EVOLUTION IN THE GENUS RHINELLA: A TOTAL EVIDENCE PHYLOGENETIC ANALYSIS OF NEOTROPICAL TRUE TOADS (ANURA: BUFONIDAE) MARTIN O. PEREYRA, BORIS L. BLOTTO, DIEGO BALDO, JUAN C. CHAPARRO, SANTIAGO R. RON, AGUSTIN J. ELIAS-COSTA, PATRICIA P. IGLESIAS, PABLO J. VENEGAS, MARIA TEREZA C. THOME, JHON JAIRO OSPINA-SARRIA, NATAN M. MACIEL, MARCO RADA, FRANCISCO KOLENC, CLAUDIO BORTEIRO, MAURICIO RIVERA-CORREA, FERNANDO J.M. ROJAS-RUNJAIC, JIR/ MORAVEC, IGNACIO DE LA RIVA, WARD C. WHEELER, SANTIAGO CASTROVIEJO-FISHER, TARAN GRANT, CELIO F.B. HADDAD, AND JULIAN FAIVOVICH BULLETIN OF THE AMERICAN MUSEUM OF NATURAL HISTORY EVOLUTION IN THE GENUS RHINELLA: A TOTAL EVIDENCE PHYLOGENETIC ANALYSIS OF NEOTROPICAL TRUE TOADS (ANURA: BUFONIDAE) MARTIN O. PEREYRA, BORIS L. BLOTTO, DIEGO BALDO, JUAN C. CHAPARRO, SANTIAGO R. RON, AGUSTIN J. ELIAS-COSTA, PATRICIA P. IGLESIAS, PABLO J. VENEGAS, MARIA TEREZA C. THOME, JHON JAIRO OSPINA-SARRIA, NATAN M. MACIEL, MARCO RADA, FRANCISCO KOLENC, CLAUDIO BORTEIRO, MAURICIO RIVERA-CORREA, FERNANDO J.M. ROJAS-RUNJAIC, JIRI MORAVEC, IGNACIO DE LA RIVA, WARD C. WHEELER, SANTIAGO CASTROVIEJO-FISHER, TARAN GRANT, CELIO EB. HADDAD, JULIAN FAIVOVICH BULLETIN OF THE AMERICAN MUSEUM OF NATURAL HISTORY Number 447, 155 pp., 15 figures, 11 tables Issued March 31, 2021 Copyright © American Museum of Natural History 2021 ISSN 0003-0090 CONTENTS AUICNOTS < HU Ah ad ath eon ES eh Seay aed ae lary aa ehh Tia aud leery 4 PROSUTA CU nr Race Rabie snd, Srna. Rie swt OU ul Foes Aa Bate Ona, SNe! Ree ec UA Rees heel OT tn 6 PT ECUAUIGEI OU ies i Se REE hac oe OR Senay Sa, ek RE tach oe LO ce cera Sa, 6 Genetal Overview «1.6.2 sateen Neue asada saliten Sayin, f tags) eteanne Seu aagbe halen Sous 6 SYSLOMIALICS OL MRICS ut nd Pn RAY a UR as acai aa Dien aes tela analy ee Oe a V4 Miate rial ate eMetnO dse yx tice etn neh Aen m8 tele haere Arg Mn eel heehee te Ae, co met lnh e 8 TAX OMOMmic: Sam Pl Ness ates Sea». option Ra a6 oad eae She Sieg hig «epics Rah aue sod cele 8 COPS TOUS xy. ba a ae ottes Porat sad-puPrs dt a asa tyme RE aes nan a pee Ore aad ee eee sa woe a etary Aaersnaen es 8 MeN STOUD: RMAC U Ar: LS Meade kW, cS eels ieee Cm eaeks Aaah Aten A bem eee Be ACe 10 Thertanella. acroloped Groupes Na, uh cedsecele aoe p he tetenreeeloersis Mins ook wav see tale os 11 hes Ri nell a-CMiclersGrOup t.: + castes speck 5.c-tgewetety maria tpoek a:detnurind yarantode-tgeyt a.d-tewawe caf 11 te Rita elia erantlosa, GLOUD ane. yt n ee Pe al eesti et RB ee ey a Tey R. «POMC le ie ihe: RhinellamiargaritiferasGLoum, oh el istatia sats week Te We adn pa ene we aa 13 ite RHMelGaIHOTINAGLOUDN.., 44. 28 Soka ek eR a eee ig 254, OM horace 13 The Reinella spinulosa Groupe. % s.5 hn Wek car esd ey MS sa ued mur Ran cata 14 The GRhivellaiverneuensis Groups ea. :8 aaron, sehr cs Se aden RSE, 4 14 Spécies unassigned to-anyoroupin 2 ction Aslng Sole. bonis leh noun ate tea elena « 15 Motea ular abate at ate cepted reine eed ae anaes Mia A TAR ty bo) Besa Metin aa Ree) Bins U5: ML PSSHE SS ANAND ert, omer Wet he as et ee. RE le en cP c MWY, Decca eee cee el oh 15 Laboratory-protocols?.. 