Evolutionary History of the Asian Horned Frogs (Megophryinae): Integrative Approaches to Timetree Dating in the Absence of a Fossil Record Stephen Mahony,*,1,2 Nicole M. Foley,1 S.D. Biju,2 and Emma C. Teeling*,1 1School of Biology and Environmental Science, University College Dublin, Belfield, Dublin, Ireland 2Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi, India *Corresponding authors: E-mails: [email protected]; [email protected]. Associate editor: Beth Shapiro

Abstract Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 Molecular dating studies typically need fossils to calibrate the analyses. Unfortunately, the fossil record is extremely poor or presently nonexistent for many species groups, rendering such dating analysis difficult. One such group is the Asian horned frogs (Megophryinae). Sampling all generic nomina, we combined a novel 5 kb dataset composed of four nuclear and three mitochondrial gene fragments to produce a robust phylogeny, with an extensive external morpho- logical study to produce a working taxonomy for the group. Expanding the molecular dataset to include out-groups of fossil-represented ancestral anuran families, we compared the priorless RelTime dating method with the widely used prior-based Bayesian timetree method, MCMCtree, utilizing a novel combination of fossil priors for anuran phylogenetic dating. The phylogeny was then subjected to ancestral phylogeographic analyses, and dating estimates were compared with likely biogeographic vicariant events. Phylogenetic analyses demonstrated that previously proposed systematic hypotheses were incorrect due to the paraphyly of genera. Molecular phylogenetic, morphological, and timetree results support the recognition of Megophryinae as a single genus, Megophrys, with a subgenus level classification. Timetree results using RelTime better corresponded with the known fossil record for the out-group anuran tree. For the priorless in-group, it also outperformed MCMCtree when node date estimates were compared with likely influential historical biogeographic events, providing novel insights into the evolutionary history of this pan-Asian anuran group. Given a relatively small molecular dataset, and limited prior knowledge, this study demonstrates that the computationally rapid RelTime dating tool may outperform more popular and complex prior reliant timetree methodologies. Key words: amphibian, phylogenetics, taxonomy, biogeography, timetree, Megophryidae, India, Asia.

Introduction considered, and regularly revised before application. For an- Article urans, all previously published molecular tree dating studies Higher-level amphibian taxonomy and systematic knowledge have relied on relatively complex analytical techniques that has rapidly advanced over the past decade due to evolving require the calibration of nodes using various combinations of and ever improving molecular phylogenetic tools. This has led priors (e.g., fossils, geological events, etc.), molecular markers to a near complete revision of many extant amphibian sys- (mtDNA, or nuDNA only, or a combination of the two), and tematic groups from the traditional, morphology dependent analytical programs (e.g., r8s, MultiDivTime, BEAST, etc.) (e.g., system (e.g., Frost et al. 2006; Pyron and Wiens 2011; Padial Roelants and Bossuyt 2005; San Mauro et al. 2005; Bossuyt et al. 2014; Peloso et al. 2015). Deep molecular evolutionary et al. 2006; Roelants et al. 2007; Kurabayashi et al. 2011; Zhang divergence in superficially morphologically homogenous et al. 2013). Divergence estimates are typically unique to each groups often initially comes as a surprise, but typically reflects particular study, and can differ considerably from one study the absence of extensive taxonomic review, or published to another. The amphibian fossil record is by no means com- knowledge with regard to the systematics, biology, and evo- plete in terms of chronological and taxonomic representa- lution of poorly studied amphibian groups (e.g., McLeod 2010; tion, and until quite recently the systematic associations of Biju et al. 2011; Biju, et al. 2014a; 2014b; Biton et al. 2013; many fragmentary fossils with extant taxa has been relatively Kamei et al. 2012). Beyond taxonomy and systematics, ad- subjective (Marjanovic and Laurin 2007). To reduce the sub- vances in molecular phylogenetic analyses have driven new jectivity of fossil identifications, more rigorous statistical/phy- and improved methodologies to estimate divergence times logenetic methods are now more commonly applied to fossil and historical biogeography by applying various “known” morphological data, such as maximum parsimony analyses time-constraints to nodes of phylogenetic trees. The resulting (e.g., Sanchiz et al. 2002; Baez et al. 2009; Baez 2013; Evan et al. date estimations rely heavily on the applied time-constraints, 2014). However, similar to dating priors, errors in scoring therefore, the choice of constraints must be very carefully morphological characters and incomplete datasets due to

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744 Mol. Biol. Evol. 34(3):744–771 doi:10.1093/molbev/msw267 Advance Access publication January 18, 2017 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE partial specimens, have resulted in misleading and incongru- MCMCtree (Yang 2007). RelTime is essentially a calibration ent taxonomic conclusions (e.g., Faivovich et al. 2014). Using free approach to divergence time estimation, which provides prehistoric geological events, such as tectonic movements to the option to include internal node priors, and a root age constrain divergences, by necessity makes a series of assump- prior that translates relative times to absolute times (Tamura tions, such as the dispersal abilities of the animals, that vicar- et al. 2012). This program has been found to produce com- iance was the cause of divergence, and that temporary land parable estimates with other analytical techniques that are connections did not exist between land masses (Hipsley and more reliant on the accuracy of calibration priors (Tamura Mu¨ller 2014; Marjanovic and Laurin 2014). et al. 2012; Battistuzzi et al. 2015). Since RelTime has never Relative time dating offers an alternative to using priors, as been used on an amphibian tree, the method was rigorously it typically does not require knowledge of the distribution of tested using a variety of constraints and datasets to assess its the lineage rate heterogeneity, clock calibrations and associ- potential as a tree-dating tool for anuran phylogenies that ated distributions, and therefore may be suitable for groups lack a fossil record. Alternatively, MCMCtree (part of the Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 with missing fossil data (Tamura et al. 2012). The Asian en- PAML package) is a Bayesian analytical approach which esti- demic anuran family Megophryidae Bonaparte, 1850 has no mates divergence times utilising fossil priors under various known fossil record (Sanchiz and Rocek 1996). Within this nucleotide evolution models. Using these different tree dating familytherehavebeentwoorthreepreviouslyrecognised methodologies, the following were assessed: 1) whether sim- subfamilies (or tribes), Leptolalaginae Delorme, Dubois, ilar divergence times can be obtained using differing analytical Grosjean and Ohler, 2006 and Leptobrachiinae Dubois, approaches on a dataset with deep time out-group expansion 1983 (together as the tribe Leptobrachiini Dubois, 1980), to accommodate fossil priors; 2) compared and contrasted and Megophryinae Bonaparte, 1850 (¼tribe Megophryini the dating estimates with others that have performed mo- sensu Dubois, 1980—Dubois 1983; Dubois 2005; Delorme lecular phylogenetic tree dating for anurans; 3) determined et al. 2006), and molecular phylogenetic studies on amphib- whether the use of a novel combination of fossil priors for ians have demonstrated that at least the latter two subfami- dating nodes improves comparability between molecular lies are monophyletic (e.g., Frostetal.2006; Pyron and Wiens based estimations, and the known fossil record. The phyloge- 2011; Pyron 2014). Individual genera (or clades thereof) from netic and timetree dating results provide compelling insights within these subfamilies have been subject to preliminary into the diversification patterns and ancestral biogeography molecular analyses, primarily with the purpose of exploring of the subfamily Megophryinae. hidden taxonomic diversity (e.g., Zheng et al. 2008; Chen et al. 2009; Brown et al. 2010; Matsui et al. 2010; Ohler et al. 2011; Results Wang et al. 2012, 2014; Nguyen et al. 2013; Li et al. 2014; Zhao et al. 2014; Poyarkov et al. 2015),butnoattempthasyetbeen Megophryinae Phylogeny made to resolve the deeper systematic relationships within The Megophryinae phylogeny constructed using varying rate these groups. As currently understood, Megophryinae con- heterogeneity models (selected by jModelTest) in Maximum tains ca. 68 species variously assigned to five poorly diagnosed Likelihood (ML) GTR GAMMA (MGG) and Bayesian genera, based primarily on limited molecular, karyological and Inference (BI) GTR GAMMA (BGG) and BI GTR CAT morphological studies that sampled a small number of spe- (BGC) analyses, mostly resolved seven primary species groups. cies (Rao and Yang 1997a, 1997b; Delorme et al. 2006). These groups can be associated with previously named However, this taxonomic arrangement is not widely accepted, genus-level taxa. Megophrys Kuhl and Van Hasselt, 1822b and some have cautioned against the premature recognition and Pelobatrachus Beddard, 1908 formedasistertaxaclade of multiple genus level species groups within the subfamily, (hereafter the M–P clade: fig. 1), as did Atympanophrys Tian prior to a more comprehensive molecular phylogenetic anal- and Hu, 1983 and Brachytarsophrys Tian and Hu, 1983 (here- ysis that at least includes the type species of all genus level after the A–B clade) in most analyses. Across all datasets nomina (e.g., Stuart, et al. 2006b; Mahony 2011; Mahony et al. Ophryophryne Boulenger, 1903b and Panophrys Rao and 2011, 2013). Yang, 1997a formed a sister taxa clade (hereafter the O–P Here, we elucidate the evolutionary history of the subfam- clade). The group directly related to the most recent com- ily Megophryinae using a combination of mitochondrial and mon ancestor (MRCA) of Megophryinae could not be re- nuclear genes (5 kb) sampling across all valid and synony- solved in this study, but is clearly represented by either the mised genus-level nomina (supplementary table S1, A–B clade, or the M–P clade. The remaining group, Supplementary Material online), to evaluate the monophyly Xenophrys Gu¨nther, 1864, contains five subclades (hereafter of the genus-level groups currently recognised. To estimate regarded as species groups), of which three species groups divergence dates on the tree, the molecular dataset was ex- (megacephala, lekaguli,andmajor) consistently formed a panded to include other major anuran families thus enabling clade, though their relationships within this clade varied the application of fossils as constraints. The relevance of con- across analytical datasets, and when utilising different substi- straints previously used to date amphibian trees is reviewed, tution models (fig. 2A–D). Trees produced from the mtDNA and a novel combination of fossil constraints is assembled. only alignment dataset (1a) could not consistently resolve the Two fundamentally different molecular phylogenetic tree- systematic position of the remaining two Xenophrys species dating techniques were used to estimate the age of nodes groups (vegrandis and aceras),whichwereeachcomposedof within Megophryinae, RelTime (Tamura et al. 2012)and independent long branches, however, these taxa formed a

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FIG.1.Combined phylogenetic and RelTime timetree based on the topology obtained from the “best” tree (Bayesian, GTR Gamma, 54 taxa mt þ nuDNA alignment, Dataset 1c), and the time estimation using the root age range of 247.2–350 MY and Fossil 2 as priors on the 70 taxa mt þ nuDNA alignment dataset (3a). Non-Megophryinae taxa are not shown. Refer to online version for colour figure. Taxa highlighted in orange representtypespeciesforeachsubgenus.Bracketsdenotingsubgenericandspeciesgroupsarecolourcodedbasedontheirpreviousgenericassignment by Delorme et al. (2006):orange¼ Megophrys, blue ¼ Brachytarsophrys,green¼ Ophryophryne,red¼ Xenophrys. CI error bars are coloured based on phylogenetic support.Node information to the left ofthe treeincludes RelTime absolutetime (AT) age estimates, ancestraldistribution estimate from BioGeoBEARS (DEC þ J model), and support values from RAxML, MrBayes, and PhyloBayes phylogenetic analyses. p. represents polytomy. clade along with the remaining Xenophrys species groups on provided intragroup relationships that were mostly identical all trees generated from datasets that either included or were between trees generated using BI and ML analyses under the composed exclusively of nuDNA (Datasets 1b–d). Trees re- different models, with the exceptions of the Pelobatrachus sulting from the combined mtDNA and nuDNA dataset (1c), group and the X. megacephala species group, where node composing the maximum number of taxa and gene coverage, support was generally low between some species. The BGG

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FIG.2.Alternative Megophryinae topologies: (A) the “best” tree (Bayesian, GTR Gamma, 54 taxa mt þ nuDNA alignment, Dataset 1c); (B–D) likely alternative topologies observed from our analyses. Alternative branching is coloured for convenience (refer to online version for colour figure). (E– H) Alternative topologies observed in other studies: (E) Jiang et al. (2003);(F) Pyron and Wiens (2011);(G) Pyron (2014);(H) Zheng et al. (2004b). tree from this dataset (1c) is considered here to represent the were not represented by the 54 taxa dataset (1c), were dis- best tree from the different analyses (fig. 1), providing highest tributed amongst the Atympanophrys, Brachytarsophrys, inter- and intraclade support for most nodes. Refer to the Panophrys and Pelobatrachus groups, and the Xenophrys ace- supplementary text S1, Supplementary Material online for a ras, X. lekaguli,andX. major species groups. Atympanophrys more detailed comparison of trees obtained from alignment shapingensis Liu, 1950, the type species of Atympanophrys Datasets 1a–d, and the results of outlier analyses, and formed the sister taxon to A. nankiangensis Liu & Hu, 1966 supplementary table S2:col.I–V,Supplementary Material on- (Hu et al. 1966) demonstrating that our use of the latter line for support values for major nodes across Datasets 1a–d. species in the phylogenetic analyses was representative of this taxon. M. dringi formed a poorly supported (bs ¼ 43) Expanded Phylogeny sister taxon relationship with the A–B clade, however, its sys- The ML analyses of the expanded taxon dataset that included tematic position was found to be unstable (see supplementary GenBank sequences, produced a tree (fig. 3: supplementary textS2,SupplementaryMaterialonline).Thisanalysesalsohigh- table S2: col. VIII, Supplementary Material online) that had an lighted a number of errors associated with GenBank sequences, equivalent intragroup/species group topology as the MGG and related errors in some recent publications citing data from and BGG trees obtained from the phylogenetic analyses of GenBank (supplementary table S3, Supplementary Material the alignment Dataset 1c (supplementary table S2:col.V, online). Supplementary Material online). Most groups and species groups received high support (bs [bootstrap support] >95). Topology Tests Exceptions are the M–P clade, where the split between the Alternative topologies estimated during phylogenetic analy- Megophrys and Pelobatrachus groups received low support ses of the aligned datasets suggest that either the M–P clade, (bs ¼ 50), and the monophyly of Xenophrys, which received or the A–B clade should form the sister taxon to all other moderately high support (bs ¼ 86). All additional taxa (ex- Megophryinae. Within Xenophrys, alternative trees have been cept M. dringi; Inger et al. 1995) from GenBank sequences that obtained, estimating a sister relationship between the

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FIG.3.Maximum likelihood tree for the expanded alignment (Dataset 2) containing all available Megophryinae taxa with unique sequences, both from this study, and those obtained from GenBank. Species names in orange text (refer to online version for colour figure) represent sequences not used in the 54 taxa analyses (Dataset 1c). Values for nodes with low bootstrap support (<80) are provided adjacent the nodes.

