae Eurasia 46 Duttaphrynus melanostictus Bufonidae Eurasia, India-Seychelles 47 Duttaphrynus stomaticus Bufonidae Eurasia, India-Seychelles 48 Adenomus kelaartii Bufonidae India-Seychelles 49 Amazophrynella minuta Bufonidae South America 50 Atelopus peruensis Bufonidae South America 51 Incilius luetkenii Bufonidae South America 52 Incilius nebulifer Bufonidae South America 53 Melanophryniscus stelzneri Bufonidae South America 54 cophotis Bufonidae South America 55 Nannophryne variegata Bufonidae South America 56 Peltophryne lemur Bufonidae South America 57 Peltophryne longinasus Bufonidae South America 58 Peltophryne peltocephala Bufonidae South America 59 Rhaebo glaberrimus Bufonidae South America 60 Rhaebo nasicus Bufonidae South America 61 Rhinella marina Bufonidae South America 62 Rhinella ocellata Bufonidae South America 63 Anaxyrus canorus Bufonidae North America

Feng et al. www.pnas.org/cgi/content/short/1704632114 8of14 Table S6. Cont. No. Species Taxonomy Distribution

64 Anaxyrus punctatus Bufonidae North America 65 Calyptocephalella gayi Calyptocephalellidae South America 66 bacatum Centrolenidae South America 67 Centrolene daidaleum Centrolenidae South America 68 Chimerella mariaelenae Centrolenidae South America 69 Cochranella granulosa Centrolenidae South America 70 prosoblepon Centrolenidae South America 71 Hyalinobatrachium aureoguttatum Centrolenidae South America 72 Hyalinobatrachium colymbiphyllum Centrolenidae South America 73 Hyalinobatrachium ibama Centrolenidae South America 74 Ikakogi tayrona Centrolenidae South America 75 Nymphargus grandisonae Centrolenidae South America 76 adiazeta Centrolenidae South America 77 Rulyrana flavopunctata Centrolenidae South America 78 Sachatamia ilex Centrolenidae South America 79 Teratohyla spinosa Centrolenidae South America 80 Vitreorana helenae Centrolenidae South America 81 Platymantis hazelae Ceratobatrachidae Eurasia 82 Cornufer pelewensis Ceratobatrachidae Eurasia 83 Ceratophrys cornuta Ceratophryidae South America 84 Ceratophrys ornata Ceratophryidae South America 85 Lepidobatrachus laevis Ceratophryidae South America 86 Lepidobatrachus sp. Ceratophryidae South America 87 Ceuthomantis smaragdinus Ceuthomantidae South America 88 Conraua crassipes Conrauidae Africa 89 Craugastor fitzingeri Craugastoridae South America 90 Craugastor podiciferus Craugastoridae South America 91 Craugastor augusti Craugastoridae North America 92 Thoropa taophora Cycloramphidae South America 93 Allobates femoralis Dendrobatidae South America 94 Hyloxalus jacobuspetersi Dendrobatidae South America 95 Dendrobates auratus Dendrobatidae South America 96 Epipedobates tricolor Dendrobatidae South America 97 Phyllobates vittatus Dendrobatidae South America 98 Ranitomeya imitator Dendrobatidae South America 99 Euphlyctis cyanophlyctis Dicroglossidae: Dicroglossinae Eurasia 100 Fejervarya limnocharis Dicroglossidae: Dicroglossinae Eurasia 101 Fejervarya multistriata Dicroglossidae: Dicroglossinae Eurasia 102 Hoplobatrachus tigerinus Dicroglossidae: Dicroglossinae Eurasia 103 Limnonectes fujianensis Dicroglossidae: Dicroglossinae Eurasia 104 Limnonectes laticeps Dicroglossidae: Dicroglossinae Eurasia 105 Limnonectes magnus Dicroglossidae: Dicroglossinae Eurasia 106 Quasipaa spinosa Dicroglossidae: Dicroglossinae Eurasia 107 Fejervarya granosa Dicroglossidae: Dicroglossinae India-Seychelles 108 Liurana xizangensis Dicroglossidae: Occidozyginae Eurasia 109 Occidozyga laevis Dicroglossidae: Occidozyginae Eurasia 110 Occidozyga lima Dicroglossidae: Occidozyginae Eurasia 111 Diasporus diastema Eleutherodactylidae South America 112 Eleutherodactylus coqui Eleutherodactylidae South America 113 Eleutherodactylus planirostris Eleutherodactylidae South America 114 Eleutherodactylus marnockii Eleutherodactylidae North America 115 Heleophryne purcelli Heleophrynidae Africa 116 Cryptobatrachus boulengeri Hemiphractidae South America 117 Flectonotus fitzgeraldi Hemiphractidae South America 118 Gastrotheca pseustes Hemiphractidae South America 119 Gastrotheca weinlandii Hemiphractidae South America 120 Hemiphractus bubalus Hemiphractidae South America 121 ginesi Hemiphractidae South America 122 Hemisus marmoratus Hemisotidae Africa 123 Hyla chinensis Hylidae: Hylinae Eurasia 124 Aplastodiscus perviridis Hylidae: Hylinae South America 125 Dendropsophus parviceps Hylidae: Hylinae South America

Feng et al. www.pnas.org/cgi/content/short/1704632114 9of14 Table S6. Cont. No. Species Taxonomy Distribution

126 Hyloscirtus lindae Hylidae: Hylinae South America 127 Hypsiboas fasciatus Hylidae: Hylinae South America 128 Osteocephalus taurinus Hylidae: Hylinae South America 129 Pseudis paradoxa Hylidae: Hylinae South America 130 Scinax ruber Hylidae: Hylinae South America 131 Trachycephalus typhonius Hylidae: Hylinae South America 132 Acris crepitans Hylidae: Hylinae North America 133 Hyla arenicolor Hylidae: Hylinae North America 134 Hyla cinerea Hylidae: Hylinae North America 135 Cyclorana maini Hylidae: Pelodryadinae Australia 136 Litoria caerulea Hylidae: Pelodryadinae Australia 137 Nyctimystes kubori Hylidae: Pelodryadinae Australia 138 Nyctimystes pulcher Hylidae: Pelodryadinae Australia 139 Agalychnis callidryas Hylidae: Phyllomedusinae South America 140 Agalychnis lemur Hylidae: Phyllomedusinae South America 141 Agalychnis saltator Hylidae: Phyllomedusinae South America 142 Cruziohyla calcarifer Hylidae: Phyllomedusinae South America 143 Phyllomedusa hypochondrialis Hylidae: Phyllomedusinae South America 144 Phyllomedusa tomopterna Hylidae: Phyllomedusinae South America 145 nasus Hylodidae South America 146 Afrixalus dorsalis Hyperoliidae Africa 147 Hyperolius bolifambae Hyperoliidae Africa 148 Hyperolius viridiflavus Hyperoliidae Africa 149 Phlyctimantis boulengeri Hyperoliidae Africa 150 Leiopelma hochstetteri Leiopelmatidae Australia 151 Physalaemus pustulosus Leptodactylidae South America 152 Leptodactylus albilabris Leptodactylidae South America 153 Leptodactylus latrans Leptodactylidae South America 154 Leptodactylus melanonotus Leptodactylidae South America 155 Lithodytes lineatus Leptodactylidae South America 156 Physalaemus cuvieri Leptodactylidae South America 157 Pleurodema thaul Leptodactylidae South America 158 Pleurodema somuncurensis Leptodactylidae South America 159 Boophis madagascariensis Mantellidae: Boophinae Madagascar 160 Boophis xerophilus Mantellidae: Boophinae Madagascar 161 Aglyptodactylus madagascariensis Mantellidae: Laliostominae Madagascar 162 Blommersia wittei Mantellidae: Mantellinae Madagascar 163 Mantella madagascariensis Mantellidae: Mantellinae Madagascar 164 Brachytarsophrys feae Megophryidae Eurasia 165 Leptobrachium chapaense Megophryidae Eurasia 166 Leptolalax alpinus Megophryidae Eurasia 167 Megophrys nasuta Megophryidae Eurasia 168 Ophryophryne microstoma Megophryidae Eurasia 169 Oreolalax jingdongensis Megophryidae Eurasia 170 Scutiger gongshanensis Megophryidae Eurasia 171 Xenophrys omeimontis Megophryidae Eurasia 172 Micrixalus sp. Micrixalidae India-Seychelles 173 Gastrophrynoides immaculatus Microhylidae: Asterophryinae Eurasia 174 Callulops wilhelmanus Microhylidae: Asterophryinae Australia 175 Cophixalus cheesmanae Microhylidae: Asterophryinae Australia 176 Cophixalus cryptotympanum Microhylidae: Asterophryinae Australia 177 Cophixalus sp. A Microhylidae: Asterophryinae Australia 178 Cophixalus sp. B Microhylidae: Asterophryinae Australia 179 Hylophorbus rufescens Microhylidae: Asterophryinae Australia 180 Liophryne schlaginhaufeni Microhylidae: Asterophryinae Australia 181 Mantophryne lateralis Microhylidae: Asterophryinae Australia 182 Oreophryne sp. A Microhylidae: Asterophryinae Australia 183 Oreophryne sp. B Microhylidae: Asterophryinae Australia 184 Xenorhina obesa Microhylidae: Asterophryinae Australia 185 Xenorhina sp. Microhylidae: Asterophryinae Australia 186 Anodonthyla boulengerii Microhylidae: Cophylinae Madagascar 187 Platypelis tuberifera Microhylidae: Cophylinae Madagascar

Feng et al. www.pnas.org/cgi/content/short/1704632114 10 of 14 Table S6. Cont. No. Species Taxonomy Distribution

188 Plethodontohyla inguinalis Microhylidae: Cophylinae Madagascar 189 Stumpffia pygmaea Microhylidae: Cophylinae Madagascar 190 Dyscophus antongilii Microhylidae: Dyscophinae Madagascar 191 Chiasmocleis ventrimaculata Microhylidae: Gastrophryninae South America 192 Ctenophryne geayi Microhylidae: Gastrophryninae South America 193 Elachistocleis ovalis Microhylidae: Gastrophryninae South America 194 Stereocyclops incrassatus Microhylidae: Gastrophryninae South America 195 Gastrophryne olivacea Microhylidae: Gastrophryninae North America 196 rogersi Microhylidae: Hoplophryninae Africa 197 Kalophrynus interlineatus Microhylidae: Kalophryninae Eurasia 198 Kalophrynus pleurostigma Microhylidae: Kalophryninae Eurasia 199 Melanobatrachus indicus Microhylidae: Melanobatrachinae India-Seychelles 200 Calluella guttulata Microhylidae: Microhylinae Eurasia 201 Chaperina fusca Microhylidae: Microhylinae Eurasia 202 Kaloula conjuncta Microhylidae: Microhylinae Eurasia 203 Kaloula pulchra Microhylidae: Microhylinae Eurasia 204 pollicaris Microhylidae: Microhylinae Eurasia 205 Microhylidae: Microhylinae Eurasia 206 Microhyla annectens Microhylidae: Microhylinae Eurasia 207 Microhyla heymonsi Microhylidae: Microhylinae Eurasia 208 Microhyla marmorata Microhylidae: Microhylinae Eurasia 209 Microhyla okinavensis Microhylidae: Microhylinae Eurasia 210 Micryletta inornata Microhylidae: Microhylinae Eurasia 211 pulchra Microhylidae: Microhylinae Eurasia 212 montanus Microhylidae: Microhylinae India-Seychelles 213 Uperodon variegatus Microhylidae: Microhylinae India-Seychelles 214 Microhylidae: Microhylinae India-Seychelles 215 Otophryne pyburni Microhylidae: Otophryninae South America 216 Synapturanus sp. Microhylidae: Otophryninae South America 217 Phrynomantis microps Microhylidae: Phrynomerinae Africa 218 Paradoxophyla palmata Microhylidae: Scaphiophryninae Madagascar 219 Scaphiophryne boribory Microhylidae: Scaphiophryninae Madagascar 220 Scaphiophryne madagascariensis Microhylidae: Scaphiophryninae Madagascar 221 Scaphiophryne marmorata Microhylidae: Scaphiophryninae Madagascar 222 Limnodynastes salmini Myobatrachidae: Limnodynastinae Australia 223 Mixophyes coggeri Myobatrachidae: Limnodynastinae Australia 223 Mixophyes coggeri Myobatrachidae: Limnodynastinae Australia 224 Crinia signifera Myobatrachidae: Myobatrachinae Australia 225 Myobatrachus gouldii Myobatrachidae: Myobatrachinae Australia 226 Uperoleia laevigata Myobatrachidae: Myobatrachinae Australia 227 Nasikabatrachus sahyadrensis Nasikabatrachidae India-Seychelles 228 Lankanectes corrugatus Nyctibatrachidae India-Seychelles 229 Nyctibatrachus sp. Nyctibatrachidae India-Seychelles 230 Odontobatrachus natator Odontobatrachidae Africa 231 Odontophrynus occidentalis Odontophrynidae South America 232 Odontophrynidae South America 233 Pelobates cultripes Pelobatidae Eurasia 234 Pelobates syriacus Pelobatidae Eurasia 235 Pelodytes ibericus Pelodytidae Eurasia 236 Petropedetes euskircheni Petropedetidae Africa 237 Petropedetes parkeri Petropedetidae Africa 238 Phrynobatrachus africanus Phrynobatrachidae Africa 239 Phrynobatrachus krefftii Phrynobatrachidae Africa 240 Phrynobatrachus natalensis Phrynobatrachidae Africa 241 Hymenochirus boettgeri Pipidae Africa 242 Pseudhymenochirus merlini Pipidae Africa 243 Xenopus epitropicalis Pipidae Africa 244 Xenopus kobeli Pipidae Africa 245 Pipa parva Pipidae South America 246 Pipa pipa Pipidae South America 247 Ptychadena cooperi Ptychadenidae Africa 248 Ptychadena mascareniensis Ptychadenidae Africa