748 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE

X. megacephala,orX. lekaguli and X. major species groups mtDNA þ reduced nuDNA datasets (3a and 3c, respec- (fig. 2A–D). The alternative topology tests, Kishino-Hasegawa tively) were considerably more realistic relative to the fossil (KH), Shimodaira-Hasegawa (SH), and approximately unbi- record for most nodes on which the minimum divergence ased (AU) were performed on the four conflicting topologies age is considered “known” by fossils (table 1; supplementary (fig. 2A–D). Topology tests found all four topologies statisti- table S5A and C, Supplementary Material online). However, cally likely (AU P > 0.438, KH P > 0.359, SH P > 0.857), but in AT estimates for some nodes were still dated considerably contrast with MGG and BGG phylogenetic trees that placed more recent when using the root age range of 247.2–300 the M–P clade as the sister taxon to all other Megophryinae, MY (million years before present) (supplementary table S5A the tree with the A–B clade as sister to remaining taxa was and C: Analyses 2 and 11, Supplementary Material online). selected as the most likely tree with marginally better support Dataset 3c produced dating estimates only slightly older (supplementary table S4, Supplementary Material online). (<5 MY for out-group anuran nodes) than those ob- Since the dataset used in the tree reconstruction includes tained from the complete mt nuDNA dataset (3a) þ Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 representatives of all taxon groups (except “Borneophrys”), (supplementary table S5A and C: compare Analyses 1–4 our topology could be compared with previously published to 10–13, Supplementary Material online), indicating that hypotheses (Jiang et al. 2003; Zheng et al. 2004b; Pyron and extensive missing data for out-group taxa had little effect Wiens 2011; Pyron 2014) for Megophryinae to explore topo- on dating estimates. B) Choosing the optimal root age logical conflicts where they occur. Of the previously published range: In the absence of a specified root age prior but phylogenetic trees (fig. 2E–H), two (Jiang et al. 2003; Pyron including Fossils 2–8, older nodes without determined pri- 2014) could not be statistically rejected based on the dataset ors were collapsed and the root age estimate of 180 MY (fig. 2E and F), though both received lower support than was considered unreasonable (Analysis 27, results not results obtained in this study (AU P > 0.069, KH P > 0.097, shown). Therefore, successful RelTime analyses requires SH P > 0.260). The topologies obtained by both Zheng et al. the specification of an estimated root age. When using a (2004b) and Pyron and Wiens (2011) were rejected (fig. 2G minimum constraint only for the root, RelTime sets this as and H). Zheng et al. (2004b) used only a single short sequence the global time factor [f](Tamura et al. 2013), used for of the 16S mitochondrial gene, which may account for the translating the relative time estimates of remaining nodes poor systematic correspondence with the trees generated in to ATs (supplementary table S5A–C: analyses 1, 7, and 10, the present study. Alternatively, analyses by Pyron and Wiens Supplementary Material online). Considering accurately (2011) using available GenBank sequences, suffered signifi- identified fossils should postdate the MRCA of the relevant cantly perhaps due to large amounts of missing data for sev- node, a minimum constraint only cannot be used to date eral megophryine species groups. the root for these analyses. When only specifying a calibra- tion range for the root (i.e., no internal priors), RelTime Dating the Anuran Tree selects the midpoint to represent f, but calculates the CI A review of literature revealed that eight fossils, and values independent of the size of the age range provided three major tectonic events that may have had vicariant for the anuran root (Tamura et al. 2013). This explains why influence, could be suitable to use as priors in our dating the CI values for the root usually exceeded the maximum analyses. These dates were used as soft-bound minimum root age (e.g., supplementary table S5A–D:Analyses2,3, priors to estimate divergence times for the root and 11, 12, 14, and 15, Supplementary Material online). Given a significant internal nodes. They were also used to com- root age range only, the closest dating estimate of the tree pare with estimated node dates generated from analyses relative to the fossil record and geological vicariant events, that did not include internal node priors (see Materials was obtained using the root age range 247.2–350 MY (ta- and Methods). ble 1; supplementary table S5A and C: Analyses 3 and 12, RelTime: To assess the suitability of RelTime to date the Supplementary Material online). C) Adding internal priors: anuran tree, various combinations of priors were tested on When an internal prior was considerably older than the the three alignment datasets (3a–c) to determine which estimated AT for the node, given a certain root range, the f produced results that were most complimentary with the estimation was forced to be the maximum root age and fossil record and possible geographic priors. A) Choosing the the constrained node was forced to be the minimum prior optimal alignment dataset: The nuDNA only and the com- ageprovidedforthenode(supplementary table S5E: bined mt þ nuDNA datasets (3b and 3a, respectively) pro- Analyses 21 and 22, Supplementary Material online), or duced considerably different estimations of divergence the node was collapsed (Analyses 25 and 26, results not dates, which would be expected due to the difference in shown). Both of these circumstances are not optimal for mutation rates between nuDNA and mtDNA (supplemen timetree estimations. Including minimum constraints to tary table S5A–C: compare Analyses 7–9 to 1–3 and 10–12, internal nodes that are close to, or slightly older than their Supplementary Material online). The nuDNA dataset abso- respective node ages estimated based on the root priors (i. lute times (ATs) were not considered reliable since they e., Fossil 2 and/or 3, or Geological 1 and 3—see Materials were more recent than phylogenetically associated fossils and Methods) forced the f estimation towards the maxi- for relevant nodes (supplementary table S5B:Analyses8– mum age provided for the root range (e.g., supplementary 9, Supplementary Material online). RelTime results for AT table S5A–E: compare Analyses 3 to 4–6, 16, and 23; 2–20; estimates using the combined mt þ nuDNA and 12–13, Supplementary Material online). These internal

749 750 Table 1. Timetree Results for the Expanded Amphibian Alignment (Dataset 3), Comparing the Four Most Optimal RelTime Runs to Results from MCMCtree Analyses. al. et Mahony

RelTime MCMCtree

Alignment Dataset 3a 3c 3a 3b 3c

Root Priors 247.2–350 247.2–350 247.2–350 247.2–350 247.2–350 . doi:10.1093/molbev/msw267 Internal Priors none/F2/F2–8 none/F2/F2–8 F2–8 F2–8 F2–8

Run No. 3/4/6 12/13/24 1 2 3

Description Taxon/Event Min [MY] Max [MY] p. >95% CI p. >95% CI p. >95% CI p. >95% CI p. >95% CI Anura–Caudata 1. T. massinoti 247.2 251.2 299–322 152–479 299–318 147–480 300 249–350 319 269–355 294 246–348 Discoglossus–Alytes 2. I. angelae 125 129.4 127–137 63–206 130–139 62–211 179 123–233 158 117–208 180 125–233 Pipoid root 3. R. parvus 152.1 157.3 164–177 84–263 168–179 84–270 236 184–293 240 195–287 234 184–290 Hymenochirus–Pipa 4. P. taqueti 83.6 89.8 103–111 52–167 106–113 51–171 125 82–174 119 80–166 127 82–175 Ceratophrys–Nasikabatrachus 5. B. ampinga 66 72.1 81–87 40–131 83–88 40–135 113 69–158 116 70–164 115 72–163 Pelodytidae–Pelobatidae 6. A.Paulus 50.3 55.4 98–106 50–158 102–109 51–166 170 128–218 163 124–205 166 125–212 Scaphiopus–Spea 7. S. skinneri 33.3 33.9 44–48 20–73 45–48 20–75 44 30–60 36 30–44 47 32–63 Pelobatidae–Megophryidae 8. E. deani 46.2 50.3 77–83 39–123 80–86 40–129 141 104–183 131 96–166 136 99–176

Neobatrachia root unassigned fossils 93.9 125 148–160 76–238 153–163 75–246 217 167–270 222 179–268 215 168–268 pelobatoid crown unassigned fossils 66 86.3 109–117 56–175 113–121 56–182 183 138–232 177 138–220 166 125–213

As Cryptobranchidae root Chunerpeton tianyiense 166.1 168.3 117–126 57–190 116–123 55–189 58 26–103 33 19–65 54 24–102 As Discoglossoidea root Eodiscoglossus oxoniensis 166.1 168.3 173–187 89–277 177–189 88–284 248 194–307 253 206–301 246 196–306 As Rhinophrynidae root Rhadinosteus parvus 152.1 157.3 139–149 69–224 143–152 69–231 201 149–257 202 151–254 201 150–255

Rhinophrynidae–Pipidae N. America/Africa split 150 190 139–149 69–224 143–152 69–231 201 149–257 202 151–254 201 150–255 Pipa–Hymenochirus S. America/Africa split 80 110 103–111 52–167 106–113 51–171 125 82–174 119 80–166 127 82–175 Neobatrachia crown India split 90 130 81–87 40–131 83–88 40–135 113 69–158 116 70–164 115 72–163

Colours of divergence date estimates relative to the oldest known fossil, or likely vicariant event assigned to each node, are as follows: green are between the minimum prior age to 30% older than the maximum prior age, black are 30–50% older than the maximum prior age, and blue are >50% older than the maximum prior age. Red date estimates are more recent than the minimum divergence ages based on the oldest known fossil assigned to that node, or likely vicariant event. Refer to online version for coloured text. Fossil numbers used as internal priors refer to the listed Fossils 2–8 in the taxon column. Alignment dataset numbers represent: 3a) complete mtDNA þ nuDNA; 3b) nuDNA only; 3c) mtDNA þ RAG-1 þ CXCR-4. All numbers referring to divergence dates are in MY (million years before present). p. represents peak divergence time, CI represents confidence interval. For results of all timetree analyses refer to supplementary table S5, Supplementary Material online.

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FIG.4.Comparison of the timetree results from RelTime and MCMCtree analyses for the non-Megophryinae amphibians: (A) RelTime tree from using the root age range of 247.2–350 MY and Fossil 2 (or all eight fossils) as an internal prior on the 70 taxa mt þ nuDNA alignment (Dataset 3a); (B) MCMCtree using the same dataset, and all eight fossils as priors. Fossils (1)–(8) are mapped onto the tree as blue bars. Green bars [1]–[3] represent the following fossils used in other studies: [1] Albian–Aptian Neobatrachia; [2] Oldest suspected pelobatoid fossils; [3] Eodiscoglossus. Grey bar with an X is Chunerpeton. Maroon bars {A}–{C} represent possible geological “vicariance” events: {A} Gondwana–Laurasia split; {B} Africa–South America split; {C} India split. Refer to online version for colour figure. priors were therefore considered more informative. Using in- 247.2–350 MY and Fossil 2 as an internal prior, ATs were ternal fossil constraints that were considerably more recent mostly estimated to fall within a range that is less than than node ages estimated based on the root range (and þ30% of the earliest estimated date of the oldest known inclusion of older fossils), did not affect AT estimations of fossil for each node (table 1; supplementary table S5A and the remaining nodes (i.e., supplementary table S5A and D: C:Analyses4[fig. 4A] and 13, Supplementary Material online). Analyses 3 ¼ 17 and 18; 4 ¼ 5and6,Supplementary Material These AT estimates also corresponded well with the oldest online). These internal priors were therefore considered unin- known unassigned pelobatoid and neobatrachian fossils (not formative. Our analyses demonstrated that the inclusion of used as priors in this study), as well as estimated timings for the recently described fossil discoglossid (Fossil 2) alone was three major tectonic events likely to have influenced divergence responsible for the oldest age estimates for all nodes on the on relevant nodes (fig. 4A;table1). Using the narrower root age tree, regardless of the inclusion of other fossil constraints range of 247.2–300 MY and Fossil 2 as an internal prior provided (supplementary table S5A:Analyses4–6,Supplementary near identical results to the analyses using a root age range of Material online). Therefore, Fossils 3–8 were uninformative 247.2–350 MY, with no internal prior (supplementary table S5A in the presence of Fossil 2. Given the root age range of and E: compare Analyses 3 with 20, Supplementary Material