Feng et al. www.pnas.org/cgi/content/short/1704632114 11 of 14 Table S6. Cont. No. Species Taxonomy Distribution

249 Ptychadena oxyrhynchus Ptychadenidae Africa 250 Amietia lubrica Pyxicephalidae: Cacosterninae Africa 251 Strongylopus grayii Pyxicephalidae: Cacosterninae Africa 252 Aubria subsigillata Pyxicephalidae: Africa 253 edulis Pyxicephalidae: Pyxicephalinae Africa 254 Amolops loloensis Ranidae Eurasia 255 Amolops ricketti Ranidae Eurasia 256 Babina chapaensis Ranidae Eurasia 257 Babina pleuraden Ranidae Eurasia 258 Sylvirana guentheri Ranidae Eurasia 259 Papurana latouchii Ranidae Eurasia 260 Hydrophylax leptoglossa Ranidae Eurasia 261 Chalcorana macrops Ranidae Eurasia 262 Papurana sp. Ranidae Eurasia 263 Meristogenys kinabaluensis Ranidae Eurasia 264 Odorrana hosii Ranidae Eurasia 265 Odorrana schmackeri Ranidae Eurasia 266 Pelophylax nigromaculatus Ranidae Eurasia 267 Rana amurensis Ranidae Eurasia 268 Rana chensinensis Ranidae Eurasia 269 Rana japonica Ranidae Eurasia 270 Rana temporaria Ranidae Eurasia 271 Sanguirana luzonensis Ranidae Eurasia 272 Staurois latopalmatus Ranidae Eurasia 273 Rana catesbeiana Ranidae North America 274 Rana draytonii Ranidae North America 275 Rana pipiens Ranidae North America 276 Rana virgatipes Ranidae North America 277 Rana berlandieri Ranidae North America, South America 278 sp. A Ranixalidae India-Seychelles 279 Indirana sp. B Ranixalidae India-Seychelles 280 Buergeria buergeri Rhacophoridae: Buergeriinae Eurasia 281 Buergeria oxycephala Rhacophoridae: Buergeriinae Eurasia 282 Kurixalus odontotarsus Rhacophoridae: Rhacophorinae Eurasia 283 Polypedates megacephalus Rhacophoridae: Rhacophorinae Eurasia 284 Rhacophorus dennysi Rhacophoridae: Rhacophorinae Eurasia 285 Pseudophilautus wynaadensis Rhacophoridae: Rhacophorinae India-Seychelles 286 Insuetophrynus acarpicus Rhinodermatidae South America 287 Rhinoderma darwinii Rhinodermatidae South America 288 Rhinophrynus dorsalis Rhinophrynidae North America 289 Scaphiopus couchii Scaphiopodidae North America 290 Scaphiopus holbrookii Scaphiopodidae North America 291 Spea intermontana Scaphiopodidae North America 292 Spea multiplicata Scaphiopodidae North America 293 Sooglossus thomasseti Sooglossidae India-Seychelles 294 Barycholos pulcher Strabomantidae South America 295 Hypodactylus brunneus Strabomantidae South America 296 Phrynopus bracki Strabomantidae South America 297 Pristimantis thymelensis Strabomantidae South America 298 Strabomantis biporcatus Strabomantidae South America 299 Strabomantis sulcatus Strabomantidae South America 300 Batrachophrynus macrostomus Telmatobiidae South America 301 Telmatobius vellardi Telmatobiidae South America 302 Andrias davidianus Cryptobranchidae Eurasia 303 Batrachuperus yenyuanensis Hynobiidae Eurasia

Fig. S1. ML analysis result of the 164-species data set (95 loci, 88,302 bp). The tree was inferred by concatenation ML analysis (i.e., RAxML) using a partitioned scheme (codon position partitioning across 95 loci). Values beside nodes are bootstrap support. Nodes without support values indicated all have >90% bootstrap support.

Fig. S1

Feng et al. www.pnas.org/cgi/content/short/1704632114 12 of 14 Fig. S2. Bayesian analysis result of the 164-species data set (95 loci, 88,302 bp). The tree was inferred by Bayesian method [using a partitioned scheme (codon position partitioning across 95 loci)]. Values beside nodes are Bayesian posterior probabilities. Nodes without support values indicated all have Bayesian posterior probabilities of 1.0.

Fig. S2

Fig. S3. Species tree result of the 164-species data set (95 loci, 88,302 bp). The tree was inferred by species-tree analysis using the program ASTRAL based on the 95 nuclear genes without data concatenation. Branch support values besides nodes are bootstrap supports.

Fig. S3

Fig. S4. Skeletal representation of the 309-species tree from ML analysis, with tips representing families and subfamilies. Numbers at nodes are bootstrap support values. Note that the species names colored by blue indicated that sequence data of these species was obtained from GenBank.

Fig. S4

Fig. S5. MCMCTree result of 164 species data set with node number at each node. The detail information of each node is provided in Table S2.

Fig. S5

Fig. S6. MCMCTree result of 309 species data set with mean node ages and 95% highest posterior density intervals depicted as a bar at each node.

Fig. S6

Fig. S7. Placement and information on all 20 calibration points used in this study.

Fig. S7

Fig. S8. Effect of excluding each calibration point on the resulting divergence time estimates. Divergence times were recalculated after a given calibration point was removed and compared with the times estimated with all 20 calibrations. Calibration numbers are provided in Fig. S7. (A) The overall relative age difference, summarized from 143 nodes of the 163 total nodes (excluding the 20 calibrated nodes). (B) Mean ages and 95% CIs of the three critical nodes (Hyloidea, Microhylidae, and Natatanura). The colored dashed lines are the mean ages estimates with all 20 calibrations. Note that the removal of individual calibration points has a minor effect on the divergence time estimation.

Fig. S8

Fig. S9. Comparison of age estimates of major clades in Anura node ages and 95% credible intervals from selected studies for five selected clades (credible intervals were not provided in some). The dark gray vertical bar marks the KPB. The white circles indicate ages estimated in this study for two different datasets; note in particular the short duration of 95% credible intervals and the overlap of the KPB by the intervals of Hyloidea, Microhylidae, and Natatanura.

Fig. S9

Fig. S10. Evolutionary chronogram of frogs based on 95 nuclear genes and 7 constraints (without all constraints in frogs). Nodal values are mean time es- timates, and the blue bars represent the 95% CI of the node age. MCMCTree (uncorrelated model) was used to infer the times by using only calibration points outside frrgs (shown as red triangles in the lower left chronogram). Note that the synchronicity of the three major frog clades near the KPB still exists.

Fig. S10

Feng et al. www.pnas.org/cgi/content/short/1704632114 13 of 14 Fig. S11. Biogeographic analysis results from BioGeoBEARS under the best fitting model (DEC+J). The pie charts at each node represent the respective percentage ancestral state likelihoods.

Fig. S11

Feng et al. www.pnas.org/cgi/content/short/1704632114 14 of 14 Outgroup Leiopelma hochstetteri Leiopelmatidae Ascaphus truei Ascaphidae Leiopelmatoidea Discoglossus pictus Alytidae Alytes obstetricans Barbourula busuangensis Bombina orientalis Bombinatoridae Discoglossoidea Bombina fortinuptialis Anura Rhinophrynus dorsalis Rhinophrynidae Pipa pipa Pipa parva Pseudhymenochirus merlini Pipidae Pipoidea Hymenochirus boettgeri Xenopus kobeli Xenopus epitropicalis Scaphiopus couchii Spea multiplicata Scaphiopodidae Spea intermontana Pelodytes ibericus Pelodytidae Pelobates syriacus Pelobatidae Brachytarsophrys feae Pelobatoidea Xenophrys omeimontis Ophryophryne microstoma Leptolalax alpinus Megophryidae Scutiger gongshanensis Oreolalax jingdongensis Leptobrachium chapaense Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae Mixophyes coggeri Myobatrachoidea Crinia signifera Myobatrachidae Limnodynastes salmini Insuetophrynus acarpicus Rhinodermatidae Rhinoderma darwinii Alsodes gargola Eupsophus calcaratus Alsodidae Atelognathus reverberii Batrachyla taeniata Batrachylidae Batrachyla leptopus Batrachophrynus macrostomus Telmatobiidae Telmatobius vellardi Lepidobatrachus sp. Ceratophryidae Ceratophrys cornuta Cryptobatrachus boulengeri Gastrotheca pseustes Hemiphractidae Gastrotheca weinlandii Nyctimystes kubori Phyllomedusa tomopterna Neobatrachia Agalychnis callidryas Agalychnis lemur Hyloscirtus lindae Aplastodiscus perviridis Hypsiboas fasciatus Hylidae Scinax ruber Osteocephalus taurinus Hyla chinensis Hyloidea Acris crepitans Dendropsophus parviceps Pseudis paradoxa Allobates femoralis Hyloxalus jacobuspetersi Dendrobatidae Ranitomeya imitator Eleutherodactylus planirostris Eleutherodactylidae Craugastor augusti Craugastoridae Craugastor fitzingeri Pristimantis thymelensis Barycholos pulcher Strabomantis sulcatus Strabomantidae 81 Hypodactylus brunneus Proceratophrys boiei Odontophrynidae 67 Odontophrynus occidentalis 36 Leptodactylus albilabris Lithodytes lineatus Physalaemus pustulosus Leptodactylidae Anura Physalaemus cuvieri 81 Pleurodema somuncurensis Pleurodema thaul Melanophryniscus stelzneri Amazophrynella minuta Peltophryne peltocephala Schismaderma carens Rentapia hosii Bufo gargarizans Bufonidae 87 Duttaphrynus melanostictus Rhinella marina Incilius nebulifer Anaxyrus canorus Anaxyrus punctatus Sooglossus thomasseti Sooglossidae Phrynomantis microps Otophryne pyburni Chiasmocleis ventrimaculata Stereocyclops incrassatus Elachistocleis ovalis Gastrophryne olivacea Stumpffia pygmaea 39 Platypelis tuberifera 57 Anodonthyla boulengerii Paradoxophyla palmata Scaphiophryne boribory 40 Scaphiophryne marmorata Kalophrynus pleurostigma Microhylidae Dyscophus antongilii Microhyla heymonsi 35 Kaloula conjuncta Kaloula pulchra Cophixalus cheesmanae Oreophryne sp. Liophryne schlaginhaufeni 42 Xenorhina sp. Callulops wilhelmanus Mantophryne lateralis Afrobatrachia Cophixalus sp. Hemisus marmoratus Hemisotidae Breviceps macrops Brevicipitidae Callulina kreffti Phlyctimantis boulengeri Hyperoliidae Hyperolius bolifambae Leptopelis parkeri Ranoidea Leptodactylodon ovatus Arthroleptis poecilonotus Nyctibates corrugatus Arthroleptidae Scotobleps gabonicus Trichobatrachus robustus Astylosternus diadematus 72 64 Odontobatrachus natator Odontobatrachidae Ptychadena oxyrhynchus Ptychadenidae 56 Phrynobatrachus natalensis Phrynobatrachidae Petropedetes euskircheni Petropedetidae 80 Conraua crassipes Conrauidae Aubria subsigillata Strongylopus grayii Pyxicephalidae Amietia lubrica Liurana xizangensis Ceratobatrachidae Natatanura Cornufer pelewensis Quasipaa spinosa Limnonectes fujianensis Hoplobatrachus tigerinus Dicroglossidae Fejervarya limnocharis Boophis madagascariensis Aglyptodactylus madagascariensis Mantellidae Buergeria oxycephala Kurixalus odontotarsus Rhacophoridae Polypedates megacephalus Rhacophorus dennysi Pelophylax nigromaculatus Amolops ricketti Amolops loloensis Sylvirana guentheri Papurana sp. Papurana latouchii Ranidae Odorrana schmackeri Rana berlandieri Rana virgatipes 0.2 Rana draytonii Rana amurensis Rana chensinensis Outgroup Leiopelma hochstetteri Leiopelmatidae Ascaphus truei Ascaphidae Leiopelmatoidea Discoglossus pictus Alytidae Alytes obstetricans Barbourula busuangensis Bombina orientalis Bombinatoridae Discoglossoidea Bombina fortinuptialis Rhinophrynus dorsalis Rhinophrynidae Pipa pipa Anura Pipa parva Pseudhymenochirus merlini Pipidae Pipoidea Hymenochirus boettgeri Xenopus kobeli Xenopus epitropicalis Scaphiopus couchii Spea multiplicata Scaphiopodidae Spea intermontana Pelodytes ibericus Pelodytidae Pelobates syriacus Pelobatidae Brachytarsophrys feae Pelobatoidea Xenophrys omeimontis Ophryophryne microstoma Leptolalax alpinus Megophryidae Scutiger gongshanensis Oreolalax jingdongensis Leptobrachium chapaense Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae Mixophyes coggeri Myobatrachoidea Crinia signifera Myobatrachidae Limnodynastes salmini Insuetophrynus acarpicus Rhinodermatidae Rhinoderma darwinii Alsodes gargola Eupsophus calcaratus Alsodidae Atelognathus reverberii Batrachyla taeniata Batrachylidae Batrachyla leptopus Batrachophrynus macrostomus Telmatobiidae Telmatobius vellardi Lepidobatrachus sp. Ceratophryidae Ceratophrys cornuta Cryptobatrachus boulengeri Gastrotheca pseustes Hemiphractidae Gastrotheca weinlandii Nyctimystes kubori Phyllomedusa tomopterna Neobatrachia Agalychnis callidryas Agalychnis lemur Hyloscirtus lindae Aplastodiscus perviridis Hypsiboas fasciatus Hylidae Scinax ruber Osteocephalus taurinus Acris crepitans Hyloidea Hyla chinensis Dendropsophus parviceps Pseudis paradoxa Allobates femoralis Hyloxalus jacobuspetersi Dendrobatidae Ranitomeya imitator Eleutherodactylus planirostris Eleutherodactylidae Craugastor augusti Craugastoridae Craugastor fitzingeri Pristimantis thymelensis 0.84 Barycholos pulcher Strabomantis sulcatus Strabomantidae 0.84 Hypodactylus brunneus Proceratophrys boiei Odontophrynidae Odontophrynus occidentalis Leptodactylus albilabris Lithodytes lineatus Physalaemus pustulosus Leptodactylidae Anura Physalaemus cuvieri Pleurodema somuncurensis Pleurodema thaul Melanophryniscus stelzneri Amazophrynella minuta Peltophryne peltocephala Schismaderma carens Rentapia hosii Bufo gargarizans Bufonidae Duttaphrynus melanostictus Rhinella marina Hymenochirus boettgeri Anaxyrus canorus Anaxyrus punctatus Sooglossus thomasseti Sooglossidae Phrynomantis microps Otophryne pyburni Chiasmocleis ventrimaculata Stereocyclops incrassatus Elachistocleis ovalis Gastrophryne olivacea Anodonthyla boulengerii Platypelis tuberifera 0.59 Stumpffia pygmaea Paradoxophyla palmata Scaphiophryne boribory Scaphiophryne marmorata Kalophrynus pleurostigma Microhylidae Dyscophus antongilii Microhyla heymonsi Kaloula conjuncta Kaloula pulchra Liophryne schlaginhaufeni Cophixalus cheesmanae Oreophryne sp. 0.68 Xenorhina sp. Callulops wilhelmanus Mantophryne lateralis Afrobatrachia Cophixalus sp. Hemisus marmoratus Hemisotidae Breviceps macrops Brevicipitidae Callulina kreffti Phlyctimantis boulengeri Hyperoliidae Hyperolius bolifambae Leptopelis parkeri Ranoidea Leptodactylodon ovatus Arthroleptis poecilonotus Nyctibates corrugatus Arthroleptidae Scotobleps gabonicus Trichobatrachus robustus Astylosternus diadematus Odontobatrachus natator Odontobatrachidae Ptychadena oxyrhynchus Ptychadenidae Phrynobatrachus natalensis Phrynobatrachidae Petropedetes euskircheni Petropedetidae Conraua crassipes Conrauidae Aubria subsigillata Strongylopus grayii Pyxicephalidae Amietia lubrica Liurana xizangensis Ceratobatrachidae Natatanura Cornufer pelewensis Quasipaa spinosa Limnonectes fujianensis Hoplobatrachus tigerinus Dicroglossidae Fejervarya limnocharis Boophis madagascariensis Aglyptodactylus madagascariensis Mantellidae Buergeria oxycephala Kurixalus odontotarsus Rhacophoridae Polypedates megacephalus Rhacophorus dennysi Pelophylax nigromaculatus Amolops ricketti Amolops loloensis Sylvirana guentheri Papurana sp. Papurana latouchii Ranidae Odorrana schmackeri Rana berlandieri Rana virgatipes Rana draytonii 0.09 Rana amurensis Rana chensinensis Outgroup 100 Ascaphus truei Ascaphidae Leiopelma hochstetteri Leiopelmatidae Leiopelmatoidea 100 Discoglossus pictus Alytes obstetricans Alytidae 100 Barbourula busuangensis Discoglossoidea 100 Bombinatoridae 100 Bombina orientalis 100 Bombina fortinuptialis Anura Rhinophrynus dorsalis Rhinophrynidae 100 100 Pipa pipa Pipa parva 100 100 Hymenochirus boettgeri Pipoidea Pseudhymenochirus merlini Pipidae 100 100 100 Xenopus epitropicalis Xenopus kobeli Scaphiopus couchii 100 Scaphiopodidae 100 Spea multiplicata Spea intermontana 100 Pelodytes ibericus Pelodytidae 100 Pelobates syriacus Pelobatidae Brachytarsophrys feae 100 100 100 Pelobatoidea 100 Xenophrys omeimontis 100 Ophryophryne microstoma Leptolalax alpinus Megophryidae 100 Scutiger gongshanensis 100 59 Leptobrachium chapaense Oreolalax jingdongensis Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae 100 Mixophyes coggeri 100 Myobatrachoidea 67.5 Limnodynastes salmini Myobatrachidae Crinia signifera 100 100 Insuetophrynus acarpicus Rhinodermatidae 100 Rhinoderma darwinii 100 Alsodes gargola Alsodidae Eupsophus calcaratus 100 Atelognathus reverberii 100 Batrachylidae 100 100 Batrachyla leptopus Batrachyla taeniata 100 Telmatobius vellardi Telmatobiidae Batrachophrynus macrostomus 100 Lepidobatrachus sp. Ceratophryidae 96.5 Ceratophrys cornuta 24 Cryptobatrachus boulengeri 100 Hemiphractidae 100 Gastrotheca pseustes Gastrotheca weinlandii 100 60 Nyctimystes kubori 100 Phyllomedusa tomopterna Neobatrachia 65.5 100 100 Agalychnis lemur Agalychnis callidryas 100 Hyloscirtus lindae 100 Hypsiboas fasciatus 100 Hylidae 100 Aplastodiscus perviridis Scinax ruber 100 Osteocephalus taurinus Acris crepitans 82.5 95 100 99 Hyla chinensis 99 Dendropsophus parviceps Pseudis paradoxa Hyloidea Eleutherodactylus planirostris Eleutherodactylidae 100 100 Craugastor augusti Craugastor fitzingeri Craugastoridae 100 Strabomantis sulcatus 96 Hypodactylus brunneus 14 Strabomantidae 34.5 Pristimantis thymelensis 100 88 Barycholos pulcher Allobates femoralis 100 Dendrobatidae 100 Ranitomeya imitator Hyloxalus jacobuspetersi 100 Odontophrynus occidentalis Odontophrynidae 30.5 Proceratophrys boiei 100 Lithodytes lineatus Leptodactylus albilabris 85.5 100 100 Pleurodema somuncurensis Leptodactylidae 100 Pleurodema thaul 100 Physalaemus pustulosus 19.5 Physalaemus cuvieri Melanophryniscus stelzneri 100 Amazophrynella minuta 100 Peltophryne peltocephala Schismaderma carens 100 100 Bufo gargarizans 94.5 Bufonidae 35 Rentapia hosii 100 Duttaphrynus melanostictus Rhinella marina 98 Incilius nebulifer 100 100 Anaxyrus punctatus Anaxyrus canorus Sooglossus thomasseti Sooglossidae Otophryne pyburni 94.5 Chiasmocleis ventrimaculata 100 Stereocyclops incrassatus 100 100 Gastrophryne olivacea 100 Elachistocleis ovalis Phrynomantis microps Stumpffia pygmaea 100 40.5 Anodonthyla boulengerii 20.5 97.5 Platypelis tuberifera 46 Paradoxophyla palmata 99.5 100 Scaphiophryne boribory 35 Scaphiophryne marmorata Microhylidae Kalophrynus pleurostigma Dyscophus antongilii 23 82 Microhyla heymonsi 99.5 100 Kaloula pulchra 100 Kaloula conjuncta 100 Cophixalus cheesmanae Oreophryne sp. 100 100 Liophryne schlaginhaufeni 49.5 Xenorhina sp. 100 Callulops wilhelmanus 100 99.5 Mantophryne lateralis Cophixalus sp. Hemisus marmoratus Hemisotidae 100 100 Callulina kreffti Brevicipitidae Afrobatrachia Breviceps macrops 100 100 Hyperolius bolifambae Hyperoliidae Phlyctimantis boulengeri Leptodactylodon ovatus 100 54 15.5 Arthroleptis poecilonotus 100 Leptopelis parkeri Ranoidea Nyctibates corrugatus Arthroleptidae 100 Scotobleps gabonicus 62.5 91 100 Trichobatrachus robustus Astylosternus diadematus 38 Phrynobatrachus natalensis Phrynobatrachidae Ptychadena oxyrhynchus Ptychadenidae Petropedetes euskircheni Petropedetidae 43.5 13.5 Conraua crassipes Conrauidae 100 Odontobatrachus natator Odontobatrachidae 9.5 Aubria subsigillata Natatanura 100 100 Amietia lubrica Pyxicephalidae 20 Strongylopus grayii 100 Liurana xizangensis Ceratobatrachidae Cornufer pelewensis Quasipaa spinosa 100 Limnonectes fujianensis 99.5 62.5 Dicroglossidae 100 Fejervarya limnocharis Hoplobatrachus tigerinus 100 Boophis madagascariensis Mantellidae 89.5 Aglyptodactylus madagascariensis 100 Buergeria oxycephala 100 Kurixalus odontotarsus 100 Rhacophoridae 100 Rhacophorus dennysi 89.5 Polypedates megacephalus Pelophylax nigromaculatus 100 Amolops ricketti 100 Amolops loloensis Sylvirana guentheri 100 100 100 Papurana latouchii 100 Papurana sp. Ranidae Odorrana schmackeri 100 100 Rana virgatipes Rana berlandieri 100 Rana draytonii 100 100 Rana chensinensis Rana amurensis Latimeriidae Protopteridae Hominidae 100 Crocodylidae 100 Outgroup Phasianidae 100 Ichthyophiidae Hynobiidae 100 100 Cryptobranchidae Ascaphidae 100 Leiopelmatoidea 100 Leiopelmatidae Alytidae 100 100 Discoglossoidea Bombinatoridae Anura Rhinophrynidae 100 Pipoidea Pipidae 100 Scaphiopodidae 100 Pelodytidae 100 Pelobatidae Pelobatoidea 100 100 Megophryidae Heleophrynidae Calyptocephalellidae 100 Myobatrachoidea Myobatrachidae