751 Mahony et al. . doi:10.1093/molbev/msw267 MBE online). In this analysis the f value was estimated at 297 MY Eocene (ca. 57.7–51.5 MY) (supplementary table S6, (almost at the maximum), demonstrating that in the presence Supplementary Material online). Interestingly, all dating esti- of an internal prior, the upper limit of the root age range pro- mates implied that the MRCA of Megophryinae (during the vided must only be broad enough to prevent ffrom being forced late Oligocene ca. 28.9–24.5 MY) was slightly more recent as the maximum, but narrow enough that the internal prior still than the split between the two currently recognised influences f(i.e., fis not equal to the midrange of the root). Given Leptolalax Dubois, 1980 subgenera, Lalos Dubois, Grosjean, the root age range of 247.2–350 MY and three significant tec- Ohler, Adler and Zhao, 2010,andLeptolalax Dubois, 1980 tonic events as internal priors, ATs for internal nodes were (represented L. khasiorum Das, Tron, Rangad and Hooroo, slightly (<11 MY) older than those obtained from analyses 2010 and L. pictus Malkmus, 1992, respectively), included in that included Fossil 2 as an internal prior (supplementary table our Leptobrachiinae out-group. Diversification within S5E, Supplementary Material online). Megophryinae appears to have occurred during four distinct : Age estimates for the three alignment datasets narrow time frames: 1) lasting 2.1–2.5 MY during the late MCMCtree ca. Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 (3a–c)usingeightfossilpriorsprovidedcomparabledivergence Oligocene to early Miocene (between ca. 28.9 and 22.4 MY) dates, with most major nodes fluctuating by <11 MY between the Megophryinae crown group diversification began, the runs (table 1). No apparent trend of earlier age estimates was ancestor of the A–B clade originated, and Megophrys and observedfromthedatasetsthatincludedthemtDNA(Datasets Pelobatrachus split from their MRCA; 2) lasting ca. 3.1–3.7 3aand3c)comparedwiththenuDNAonlydataset(3b),except MY during the early to mid Miocene (between 23.8 and 15.7 for the two most recent nodes (MRCA of Spea–Scaphiopus, MY) Atympanophrys and Brachytarsophrys split from their and Cryptobranchidae–Hynobiidae) (table 1). Analyses of MRCA, the O–P clade and the Xenophrys clade originated, Datasets 3a and 3c produced very similar estimates of average Ophryophryne and Panophrys split from their MRCA, and the age(<6MYdifference)andCIrangesforallmajornodes,which crown group Xenophrys diverged leading to the origins of the might be expected since all out-group (non Megophryinae) X. vegrandis and X. aceras species groups; 3) lasting ca. 1.9–2.7 taxa had similar sequence coverage. Analyses of Datasets 3a– MY during the mid Miocene (between ca. 15.1 and 9.6 MY), cproducedaverageageestimatesthatweregreaterthan þ30% the Xenophrys lekaguli and X. major speciesgroupsdiverged earlier than the oldest fossil used as priors for many taxa and from their MRCA, this clade split from its MRCA with the X. greater than þ50% earlier than the oldest provisionally identi- megacephala species group, and the more ancestrally di- fied fossil crown pelobatoids (table 1). The crown group of verged megophryine groups, Megophrys, Pelobatrachus,and Neobatrachia was estimated to have diverged close to the Brachytarsophrys, began diversifying; 4) lasting ca. 1.4–2.2 MY time period of the oldest known (presumably stem group) during the late Miocene (between ca. 9.7 and 6.1 MY), the fossils attributed to Neobatrachia, however, the node repre- primary lineages within Ophryophryne and Panophrys,and senting the MRCA of Cryptobranchidae–Hynobiidae was esti- three of the Xenophrys speciesgroups(megacephala, lekaguli matedtohavebeenmorethan100 MY more recent than the and major)begandiversifying(supplementary table S6, oldest known fossil assigned to Cryptobranchidae. Also, those Supplementary Material online). Two node date estimates divergences widely assumed to have resulted from major tec- (the root of Atympanophrys and “Pelobatrachus” baluensis) tonic vicariant events (see Materials and Methods) were esti- from analyses of Dataset 3a (mt þ nuDNA) provided impos- matedtohaveoccurredsignificantlyearlierthan,or closetothe sible CI values that range into negative figures (i.e., million oldestestimatesof terrestrial separation of thesecontinen- years in the future). The associated taxa representing these tal plates (fig. 4B;table1). Age estimates for the three align- nodes consisted of comparatively less sequence data than ment datasets (3a–c) using the root fossil only, or the root most other in-group taxa (i.e., missing CXCR-4, SACS and and Fossil 2 as priors, produced divergence date estimates TTN for “A.” nankiangensis; and part of the mtDNA for considerably older than analyses using eight fossil priors “Pel.” baluensis). However, “Panophrys” omeimontis,forexam- (some nodes in excess of 50 MY older: supplementary table ple, had the same amount of missing data as “A.” nankian- S5F–G, Supplementary Material online). These results are gensis, but its relevant node had a CI range comparable to not comparable with other analyses and thus are not fur- other nodes for taxa with complete sequences, so missing ther discussed. data are not suspected to have caused these erroneous CI estimates. The absence of these erroneous CI values in anal- Dating the Megophryinae Tree yses of Dataset 3c indicates that either TTN and/or SACS RelTime: From the variety of different analyses run on caused the issue. Despite this, the AT estimates for the afore- RelTime, the analyses performed on alignment Datasets 3a mentioned nodes were comparable to those obtained from and 3c, with root age set at 247.2–350 MY, both with and Dataset 3c. without the inclusion of Fossil 2 (see Materials and Methods) MCMCtree: Analyses using MCMCtree, as observed for the as an internal prior, were selected to represent the four most out-group taxa, estimated the more ancestral nodes to be realistic age estimates from which to discuss date estimates considerably older than results obtained from RelTime anal- for the in-group nodes (fig. 1). Across these four timetrees yses (supplementary table S6, Supplementary Material on- there was little overall difference for the AT dating estimates line). Also notable is that crown Megophryinae for the major in-group nodes (though slightly older for diversification was estimated to have begun ca.4–10MY Dataset 3c analyses), i.e., the AT estimate for the crown group earlier (Datasets 3a–c) than the aforementioned split be- Megophryidae varied between the late Paleocene and early tween the Leptolalax subgenera, Leptolalax,andLalos.As

752 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE with RelTime, analyses of Datasets 3a and 3c produced similar (DEC þ J—Matzke2014)modeloutperformedtheDECmodel estimates. However, the nuDNA only dataset (3b) consis- implemented by LAGRANGE (Ree and Smith 2008) for bio- tently estimated divergence dates for major nodes to be no- geographic reconstruction (P-value <0.0005: supplementary tably more recent than those obtained from the combined table S7, Supplementary Material online). Current phyloge- mt þ nuDNA datasets (3a and 3c) (supplementary table S6, neticreconstructionsinthisstudydid notunanimously resolve Supplementary Material online), indicating that the overall whether the M–P clade (from southern peninsular Thailand, difference in substitution rates between the datasets had a Malaysia, Singapore, Indonesia, and the Philippines), or the A– larger effect on the in-group portion of the tree that is free B clade (from China and Indochina) formed the more ances- from the constraints of node priors. Estimated node ages trally diverged radiation of Megophryinae, thus it is unclear from Dataset 3b that were closer to the tips were mostly whether the subfamily originated in the south, or north of its only slightly older than estimates from optimum RelTime distribution (fig. 1). Though, in this study there were some analyses of Dataset 3c using identical priors, however, the poorly sampled regions (e.g., Myanmar), and one significantly Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 difference between estimates increased considerably towards under-sampled group (Panophrys), there appears to be a dis- the root of Megophryidae (supplementary table S6, tinct region specific structuring of the sampled groups and Supplementary Material online). The primary differences be- species groups that might reflect previously recognised biogeo- tween the RelTime and MCMCtree estimates were most ob- graphic regions. The MRCA of the O–P clade is considered to vious in the considerably different pattern of diversification have originated in a region encompassing northern Myanmar- observed within and between the primary megophryine China-northern Vietnam-northern Laos, with Ophryophryne groups. In MCMCtree, Datasets 3a, and 3c (in parentheses), originating in a region encompassing central and southern produced similar patterns of diversification. Early diversifica- Vietnam and Laos-northern Cambodia, and Panophrys pri- tions between the primary Megophryinae clades were broadly marilydistributed in China (fig. 1). The three primary radiations spread between the early Eocene to early Oligocene (53.6– within Xenophrys (megacephala, lekaguli and major species 30.8 MY). Crown diversification for Megophrys, Pelobatrachus, groups) also showed some logical geographic structuring. Brachytarsophrys and Ophryophryne,andtheX. megacephala The X. megacephala species group (as currently understood) species group, and the split between the X. lekaguli and is endemic to the Indo-Myanmar extension of the Himalayas, X. major species groups were estimated to have begun during west of the Irrawaddy River, and east of the Brahmaputra. The thelateOligocenetoearlyMiocenebetweenca. 24.6–17.1 X. lekaguli species group is found in Indochina east of the MY (or ca. 26.6–21.7 MY). Within Panophrys,andthe Irrawaddy River. It appears to have diversified in the hills of X. lekaguli and X. major species groups, crown group diversi- central Thailand, and dispersed west and north, though further fication is estimated to have begun during the early to mid- samplinginthisspeciesgroupwould be essential to confirm Miocene between ca. 15.3 and 13.8 MY (ca. 17.7–14.8 MY) this. The X. major species group appears to have its origins in (supplementary table S6, Supplementary Material online). the southern Himalayas with a clear west to east dispersal into Similar to the RelTime results, MCMCtree estimates from Southeast Asia. Low topological support among taxa in the Dataset3bdepictfourdistinctandnarrowtimeframesdur- Pelobatrachus group, particularly between Philippine and ing which most divergence/diversification occurred: 1) during Bornean species did not allow a robust hypotheses for the the late Eocene to early Oligocene ca. 36.1–30.0 MY, crown dispersal of taxa between the island groups. It is however ap- group diversification of Megophryinae occurred resulting in parent that Pelobatrachus diversified in the Sundas/Philippines the divergence of the ancestral lineages of the A–B clade, M– region, but most likely Borneo (where four of the six currently Pclade,O–PcladeandtheXenophrys clade, and Megophrys recognised taxa are extant), and later dispersed to neighbour- and Pelobatrachus split from their MRCA; 2) during the late ing islands. Oligocene to early Miocene ca. 26.8–22.2 MY, Atympanophrys and Brachytarsophrys split from their Taxonomic Proposal MRCA, Ophryophryne and Panophrys split from their The results obtained from the molecular phylogenetic inves- MRCA, crown group Xenophrys diverged leading to the ori- tigations alone cannot address whether genus-level species gins of the X. vegrandis, X. aceras,andX. megacephala species groups should be recognised within the subfamily groups; 3) during the early Miocene ca. 18.3–15.7 MY, crown Megophryinae, simply due to the subjectivity of recognising group diversification within the Pelobatrachus and taxonomic units above the level of species. To determine Ophryophryne clades, and the X. megacephala species group whether Megophryinae “should” be subdivided into genus- began, and the X. lekaguli and X. major speciesgroupssplit level species groups, based on nonmolecular evidence, type from their MRCA; 4) during the mid to late Miocene ca. 12.1– series and referred specimens were examined, extensively cov- 9.5 MY, crown group diversification is estimated to have be- ering all currently valid species groups, and for some groups, gunintheMegophrys, Brachytarsophrys and Panophrys most or all recognised species (supplementary text S3, clades, and the X. lekaguli and X. major species groups Supplementary Material online).Itwasfoundthatonlytwo (supplementary table S6, Supplementary Material online). molecularly resolved clades are externally morphologically di- agnosable from all others based on a single synapomorphic Phylogeography character, i.e., Atympanophrys appears to be the only group Phylogeographic analyses using BioGeoBEARS (Matzke 2013) with a completely concealed tympanum below a thick supra- demonstrated that the Dispersal-Extinction-Cladogenesis þ J tympanic fold, and Ophryophryne appears to be the only

753 Mahony et al. . doi:10.1093/molbev/msw267 MBE group without maxillary teeth. Despite the sister taxa rela- platyparietus Rao and Yang, 1997b); !Megophrys tionship between Atympanophrys and the heavy set mem- (Brachytarsophrys) chuannanensis comb. nov. (Fei, Ye and bers of the Brachytarsophrys group, they are otherwise Huang, 2001 [Fei and Ye 2001]); ^Megophrys morphologically more similar to some members of the (Brachytarsophrys) feae Boulenger, 1887;*Megophrys Xenophrys and Panophrys groups, which formed the basis (Brachytarsophrys) intermedia Smith, 1921; Megophrys for the proposal of its synonymisation with Xenophrys by (Brachytarsophrys) popei comb. nov. (Zhao, Yang, Chen, Delorme et al. (2006). Loss and gain of maxillary teeth within Chen and Wang, 2014). other anuran genera (e.g., Engystomops Jime´nez de la Espada, 1872 and Ameererga Bauer, 1986) has not been considered Megophrys (Megophrys) noteworthy enough to justify splitting those genera. When —Megophrys Kuhl and Van Hasselt, 1822b: Type species: accounting for morphological variation within molecular Mogophrys montana Kuhl and Van Hasselt, 1822a,bymono- clades of species, previous generalisations about the morpho- typy for the genus-level rank. Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 logical diagnosability of genus-level groups become increas- Distribution: Endemic to Indonesia: Sumatra Is.; Java Is ingly vague (e.g., see supplementary text S4, Supplementary (Inger and Iskandar 2005). Material online). Considering, the crown group diversification Inclusion (2 species): ^Megophrys (Megophrys) montana of Megophryinae postdates the division between the two Kuhl and Van Hasselt, 1822a;*Megophrys (Megophrys) recognised subgenera of Leptolalax (according to RelTime parallela Inger and Iskandar, 2005. results): we propose to regard the entire Megophryinae sub- family as a single genus, with subgenus level groups to reflect Megophrys (Pelobatrachus) comb. nov. primary molecular clades. —Pelobatrachus Beddard, 1908:Typespecies:Cer.[atophryne] The following list of species assigned to subgenus level is nasu¨ta Schlegel, 1858, by monotypy for the genus-level rank. based largely on the results of the expanded phylogeny (fig. 3). —Borneophrys Delorme, Dubois, Grosjean and Ohler, 2006: Species not included in molecular analyses are underlined, Type species: Megophrys edwardinae Inger, 1989,bymono- and are provisionally assigned to clades based on general typy for the genus-level rank. morphological similarity with other members either based Remarks: The oldest name previously considered avail- on published morphological descriptions of the species, or able for this clade is Ceratophryne Schlegel, 1858 (Gorham examination of specimens. Species for which examined speci- 1966), however, a review of historical literature indicates mens included types are preceded by *, morphological exam- that this name should be considered nomenclaturally ination of species that included topotypic specimens are unavailable and thus the oldest available name, preceded by ^, and those only based on referred specimens Pelobatrachus Beddard, 1908 takes priority for this taxo- from areas other than the type locality are preceded by ! nomic group (for discussion, see supplementary text S5, Supplementary Material online). The genus level taxon Megophrys (Atympanophrys) Borneophrys is not found to be sufficiently morphologically —Atympanophrys Tian and Hu, 1983: Type species: diagnosable from Pelobatrachus based on the originally pro- Megophrys shapingensis Liu, 1950, by original designation posed definition, and is here regarded a junior subjective for the genus-level rank. synonym (for discussion see supplementary text S6, Distribution: Endemic to China: Yunnan Province, Supplementary Material online). through much of Sichuan Province, to bordering Gansu Distribution: Thailand: south peninsular; Singapore; Province (1,600–3,200 m) (Fei et al. 2009). Malaysia; Indonesia: Natuna Is., Sumatra Is. and Inclusion(3species):Megophrys (Atympanophrys)gigan- Kalimantan; Brunei; Philippines (Boulenger 1912; Taylor tica Liu, Hu and Yang, 1960; Megophrys (Atympanophrys) 1920; Inger 1954). nankiangensis Liu and Hu, 1966 (Hu et al. 1966); Inclusion(6species):*Megophrys(Pelobatrachus)baluen- *Megophrys (Atympanophrys) shapingensis Liu, 1950. sis (Boulenger, 1899b); *Megophrys (Pelobatrachus) edwardi- nae Inger, 1989;^Megophrys (Pelobatrachus) kobayashii Megophrys (Brachytarsophrys) Malkmus and Matsui, 1997;^Megophrys (Pelobatrachus) —Brachytarsophrys Tian and Hu, 1983: Type species: ligayae Taylor, 1920;!Megophrys (Pelobatrachus) nasuta Leptobrachium carinense Boulenger, 1889,byoriginaldesig- (Schlegel, 1858)[synonymMegalophrys chysii Edeling, nation for the genus-level rank. 1864]; *Megophrys (Pelobatrachus) stejnegeri Taylor, 1920. Distribution: Throughout much of southern China (Sichuan, Chongqing, Yunnan, Guizhou, Guangxi, Hunan, Megophrys (Ophryophryne) Jiangxi and Guangdong) (Fei et al. 2009); in the west, northern —Ophryophryne Boulenger, 1903b:Typespecies: and eastern Myanmar, and northern and western Thailand Ophryophryne microstoma Boulenger, 1903b,bymonotypy (as far south as Phang Nga Province) (Boulenger 1908; for the genus-level rank. Delorme et al. 2006); in the east, south through Laos and Remarks: The recognition of Ophryophryne as a subgenus- Vietnam (as far south as Lam Dong Province) (Smith 1921; level species group within Megophrys creates a nomenclatural Inger et al. 1999; Stuart 2005). issue, where Ophryophryne pachyproctus Kou, 1985 becomes Inclusion (5 species): *Megophrys (Brachytarsophrys) car- a junior secondary homonym of Megophrys pachyproctus inense (Boulenger, 1889) (synonym Brachytarsophrys Huang, 1981 (Huang and Fei 1981) according to the