100 100 Rhinodermatidae Alsodidae 100 35 Hylodidae 63 Hyloidea Batrachylidae Telmatobiidae 100 100 Ceratophryidae Hemiphractidae Neobatrachia 78 77 34 Hylinae 90 Hylidae 100 Pelodryadinae Phyllomedusinae 33 Dendrobatidae Ceuthomantidae 94 Brachycephalidae 90 Eleutherodactylidae 25 68 Craugastoridae 100 82 Strabomantidae Cycloramphidae 19 29 Odontophrynidae 13 Leptodactylidae 9 Allophrynidae 77 22 Centrolenidae Bufonidae Nasikabatrachidae 100 Sooglossoidea Sooglossidae 41 Hoplophryninae Phrynomerinae 100 Otophryninae 79 Gastrophryninae Scaphiophryninae 100 97 Microhylidae 21 Cophylinae

17 Kalophryninae Dyscophinae 71 20 Microhylinae 100 50 Melanobatrachinae Ranoidea 34 Asterophryinae Hemisotidae 100 Brevicipitidae 100 Hyperoliidae 100 Arthroleptidae Afrobatrachia 28 Odontobatrachidae Phrynobatrachidae 79 49 20 Ptychadenidae Pyxicephalidae 84 Conrauidae 100 74 Petropedetidae Natatanura Ceratobatrachidae Dicroglossidae 25 Micrixalidae 56 Ranixalidae 14 33 Mantellidae 94 Rhacophoridae 0.2 subst./site 13 Nyctibatrachidae 17 Ranidae Latimeria chalumnae

Protopterus annectens

Homo sapiens 100 Crocodylus siamensis Outgroup 100 Gallus gallus Ichthyophis bannanicus 100

100 Batrachuperus yenyuanensis Andrias davidianus 100 Ascaphus truei Ascaphidae 100 Leiopelma hochstetteri Leiopelmatidae

Alytes obstetricans 100 100 Alytidae Discoglossus pictus 100 Barbourula busuangensis 100 100 Bombina orientalis Bombinatoridae 100 Anura Bombina fortinuptialis Rhinophrynus dorsalis Rhinophrynidae

Pipa parva 100 100 Pipa pipa

100 100 Xenopus epitropicalis 100 Pipidae Xenopus kobeli 100 Hymenochirus boettgeri 100 Pseudhymenochirus merlini

Spea intermontana 100

100 Spea multiplicata Scaphiopodidae Scaphiopus holbrookii 100 100 100 Scaphiopus couchii Pelodytes ibericus Pelodytidae

Pelobates syriacus 100 Pelobatidae 100 Pelobates cultripes Brachytarsophrys feae 100 100 Megophrys nasuta

99 Ophryophryne microstoma 98 100 Xenophrys omeimontis Megophryidae 100 Leptolalax alpinus

100 Scutiger gongshanensis

100 Oreolalax jingdongensis 100 Leptobrachium chapaense

Heleophryne purcelli Heleophrynidae

Calyptocephalella gayi Calyptocephalellidae 100 Mixophyes coggeri 100 100 Limnodynastes salmini Neobatrachia 100 Crinia signifera Myobatrachidae

100 Myobatrachus gouldii 100 100 Uperoleia laevigata 100 B-C

A 0.09 D-E 100 Insuetophrynus acarpicus Rhinodermatidae Rhinoderma darwinii 100 Alsodes gargola Hyloidea Alsodidae 35 Eupsophus calcaratus Hylodes nasus Hylodidae Atelognathus reverberii 63 100 100 Batrachyla taeniata Batrachylidae 100 Batrachyla leptopus 100 Batrachophrynus macrostomus Telmatobiidae Telmatobius vellardi 100 Lepidobatrachus laevis 100 Lepidobatrachus sp. Ceratophrys ornata Ceratophryidae 100 100 Ceratophrys cornuta Cryptobatrachus boulengeri 100 Flectonotus fitzgeraldi

74 Hemiphractus bubalus Hemiphractidae 77 Stefania ginesi 77 76 100 Gastrotheca weinlandii Gastrotheca pseustes 100 Nyctimystes pulcher

34 100 Nyctimystes kubori 100 Cyclorana maini 78 100 Litoria caerulea Cruziohyla calcarifer 100 100 Phyllomedusa hypochondrialis Phyllomedusa tomopterna 82 Agalychnis lemur

90 100 100 Agalychnis saltator Agalychnis callidryas

100 Hyloscirtus lindae Hylidae Hypsiboas fasciatus 100 100 Aplastodiscus perviridis Scinax ruber 33 100 Trachycephalus typhonius 100 Osteocephalus taurinus 100 Pseudis paradoxa 87 Dendropsophus parviceps 100 Acris crepitans 100 Hyla chinensis 100 100 Hyla cinerea Hyla arenicolor Allobates femoralis 100 19 Epipedobates tricolor Hyloxalus jacobuspetersi Dendrobatidae 67 Phyllobates vittatus 75 96 Ranitomeya imitator Dendrobates auratus Ceuthomantis smaragdinus Ceuthomantidae 93 Brachycephalus ephippium Brachycephalidae 94 Ischnocnema guentheri 25 Eleutherodactylus marnockii 100 Eleutherodactylus planirostris 90 Eleutherodactylidae 65 80 Diasporus diastema Eleutherodactylus coqui

68 100 Craugastor augusti 99 Craugastor fitzingeri Craugastoridae Craugastor podiciferus 19 Barycholos pulcher 82 80 Pristimantis thymelensis 100 Strabomantis sulcatus 78 Strabomantidae Strabomantis biporcatus 52 Phrynopus bracki 32 Hypodactylus brunneus

B C 0.09 B Thoropa taophora Cycloramphidae 29 100 Proceratophrys boiei Odontophrynidae Odontophrynus occidentalis

100 Pleurodema somuncurensis 13 99 Pleurodema thaul 100 Physalaemus cuvieri

98 Physalaemus pustulosus Leptodactylidae Lithodytes lineatus 100 Leptodactylus albilabris 9 100 100 Leptodactylus latrans Leptodactylus melanonotus Allophryne ruthveni Allophrynidae 65 Cochranella granulosa

77 21 Hyalinobatrachium ibama Hyalinobatrachium colymbiphyllum 52 Hyalinobatrachium aureoguttatum 83 Nymphargus grandisonae 47 Ikakogi tayrona Centrolene daidaleum 22 25 86 Centrolene bacatum Centrolenidae

62 Teratohyla spinosa 97 Rulyrana flavopunctata 59 Rulyrana adiazeta 61 Chimerella mariaelenae

36 Sachatamia ilex 32 Vitreorana helenae 47 Espadarana prosoblepon Melanophryniscus stelzneri 94 Atelopus peruensis 94 Amazophrynella minuta Nannophryne cophotis 98 Nannophryne variegata Rhaebo nasicus 63 92 81 Rhaebo glaberrimus Peltophryne lemur 100 38 Peltophryne longinasus 100 Peltophryne peltocephala

93 Rhinella ocellata Rhinella marina 99 92 94 Incilius nebulifer Incilius luetkenii 99 Anaxyrus punctatus 95 Anaxyrus canorus 98 Schismaderma carens 19 Nectophrynoides tornieri 41 Bufonidae Phrynoidis aspera 70 Phrynoidis juxtaspera 92 Rentapia hosii Bufotes viridis 16 Adenomus kelaartii 43 Duttaphrynus stomaticus 19 85 Duttaphrynus melanostictus Bufo bufo 12 100 Bufo japonicus 100 Bufo gargarizans Epidalea calamita 38 55 Leptophryne borbonica 19 Ingerophrynus divergens 98 Ingerophrynus galeatus 45 Mertensophryne micranotis 85 Sclerophrys maculata 98 Sclerophrys brauni

C 0.09 A 100 Nasikabatrachus sahyadrensis Nasikabatrachidae Sooglossus thomasseti Sooglossidae

41 Hoplophryne rogersi Hoplophryninae Phrynomantis microps Phrynomerinae Synapturanus sp. 94 Otophryne pyburni Otophryninae 79 Ctenophryne geayi 78 Microhylidae Chiasmocleis ventrimaculata 96 100 Stereocyclops incrassatus Gastrophryninae 100 Gastrophryne olivacea 100 Elachistocleis ovalis Paradoxophyla palmata 100 Scaphiophryne boribory 100 Scaphiophryninae 21 Scaphiophryne madagascariensis 97 100 Scaphiophryne marmorata Anodonthyla boulengerii 24 100 Stumpffia pygmaea Plethodontohyla inguinalis Cophylinae 96 Platypelis tuberifera 100 100 Kalophrynus interlineatus Kalophryninae 17 Kalophrynus pleurostigma Melanobatrachus indicus Melanobatrachinae 34 Gastrophrynoides immaculatus Liophryne schlaginhaufeni 100 Oreophryne sp. B 99 100 20 Oreophryne sp. A 99 Cophixalus cryptotympanum 100 Cophixalus cheesmanae 98 Xenorhina obesa Asterophryinae 69 Xenorhina sp. 100 Callulops wilhelmanus 50 78 Mantophryne lateralis 50 Cophixalus sp. A 48 Hylophorbus rufescens 93 Cophixalus sp. B Dyscophus antongilii Dyscophinae 100 Micryletta inornata 51 Calluella guttulata 100 71 100 Microhyla heymonsi Microhyla okinavensis 94 100 Microhyla annectens 81 Microhyla marmorata Chaperina fusca

100 Phrynella pulchra Microhylinae 13 Metaphrynella sundana 100 Metaphrynella pollicaris 100 Kaloula pulchra 100 Kaloula conjuncta 78 Uperodon systoma Uperodon montanus 99 52 Afrobatrachia Uperodon variegatus Hemisus marmoratus Hemisotidae 100 100 Breviceps macrops 100 Breviceps mossambicus Spelaeophryne methneri Brevicipitidae 100 Probreviceps durirostris 63 Callulina laphami 100 Callulina kreffti 100 Phlyctimantis boulengeri 100 Afrixalus dorsalis 100 Hyperoliidae 100 Hyperolius bolifambae 100 Hyperolius viridiflavus 96 Leptopelis parkeri Leptopelis kivuensis 100 Leptodactylodon ovatus 79 94 100 Arthroleptis variabilis Arthroleptis poecilonotus Arthroleptidae 87 Nyctibates corrugatus 100 Scotobleps gabonicus 100 Trichobatrachus robustus 100 Astylosternus diadematus

E D 0.09 D Odontobatrachus natator Odontobatrachidae

28 100 Phrynobatrachus krefftii 100 Phrynobatrachus africanus Phrynobatrachidae 49 Phrynobatrachus natalensis

100 Ptychadena mascareniensis 20 Ptychadena cooperi 91 Ptychadenidae Ptychadena oxyrhynchus Conraua crassipes Conrauidae 74 100 Petropedetes euskircheni Petropedetidae Petropedetes parkeri 84 100 Pyxicephalus edulis Aubria subsigillata 100 100 Strongylopus grayii Pyxicephalidae 100 Amietia lubrica Liurana xizangensis Natatanura 100 Platymantis hazelae Ceratobatrachidae 100 Cornufer pelewensis 100 Occidozyga laevis Occidozyga lima 100 Quasipaa spinosa

100 Limnonectes laticeps Limnonectes magnus 28 65 25 Limnonectes fujianensis Dicroglossidae 97 100 Euphlyctis cyanophlyctis

100 Hoplobatrachus tigerinus Fejervarya granosa 100 100 Fejervarya multistriata Fejervarya limnocharis Micrixalus sp. Micrixalidae 56 14 100 Indirana sp. B Ranixalidae Indirana sp. A Boophis xerophilus 98 33 100 Boophis madagascariensis Aglyptodactylus madagascariensis Mantellidae 44 Mantella madagascariensis 100 94 Blommersia wittei 100 Buergeria oxycephala Buergeria buergeri Rhacophorus dennysi 13 100 99 Polypedates megacephalus Rhacophoridae 100 Pseudophilautus wynaadensis 99 Kurixalus odontotarsus Nyctibatrachus sp. 26 Nyctibatrachidae Lankanectes corrugatus

17 Staurois latopalmatus Meristogenys kinabaluensis 100 Pelophylax nigromaculatus Amolops ricketti 100 100 Amolops loloensis 78 Sanguirana luzonensis 33 Babina pleuraden 100 Babina chapaensis 84 78 Chalcorana macrops Hydrophylax leptoglossa 43 Sylvirana guentheri 84 77 Papurana latouchii 76 Ranidae 99 Papurana sp. 100 Odorrana hosii Odorrana schmackeri 100 Rana pipiens Rana berlandieri 91 100 Rana catesbeiana 100 Rana virgatipes 100 Rana draytonii 41 Rana chensinensis 100 Rana temporaria 100 Rana japonica 73 Rana amurensis

E 0.09 450 400 350 300 250 200 150 100 50 0 (Mya)

Silurian Devonian Carboniferous Permian Triassic Jurassic Cretaceous Paleogene Neogene Latimeria chalumnae Latimeriidae 1 Protopterus annectens Protopteridae Homo sapiens Hominidae 4 2 5 Crocodylus siamensis Crocodylidae Gallus gallus Phasianidae Outgroup 3 Ichthyophis bannanicus Ichthyophiidae 8 Batrachuperus yenyuanensis Cryptobranchidae Andrias davidianus Hynobiidae 6 10 Leiopelma hochstetteri Leiopelmatidae Lissamphibia Ascaphus truei Ascaphidae Leiopelmatoidea 7 13 Discoglossus pictus Alytes obstetricans Alytidae Batrachia 12 Barbourula busuangensis 14 Bombinatoroidea 15 Bombina orientalis Bombinatoridae 9 Bombina fortinuptialis Anura Rhinophrynus dorsalis Rhinophrynidae 17 19 Pipa pipa Pipa parva 18 Pseudhymenochirus merlini 21 Pipoidea Hymenochirus boettgeri Pipidae 11 20 22 Xenopus kobeli Bombinanura Xenopus epitropicalis Scaphiopus couchii 25 26 Spea multiplicata Scaphiopodidae 24 Spea intermontana Pelodytes ibericus Pelodytidae 27 Pelobates syriacus Pelobatidae Brachytarsophrys feae 16 28 30 31 Xenophrys omeimontis Pelobatoidea Pipanura 29 Ophryophryne microstoma Leptolalax alpinus Megophryidae 32 Scutiger gongshanensis 33 34 Leptobrachium chapaense Oreolalax jingdongensis Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae 38 Mixophyes coggeri 39 40 Crinia signifera Myobatrachidae Limnodynastes salmini 23 42 Insuetophrynus acarpicus Acosmanura 37 Rhinoderma darwinii Rhinodermatidae Alsodes gargola 45 Alsodidae Eupsophus calcaratus 44 Atelognathus reverberii 41 46 47 Batrachyla taeniata Batrachylidae Batrachyla leptopus 49 Batrachophrynus macrostomus Telmatobius vellardi Telmatobiidae 43 52 Lepidobatrachus sp. Ceratophrys cornuta Ceratophryidae Cryptobatrachus boulengeri 54 51 55 Gastrotheca pseustes Hemiphractidae Gastrotheca weinlandii 35 Nyctimystes kubori 48 53 57 Phyllomedusa tomopterna Neobatrachia 58 59 Agalychnis callidryas Agalychnis lemur 56 Hyloscirtus lindae 61 62 Aplastodiscus perviridis 60 Hypsiboas fasciatus Hylidae Scinax ruber 50 63 Osteocephalus taurinus 64 66 Hyla chinensis 65 Acris crepitans 67 Dendropsophus parviceps Pseudis paradoxa Allobates femoralis 69 70 Hyloxalus jacobuspetersi Dendrobatidae Hyloidea Ranitomeya imitator Eleutherodactylus planirostris Eleutherodactylidae Craugastor augusti 72 74 Craugastoridae 68 Craugastor fitzingeri 73 36 76 Pristimantis thymelensis Barycholos pulcher 75 Strabomantidae 77 Strabomantis sulcatus Hypodactylus brunneus 71 80 Proceratophrys boiei Odontophrynus occidentalis Odontophrynidae 79 82 Leptodactylus albilabris Lithodytes lineatus 81 84 Physalaemus pustulosus Physalaemus cuvieri Leptodactylidae 78 83 85 Pleurodema somuncurensis Pleurodema thaul Melanophryniscus stelzneri 86 Amazophrynella minuta 87 Peltophryne peltocephala Schismaderma carens 88 90 Rentapia hosii 91 92 Bufo gargarizans Bufonidae 89 Duttaphrynus melanostictus Rhinella marina 93 Incilius nebulifer 94 95 Anaxyrus canorus Anaxyrus punctatus Sooglossus thomasseti Sooglossidae Phrynomantis microps Otophryne pyburni 100 Chiasmocleis ventrimaculata 98 101 Stereocyclops incrassatus 102 103 Elachistocleis ovalis Gastrophryne olivacea Stumpffia pygmaea 99 106 107 Platypelis tuberifera 105 Anodonthyla boulengerii 96 Paradoxophyla palmata 108 109 Scaphiophryne boribory 104 Scaphiophryne marmorata Kalophrynus pleurostigma Microhylidae Dyscophus antongilii 110 112 Microhyla heymonsi 113 114 Kaloula conjuncta 111 Kaloula pulchra 116 Cophixalus cheesmanae Oreophryne sp. 115 97 Liophryne schlaginhaufeni 117 Xenorhina sp. 118 Callulops wilhelmanus 119 120 Mantophryne lateralis Afrobatrachia Cophixalus sp. Hemisus marmoratus 123 Hemisotidae 124 Breviceps macrops Callulina kreffti Brevicipitidae 122 126 Phlyctimantis boulengeri Hyperolius bolifambae Hyperoliidae 125 Leptopelis parkeri 127 129 Leptodactylodon ovatus Arthroleptis poecilonotus 128 130 Nyctibates corrugatus Arthroleptidae Scotobleps gabonicus 131 132 Trichobatrachus robustus 121 Astylosternus diadematus Ranoidea Odontobatrachus natator Odontobatrachidae 135 136 Ptychadena oxyrhynchus Ptychadenidae Phrynobatrachus natalensis 134 Phrynobatrachidae 138 Petropedetes euskircheni Petropedetidae Conraua crassipes 137 Conrauidae Aubria subsigillata 139 140 Strongylopus grayii Pyxicephalidae 133 Amietia lubrica Natatanura 142 Liurana xizangensis Cornufer pelewensis Ceratobatrachidae 144 Quasipaa spinosa Limnonectes fujianensis 141 145 Dicroglossidae 146 Hoplobatrachus tigerinus Fejervarya limnocharis 149 Boophis madagascariensis 143 Aglyptodactylus madagascariensis Mantellidae 148 Buergeria oxycephala 150 Kurixalus odontotarsus 151 152 Polypedates megacephalus Rhacophoridae 147 Rhacophorus dennysi Pelophylax nigromaculatus 153 155 Amolops ricketti Amolops loloensis Sylvirana guentheri 154 157 158 Papurana sp. 156 Papurana latouchii Odorrana schmackeri Ranidae 159 161 Rana berlandieri 160 Rana virgatipes Rana draytonii 162 163 Rana amurensis Rana chensinensis

450 400 350 300 250 200 150 100 50 0 450 400 350 300 250 200 150 100 50 0 (Mya)

Silurian Devonian Carboniferous Permian Triassic Jurassic Cretaceous Paleogene Neo

Latimeria chalumnae Latimeriidae Protopterus annectens Protopteridae 432.24 Homo sapiens Hominidae 319.32 419.65 Crocodylus siamensis Crocodylidae 253.09 Gallus gallus Phasianidae 348.26 Ichthyophis bannanicus Ichthyophiidae Batrachuperus yenyuanensis Cryptobranchidae 156.14 Andrias davidianus Hynobiidae 297.31 Ascaphus truei Leiopelmatidae 204.25 Leiopelma hochstetteri Ascaphidae 270.72 Alytes obstetricans 114.63 Alytidae Discoglossus pictus 148.63 Barbourula busuangensis 42.02 Bombina orientalis Bombinatoridae 219.9 9.54 Bombina fortinuptialis Rhinophrynus dorsalis Rhinophrynidae Pipa parva 165.49 11.51 Pipa pipa 117.67 Xenopus epitropicalis 203.26 43.99 Xenopus kobeli Pipidae 103.74 Hymenochirus boettgeri 54.11 Pseudhymenochirus merlini Spea intermontana 9.04 Spea multiplicata 42.8 Scaphiopodidae Scaphiopus holbrookii 14.43 191.91 Scaphiopus couchii 137.01 Pelodytes ibericus Pelodytidae Pelobates syriacus 6.52 Pelobatidae 122.18 Pelobates cultripes Brachytarsophrys feae 87.94 25.33 Megophrys nasuta 22.45 Ophryophryne microstoma 16.22 52.18 Xenophrys omeimontis Megophryidae 181.66 Leptolalax alpinus 33.07 Scutiger gongshanensis 18.53 Oreolalax jingdongensis 15.45 Leptobrachium chapaense Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae 100.58 Mixophyes coggeri 76.34 Limnodynastes salmini 141.7 69.75 Crinia signifera Myobatrachidae 118.28 37.96 Myobatrachus gouldii 24.2 B-C Uperoleia laevigata 128.44 D-E

450 400 350 300 250 200 150 100 50 0 (Mya)

A 150 100 50 0 (Mya)

Cretaceous Paleogene Neogene

Insuetophrynus acarpicus A 60.75 Rhinodermatidae Rhinoderma darwinii Alsodes gargola 19.28 Alsodidae Eupsophus calcaratus 60.65 Hylodes nasus Hylodidae 52.04 Atelognathus reverberii 26.63 Batrachyla taeniata Batrachylidae 71.93 13.11 Batrachyla leptopus Batrachophrynus macrostomus 4.69 Telmatobiidae Telmatobius vellardi Lepidobatrachus laevis 4.52 Lepidobatrachus sp. 19.16 Ceratophryidae Ceratophrys ornata 11.29 67.29 Ceratophrys cornuta Cryptobatrachus boulengeri 43.81 Flectonotus fitzgeraldi 40.7 Hemiphractus bubalus 63.26 35.65 Hemiphractidae Stefania ginesi 31.58 Gastrotheca weinlandii 17.53 Gastrotheca pseustes Nyctimystes pulcher 9.29 Nyctimystes kubori 61.99 36.19 Pelodryadinae Cyclorana maini 65.63 25.04 Litoria caerulea 47.38 Cruziohyla calcarifer Phyllomedusa hypochondrialis 27.24 11.25 Phyllomedusa tomopterna 24.54 Phyllomedusinae Agalychnis lemur 17.09 58.21 Agalychnis saltator 3.54 Agalychnis callidryas Hylidae Hyloscirtus lindae 31.18 Hypsiboas fasciatus 24.27 Aplastodiscus perviridis 48.54 Scinax ruber Trachycephalus typhonius 23.52 45.32 Osteocephalus taurinus 64.17 Hylinae 43.14 Pseudis paradoxa 34.48 Dendropsophus parviceps 40.4 Acris crepitans 26.81 Hyla chinensis 17.31 Hyla cinerea 8.6 Hyla arenicolor Allobates femoralis