754 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE

Principle of Priority (International Commission of Zoological lini comb. nov. (Wang and Yang, 2014 [Wang et al. 2014]); Nomenclature 1999: articles 52.3, 57.3). We therefore propose ^Megophrys (Panophrys) minor Stejneger, 1926; Megophrys the replacement name Megophrys (Ophryophryne) koui nom. (Panophrys) obesa Wang,LiandZhao,2014(Li et al. 2014); nov. for Ophryophryne pachyproctus Kou, 1985 in compli- *Megophrys (Panophrys) omeimontis Liu, 1950;!Megophrys ance with article 60.3 (International Commission of (Panophrys) palpebralespinosa Bourret, 1937; Zoological Nomenclature 1999), where the specific epithet Megophrys (Panophrys) sangzhiensis Jiang, Ye and Fei, is a patronym for the author of the original name, out of 2008; Megophrys (Panophrys) shuichengensis Tian and respect for his taxonomic contribution. Sun, 1995; Megophrys (Panophrys) spinata Liu and Hu, Distribution: From Guangxi and eastern Yunnan, of 1973 (Hu et al. 1973); Megophrys (Panophrys) tuberogranu- southern China, south through northwestern Thailand, latus Shen, Mo and Li, 2010 (Mo et al. 2010); Megophrys much of Laos and Vietnam, entering eastern Cambodia (Panophrys) wawuensis Fei, Jiang and Zheng, 2001 (Fei and (Ohler 2003; Stuart et al. 2006a; Fei et al. 2009). Ye 2001); * ( ) Ye Megophrys Panophrys wuliangshanensis Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 Inclusion (6 species): *Megophrys (Ophryophryne) gerti and Fei, 1995; Megophrys (Panophrys) wushanensis Ye comb. nov. (Ohler, 2003); *Megophrys (Ophryophryne) hansi and Fei, 1995. comb. nov. (Ohler, 2003); *Megophrys (Ophryophryne) micro- stoma (Boulenger, 1903b); Megophrys (Ophryophryne) koui Megophrys (Xenophrys) nom. nov.;!Megophrys (Ophryophryne) poilani (Bourret, —Xenophrys Gu¨nther, 1864:Typespecies:Xenophrys monti- 1937); *Megophrys (Ophryophryne) synoria comb. nov. cola Gu¨nther, 1864,bymonotypyforthegenus-levelrank. (Stuart, Sok and Neang, 2006). Remarks: The type series of Xenophrys monticola Gu¨nther, 1864 is composed of two syntype specimens currently in the Megophrys (Panophrys) Natural History Museum, London (BMNH), one from Sikkim —Panophrys Rao and Yang, 1997a:Typespecies:Megophrys (BMNH 1947.2.25.13), and one from “Khasya” (BMNH omeimontis Liu, 1950, by original designation for the genus- [18]53.8.12.52). Though it appears that both specimens rep- level rank. resent members of the Megophrys (Xenophrys)subgenus, Remarks: Some of the below listed taxa that were not morphologically they do not appear to represent the same examined morphologically, or included in molecular analyses biological species. In accordance with Article 74.7 of the Code in this study are provisionally included in this subgenus based (International Commission on Zoological Nomenclature on superficial similarities with sampled congeners, however it 1999), through lectotype designation we restrict the name- is likely that at least some of these taxa may be found to be bearing type of Xenophrys monticola Gu¨nther, 1864 to the part of the M. (Xenophrys) clade. A sequence of M. jingdon- adult female specimen (BMNH 1947.2.25.13 [formerly BMNH gensis (based on specimens from Vietnam) from Grosjean 60.3.19.1336]: illustrated in Gu¨nther 1864:pl.xxvi,fig.H:snout et al. (2015) was included in the expanded phylogenetic align- to vent length 40.5 mm) from Sikkim state, northeast India. ment (Dataset 2—tree not shown) and was found to be This action is essential to provide nomenclatural stability to nested in the M. (Panophrys)subgenus. both the species and subgenus level taxon. Distribution: Throughout much of mainland China south Distribution: From western Nepal, east along the south- of approximately 33 latitude, ranging as far west as Sichuan ern Himalayas, through Bhutan and northeastern India, hilly and Yunnan, and south into northern Laos, Vietnam and eastern Bangladesh, Myanmar, and throughout hilly regions Thailand (Stuart 2005; Delorme et al. 2006; Fei et al. 2009; of mainland Southeast Asia (Thailand, Laos, Vietnam, Orlov et al. 2015). Possibly into bordering areas in northern Cambodia, West Malaysia) (Boulenger 1893, 1908; Stuart and northeastern Myanmar. A questionably identified speci- 2005; Mahony and Ali Reza 2008, Mahony et al. 2013; men of M. (P.) minor was reported from Arunachal Pradesh, Mahony 2011). In southern China, with isolated pockets in northeast India (Mahony et al. 2013). southern Xizang, throughout Yunnan, Guangxi, Guangdong, Inclusion (25 species): Megophrys (Panophrys) acuta and southern Hunan (Fei et al. 2009). Unconfirmed reports of Wang, Li and Jin, 2014 (Li et al. 2014); Megophrys X. aceras from Sumatra (IUCN SSC Amphibian Specialist (Panophrys) baolongensis Ye, Fei and Xie, 2007; Megophrys Group 2014), and reports of megophryine tadpoles in north- (Panophrys) binchuanensis Ye and Fei, 1995; Megophrys western India (Annandale 1911; Ray 1997) require further (Panophrys) binlingensis Jiang, Fei and Ye, 2009 (Fei et al. verification. 2009); *Megophrys (Panophrys) boettgeri (Boulenger, 1899a); Inclusion (21 species): M. aceras clade: *Megophrys *Megophrys (Panophrys) brachykolos Inger and Romer, 1961; (Xenophrys) aceras Boulenger, 1903a;*Megophrys Megophrys (Panophrys) caudoprocta Shen, 1994; Megophrys (Xenophrys) longipes Boulenger, 1886. M. lekaguli clade: (Panophrys) cheni comb. nov. (Wang and Liu, 2014 [Wang et *Megophrys (Xenophrys) lekaguli Stuart, Chuaynkern, Chan- al. 2014]); Megophrys (Panophrys) daweimontis Rao and Yang, ard and Inger, 2006;^Megophrys (Xenophrys) parva 1997a; Megophrys (Panophrys) huangshanensis Fei and Ye, (Boulenger, 1893); *Megophrys (Xenophrys) takensis Mahony, 2005;*Megophrys (Panophrys) jingdongensis Fei and Ye, 2011. M. major clade: *Megophrys (Xenophrys) glandulosa Fei, 1983; Megophrys (Panophrys) jinggangensis comb. nov. Ye and Huang, 1990;*Megophrys (Xenophrys) major (Wang, 2012 [Wang et al. 2012]); *Megophrys (Panophrys) Boulenger, 1908 (synonym ^Megophrys longipes maosonensis kuatunensis Pope, 1929; Megophrys (Panophrys) latidactyla Bourret, 1937); Megophrys (Xenophrys) mangshanensis Fei and Orlov, Poyarkov and Nguyen, 2015; Megophrys (Panophrys) Ye, 1990 (Fei et al. 1990); *Megophrys (Xenophrys) monticola

755 Mahony et al. . doi:10.1093/molbev/msw267 MBE comb. nov. (Gu¨nther, 1864); *Megophrys (Xenophrys) robusta samples of Atympanophrys, Ophryophryne,andPanophrys Boulenger, 1908. M. megacephala clade: *Megophrys formed a morphologically distinct clade from the single (Xenophrys) ancrae Mahony, Teeling, and Biju, 2013; Brachytarsophrys species they examined. They noted slight *Megophrys (Xenophrys) megacephala Mahony, Sengupta, differences in the degree of enlargement of the oral disc, Kamei and Biju, 2011;*Megophrys (Xenophrys) oropedion whether eyes were orientated dorsolaterally or laterally, and Mahony, Teeling, and Biju, 2013;*Megophrys (Xenophrys) whether the oral disc orientation was dorsal or “antero-up- serchhipii comb. nov. (Mathew and Sen, 2007); *Megophrys ward”, however no substantial morphological differences (Xenophrys) zunhebotoensis comb. nov. (Mathew and Sen, were found. Grosjean (2003) also found no significant differ- 2007). M. vegrandis clade: *Megophrys (Xenophrys) vegrandis ences in tadpole morphology to support the recognition of Mahony, Teeling, and Biju, 2013. Clade unassigned: genera in Megophryinae. Our extensive study of post meta- *Megophrys (Xenophrys) auralensis Ohler, Swan and Daltry, morphosed frogs, covering all molecularly resolved clades 2002;* ( ) Mahony, 2011; found that external morphological characters previously Megophrys Xenophrys damrei Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 *Megophrys (Xenophrys) medogensis Fei, Ye and Huang, regarded diagnostic at the genus level, were ambiguous 1983; Megophrys (Xenophrys) pachyproctus Huang, 1981 when considering all species in most molecular clades (Huang and Fei 1981); *Megophrys (Xenophrys) zhangi Ye (supplementary text S4 and S6, Supplementary Material on- and Fei, 1992. line). Thus, as a resolution to this age-old taxonomic conun- drum, it is here proposed to simply recognise the moderately Megophrys Incertae Sedis recently diverged Megophryinae clade as a single-genus level Remarks: Megophrys dringi Inger, Stuebing and Tan, 1995 was species group, Megophrys. This relegates the previously rec- previously assigned to the genus Xenophrys (Dubois and ognised genus-level ranks, to molecularly resolved subgenus- Ohler 1998; Ohler 2003; Delorme et al. 2006). Having level clades, therefore satisfying biologists’ requirements to examined the type series of Megophrys dringi as part of this have more refined species groups to work on, without the study, it appears to be morphologically most similar to some need for unnecessary large-scale nomenclatural rearrange- members of the M. (Xenophrys)subgenus.Refertothe ments at the genus level. Our phylogenetic study has also supplementary text S2, Supplementary Material online for identified several potentially undescribed species-level taxa in phylogenetic results related to the molecular systematic is- the M. (Xenophrys), M. (Panophrys), and M. (Ophryophryne) sues associated with this species. subgenera that require further taxonomic attention in order Distribution: Malaysian Borneo. to provide a better understanding of conservation risks faced Inclusion (1 species): *Megophrys dringi Inger, Stuebing by species with potentially limited geographic distributions. and Tan, 1995.

Discussion Phylogeny This study represents the first extensive phylogenetic analyses Higher Taxonomy of the genus Megophrys (as redefined here), covering all The highly supported molecular tree shows that several of the known previously named genus-level species groups and their currently recognised genera within Megophryinae are para- type species (bar Borneophrys). Also, for the first time, nuclear phyletic and recently diverged. Rao and Yang (1997) data have been used to produce a systematically stable mo- recognised five genera, and considered karyotype data, broad lecular framework elucidating the relationships of most sub- generalisations of morphology (e.g., head proportions and genera within this genus. However, the phylogenetic whether digit tips are rounded or flat), and a single behav- relationships within the group are not yet fully resolved as ioural trait (whether or not frogs were typically found under evidenced by several disagreements between trees generated stones) as useful for diagnosing genus-level groups. However, using different tree building algorithms and models of se- the karyotype data they presented overlaps significantly be- quence evolution. Relationships within the clade containing tween “genera”, and the other generalisations are also too three M. (Xenophrys) species groups (megacephala, lekaguli, ambiguous for diagnosing genus-level species groups. Zheng and major), and intraspecies relationships within M. et al. (2002) described differences between the sperm mor- (Pelobatrachus), and the M. (X.) megacephala species group phology of some named genus-level groups. Atympanophrys often agreed between the ML and BI (GAMMA) analyses, but and Brachytarsophrys had distinctly longer spermatozoa than sometimes differed significantly when using the BI (CAT) members of Ophryophryne and their “Megophrys”(which model. Lartillot et al. (2007) demonstrated that compared consisted of members of both Xenophrys and Panophrys). with other models, the CAT model can be less prone to However, insufficient morphological differences between mo- systematic errors such as long branch attraction, which might lecular clades (as resolved in this study, i.e., Xenophrys and account for the observed topological differences since the Panophrys) conflicts with the use of spermatozoa morphol- aforementioned groups either contain, or are adjacent to ogy as a significant diagnostic character for the recognition of long branch taxa. The low support values obtained for these these genera. Li et al. (2011) attempted to identify tadpole nodes might also be due to incomplete taxon sampling (in- morphological characters that may diagnose different groups cluding regional sampling bias), insufficient phylogenetic sig- (Atympanophrys, Brachytarsophrys, Ophryophryne, nal in the sequence dataset, incomplete lineage sorting due to Panophrys [as “Xenophrys”]). They concluded that their rapid diversification and/or even loss of genetic signal due to

756 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE extinctions. The inclusion of additional molecular markers supplementary table S5E, Supplementary Material online). and taxa in future studies might resolve these “hard” nodes. The optimum RelTime trees correspond very well with esti- matesofmajorvicariantevents(table 1), e.g., the split be- Timetree tween the Gondwanan Pipidae and Laurasian Rhinophrynidae For anurans: The four optimum RelTime trees correlated is estimated towards the more recent approximation of relatively well with both the fossil record and major tectonic around 150 MY for the terrestrial split between these land events that may have acted as vicariant events in amphibian masses (e.g., Ezcurra and Agnolın 2012), rather than older evolution (table 1; fig. 4A; supplementary table S5A and C, estimates of up to 190 MY (e.g., Bewick et al. 2012), which Supplementary Material online). RelTime estimates are also corresponds more closely with results obtained from the considerably more recent for most nodes than those ob- MCMCtree analyses (fig 1; table 1). tained using other, more complex Bayesian methods, which The optimal RelTime trees estimated the age of the MRCA of Pelobatidae–Pelodytidae, and Pelobatidae–Megophryidae rely more heavily on multiple internal calibrations. The Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 MCMCtree analyses using the novel combination of fossil at more than þ50% earlier than the oldest known fossils for priors produced several node age estimates comparable those nodes, with the latter estimated to be close to the with previous studies using similar Bayesian methods (San oldest unassigned pelobatoid fossils. This implies that despite Mauro et al. 2005; Roelants et al. 2007; Kurabayashi et al. Aerugoamnis paulus Henrici, Baez and Grande, 2013 and 2011; Pyron 2014)(comparetable 1 with supplementary table Eopelobates deani Rocek, Wuttke, Gardner and Bhullar, S5H, Supplementary Material online), exceptions being older 2014 representing the earliest phylogenetically assigned fossils, dates obtained here for the root ages of Discoglossidae (180– theymaybeconsiderablymorerecentthantheMRCAsof 158 vs. 152–110 MY), Pelodytidae (170–163 vs. 143–130 MY), their respective families, and thus are likely to be unsuitable Pelobatidae (141–131 vs. 126–101 MY), Scaphiopodidae for use as fossil priors in dating analyses of the anuran tree. (183–177 vs. 167–154 MY), and Costata (253–246 vs. 240– The node age averages for most family level MRCA in 210 MY), and the considerably younger estimates for the MCMCtree analyses, and most compared published studies crown divergence of Neobatrachia (116–113 vs. 161–125 (bar Marjanovic and Laurin 2014), were estimated to be more MY). than þ30% earlier than the oldest known fossils for most Chunerpeton tianyiense Gao and Shubin, 2003 is an am- nodes, suggesting extensive ghost lineages within Anura. phibian fossil often used to date the root of Cryptobranchidae However, the better concordance between the fossil record at a minimum of 145, 161, or 166 MY, depending on the study and major tectonic events, and the RelTime AT estimates (Roelants and Bossuyt 2005; Bossuyt et al. 2006; Roelants et al. imply that RelTime might provide more realistic estimates 2007; San Mauro et al. 2005; Kurabayashi et al. 2011; Zhang of divergence times than other dating methods. et al. 2013). This age is considerably older than the estimates For Megophrys: Intheabsenceofaknownfossilrecord,it produced using MCMCtree (58–33 MY, CI 103–19 MY), and is difficult to evaluate the accuracy, success or failure of either all RelTime (AT ¼ 134–96 MY) dating, except when the root RelTime or MCMCtree’s ability to produce reliable timetree age range was set to a very broad (and unrealistic) 247.2–600 estimates. One of the benefits of using a relative time estima- MY in RelTime (supplementary table S5A–E, Supplementary tion method is that results can be used to observe the order Material online). This further questions the phylogenetic as- and patterns of divergence in a phylogenetic tree (Loader signment of C. tianyiense to Cryptobranchidae (e.g., et al. 2007; Tamura et al. 2012). Our four optimum RelTime Marjanovic and Laurin 2014). The regular use of trees and the MCMCtree tree for the nuDNA dataset (3b) Eodiscoglossus oxoniensis Evans, Milner and Mussett, 1990 demonstrate that the primary Megophrys clades were influ- (minimum 164 MY) to constrain the root of discoglossoids enced by four distinct diversification events that each (e.g., Bossuyt et al. 2006; Roelants et al. 2007; Kurabayashi et al. lasted <3.7 MY for RelTime and <6.1 MY for MCMCtree 2011; Zhang et al. 2013) fits well with our more reliable Dataset 3b. These results indicate that most major clades RelTime estimations (AT 189–173 MY), and is considerably originated during an extended cool and dry period in SE more recent than MCMCtree estimates (253–246 MY). Asia lasting between 34 and 20 MY and in the beginning of However, its phylogenetic relationship with the significantly a primarily warm and wet period that lasted between 20 more recent E. santonjae Villalta, 1957 requires further inves- and 12 MY (Bain and Hurley 2011, and references therein). tigation before it should be continued to be used as a con- Most clades are estimated to have begun crown group diver- straint for this node (Marjanovic and Laurin 2007, 2014; Baez sification during a period that exactly coincides with the pe- 2013). The use of Rhadinosteus parvus Henrici, 1998 by others riod of climatic cooling and drying in Southeast Asia (12–6 to date the root of Rhinophrynidae at a minimum of 152 MY MY) (supplementary table S6, Supplementary Material on- (e.g., Bossuyt et al. 2006; Blackburn et al. 2010; Kurabayashi line). During this period previously widespread humid forests et al. 2011; Zhang et al. 2013) is found to be older than our contracted, and dry forest expanded (Bain and Hurley 2011, RelTime estimates based on setting the root age alone at 247. and references therein), which could have driven diversifica- 2–350 MY. However, it is very close to estimates for that node tion through vicariance. By contrast, MCMCtree date estima- obtained from RelTime analyses that included the prior tions for the two datasets containing mitochondrial Iberobatrachus angelae Baez, 2013 (Fossil 2), and those based sequences (3a and 3c) present a distinctly less structured on vicariant events (158–149 MY), whereas MCMCtree diversification history for Megophryinae with crown group estimates this node age to be 202–201 MY (fig. 4; table 1; diversifications of major clades occurring throughout the

757 Mahony et al. . doi:10.1093/molbev/msw267 MBE warm and wet, and cool and dry periods during the past 34 optimum RelTime and nuDNA only MCMCtree analyses MY. Simulation studies demonstrated that MCMCtree suf- date the split between these two pairs of sister taxa at be- fered from lower accuracy than RelTime in datasets where tween 5.1–3.3 MY and 2.7–2.3 MY respectively, suggesting expected evolutionary rates varied randomly on each branch themorelikelyscenarioofasingle species dispersal during at 6100% of the overall rate, and when there were clade- early surface level uplift of the plateau, followed by an ex- specific rate accelerations (Tamura et al. 2012). This might tended isolation of M.(X.) oropedion and M.(X.)cf.major[1] explain the observed differences between estimates from from their ancestral lineages in the Naga Hills. The MCMCtree MCMCtree analyses of the datasets containing mtDNA (3a estimates from the nuDNA only dataset (3b) for the M.(X.)cf. and 3c) and the nuclear only dataset (3b). major[1]–M.(X.)cf.major[3] split (ca. 4.6 MY) corresponds The question remains, should the considerably older date well with RelTime, however, the split between M.(X.) orope- estimates obtained from software such as MCMCtree, dion and M.(X.)cf.oropedion (ca. 6.9 MY) still considerably MultiDivTime and R8s on mito-nuclear datasets, be consid- predates surface uplift of the Shillong Plateau. Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 ered more reliable than estimates using RelTime? The results All of these alternative divergence-dispersal scenarios are presented here suggest the answer is simply no, however possible, and it should be kept in mind that practically nothing these results may be specific to this study. The MCMCtree is known of amphibian extinction events in the eastern analyses of Datasets 3a and 3b date the divergence between Himalayas and Southeast Asia to favour one over the other. the Palawan endemic M.(P.) ligayae and its Bornean conge- In the above highlighted examples, date estimates from ners at ca. 20 MY (supplementary table S6, Supplementary MCMCtree would require either a minimum three step pro- Material online), at which time the Palawan plate would have cess as a means to explain such dispersal-divergence events been situated far to the north of Borneo in the south China between these sister taxa, that includes a dispersal across land, Sea (Blackburn et al. 2010; de Bruyn et al. 2014). These dates followed by a vicariantevent,and subsequentextinction of one would suggest that the ancestor of this species (assuming lineage. Or, a two step process in the case of the long-distance geographical vicariance was responsible for divergence) oceanic dispersal hypothesis. However, RelTime was found to would have had to undergo an oceanic dispersal of hundreds provide dating estimates that appear to correspond with a of kilometers. By contrast, the date estimates provided by significantly less complicated and presumably more likely ex- RelTime (ca. 11.4–10.1 MY) and the slightly older planation involving a minimum two step process involving a MCMCtree of Dataset 3b (ca. 13.3 MY) (supplementary table short-distance dispersal, followed by a vicariant event. S6, Supplementary Material online) put this divergence at RelTime has recently been described as a fourth generation approximately the same time that Palawan either formed a timetree methodology (Kumar and Hedges 2016), and has temporary land connection with, or at least was situated in been demonstrated to produce comparable time estimations very close proximity to the island of Borneo (Blackburn et al. to third generation methods (e.g., MCMCtree) when applied 2010). These date estimates also corresponded well with sug- to simulated and empirical datasets (Tamura et al. 2012). It is gested land bridge connections and dispersal events observed notclearwhyRelTimeestimationsinourstudydiffersocon- by the bombinatorid genus, Barbourula Taylor and Noble, siderably from results obtained from Bayesian methods (e.g., 1924 (Blackburn et al. 2010). San Mauro et al. 2005; Roelants et al. 2007; Kurabayashi et al. Other examples of where MCMCtree date estimates 2011; Pyron 2014; results herein). Using a large number of (Datasets 3a–3c) appear to be excessively old in comparison internal node priors has been found to improve the reliability with the RelTime estimates, are for species that are currently of Bayesian timetree estimations by reducing the influence of distributed primarily (or only) on the Shillong Plateau, north- individual erroneous priors (Meredith et al. 2011; Zheng and east India. MCMCtree of the mito-nuclear datasets (3a and Wiens 2015): the mammal dataset used by Tamura et al. 3c) dated the divergence of M.(X.)cf.major[1], sampled from (2012) to compare methods, used 82 calibrations (64 con- the Garo Hills, a western extension of the Shillong Plateau, straining nodes within the placental mammals) in their from its sister taxon M.(X.)cf.major[3], sampled from the MCMCtree analyses. However, amphibian timetree studies adjacent Naga Hills, at ca. 8.9–7.4 MY: and similarly the sister have used comparatively fewer (7–20), and often similar taxa M.(X.) oropedion and M.(X.)cf.oropedion also endemic sets of internal priors for estimating divergence times over a to the Shillong Plateau and the immediately adjacent Naga considerably deeper time scale than the mammal tree. Hills respectively, at ca.9.3MY(supplementary table S6, RelTime has also been found to out-perform MCMCtree Supplementary Material online). The uplift of the Shillong (and R8s) under circumstances where evolutionary rates Plateau is estimated to have begun 15–8 MY, however, due varied extensively among branches (Tamura et al. 2012), a to the erosion of the overlying comparatively soft sedimen- circumstance that might be expected in datasets covering a tary rock, actual surface level uplift of the plateau may have broad range of distantly related lineages. These issues alone, or been as recent as 4–3 MY (Biswas et al. 2007; Clark and combined, may have resulted in lower accuracy, while still Bilham 2008). For the mito-nuclear MCMCtree estimations maintaining a degree of apparent consistency towards older to be explained biogeographically, it would imply that these divergence estimations between different studies. lineages were already deeply diverged in the Naga Hills (or adjacent regions), followed by the more recent dispersal of Biogeography of Megophrys one lineage onto the plateau and subsequent local extinction The program BioGeoBEARS, used to infer the possible histor- of that taxon from its ancestral range. In contrast, our ical biogeography of Megophrys was not found to be

758 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE particularly insightful on this dataset, especially where extant patches surrounded by drier seasonal forest (Bain and taxa on adjacent branches are geographically distant from Hurley 2011), which may have resulted in vicariance due one another. At the root, for example, the more ancestrally to habitat restriction. This period saw the divergence of diverged lineages are either the M–P clade from south of the the ancestors of three primary M. (Xenophrys) radiations Isthmus of Kra, or the northern A–B clade from China and (major, lekaguli,andmegacephala species groups), as well Indochina north of the Isthmus of Kra. In this situation, the as the initial divergences within all subgenus-level groups of group could have originated from almost any part of the megophryines. Megophrys geographic distribution. Limiting the analysis to Though most subgenus-level groups and species groups allow for a maximum of three ancestral regions per node are restricted to limited geographic ranges, e.g., the northeast resulted in an understandably vague and geographically dis- Indian endemic M. (X.) megacephala group, and Chinese en- connected ancestral range estimate as China-Himalaya-Java/ demic M. (Atympanophrys), etc., interestingly the three clades Sumatra. Other problematic nodes are the split between the that demonstrate the most considerable dispersal abilities, are Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 Himalayan M. (X.) vegrandis and all remaining M. (Xenophrys) comprised of the physically largest species with members species groups, since the second most ancestrally diverged tolerant of low to mid-elevation habitats, e.g., M. branch of the M. (Xenophrys) clade represents a geographi- (Brachytarsophrys) (Thai-Burma, China, Indochina), M. cally disparate taxon that is endemic to the southern half of (Pelobatrachus) (Borneo, Philippines, Thai-Malaya peninsula, the Thai-Malaya peninsula (M. [X.] aceras). These two taxa sit Sumatra), and the M. (X.) major species group (Himalaya, on long branches with presumably a long history of extinc- China, Thai-Burma, Indochina), implying that large body tions and dispersal events. A significant number of unde- size may have provided dispersal advantages within scribed taxa are suspected to occur in Myanmar (assuming Megophrys. Branching patterns within M. (Xenophrys)indi- a similar pattern of diversity as observed for northeast India), cate that at least two into, and one out-of-India dispersal indicating that a major regional bias exists in our dataset due events have occurred during the early and late Miocene. to the poor sampling of this region. Further exploration of The oldest extant lineage within M. (Ophryophryne)(M. intervening regions in Myanmar may reveal additional mem- (O.) synoria) is currently considered endemic to the southern bers of these species groups that will help resolve the histor- limits of the geographic range for the genus in northeastern ical biogeography of the M. (Xenophrys)subgenus.Thelackof Cambodia, with a clear south to north dispersal and diversi- topological resolution for the positions of the M. (Xenophrys) fication of the subgenus through Vietnam (and presumably species groups, major, lekaguli and megacephala, relative to Laos), into southern China, and west into northern Thailand. each other, or the intertaxa relationships within M. Furthermore, if RelTime AT estimates within Megophrys are (Pelobatrachus) also prevents a reliable interpretation of his- to be considered reliable, significant marine incursions into torical biogeography, without attempting to test all possible the Sunda Shelf in the late Oligocene to early Miocene (be- topologies. tween 25 and 20 MY) are suggested to have severed terrestrial The intricate and dynamic biogeography of southern and connections between the islands of Sumatra/Java and penin- southeastern Asia requires careful interpretation of many still sular Malaysia/Borneo (Hall 2009, 2012; de Bruyn et al. 2014), poorly understood prehistoric events (e.g., climatic, geological, which may account for the divergence between M. habitat, and sea level fluctuations through time) that may (Megophrys)andM. (Pelobatrachus) dated at around this have led to vicariance, dispersal, and diversification within time (fig. 1; supplementary table S6, Supplementary fauna with limited oversea dispersal abilities. Some interesting Material online). A short temporary land bridge between dispersal patterns can, however, be interpreted from the phy- Sumatra and Java at around 10 MY (Hall 2009, 2012; de logenetic analyses, in combination with the results obtained Bruyn et al. 2014) could account for the deep divergence from the RelTime dating analyses, considered in this study to between the Sumatran M.(M.) sp. and Javan M.(M.) mon- be more reliable than the MCMCtree estimates (as demon- tana estimated to have occurred sometime between 13.6 and strated above). Between approximately 28.9–24.5 MY the 9.6 MY (fig. 1; supplementary table S6, Supplementary MRCA of the Megophrys clade underwent a major south to Material online). north (or vice versa) long distance dispersal, which corre- sponds well with the primary Himalayan uplift during the early Miocene (see Aitchison et al. 2007; Adlakha et al. Conclusions 2013, for review). The rise of mountain ranges connecting By integrating multiple molecular analytical techniques, this the highlands of the Sundas in the south (Hall 2012), with study has led to a dramatic improvement on the sparsely China, coinciding with the warm wet climate from around 20 known, and largely misunderstood evolutionary history of to 12 MY (Bain and Hurley 2011) may have provided ideal one of the most geographically widespread Asia anuran gen- conditions for range expansion throughout southern China era—Megophrys. Despite not resolving the basal topology of and Indochina, and during this time, several major clades the phylogenetic tree, the remaining systematic relationships originated, M. (Atympanophrys), M. (Brachytarsophrys), M. between most clades of species is well resolved, and for the (Ophryophryne), M. (Panophrys), and the M. (Xenophrys) first time, a working taxonomy is available for this group. The vegrandis and M. (X.) aceras species groups. During the long relatively recent ancestral radiation of Megophrys in the late dry period that followed from about 12 to 6 MY, the contin- Oligocene, postdate the divergence of subgeneric level taxa in uous tropical wet forests receded into smaller isolated related genera, so, coupled with the absence of morphological