36.85 Epipedobates tricolor 27.13 Hyloxalus jacobuspetersi 31.32 Dendrobatidae Phyllobates vittatus 22.36 Ranitomeya imitator 17.6 Dendrobates auratus Ceuthomantis smaragdinus Ceuthomantidae Brachycephalus ephippium 35.08 Brachycephalidae 53.92 Ischnocnema guentheri Eleutherodactylus marnockii 33.54 Eleutherodactylus planirostris 50.14 32.14 Eleutherodactylidae Diasporus diastema 26.08 Eleutherodactylus coqui 48.21 63.07 Craugastor augusti 32.47 Craugastor fitzingeri Craugastoridae 17.83 Craugastor podiciferus 45.11 Barycholos pulcher 39.22 Pristimantis thymelensis 43.19 Strabomantis sulcatus 10.26 Strabomantidae Strabomantis biporcatus 61.74 40.78 Phrynopus bracki 35.59 Hypodactylus brunneus C B 150 100 50 0 (Mya) 50 0 (Mya)

Cretaceous Paleogene Neogene B Thoropa taophora Cycloramphidae 50.7 Proceratophrys boiei 32.89 Odontophrynidae Odontophrynus occidentalis Pleurodema somuncurensis 57.28 6.01 Pleurodema thaul 38.47 Physalaemus cuvieri 25.31 52.02 Physalaemus pustulosus Leptodactylidae Lithodytes lineatus 37.46 Leptodactylus albilabris 57.9 25.74 Leptodactylus latrans 14.2 Leptodactylus melanonotus Allophryne ruthveni Allophrynidae Cochranella granulosa 8.89 Hyalinobatrachium ibama 40.76 17.73 Hyalinobatrachium colymbiphyllum 11.6 Hyalinobatrachium aureoguttatum 25.7 Nymphargus grandisonae 16.79 Ikakogi tayrona Centrolene daidaleum 59.66 21.99 12.06 Centrolene bacatum Centrolenidae 17.53 Teratohyla spinosa Rulyrana flavopunctata 15.09 6.15 Rulyrana adiazeta 11.91 Chimerella mariaelenae 10.07 Sachatamia ilex 8.45 Vitreorana helenae 7.12 Espadarana prosoblepon Melanophryniscus stelzneri Atelopus peruensis 48.93 Amazophrynella minuta 42.66 Nannophryne cophotis 35.55 Nannophryne variegata Rhaebo nasicus 32.75 17.96 Rhaebo glaberrimus 23.11 Peltophryne lemur 31.1 11.76 Peltophryne longinasus 6.14 Peltophryne peltocephala Rhinella ocellata 17.19 Rhinella marina 25.24 19.97 Incilius nebulifer 10.23 Incilius luetkenii 16.85 Anaxyrus punctatus 12.52 Anaxyrus canorus 21.56 Schismaderma carens 15.29 Nectophrynoides tornieri Bufonidae 12.73 Phrynoidis aspera 7.34 18.84 Phrynoidis juxtaspera Rentapia hosii Bufotes viridis 18.03 Adenomus kelaartii 14.12 Duttaphrynus stomaticus 17.47 11.16 Duttaphrynus melanostictus

17.12 Bufo bufo 9.23 Bufo japonicus 7.16 Bufo gargarizans 16.46 Epidalea calamita 12.16 Leptophryne borbonica 15.9 Ingerophrynus divergens 7.45 Ingerophrynus galeatus 15.05 Mertensophryne micranotis 13.66 Sclerophrys maculata 7.58 Sclerophrys brauni 50 0 (Mya) C 150 100 50 0 (Mya) Jurassic A Cretaceous Paleogene Neogene Nasikabatrachus sahyadrensis Nasikabatrachidae 65.96 Sooglossus thomasseti Sooglossidae Hoplophryne rogersi Hoplophryninae 58.52 Phrynomantis microps Phrynomerinae Synapturanus sp. 44.19 Otophryninae Otophryne pyburni 62.96 Ctenophryne geayi 44.57 Chiasmocleis ventrimaculata 50.47 70.41 Stereocyclops incrassatus Gastrophryninae 28.9 Gastrophryne olivacea 21.8 Elachistocleis ovalis Paradoxophyla palmata 50.71 Scaphiophryne boribory 9.23 Scaphiophryninae 67.24 Scaphiophryne madagascariensis 4.78 59.93 Scaphiophryne marmorata Anodonthyla boulengerii 33.82 Stumpffia pygmaea 36.08 Cophylinae Plethodontohyla inguinalis 25.11 Platypelis tuberifera Kalophrynus interlineatus 3.66 Kalophryninae 121.46 65.84 Kalophrynus pleurostigma Melanobatrachus indicus Melanobatrachinae

54.61 Gastrophrynoides immaculatus Liophryne schlaginhaufeni 38.75 Oreophryne sp. B Microhylidae 11.14 64.83 Oreophryne sp. A 21.81 17.55 Cophixalus cryptotympanum 9.48 Cophixalus cheesmanae 20.83 Xenorhina obesa Asterophryinae 13.64 Xenorhina sp. 17.54 60.03 Callulops wilhelmanus 14.89 Mantophryne lateralis 13.2 Cophixalus sp. A 12.72 Hylophorbus rufescens 8.68 Cophixalus sp. B Dyscophus antongilii Dyscophinae Micryletta inornata 50.07 Calluella guttulata 58.17 Microhyla heymonsi 41.26 15 100.68 Microhyla okinavensis 34.6 Microhyla annectens 53.12 16.27 Microhyla marmorata Chaperina fusca Phrynella pulchra Microhylinae 49.21 17.63 Metaphrynella sundana 7.01 Metaphrynella pollicaris 32.48 Kaloula pulchra 16.79 Kaloula conjuncta 26.43 Uperodon systoma 18.98 Uperodon montanus 14.02 Uperodon variegatus Hemisus marmoratus Hemisotidae Breviceps macrops 55.3 18.96 Breviceps mossambicus 37.57 Spelaeophryne methneri 26.73 Brevicipitidae Probreviceps durirostris 24.51 Callulina laphami 10.64 82.42 Callulina kreffti Phlyctimantis boulengeri 43.87 Afrixalus dorsalis Hyperoliidae 24.61 Hyperolius bolifambae 10.8 Hyperolius viridiflavus 65.45 Leptopelis parkeri 39.67 Leptopelis kivuensis Leptodactylodon ovatus 50.4 44.08 Arthroleptis variabilis 9.93 47.69 Arthroleptis poecilonotus Arthroleptidae Nyctibates corrugatus 37.73 Scotobleps gabonicus 35.64 Trichobatrachus robustus 20.84 Astylosternus diadematus 96.48 E

150 100 50 0 (Mya) D 100 50 0 (Mya)

Cretaceous Paleogene Neogene D Odontobatrachus natator Odontobatrachidae Phrynobatrachus krefftii 62.94 43.29 Phrynobatrachus africanus 15.17 Phrynobatrachidae 56.88 Phrynobatrachus natalensis Ptychadena mascareniensis 24.88 Ptychadena cooperi Ptychadenidae 64.94 19.26 Ptychadena oxyrhynchus Conraua crassipes Conrauidae 59.95 Petropedetes euskircheni 15.22 Petropedetidae Petropedetes parkeri Pyxicephalidae 62.98 Pyxicephalus edulis 22.36 Pyxicephalinae Aubria subsigillata 49.56 Strongylopus grayii 7.22 Cacosterninae Amietia lubrica 66.28 Liurana xizangensis 40.24 Platymantis hazelae Ceratobatrachidae 31.27 Cornufer pelewensis Occidozyga laevis 21.53 Occidozyginae Occidozyga lima 52.32 Quasipaa spinosa Dicroglossidae Limnonectes laticeps 45.43 20.88 Limnonectes magnus 16.24 64.46 Limnonectes fujianensis Dicroglossinae 42.48 Euphlyctis cyanophlyctis 18.13 Hoplobatrachus tigerinus 28.53 Fejervarya granosa 22.16 Fejervarya multistriata 5.41 Fejervarya limnocharis Micrixalus sp. Micrixalidae 63.05 51.35 Indirana sp. B 27.99 Ranixalidae Indirana sp. A

Boophis xerophilus Mantellidae 21.86 Boophinae 58.93 Boophis madagascariensis 43.35 Aglyptodactylus madagascariensis Laliostominae 41.44 Mantella madagascariensis 24.71 Mantellinae 54.07 Blommersia wittei

Buergeria oxycephala Rhacophoridae 22.18 Buergeriinae Buergeria buergeri 42.21 Rhacophorus dennysi 60.89 23.45 Polypedates megacephalus 27.22 Rhacophorinae Pseudophilautus wynaadensis 22.96 Kurixalus odontotarsus Nyctibatrachus sp. 33.77 Nyctibatrachidae Lankanectes corrugatus 56.8 Staurois latopalmatus Meristogenys kinabaluensis 42.86 Pelophylax nigromaculatus 35.35 Amolops ricketti 19.01 Amolops loloensis 30.29 Sanguirana luzonensis 19.86 Babina pleuraden 3.96 Babina chapaensis 22.48 27.08 Chalcorana macrops 19.21 Hydrophylax leptoglossa 13.79 Sylvirana guentheri 16.05 Papurana latouchii 12.9 Ranidae 24.8 Papurana sp. Odorrana hosii 7.19 Odorrana schmackeri Rana pipiens 3.66 22.72 Rana berlandieri 12.38 Rana catesbeiana 4.51 17.58 Rana virgatipes Rana draytonii Rana chensinensis 14.09 5.8 Rana temporaria 8.83 Rana japonica 5.22 Rana amurensis

100 50 0 (Mya) E Latimeriidae Protopteridae Hominidae 1 3 Crocodylidae 4 Phasianidae 2 Ichthyophiidae Tetrapoda Hynobiidae 7 5 Cryptobranchidae Amphibia Leiopelmatoidea Leiopelmatidae 6 Ascaphidae Batrachia Alytoidea Alytidae 9 8 Bombinatoridae Anura Pipoidea Rhinophrynidae 11 Pipa 12 Xenopus Bombinanura Pseudhymenochirus Hymenochirus Pelobatoidea Scaphiopodidae 10 13 Pipanura Pelodytidae 14 Pelobatidae 15 Megophryidae Heleophrynidae Calyptocephalellidae 16 18 Acosmanura Myobatrachidae Rhinodermatidae Alsodidae Batrachylidae 17 Neobatrachia Telmatobiidae Ceratophryidae Hyloidea Hemiphractidae Hylidae Dendrobatidae Eleutherodactylidae Phthanobatrachia Craugastoridae Strabomantidae Bufonidae Odontophrynidae Leptodactylidae Sooglossidae Microhylidae Hemisotidae Diplasiocoela Afrobatrachia Brevicipitidae 19 Hyperoliidae Ranoidea Arthroleptidae Odontobatrachidae Ptychadenidae 20 Phrynobatrachidae Pyxicephalidae Petropedetidae Natatanura Conrauidae Ceratobatrachidae Dicroglossidae Ranidae Mantellidae Rhacophoridae

Num Node to be calibrated Fossils Minimum Soft Maximum Source 1 Choanata † Youngolepis 408 427 Benton et al. (2015) 2 Tetrapoda † Lethiscus stocki 337 351 Benton et al. (2015) 3 Amniota † Hylonomus lyelli 318 332.9 Benton et al. (2015) 4 Crocodile and birds † Ctenosauriscus koeneni 247.1 260.2 Benton et al. (2015) 5 Amphibia (Liss-) † Gerobatrachus hottoni 270.6 337 Anderson (2008) 6 Batrachia † Triadobatrachus massinoti 252 272.8 Cannatella (2015); Benton et al. 2015 7 Cryptobranchoidea † Chunerpeton tianyiensis 161.2 252 Gao and Shubin (2003) 8 Anura † Liaobatrachus zhaoi 129.7 252 Chang et al. (2009) 9 Alytoidea † Iberobatrachus angelae 125 252 Gomez et al. (2016) 10 Pipanura † Rhadinosteus parvus 148.1 252 Cannatella (2015) 11 Pipoidea † Neusibatrachus wilferti 127.2 252 Gomez et al. (2016) 12 Pipidae † Pachycentra taqueti 83.6 148.1 Cannatella (2015) 13 Pelobatoidea † Elkobatrachus brocki 46.1 148.1 Henrici and Haynes (2006) 14 Pelodytes + (Pelobatidae + Megophryidae) † Miopelodytes gilmorei 38.9 148.1 Henrici and Haynes (2006) 15 Pelobatidae + Megophryidae † Macropelobates osborni 28.1 148.1 Cohen et al. (2013) 16 Acosmanura † Eurycephalella alcinae 113 252 Baez (2009) 17 Neobatrachia † Beelzebufo ampinga 66 148.1 Rogers et al. (2013) 18 Myobatrachoidea † Calyptocephalella pichileufensis 47.5 148.1 Gomez et al. (2011) 19 Ranoidea † Thamastosaurus gezei 33.9 148.1 Rage and Rocek (2007) 20 Node between Ptychadena + Phrynobatrachus Ptychadenidae fossil 25 148.1 Blackburn et al. (2015) References