759 Mahony et al. . doi:10.1093/molbev/msw267 MBE or biological synapomorphies for most species groups, almost specimens. Prior to fixation, thigh muscle tissue was excised all currently described taxa are assigned to a subgeneric rank. for molecular analysis, stored in PCR grade absolute EtOH at - Dating results demonstrate that when compared with 20 C in the collection of SDBDU (Systematics Lab, University MCMCtree divergence time estimates, RelTime estimates of Delhi, Delhi, India). Additional megophryine tissue samples are more congruent with the fossil record for the anuran (thigh muscle or liver, depending upon availability) from tree, and historical biogeographic events for both the anuran Cambodia, China, India, Indonesia, Laos, Malaysia, tree and megophryine tree. Correlating RelTime divergence Myanmar, Philippines, Thailand, and Vietnam were provided dates with known geological events has provided a wealth of from the following collections: AMCC (Ambrose Monell Cryo strong biogeographic hypotheses that will be invaluable for Collection, American Museum of Natural History, NY, USA); future testing and comparative studies. RelTime achieves this CAS (California Academy of Science, CA, USA); FMNH (Field using only an approximate root prior with or without a single Museum of Natural History, IL, USA); IRSNB (Institut Royal internal prior, whereas, similar to other Bayesian methods, des Sciences Naturelles de Belgique, Brussels, Belgium); KU Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 MCMCtree suffered from lower accuracy when reduced or (University of Kansas Museum of Natural History, Division of no internal priors were specified. The dating methodology Herpetology, KS, USA); RGK (Rachunliu G. Kamei private optimised in this study (choice of molecular markers, novel collection); UTA (University of Texas at Arlington, fossil priors, etc.) may be applied to the many other anuran Department of Biological Sciences, TX, USA). Tissue samples amphibian genera and families that are not represented in the were obtained for a total of 52 species (49 in-group, 3 out- fossil record. With appropriate taxonomic modification to the group taxa: supplementary table S1, Supplementary Material analytical template, it should also prove useful to date phy- online), including the type species for all generic level taxa bar logenies in other taxonomic groups, with equally missing fos- Atympanophrys and Borneophrys. sil records. Owing to high incidences of species misidentification within museum collections of Megophryinae: the specimens Materials and Methods from which tissues were obtained through interinstitutional loans (except KU and UTA) were examined to verify identi- Taxon Sampling fications. Tissue sampled frog specimens from UTA were In order to address the systematic relationships within identified based on photographs of specimens in life and in Megophryinae, tissue samples were obtained for as many preservation. Tissues obtained from larvae could not be iden- species as possible, across all nomenclaturally available (re- tified based on morphology alone, due to the lack of mor- gardless of validity) genus-level groups (except Borneophrys): phological descriptions of larvae assigned (through molecular Atympanophrys, Brachytarsophrys, Ophryophryne, Panophrys, comparisons) to representative species known from adult Pelobatrachus, Megophrys,andXenophrys, making a particu- frogs, thus most larvae identifications in literature currently lar effort to include type species where possible (refer to sup require verification. Perhaps the most comprehensive mu- plementary table S1, Supplementary Material online). All seum specimen study yet to have been undertaken on this populations of M. (Xenophrys) that were sampled from genus was made in an effort to define morphologically, the northeast India by the Systematics Lab members molecularly delineated Megophrys species groups. This in- (University of Delhi, Delhi, India), have associated state-wise cluded specimens from the type locality (types and/or top- collection permits: Arunachal Pradesh CWL/G/13(17)/06-07/ otypes) of 48 species, out of a total of at least 62 species PT/3878-83 dated June 5, 2009; Manipur 3/22/2006-WL dated examined (supplementary text S3, Supplementary Material March 3, 2009, 34/2/Trg/2006/Forests/451 dated May 31, online). Examining multiple specimens (up to 23) of each 2008, letter from the “Addl. Principal Chief Conservator of taxon also provided valuable information on morphological Forests cum Chief Wildlife Warden, Government of Manipur variation within species, which facilitated the revision of the Dated, and 27.04.2007” [permit number not provided]; previously proposed genus-level morphological diagnoses Meghalaya FWC. G/16 dated February 19, 2009, FWC.G/173 that were based on a much less comprehensive examination dated March 23, 2009; Nagaland FOR/GEN-42/2006 dated of specimens (Delorme et al. 2006). Museum specimens were May 29, 2007; Tripura F. 8(163)/For-WL-2001/7,168-74 dated examined from the following institutions: NHM [Natural June 27, 2009, F.22-3(6)/For-JICA/I&P/B-D/07/Vol-II/2460 History Museum, London, previously British Museum dated July 1, 2009, F.22-3(6)/FOR-JICA/I&P/B-D/07/Vol-II/ (Natural History)—specimen acronym retained as BMNH 3522-24 dated August 1, 2009; West Bengal No.1806/WL/ for comparability with historical literature]; AMNH 4R-1(P-IX) dated June 24, 2011. The Systematics Lab members (American Museum of Natural History); ZSI (Zoological carried out fieldwork during the monsoon seasons from be- Survey of India, Kolkata, West Bengal, India); ZSI-E tween 2007 and 2011. Specimens were collected primarily (Zoological Survey of India, Eastern Regional Station, during opportunistic night surveys of suitable habitats, were Shillong, Meghalaya, India), IVC (Department of Zoology, humanely euthanised using an aqueous solution of ethyl 3- Arya Vidyapeeth College, Guwahati, Assam, India), CAS, aminobenzoic methanesulfonate salt (MS 222), fixed and FMNH, IRSNB, RGK. temporarily stored in 4% formalin, and later transferred to 70% EtOH for long-term storage. Prior to euthanisation, speci- Extraction and PCR Amplification mens were photographed for documenting colouration in life All tissues were extracted using Qiagen DNeasy Blood and and soft tissue morphology, typically obscure in preserved Tissue Kits primarily following the manufacturer’s

760 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE instructions, with the exception of an extended (10 min) Raj et al. 2012; Wang et al. 2012, 2014; Shen et al. 2013; Xiang room temperature incubation step prior to the elution of et al. 2013a; Li et al. 2014; Matsui et al. 2014; Oberhummer extracted DNA from the column, and additional final elution et al. 2014; Zhao et al. 2014; Hendrix et al. 2017 (supplemen step using 40 mlH2O. Universal primers were used to amplify tary table S1, Supplementary Material online). Due to the mitochondrial markers 12S-tValine-16S (Palumbi 1996; Fu et large volume of misidentified sequences present in al. 2007; Zhang et al. 2008), and nuclear markers: CXCR-4, GenBank (Padial et al. 2014; Peloso et al. 2015;andresults RAG-1 (Biju and Bossuyt 2003); TTN, SACS (Shen et al. presented herein), specific care was taken to ensure that 2011; supplementary table S8, Supplementary Material on- genes were sequenced from a single specimen when building line). PCR reactions were prepared in 24 ml aliquots compris- concatenated multiple gene datasets. In cases where se- ing: 2.0 mlextractedDNA(10ng/ml); 2.5 mlSigma10 PCR quences from multiple specimens could be combined to buffer minus Mg; 0.5 mlMgCL;0.5ml forward and reverse maximise multi-gene dataset coverage, efforts were made primer (10 ng/ml); 0.2 mlPlatinum DNA Polymerase to ensure any overlapping sequence portions originating Taq Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 (Invitrogen); 17.8 mlPCRgradeH2O. Ratios of ingredients from multiple specimens had high homology values using were adjusted accordingly for the optimisation of problem- BLAST, to prevent creating a single taxon dataset consisting atic samples. The following Touch-Down PCR reaction pro- of multiple taxa. GenBank sequences with <100 bp overlap tocols (Murphy and O’Brien 2007)wereused:(1)Touchdown with markers amplified in this study were excluded, e.g., 12S 55: 20 at 95 C, 10 cycles of 4500 at 95 C, 4000 at 65 C(witha sequences from Li et al. (2014) and Wang et al. (2014) had reduction of 1 C each cycle), 10 at 72 C, followed by 35 cycles only a 100 bp overlap with the 30 end of the 12S sequence of 4500 at 95 C, 4000 at 55 Cand10 at 72 C, and a final step of used in this study, and caused inconsistencies/misalignments 100 at 72 C; (2) Touchdown 50: 20 at 95 C, 10 cycles of 4500 at using MUSCLE (Edgar 2004)onMEGA6.06(Tamura et al. 95 C, 4000 at 60 C (with a reduction of 1 Ceachcycle),10 at 2013). Only the longest available sequences were retained 72 C, followed by 35 cycles of 4500 at 95 C, 4000 at 50 Cand when identical sequences were available for each species 10 at 72 C, and a final step of 100 at 72 C; (3) Touchdown (or specimen). Newly generated sequences were submitted 63–57: 20 at 95 C, 6 cycles of 4500 at 95 C, 4000 at 63 C(with to GenBank KY022152–KY022410, KX773566–KX773567 areductionof1Ceachcycle),10 at 72 C, followed by 35 (supplementary table S1, Supplementary Material online). cycles of 4500 at 95 C, 4000 at 57 Cand10 at 72 Candafinal Sequences were imported into MEGA 6.06, preliminarily step of 100 at 72 C. aligned using MUSCLE and corrected when necessary to the Nonspecific amplification was typical (with occasional ex- standard 30–50 orientation. nuDNA was translated to amino ceptions) using the primer sets for CXCR-4, RAG-1, SACS,and acid sequences, adjusted for open reading frames, and TTN (Biju and Bossuyt 2003; Shen et al. 2011). To eliminate checked to ensure absence of premature stop codons. Two nonsynonymous amplicons, one or a combination of two short indels (one and two codons in length) in the nuDNA techniques were commonly employed: 1) Touch-prep sequences of two out-group taxa, and a long indel which (Murphy and O’Brien 2007) of weak electrophoretic target replaces the tRNA-Val gene in the Leptolalax species (as ob- bands from initial PCR products, along with re-amplification served by others Xiang et al. 2013b; Zhang et al. 2013), were using the same PCR protocols with amplification cycles re- removed from the alignment (further details provided in sup duced to 30; 2) When the primers amplified the target gene plementary text S7, Supplementary Material online). Due to optimally for sequencing, but in the presence of non target the presence of extensive hyper-variable regions within bands, the target DNA bands were extracted using Montage noncoding mtDNA, that are typically difficult/or impossible DNA Gel Extraction Kits (Millipore). All PCR products of to unambiguously align, mtDNA was aligned on the T-Coffee sufficient quality from initial PCR amplification, and those web-server using a combination of MAFFT, MUSCLE, following technique 1 (above) were further purified using CLUSTALW,andtheT-Coffeealgorithms(Notredame et al. TSAP (Promega), and Sanger sequenced in both directions 2000). All positions assessed by the colour coding system to by Macrogen (Europe). be of poor or average reliability were manually removed from the alignment. However, since these ambiguously alignable Sequence Editing and General Alignment Datasets regions (AARs) contain phylogenetically informative posi- Sequence chromatograms were checked for quality, edited, tions, the unedited alignment was also retained and further and assembled into contigs using CodonCode Aligner 3.7.1 manually adjusted by eye on MEGA 6.06 for obvious misalign- (CodonCode Corporation, Dedham, Massachusetts). ments, to determine whether the removal of AARs negatively Heterozygous SNPs were assigned relevant IUPAC ambiguity affects phylogenetic signal. Nucleotide positions with missing codes. Sequences for additional taxa/specimens not amplified data were represented by “N” in alignments, i.e., where in this study, were obtained from GenBank, originating from sequences for some taxa are short, or where joining two the following publications: Biju and Bossuyt 2003; Garcıa-Parıs nonoverlapping adjacent sequences. et al. 2003; Zhang et al. 2003, 2006, 2013; San Mauro et al. Three different general alignment datasets were prepared 2004; Zheng et al. 2004a, 2004b; Evans et al. 2004; Roelants to facilitate the three primary analytical questions: 1) and Bossuyt 2005; Frost et al. 2006; Gissi et al. 2006; Stuart Megophryinae phylogeny: A 54 taxa dataset, with all taxa et al. 2006b; Veith et al. 2006; Evans 2007; Fu et al. 2007; Van represented by mtDNA and 1–4 nuclear genes (alignment Bocxlaer et al. 2009; Irisarri et al. 2010, 2011; Kurabayashi et al. length 4,994 bp) was assembled for the determination of the 2011; Ohler et al. 2011; Hamidy et al. 2012; Hasan et al. 2012; systematic relationships between clades of species (subgenera