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Calibration point removed Anura Neobatrachia Hyloidea Microhylidae Natatanura 0 Neogene 50 e Paleogen 100 s Cretaceou 150 Jurassic Age (Mya) 200 c riassi T 250 n Permia 300 de Sá (2012) de Sá (2012) de Sá (2012) de Sá (2012) de Sá (2012) i et al. (2012) i et al. (2012) i et al. (2012) i et al. (2012) i et al. (2012) Wiens (2011) Wiens (2011) Wiens (2011) Wiens (2011) Wiens (2011) e et al. (2009) e et al. (2009) e et al. (2009) e et al. (2009) e et al. (2009) ashi et al. (2011) ashi et al. (2011) ashi et al. (2011) ashi et al. (2011) ashi et al. (2011) isarr isarr isarr isarr isarr az�o et al. (2015) az�o et al. (2015) az�o et al. (2015) az�o et al. (2015) az�o et al. (2015) y y y y y Cannatella (2015) Cannatella (2015) Cannatella (2015) Cannatella (2015) Cannatella (2015) dataset this study dataset this study dataset this study dataset this study dataset this study dataset this study dataset this study dataset this study dataset this study dataset this study dataset Ir Ir Ir Ir Ir Fr Fr Fr Fr Fr Bossuyt et al. (2006) Bossuyt et al. (2006) Bossuyt et al. (2006) Bossuyt et al. (2006) Bossuyt et al. (2006) Bossuyt et al. Heinick Heinick Heinick Heinick Heinick aba aba aba aba aba Roelants et al. (2007) Roelants et al. (2007) Roelants et al. (2007) Roelants et al. (2007) Roelants et al. (2007) Roelants et al. San Mauro et al. (2005) San Mauro et al. (2005) San Mauro et al. (2005) San Mauro et al. (2005) San Mauro et al. (2005) San Mauro et ur ur ur ur ur Biju and Bossuyt (2003) Biju and Bossuyt (2003) Biju and Bossuyt (2003) Biju and Bossuyt (2003) Biju and Bossuyt (2003) Biju and an Bocxlaer et al. (2006) an Bocxlaer et al. (2006) an Bocxlaer et al. (2006) an Bocxlaer et al. (2006) an Bocxlaer et al. (2006) an Bocxlaer et K K K K K v v v v v an der Meijden et al. (2007) an der Meijden et al. (2007) an der Meijden et al. (2007) an der Meijden et al. (2007) an der Meijden et al. (2007) an der Meijden Zhang et al. (2013), BEAST Zhang et al. (2013), BEAST Zhang et al. (2013), BEAST Zhang et al. (2013), BEAST Zhang et al. (2013), BEAST v v v v v 164 species 164 species 164 species 164 species 309 species 309 species 309 species 309 species s 164 specie s 309 specie Zhang et al. (2013), Multidivtime Zhang et al. (2013), Multidivtime Zhang et al. (2013), Multidivtime Zhang et al. (2013), Multidivtime Zhang et al. (2013), Multidivtime References

Biju SD, Bossuyt F (2003) New frog family from India reveals an ancient biogeographical link with the Seychelles. Nature, 425, 711–714. Bossuyt F, Brown R, Hillis D, Cannatella D, Milinkovitch M (2006) Phylogeny and biogeography of a cosmopolitan frog radiation: Late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae. Systematic Biology, 55, 579–594. Cannatella D (2015) Xenopus in space and time: fossils, node calibrations, tip-dating, and paleobiogeography. Cytogenetic and Genome Research, 145, 283–301. de Sá RO, Streicher JW, Sekonyela R et al. (2012) Molecular phylogeny of microhylid frogs (Anura: Microhylidae) with emphasis on relationships among New World genera. BMC Evolutionary Biology, 12, 241. Frazão A, Silva HR da, Russo CA de M (2015) The gondwana breakup and the history of the Atlantic and Indian oceans unveils two new clades for early neobatrachian diversification (A Rzhetsky, Ed,). PLOS ONE, 10, e0143926. Heinicke MP, Duellman WE, Trueb L et al. (2009) A new frog family (Anura: Terrarana) from South America and an expanded direct-developing clade revealed by molecular phylogeny. Zootaxa, 35, 1–35. Irisarri I, Mauro DS, Abascal F et al. (2012) The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates. BMC Genomics, 13, 626. Kurabayashi A, Matsui M, Belabut DM et al. (2011) From Antarctica or Asia? New colonization scenario for Australian-New Guinean narrow mouth toads suggested from the findings on a mysterious genus Gastrophrynoides. BMC Evolutionary Biology, 11, 175. van Bocxlaer I, Roelants K, Biju SD, Nagaraju J, Bossuyt F (2006) Late Cretaceous vicariance in Gondwanan amphibians. PLOS ONE, 1, e74. van der Meijden A, Vences M, Hoegg S et al. (2007) Nuclear gene phylogeny of narrow-mouthed toads (Family: Microhylidae) and a discussion of competing hypotheses concerning their biogeographical origins. Molecular Phylogenetics and Evolution, 44, 1017–1030. Roelants K, Gower DJ, Wilkinson M et al. (2007) Global patterns of diversification in the history of modern amphibians. Proceedings of the National Academy of Sciences, 104, 887–892. San Mauro D, Vences M, Alcobendas M, Zardoya R, Meyer A (2005) Initial diversification of living amphibians predated the breakup of Pangaea. The American Naturalist, 165, 590–599. Wiens JJ (2011) Re-evolution of lost mandibular teeth in frogs after more than 200 million years, and re-evaluating dollo’s law. Evolution, 65, 1283–1296. Zhang P, Liang D, Mao R-L et al. (2013) Efficient sequencing of anuran mtdnas and a mitogenomic exploration of the phylogeny and evolution of frogs. Molecular Biology and Evolution, 30, 1899– 1915.

200 150 100 50 0 (Mya) Triassic Jurassic Cretaceous Paleogene Neogene

201.5 Leiopelma hochstetteri Leiopelmatidae Ascaphus truei Ascaphidae Discoglossus pictus 106.4 Alytidae Alytes obstetricans 144.3 Barbourula busuangensis 43.7 10.3 Bombina orientalis Bombinatoridae 218.1 Bombina fortinuptialis Anura Rhinophrynus dorsalis Rhinophrynidae 165.7 11.9 Pipa pipa Pipa parva 121.5 60.2 Pseudhymenochirus merlini Hymenochirus boettgeri Pipidae 202.1 107.8 Xenopus kobeli Bombinanura 46.9 Xenopus epitropicalis Scaphiopus couchii 44.2 9.6 Spea multiplicata Scaphiopodidae Spea intermontana 144 Pelodytes ibericus Pelodytidae 129.5 Pelobates syriacus Pelobatidae Brachytarsophrys feae 190.3 98.6 27.2 18.6 Xenophrys omeimontis Pipanura 60.7 Ophryophryne microstoma Leptolalax alpinus Megophryidae 36.9 Scutiger gongshanensis 20.4 17 Leptobrachium chapaense Oreolalax jingdongensis Heleophryne purcelli Heleophrynidae Calyptocephalella gayi Calyptocephalellidae 104.2 Mixophyes coggeri 74.3 67.2 Crinia signifera Myobatrachidae Limnodynastes salmini 180.6 Insuetophrynus acarpicus 62.6 Rhinodermatidae Acosmanura 124.8 Rhinoderma darwinii Alsodes gargola 20.6 Alsodidae Eupsophus calcaratus 62.9 73.9 Atelognathus reverberii 27.5 13.7 Batrachyla taeniata Batrachylidae Hyloidea Batrachyla leptopus Batrachophrynus macrostomus 5.1 Telmatobiidae Telmatobius vellardi 69.1 18.7 Lepidobatrachus sp. Ceratophrys cornuta Ceratophryidae Cryptobatrachus boulengeri 44.7 65 Gastrotheca pseustes Hemiphractidae 19.7 Gastrotheca weinlandii 148.5 Nyctimystes kubori 63.7 48.3 Neobatrachia 67.5 Phyllomedusa tomopterna 25.1 Agalychnis callidryas 17.2 Agalychnis lemur 60 Hyloscirtus lindae 33.1 Aplastodiscus perviridis 25.9 50.5 Hypsiboas fasciatus Hylidae Scinax ruber 47.1 65.9 Osteocephalus taurinus 44.8 Hyla chinensis Outgroup 25.2 Acris crepitans 41.9 Dendropsophus parviceps 36 Pseudis paradoxa Allobates femoralis 35.3 Hyloxalus jacobuspetersi 29.5 Dendrobatidae Ranitomeya imitator Anura Eleutherodactylus planirostris Eleutherodactylidae 49.6 Craugastor augusti 30.8 Craugastoridae 64.7 Craugastor fitzingeri 46.2 Pristimantis thymelensis 135.8 40.2 Barycholos pulcher 44.3 Strabomantis sulcatus Strabomantidae 41.7 Hypodactylus brunneus 63.2 Proceratophrys boiei 32.7 Odontophrynus occidentalis Odontophrynidae Leptodactylus albilabris 59.1 37.1 Lithodytes lineatus 53.8 Physalaemus pustulosus 26.3 Physalaemus cuvieri Leptodactylidae 39.9 61 Pleurodema somuncurensis 6.3 Pleurodema thaul Melanophryniscus stelzneri 49 Amazophrynella minuta 36.2 Peltophryne peltocephala Schismaderma carens 18.4 26 Rentapia hosii 17.4 Bufo gargarizans 16.8 Bufonidae Duttaphrynus melanostictus 21.5 Rhinella marina 19.9 Incilius nebulifer 16.9 Anaxyrus canorus 12.6 Anaxyrus punctatus Sooglossus thomasseti Sooglossidae Phrynomantis microps Otophryne pyburni Microhylidae 61.3 Chiasmocleis ventrimaculata 49.5 69 Stereocyclops incrassatus 28.8 Elachistocleis ovalis 21.8 Gastrophryne olivacea Stumpffia pygmaea 65.5 33.3 Platypelis tuberifera 31.3 58.5 Anodonthyla boulengerii 128.4 Paradoxophyla palmata 49.1 Scaphiophryne boribory 7.4 63.8 Scaphiophryne marmorata Kalophrynus pleurostigma Microhylidae Dyscophus antongilii 54.3 62.6 Microhyla heymonsi 47.3 Kaloula conjuncta 15.4 56.5 Kaloula pulchra Cophixalus cheesmanae 17.4 Oreophryne sp. 21 104.6 Liophryne schlaginhaufeni 20.2 Xenorhina sp. Ranoidea 17.3 Callulops wilhelmanus 14.6 Mantophryne lateralis 12.8 Cophixalus sp. Hemisus marmoratus Hemisotidae 56 Breviceps macrops 35.6 Afrobatrachia Callulina kreffti Brevicipitidae 85.5 Phlyctimantis boulengeri 41.6 Hyperolius bolifambae Hyperoliidae 66.4 Leptopelis parkeri Leptodactylodon ovatus 44.2 50.8 Arthroleptis poecilonotus 48 Nyctibates corrugatus Arthroleptidae 38.4 Scotobleps gabonicus 36.2 Trichobatrachus robustus 21.9 100.1 Astylosternus diadematus Odontobatrachus natator Odontobatrachidae 63.4 Ptychadena oxyrhynchus 56.7 Ptychadenidae Phrynobatrachus natalensis Phrynobatrachidae 65.6 Petropedetes euskircheni 60.6 Petropedetidae Conraua crassipes 63.6 Conrauidae Aubria subsigillata 49 Strongylopus grayii 7.4 Pyxicephalidae 67 Amietia lubrica Liurana xizangensis Natatanura 40.8 Cornufer pelewensis Ceratobatrachidae Quasipaa spinosa 47.6 Limnonectes fujianensis 44.4 Hoplobatrachus tigerinus Dicroglossidae 65.1 27.7 Fejervarya limnocharis Boophis madagascariensis 44.5 63.6 Aglyptodactylus madagascariensis Mantellidae 55.8 Buergeria oxycephala 44.2 Kurixalus odontotarsus 27.3 Polypedates megacephalus Rhacophoridae 23.4 61.4 Rhacophorus dennysi Pelophylax nigromaculatus Amolops ricketti 31.4 19.4 Amolops loloensis Sylvirana guentheri 18.3 27.7 Papurana sp. 14.8 Papurana latouchii 25.1 Odorrana schmackeri Ranidae Rana berlandieri 11.7 22.9 Rana virgatipes 16.9 Rana draytonii 12.9 Rana amurensis 6.7 Rana chensinensis 200 150 100 50 0 BioGeoBEARS DEC+J on combined dataset M0_stratified ancstates: global optim, 4 areas max. d=0.0225; e=0; j=0.0449; LnL=-207.26;

250 200 150 100 50 0 (Mya)

Permian Triassic Jurassic Cretaceous Paleogene Neogene

E Andrias davidianus Hynobiidae

E Batrachuperus yenyuanensis Cryptobranchidae

O Leiopelma hochstetteri Leiopelmatidae

N Ascaphus truei Ascaphidae

E Discoglossus pictus Alytidae E Alytes obstetricans

E Barbourula busuangensis

E Bombina fortinuptialis Bombinatoridae E Bombina orientalis

N Rhinophrynus dorsalis Rhinophrynidae

S Pipa pipa

S Pipa parva

A Pseudhymenochirus merlini Pipidae A Hymenochirus boettgeri

A Xenopus kobeli

A Xenopus epitropicalis

N Scaphiopus couchii

N Scaphiopus holbrookii Scaphiopodidae N Spea multiplicata

N Spea intermontana

E Pelodytes ibericus Pelodytidae

E Pelobates cultripes Pelobatidae E Africa (A) Pelobates syriacus E Leptolalax alpinus Eurasia (E) E Scutiger gongshanensis E Leptobrachium chapaense

E Oreolalax jingdongensis India-Seychelles (I) Megophryidae E Brachytarsophrys feae Madagascar (M) E Megophrys nasuta E Xenophrys omeimontis North America (N) E Ophryophryne microstoma A Heleophryne purcelli Heleophrynidae

South America (S) S Calyptocephalella gayi Calyptocephalellidae

O Mixophyes coggeri

Australia (O) O Limnodynastes salmini

O Crinia signifera E + N + O Myobatrachidae O Uperoleia laevigata O N + O Myobatrachus gouldii S Rhinoderma darwinii Rhinodermatidae S Insuetophrynus acarpicus

S Eupsophus calcaratus Alsodidae S Alsodes gargola

S Hylodes nasus Hylodidae

S Atelognathus reverberii

S Batrachyla leptopus Batrachylidae

S Batrachyla taeniata

S Telmatobius vellardi Telmatobiidae S Batrachophrynus macrostomus

S Ceratophrys cornuta

S Ceratophrys ornata Ceratophryidae S Lepidobatrachus sp.

S Lepidobatrachus laevis

S Cryptobatrachus boulengeri

S Flectonotus fitzgeraldi

S Hemiphractus bubalus Hemiphractidae S Stefania ginesi

S Gastrotheca pseustes

S Gastrotheca weinlandii

O Litoria caerulea

O Cyclorana maini Pelodryadinae O Nyctimystes kubori

O Nyctimystes pulcher

S Cruziohyla calcarifer

S Phyllomedusa tomopterna

S Phyllomedusa hypochondrialis Phyllomedusinae S Agalychnis lemur

S Agalychnis callidryas ●S Agalychnis saltator Hylidae

S Hyloscirtus lindae

S Aplastodiscus perviridis

S Hypsiboas fasciatus

S Scinax ruber

S Osteocephalus taurinus

S Trachycephalus typhonius Hylinae S Dendropsophus parviceps

S Pseudis paradoxa

N Acris crepitans

E Hyla chinensis

N Hyla arenicolor

N Hyla cinerea

S Allobates femoralis

S Hyloxalus jacobuspetersi

S Epipedobates tricolor Dendrobatidae S Phyllobates vittatus

S Dendrobates auratus

S Ranitomeya imitator

S Ceuthomantis smaragdinus Ceuthomantidae

S Ischnocnema guentheri Brachycephalidae S Brachycephalus ephippium

N Eleutherodactylus marnockii

S Eleutherodactylus planirostris Eleutherodactylidae S Eleutherodactylus coqui

S Diasporus diastema

N Craugastor augusti

S Craugastor podiciferus Craugastoridae

S Craugastor fitzingeri

S Pristimantis thymelensis

S Barycholos pulcher S ● Hypodactylus brunneus Strabomantidae S Phrynopus bracki

S Strabomantis biporcatus

S Strabomantis sulcatus

S Thoropa taophora Cycloramphidae

S Odontophrynus occidentalis Odontophrynidae S Proceratophrys boiei

S Lithodytes lineatus

● S Leptodactylus albilabris

S Leptodactylus melanonotus

S Leptodactylus latrans Leptodactylidae S Physalaemus pustulosus

S Physalaemus cuvieri

● S Pleurodema thaul

S Pleurodema somuncurensis

S Allophryne ruthveni Allophrynidae

S Hyalinobatrachium aureoguttatum

S Hyalinobatrachium colymbiphyllum

S Hyalinobatrachium ibama

S Cochranella granulosa

S Ikakogi tayrona

S Nymphargus grandisonae

S Centrolene bacatum

S Centrolene daidaleum Centrolenidae ● S Teratohyla spinosa ● S Rulyrana adiazeta ●S Rulyrana flavopunctata ● S Chimerella mariaelenae ● S Sachatamia ilex ● S Espadarana prosoblepon ● S Vitreorana helenae

S Melanophryniscus stelzneri

S Atelopus peruensis

S Amazophrynella minuta

S Nannophryne cophotis

S Nannophryne variegata

S Rhaebo glaberrimus

S Rhaebo nasicus

● S Peltophryne lemur

S Peltophryne peltocephala

●S Peltophryne longinasus

S Rhinella marina

S Rhinella ocellata

N Anaxyrus canorus

N Anaxyrus punctatus

S Incilius luetkenii

S Incilius nebulifer

A Schismaderma carens

A Nectophrynoides tornieri Bufonidae E Phrynoidis juxtaspera

E Phrynoidis aspera

E Rentapia hosii

E Bufotes viridis

I Adenomus kelaartii

EI Duttaphrynus melanostictus

EI Duttaphrynus stomaticus

E Bufo bufo

E Bufo gargarizans

E Bufo japonicus

E Leptophryne borbonica

E Epidalea calamita

E Ingerophrynus galeatus

E Ingerophrynus divergens

A Mertensophryne micranotis

A Sclerophrys brauni

A Sclerophrys maculata

I Sooglossus thomasseti Sooglossidae

I Nasikabatrachus sahyadrensis Nasikabatrachidae

A Phrynomantis microps Hoplophryninae

A Hoplophryne rogersi Phrynomerinae

S Otophryne pyburni Otophryninae S Synapturanus sp.

S Chiasmocleis ventrimaculata

S Ctenophryne geayi

S Stereocyclops incrassatus Gastrophryninae

S Elachistocleis ovalis

N Gastrophryne olivacea

M Platypelis tuberifera

M Plethodontohyla inguinalis Cophylinae M Stumpffia pygmaea

M Anodonthyla boulengerii

M Paradoxophyla palmata

M Scaphiophryne boribory Scaphiophryninae M Scaphiophryne marmorata

M Scaphiophryne madagascariensis

E Kalophrynus pleurostigma Kalophryninae E Kalophrynus interlineatus

I Melanobatrachus indicus Melanobatrachinae

E Gastrophrynoides immaculatus

O Liophryne schlaginhaufeni

O Cophixalus cheesmanae Microhylidae O Cophixalus cryptotympanum

O Oreophryne sp. A

O Oreophryne sp. B

O Xenorhina sp. Asterophryinae

O Xenorhina obesa

O Callulops wilhelmanus

O Mantophryne lateralis ● O Cophixalus sp. A ● O Cophixalus sp. B ● O Hylophorbus rufescens

M Dyscophus antongilii Dyscophinae

E Micryletta inornata

E Calluella guttulata

E Microhyla marmorata

E Microhyla annectens

E Microhyla okinavensis

E Microhyla heymonsi

E Chaperina fusca

E Phrynella pulchra Microhylinae

E Metaphrynella pollicaris

E Metaphrynella sundana

E Kaloula conjuncta

E Kaloula pulchra

I Uperodon systoma

I Uperodon variegatus

I Uperodon montanus

A Hemisus marmoratus Hemisotidae

A Breviceps mossambicus

A Breviceps macrops

A Spelaeophryne methneri Brevicipitidae A Probreviceps durirostris

A Callulina kreffti

A Callulina laphami

A Phlyctimantis boulengeri A Afrixalus dorsalis Hyperoliidae A Hyperolius viridiflavus

A Hyperolius bolifambae

A Leptopelis kivuensis

A Leptopelis parkeri

A Leptodactylodon ovatus

A Arthroleptis poecilonotus

A Arthroleptis variabilis Arthroleptidae

A Nyctibates corrugatus

A Scotobleps gabonicus

A Astylosternus diadematus

A Trichobatrachus robustus

A Conraua crassipes Conrauidae

A Petropedetes parkeri Petropedetidae A Petropedetes euskircheni Pyxicephalidae A Amietia lubrica Cacosterninae A Strongylopus grayii

A Aubria subsigillata Pyxicephalinae A Pyxicephalus edulis

A Odontobatrachus natator Odontobatrachidae

A Ptychadena mascareniensis

A Ptychadena oxyrhynchus Ptychadenidae A Ptychadena cooperi

A Phrynobatrachus krefftii

A Phrynobatrachus natalensis Phrynobatrachidae A Phrynobatrachus africanus

E Liurana xizangensis

E Cornufer pelewensis Ceratobatrachidae

E Platymantis hazelae

E Occidozyga lima Occidozyginae E Occidozyga laevis

E Quasipaa spinosa

E Limnonectes laticeps Dicroglossidae

E Limnonectes fujianensis

E Limnonectes magnus Dicroglossinae

E Hoplobatrachus tigerinus

E Euphlyctis cyanophlyctis

I Fejervarya granosa

E Fejervarya limnocharis

E Fejervarya multistriata

I Micrixalus sp. Micrixalidae

I Indirana sp. A Ranixalidae I Indirana sp. B

M Boophis madagascariensis Boophinae Mantellidae M Boophis xerophilus

M Aglyptodactylus madagascariensis Laliostominae M Blommersia wittei Mantellinae M Mantella madagascariensis

E Buergeria buergeri Buergeriinae Rhacophoridae E Buergeria oxycephala

E Kurixalus odontotarsus

I Pseudophilautus wynaadensis Rhacophorinae E Polypedates megacephalus

E Rhacophorus dennysi

I Lankanectes corrugatus Nyctibatrachidae I Nyctibatrachus sp.

E Staurois latopalmatus

E Meristogenys kinabaluensis

E Pelophylax nigromaculatus

E Amolops loloensis

E Amolops ricketti

E Sanguirana luzonensis

E Babina chapaensis

E Babina pleuraden

E Chalcorana macrops

E Papurana sp.

E Papurana latouchii

E Sylvirana guentheri Ranidae E Hydrophylax leptoglossa

E Odorrana schmackeri

E Odorrana hosii

N Rana virgatipes

N Rana catesbeiana

NS Rana berlandieri

N Rana pipiens

N Rana draytonii

E Rana amurensis

E Rana japonica

E Rana temporaria

E Rana chensinensis

250 200 150 100 50 0 (Mya) Millions of years ago