761 Mahony et al. . doi:10.1093/molbev/msw267 MBE and species groups). This dataset represents 49 Megophryinae 10–15 million generations sampling every 1,000 generations. taxa sequenced in this study, three Leptobrachiinae out- Each run was assessed for convergence when the average group taxa, and GenBank sequences for Atympanophrys nan- standard deviation of split frequencies values were <0.05. kiangensis and Megophrys omeimontis.Foursubcategoriesof Log files were checked for adequate MCMC mixing on this alignment dataset were prepared: 1a) 54 taxa, mtDNA Tracer (Rambaut and Drummond 2013)toensureeffective only, with AARs retained, and removed (alignment lengths sample sizes (ESS) values were >200, and tree files plotted 1,989 bp and 1,796, respectively); 1b) 54 taxa, concatenated using the comparison function on AWTY web-server nuDNA markers (alignment length 3,196 bp); 1c) 54 taxa, (Nylander et al. 2008) to assess tree convergence. Analyses concatenated Datasets 1a and 1b (alignment length were either continued, or terminated when finished, discard- 4,994 bp); 1d) 46 taxa, reduced Dataset 1c, removing all ing the first 10% of trees as burn-in. PhyloBayes was used to taxa missing one or more markers, which included taxa/se- implement the GTR CAT model and a Dirichlet process to quences otherwise flagged as potential outliers (alignment describe rate variation across sites. Two simultaneous MCMC Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 length 4,994 bp). 2) Expanded phylogeny: This alignment chains were run sampling every 100 generations, and checking contains Dataset 1c, and is expanded to include unique se- for convergence after automatically discarding 20% of trees quences obtained from GenBank for Megophryinae (see sup from each independent run, and comparing the resulting 50% plementary table S1, Supplementary Material online), along majority rule consensus trees every 100 generations until split with an additional 11 sequencesofmtDNAgeneratedinthis frequency differences between chains converged on a val- study, covering a total of 114 specimens with one or more ue <0.1. Tree convergence was assumed when the same to- compatible genes (alignment length 4,894 bp). This alignment pology was obtained after repeating the analyses three times. included M. (Atympanophrys) shapingensis (the type species Prior to phylogenetic analyses, each gene was run sepa- of Atympanophrys) and was constructed for estimating the rately on RAxML to check for obvious outlier taxa, and gen- systematic position of additional taxa from GenBank in rela- erally observe whether nuclear markers provide sufficient tion to the clades determined from analyses of Dataset 1. 3) phylogenetic signal at the species level. Phylo-MCOA (de Timetree: Due to the absence of a fossil record for Vienne et al. 2012), an R package (R Core Team, 2015), was Megophryidae (Sanchiz and Rocek 1996), this alignment con- used to examine the individual alignments for outlier, or sists of Dataset 1c, with out-group taxa expanded to include rogue taxa/sequences that may be a result of natural genetic representatives of all currently recognised archaeobatrachian anomalies (e.g., incomplete lineage sorting, gene rearrange- families, along with two representative neobatrachian fami- ments, etc.), or analytical error (e.g., contamination), which lies, and two Caudata. The expansion of the out-group taxa often go unnoticed and can have detrimental effects on the was necessary for the utilisation of multiple fossil calibrations accuracy of phylogenetic tree estimation. Phylogenetic anal- in the dating analyses, rather than relying on the estimated yses can subsequently be run with identified outlier taxa/se- datesobtainedinotherstudiesaspriorsforin-groupnodes. quences removed to investigate their effect on tree Three alignments were prepared: 3a) combined estimations (de Vienne et al. 2012). Phylo-MCOA identified mt þ nuDNA dataset (alignment length 4,850 bp); 3b) two taxa (M.(A.) nankiangensis and M.(P.) omeimontis)and nuDNA only dataset (alignment length 3,196 bp); 3c) com- one sequence for another species (CXCR-4 of M.(X.)cf.ma- bined mtDNA þ RAG-1 þ CXCR-4 (alignment length jor[6]) as above the 50% threshold of being potential outliers 2,898 bp). Alignment Dataset 3c was used to determine the in our dataset (refer to supplementary text S1, effect of mostly missing data for TTN and SACS for the out- Supplementary Material online for discussion). The phyloge- group taxa. netic analyses of alignment Datasets 1a and 1b provide a direct topological comparison between the use of a Phylogenetic Analyses mtDNA only dataset, equivalent to previously published anal- Single gene alignments used in each dataset were assessed for yses (Jiang et al. 2003; Zheng, et al. 2004a, 2004b)compared their best fitting model of nucleotide evolution using with a nuDNA only alignment dataset, and the combined jModelTest2 (Darriba et al. 2012)ontheCIPRESplatform alignment dataset of (1c). Analyses of alignment Dataset 1d (Miller et al. 2010), under AIC (supplementary table S9, were intended to help determine the potential effect of miss- Supplementary Material online). The mtDNA was treated ing and outlier data on our primary (Dataset 1c) phylogenetic as a single noncoding gene/partition. Molecular phylogenetic reconstruction. All phylogenetic trees were viewed using inference was estimated using ML and Bayesian (BI) analytical FigTree (Rambaut 2009), with strong support considered methods. ML analyses were performed on RAxML-HPC2 for nodes having 95% values for bootstrap support (ML), (Stamatakis 2014) on XSEDE (CIPRES platform). All ML anal- or posterior probabilities (BI). yses used the model GTR GAMMA, treating each gene as a single partition, with 1,000 bootstrap replications, and other- Dating Calibrations wise under default parameters. BI analyses were performed on Owing to the largely incomplete anuran fossil record, con- MrBayes 3.2.3 (Ronquist et al. 2012), and PhyloBayes 3.3 flicting opinions exist as to the oldest recorded fossils for (Lartillot et al. 2009). Datasets were partitioned per gene, some taxa, or whether such fossils represent stem or crown and the nearest optimum model as assessed from taxa, so each calibration point was set as the minimum pos- jModelTest 2, was applied. Partitions were unlinked and sible age of relative nodes, to the base of the stratigraphic layer run on three hot and one cold chain over four runs, for from which the oldest assignable fossil was collected. Age

762 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE range estimates (not accounting for standard deviation) for (4) MRCA for the Hymenochirus–Pipa split at 83.6 MY chronostratigraphic stages follow Cohen et al. (2013), and based on the fossil pipid Pachycentrata taqueti (Baez when known, the North American land-mammal-age system and Rage, 1998) from the upper Cretaceous (NALMA). Of the fossil calibrations used in this study, several (Coniacian–Santonian, 89.8–83.6 MY) of Niger. This taxa recently described were used here for the first time, and is the oldest known species considered most closely re- others previously commonly used in dating analyses were not, lated to Hymenochirus (Rocek 2000; Bewick et al. 2012; creating a novel combination of divergence date estimation Gardner and Rage 2016). priors for Anura, some of which require explanation. (5) MRCA for the Ceratophrys–Nasikabatrachus split at 66 MY based on the late Cretaceous (Maastrichtian, Fossils 72.1–66.0 MY) fossil Beelzebufo ampinga Evans Jones (1) MRCA for the Anura–Caudata split at 247.2 MY based and Krause, 2008, currently debated to be a ceratophryid from Madagascar (Evans et al. 2008, 2014; Gardner and on the oldest known fossil stem group frogs, Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 Triadobatrachus massinoti (Piveteau, 1936)and Rage 2016). However, Faivovich et al. (2014) claimed that Czatkobatrachus polonicus Evans and Borsuk-Białynicka, the dataset used by Evans et al. (2014) for determining 1998 from the early Triassic (Olenekian, 251.2–247.2 the systematic position of this species contained a con- MY), of Madagascar and Poland respectively (Rage and siderable number of character scoring errors. Regardless Rocek 1989; Evans and Borsuk-Białynicka 1998; Shishkin of systematic affinities, there appears to be no doubt and Sulej 2009; Gardner and Rage 2016). that this is a crown group Neobatrachia (Gardner and (2) MRCA for the Discoglossus–Alytes splitat125MYbased Rage 2016), thus for dating this node, the genus on the oldest partially complete costatan fossils repre- Ceratophrys (Ceratophryidae) was selected to represent senting Iberobatrachus angelae from the late Cretaceous a recent hyloid. Along with its sister taxon Sooglossidae, (upper Barremian, 129.4–125.0 MY) of the La Nasikabatrachidae is one of the earliest diverged, extant Hue´rguina Formation, Spain (Baez 2013). Through phy- neobatrachian lineages, that is, either a basal hyloid logenetic analyses, Baez (2013) found it to represent a (Frost et al. 2006), a basal ranoid (San Mauro et al. discoglossid, more similar to the extant Discoglossus than 2005; Roelants et al. 2007; Wiens 2011; Zhang et al. Alytes. Older extinct taxa, such as Eodiscoglossus (Jurassic: 2013), or the sister group to all remaining neobatra- E. oxoniensis, based on isolated skeletal elements [Evans chians bar the earlier diverged heleophrynids (Pyron et al. 1990]; Cretaceous: E. santonjae, a fully articulated and Wiens 2011). Earlier, late Cretaceous neobatrachian skeleton [Villalta 1957]) are considered discoglossoid, fossils have been described, or reported, dating at least to and the former has previously been used as a dating the Cenomanian (100.5–93.9 MY: e.g., an apparent prior for the root of Discoglossidae (e.g., Pramuk et al. ranoid from the Wadi Milk Formation of Sudan [Baez 2008), the costatan crown (e.g., Blackburn et al. 2010), and Werner 1996; Gardner and Rage 2016]), and older and the costatan root (e.g., Roelants and Bossuyt 2005; still from the early Albian–late Aptian (120–105 MY) Bossuyt et al. 2006; Zhang et al. 2013; Kurabayashi et al. (Baez et al. 2009; Baez et al. 2012), however, it is unclear 2011). E. santonjae has been recently excluded from whether these taxa represent stem or crown group neo- Costata based on the resulting phylogenetic analyses batrachians (Zhang et al. 2013), and thus are not con- of Baez (2013). Assuming Eodiscoglossus is monophyletic, sidered here for dating this node. that result indicates the need for a review of the system- (6) MRCA for the Pelodytidae–Pelobatidae split at 50.3 MY atic placement of this taxon (Baez 2013; Marjanovicand based on the recently described, earliest known fossil Laurin 2014). The indeterminate costatan pelodytid, Aerugoamnis paulus from the lower Eocene Enneabatrachus hechti Evans and Milner, 1993 (late (Wasatchian, 55.4–50.3 MY) of the Green River forma- Jurassic), is known only from fragmentary bones, and a tion, Wyoming, which was characterised based on a sin- partial skeleton (Rocek et al. 2010; Gardner and Rage gle almost complete, and mostly articulated specimen 2016), so neither Enneabatrachus nor Eodiscoglossus (Henrici et al. 2013). were considered here for node age priors. (7) MRCA for the Spea–Scaphiopus split at 33.3 MY based (3) MRCA for the root of Pipoidea at 152.1 MY based on the on the oldest fossil scaphiopodid originally assigned with oldest known pipoid fossil Rhadinosteus parvus from the considerable confidence to the genus Scaphiopus (and late Jurassic (Kimmeridgian, 157.3–152.1 MY) of Utah not Spea), S. skinneri Estes, 1970, from the early (Henrici 1998). Henrici (1998) suggested that it might be Oligocene (Rupelian stage, 33.9–28.1 MY, more specif- more closely related to the Rhinophrynidae than Pipidae ically the Orellan, 33.9–33.3 MY). The oldest known based on the presence of ectochordal vertebrae. As a fossils of Spea, S. neuter (Kluge, 1966)isfromthelate result, the fossil has recently been used to date the split Oligocene (early Arikareean, 30.6–23.0 MY) of South between Rhinophrynidae and Pipidae (e.g., Bossuyt et al. Dakota. This species was originally considered to be phy- 2006; Bewick et al. 2012; Zhang et al. 2013). Rocek (2013) logenetically close to the MRCA of Scaphiopus and Spea indicates doubt as to its familial status within (Kluge 1966), and has subsequently been assigned to the Rhinophrynidae, thus the conservative approach was genus Spea based on phylogenetic analyses that in- taken to use the fossil to calibrate the minimum age of cluded the examination of additional material (Henrici Pipoidea (as have others, e.g., Roelants and Bossuyt 2005). 2009). Scaphiopus guthriei (Estes, 1970)fromthelower

763 Mahony et al. . doi:10.1093/molbev/msw267 MBE

Eocene (Ypresian, or more specifically Wasatchian, Dating Methods 55.4–50.3 MY) of the Wind River formation, was Both RelTime and MCMCtree require an input tree to either not used to date the split between Scaphiopus and improve analytical efficiency, and/or determine the place- Spea, as it was only tentatively assigned to the genus ment of calibration priors. Alignment Datasets 3a (combined level (Henrici 2000; Henrici and Haynes 2006; Rocek mt þ nuDNA) and 3b (nuDNA only) were first run on 2013). Significantly earlier (upper Cretaceous) fragmen- RAxML (GTR CAT model, 1,000 bootstrap replicates) to tary remains of pelobatoids (including Scaphiopodidae, test topological conformity of out-groups with previously Pelodytidae, Pelobatidae and Megophryidae) have been published molecular phylogenetic studies, particularly for preliminarily reported, dating from the Maastrichtian Dataset 3b, which is missing TTN and SACS for most out- through to the Santonian (86.3–66.0 MY), from nu- group taxa (supplementary table S1, Supplementary Material merous localities in Europe, United States, and India (see online). Only Dataset 3b differed in that it did not resolve a Rocek 2000; Rocek et al. 2014; Csiki-Sava et al. 2015;and sister taxa relationship between Ascaphus and Leiopelma. Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 references therein), however, unanimous identifications Within Megophryinae, topological differences between await confirmation (Venczel et al. 2016). Datasets 3a and 3b, and the “optimum” phylogenetic tree (8) MRCA for the Pelobatidae–Megophryidae split at 46.2 (MrBayes tree generated from alignment Dataset 1c) oc- MY, based on the recently described oldest unequivo- curred for poorly resolved nodes, as would be expected cally assigned pelobatid species, Eopelobates deani,from when using different out-group taxa (e.g., Luo et al. 2010) the early middle Eocene (or more specifically the (refer to supplementary table S2: col. VI-VII, Supplementary Bridgerian, 50.3–46.2 MY) of the Green River Material online for further comparison of in-group boot- Formation, Wyoming (Rocek et al. 2014). strap support between these and other phylogenetic trees). For downstream comparability of results, the guide tree corresponding with the topology of alignment Geological Constraints Dataset 3a was used to fix the out-groups, and our For the use of plate tectonic ages as constraints, the following “optimum” phylogenetic tree used to standardise the time range estimates were used to hypothetically reflect the topology for the in-groups. splits: RelTime analyses: The GUI based version of RelTime in MEGA 6.06 was utilised, using local clock type, and (1) Between Rhinophrynidae and Pipidae, the terrestrial GTR þ IþG with four discrete gamma rate categories, on split between the African–South American landmass all codon positions, for the three alignments 3a, 3b, and 3c (Gondwana) and North America (Laurasia), at a min- (not partitioned). Since internal node estimation relies heavily imum of 150 MY (Ezcurra and Agnolın 2012 [some on the root age, a number of constraint combinations were time between 161 and 130 MY]; Upchurch et al. 2002 investigated to determine: a) which root age range provided [Callovian 166–164 MY]; Sanmartin and Ronquist internal node ages in excess of those known from the fossil 2004 [180–160 MY]; Bewick et al. 2012 [190 MY]). record; b) what was the effect of adding additional internal (2) Between the African endemic Hymenochirus and the fossil constraints in various combinations; c) what was the South American endemic Pipa, the age range for the effect of using time estimates of vicariant events resulting terrestrial split between Africa, Antarctica, and South from continental plate tectonic movements as node con- America was used, at a minimum of 80 MY (Pitman straints (supplementary table S10, Supplementary Material et al. 1993; San Mauro et al. 2005; Bossuyt et al. 2006; online). To investigate the effect of root age, the Roelants et al. 2007 [86 MY]; Roelants and Bossuyt minimum–maximum age was set at: (a[i]) 247.2 MY as min- 2005; Bewick et al. 2012 [100 MY]; Van der Meijden imum, no maximum; (a[ii]) 247.2–275 MY to assume the et al. 2007; Kurabayashi et al. 2011 [110 MY]; stem fossil Triadobatrachus is close to the anuran-caudatan Sanmartin and Ronquist 2004 [110–95 MY]). A recent root, and reflecting the age estimate of MarjanovicandLaurin revised global palaeobiogeographical model, however, (2014) [254 MY]; (a[iii]) 247.2–300 MY to reflect the age considers sufficient evidence exists to imply faunal ex- estimates of Zhang et al. (2013) [280 MY]; (a[iv]) 247.2– changes between Africa and South America via island 350 MY to broadly reflect older estimates e.g., Roelants et al. chains across the south Atlantic from the Campanian (2007) [332 and 357.8 MY]; (a[v]) 247.2–600 MY accounting to Eocene times (83.5–36.6 MY) (Ezcurra and for still older estimates, e.g., Kurabayashi et al. (2011) Agnolın 2012). [“Calibration F” 376 MY]. Constraints (a[i])–(a[v]) were (3) Between the Indian endemic Nasikabatrachidae and tested on the combined mt þ nuDNA dataset (3a), and con- South American endemic Ceratophryidae, the terrestrial straints (a[i]), (a[iii]) and (a[iv]) were further tested on both split between India (including Madagascar–Seychelles) the nuDNA only dataset (3b), and the mtDNA þ CXCR- and other land masses (i.e., Antarctica, Africa, South 4 þ RAG-1 alignment dataset (3c). To test the effect of adding America) was set at a minimum of 90 MY for the pro- internal fossil calibrations, using primarily the root constraint posed “Central corridor” hypothesis (Chatterjee and (a[iv]), the following combinations of calibrations were added: Scotese 1999; Van Bocxlaer et al. 2006 [90 MY]; (b[i]) Fossil/s 2 and/or 3 only, and Fossils 2–8, to determine Sanmartin and Ronquist 2004 [121 MY]; Rabinowitz how the oldest fossil constraints affected the timetree: also et al. 1983; San Mauro et al. 2005 [130 MY]). tested on the root constraints (a[ii]) and (a[iii]); (b[ii]) all

764 Evolutionary History of the Asian Horned Frogs (Megophryinae) . doi:10.1093/molbev/msw267 MBE fossils except Fossils 2 and 3, to determine how the exclusion 2002) was used to calculate the log likelihood of each tree of the oldest fossils effected node calibrations; (b[iii]) Fossils 6 and produce the input file for the software Consel and 8 only, to determine if fossils that are assumed to be (Shimodaira and Hasegawa 2001) which performs KH, SH, considerably younger than the MRCA affect age estimation of and AU tests. Alignment Dataset 1c (54 taxa), with associ- other nodes, given a fixed root age range: also tested on the ated MrBayes tree was used as the topological standard tree root constraint (a[iii]). Analysis was also run on Dataset 3a because it was the most robust from our analyses. Tree- with no root prior but Fossils 2–8 as internal priors to deter- Puzzle requires that all trees contain the same taxa, however, mine if RelTime can estimate a reasonable root age. Refer to since each study contained different taxa, with varying levels supplementary table S10, Supplementary Material online, for of overlap with our dataset, the alternative topologies were summary of prior combinations. The results from all RelTime constructed to account for relationships between major analyses were compared with the fossil record and possible species groups, and not individual species within the species vicariant events (outlined previously), as used for calibrations groups (fig. 2). Species groups from previous studies that Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 in this study, alternative fossils either questionably assigned to contained no overlapping taxa were assigned a topological taxon rank or considered confidently assigned but based on position based on the MrBayes tree of alignment Dataset 2. fragmentary remains, and some fossils previously used as Alternative topology trees were constructed using Mesquite node constraints which were not used in this study (table 3.03 (Maddison and Maddison 2015). Tree-Puzzle was run 1; supplementary table S5, Supplementary Material online). using the “Evaluate user defined trees” tree search proce- MCMCtree analyses: For MCMCtree,baseml(inPAML4.8 dure. jModelTest 2.1.7 (Darriba et al. 2012) was used to es- package) was utilised for obtaining substitution rates per unit timate under AIC the most suitable model of substitution time for each gene under the GTR þ G substitution model for the overall alignment (i.e., not partitioned), and the six (gamma rate ¼ 0.5) for alignment Datasets 3a–c, using the different substitution rates, since Tree-Puzzle does not yet strictmolecularclockassumptionontheapproximateaverage automate substitution rate parameter estimation (Schmidt of 300 MY based on the lower (Fossil 1: 247.2 MY), and a rea- et al. 2004). Resulting parameters used for Tree-Puzzle anal- sonable estimate of the upper (350 MY) root age. Baseml failed yses were as follows: GTR þ IþG; substitution rates: A–C to reach convergence for CXCR-4 and SACS, so their substitu- 2.32; A–G 5.40; A–T 2.83; C–G 0.90; C–T 13.63; G–T 1.00; tion rates per unit time were set as 0.092 based on the four Gamma rate categories. assumption of comparable rates with the two other nuclear genes (RAG-1 ¼ 0.0902050.009665; TTN ¼ 0.093313 0.025411). The weighted average substitution rates relative to sequence length were calculated to set the overall substitu- Phylogeographic Analysis tion rate (rgene_gamma): (3a) 1 9.42; (3b) 1 10.87; (3c) 1 7.65, Biogeographic analysis was performed using the R package and rate drift parameter (sigma2_gamma: 1 3) priors for each BioGeoBEARS, which uses an ML method to estimate the alignment dataset for MCMCtree. Analyses were run using the historical distribution of internal nodes on phylogenetic approximate likelihood method (dos Reis and Yang 2011)us- trees, based on the distribution of the extant taxa (Matzke ing three combinations of priors on each alignment dataset 2013). The RelTime tree generated from alignment Dataset (3a–c) for comparability with RelTime analyses (supplemen 1c (54 taxa mt þ nuDNA), using two priors: root 247.2–350 tarytableS10,SupplementaryMaterialonline):rootprioronly; MY and Fossil 2 was used, and the DEC model of root prior and Fossil 2; root prior and Fossils 2–8. The dating LAGRANGE, and the modified DEC þ J method that allows analyses used the GTR þ G model of nucleotide substitution, for founder-event speciation (Matzke 2014), were used for with prior rates of internal nodes specified using the indepen- the phylogeographic reconstruction. Distribution data were dent rates model (Rannala and Yang 2007), and birth, death, gathered for all species based on a combination of unpub- samplingrateparametersweresetto110.1,respectively.Fossil lished data for taxonomically undescribed species included constraints were scaled so that one unit equals 100 MY, and all in the molecular dataset, and museum specimens examined bartherootpriorweretreatedasminimum(orlower)bounds. in this study (supplementary table S11, Supplementary TheMCMCchainwasrunforatotalof100,000cycles,sampling Material online), supplemented with additional literature every ten, and with an initial burn-in period of 10,000 cycles, records for China (Fei et al. 2009). Eight broad distributional providing a total of 10,000 samples. Each dataset was run twice areas were coded, each of which primarily contain their own using randomly generated seeds, and the results compared to unique assemblage of Megophryinae species, though some help identify convergence issues for MCMC chains. widespread species are found in adjacent areas; categorised as: Nepal–Indian Himalayas, Bhutan, eastern Bangladesh, Alternative Topology Tests and western Myanmar (H); Thailand, eastern Myanmar, Numerous alternative phylogenetic trees have been pro- southern Cambodia (T); northern Myanmar, China, north- posed in the past either as an attempt to hypothesise or ern Vietnam, and northern Laos (C); northern Cambodia, estimate systematic relationships within Megophryinae, or central and southern Vietnam and Laos (V); peninsular as a by-product of broader amphibian wide phylogenies, of Thailand (south of the Isthmus of Kra), peninsular which four of the more comprehensive were tested (fig. 2; Malaysia (M); Borneo (B); Philippines (P); Java–Sumatra (J). Jiang et al. 2003; Zheng, et al. 2004b; Pyron and Wiens 2011; The maximum number of areas permitted per species was Pyron 2014). The software Tree-Puzzle 5.2 (Schmidt et al. restricted to three.

765 Mahony et al. . doi:10.1093/molbev/msw267 MBE

Supplementary Material Baez AM, Gomez RO, Ribeiro LC, Martinelli AG, Teixeira VP, Ferraz ML. 2012. The diverse Cretaceous neobatrachian fauna of South Supplementary data areavailableatMolecular Biology and America: Uberabatrachus carvalhoi,anewfrogfromthe Evolution online. Maastrichtian Marılia Formation, Minas Gerais, Brazil. Gondwana Res. 22(3):1141–1150. Acknowledgments Bain RH, Hurley MM. 2011. A biogeographic synthesis of the amphibians and reptiles of Indochina. Bull Am Mus Natl Hist. 360:1–138. This work was supported by an Irish Research Council (IRC) Battistuzzi FU, Billing-Ross P, Murillo O, Filipski A, Kumar S. 2015. A Embark Scholarship to S.M. and E.C.T. for the PhD research of protocol for diagnosing the effect of calibration priors on posterior S.M., a SUSI Student Maintenance Grant to S.M., a University time estimates: a case study for the Cambrian explosion of animal College Dublin Seed Grant for Indian travel for S.M., a Field phyla. Mol Biol Evol. 32(7):1907–1912. Bauer L. 1986. A new genus and a new specific name in the dart poison Museum of Natural History Science Visiting Scholarship to frog family (Dendrobatidae, Anura, Amphibia). Ripa Netherlands. S.M. E.C.T. and N.M.F are funded by a European Research 1986:1–12. Council Starting grant (ERC-2012-StG311000) awarded to Beddard FE. 1908. “1907” Contributions to the knowledge of the anat- Downloaded from https://academic.oup.com/mbe/article-abstract/34/3/744/2919384 by guest on 06 August 2019 E.C.T. Fieldwork was funded by a Department of Science omy of the batrachian family Pelobatidae. Proc Zool Soc Lond. and Technology DU/DST Purse Grant (2009/868) from the 1907:871–911. Bewick AJ, Chain FJ, Heled J, Evans BJ. 2012. The pipid root. Syst Biol. Government of India to S.D.B., Research and Development 61(6):913–926. grants (2007/449, 2011/423) from the University of Delhi to Biju SD, Bossuyt F. 2003. New frog family from India reveals an ancient S.D.B. S.D.B. is funded by Research and Development grants biogeographical link with the Seychelles. Nature 425(6959): 711–714. (2015/9677) from University of Delhi. For forest study/collec- Biju SD, Van Bocxlaer I, Mahony S, Dinesh KP, Radhakrishnan C, tion permits and associated support to S.D.B., we thank the Zachariah A, Giri V, Bossuyt F. 2011. A taxonomic review of the Night Frog genus Nyctibatrachus Boulenger, 1882 in the Western State Forests Departments of Arunachal Pradesh, Assam, Ghats, India (Anura: Nyctibatrachidae) with description of twelve Manipur, Meghalaya, Nagaland, and West Bengal. For access new species. Zootaxa 3029:1–96. to specimen collections, associated support, and hospitality Biju SD, Garg S, Mahony S, Wijayathilaka N, Senevirathne G, during museum/collection visits, S.M. would like to thank Meegaskumbura M. 2014a. DNA barcoding, phylogeny and system- Barry Clarke, Colin McCarty, David Gower, Jeffrey Streicher, atics of Golden-backed frogs (Hylarana, Ranidae) of the Western Ghats-Sri Lanka biodiversity hotspot, with the description of seven and Mark Wilkinson (NHM), Alan Resatar (FMNH), Saibal new species. Contri Zool. 83:269–335. Sengupta (Arya Vidyapeeth College, Assam), Kaushik Deuti Biju SD, Garg S, Gururaja KV, Yogesh S, Sandeep AW. 2014b. DNA and K. Venkataraman, and staff of the amphibian section (ZSI, barcoding reveals unprecedented diversity in Dancing Frogs of Kolkata); S.D.B. would like to thank the staff of the amphibian India (Micrixalidae, Micrixalus): a taxonomic revision with descrip- section (ZSI-ERS) and Chengdu Institute of Biology (Chinese tion of 14 new species. Ceylon J Sci Biol Sci. 43(1):37–123. Biswas S, Coutand I, Grujic D, Hager C, Sto¨ckli D, Grasemann B. 2007. Academy of Sciences, China). 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