0.04. <yeerea gn ced ae a itd wo pRaet Ae She See a os VERA ae se «0 a 15 Genotypic-chatacter samplings Ss steno. we cud tae: Oe eared a ofa ears) toms: ee Sisson, Were fama 2 15 PHCHG ty IC Gat ary Wc, seen t ees: Risa BE Yo IER De Bank Dose, Gta Bose A RELVES SRR Bavek 16 Phivlogeneticranalysesic,, a5 Se terse ten ete set ce ecsar ee aw wheels, weaned a at eee ae oR ne aver die ae 16 Separate phylogenetic analyses of nuclear and mitochondrial sequences............... 17 Total-evidence Bia lysis: wii ria ctr ak nee Meet a skal eane sts ot ME een eed on oe, Mek ae 17 Resainpline: Support measures o.. i301. 5t eae tn and ne asa, « Sank gene raa ns Dawe Seaale 19 Maximum likelin@ocd-anal ysis. ncn, ayn 2 jhe esas Rotoy Bey Gee al eee, BUG A ee eal 19 TAXONOMIC eV all atiOnins © Gentoo 0 ae. 8 Leeder, (eee. en Re Den ee pone ee 8 Leelee 19 Listeandidescniption-olemaracterss: cs. spt Anis i ee, 08 erg oe 1k ee ae 3 20 PLGUIC-OStCOlO GN. sors tsedport a.d-toarmrtnd f grantetse perk grein cad aearonns, apenas tarmeare fscantone-apeah sain ont 20 PA GUL TIVG CU LALUGPS Dae PR acs othe ag eB -chnmeascs este, Be N PES eRe nee lize gst et BaS oc hecaseso atte el 26 Tympane tid dleteatc OMIp) exept Ana ot ben ae Ween ceils eee ee taba See ae ean ee eee 28 Adult visceral anatomy: <a.0 6 ons... piu t aot Balhae 3 Mig a eortielt ne Seely ik oe Pie ot Manag os ls 30 Adultsextennal an Onpme lo verse Mie Sis song Weearins Auecsn deh Aosta Wir at attraaseie ha selatd Marie Maer nth 2 30 Latvalexter tial sion nOlO Sys ters an tye one nema Reis, aber a Sense Pen able een St 32 Darya che ngroct ari unite ins se Mave shi see ae sae tee each ae 2 Mets chiar Midurdhe a le Dae, acagns tes een he ae 33 Bm bryonic-morp holo verte Ph aig A nei Rede bce Eri td pte Rae eR aly oes ee rime bes Ec oee, 33 TAIRA IS CORY. 1.305 et phan et SMe a oer yh PEED ve eg eee Nath oe MAIN a ears iyi PRM at 33 ROVLOROINGIIC Ses, AE bP aa ATU dh aes senna hT emote. tgs er es eran) ngs sates Se tna ATEN, sees 34 Results: an seas, tte ot eee eg, oe Sak Hee Re ak BA el cs, an aie Ain ethagt 34 Separate analyses of restricted nuclear (rND) and mitochondrial (rMD) datasets ......... 34 Totalpevideticealaly sis «89 ccan xo 7 wi Aaa tA sas Ser ek ata hace on 0) Lee ent eee 7 35 LiveorrechedP-GIStAN CES. os. antec etched: santas taal :detna ete sect nartne tole ganintes tus sedotns gtk Se) TOPSCUSSIOR et tes wee se Si ieee Mn eee tl USE Cen ieee, Ben eed Role Meas Wie yeh Le Eve, ee 35 Systeniatics: and tax ONOMNy: <Auires 5 yen Rice ey aoeeten ete hat eR Pe ep Pagre Apes Ra Mica iew acoplgrese a 35 Relationships among outeroups and, Rui el a px jce eta ogra n eel eory ae mrp eE oar ed Be 35 Rhinelaand its iwiterral relationships 4c) ¢2 08 vaucs ake tule A elas ke Sea 37 ire Rhinellatavaritia- Clade gos paw eee tge te eT as Meal BAO Re Md ay Lt del 43 dhexRhineliasarunco. Group wes ofa ie ele enh Aelita aa ee ARR Ga epee el 44 The: RAipella-Spinlosa ropa Fah fash 4 Geet es asses Sete atee Meera ke eM Pat Rad teen 45 he Riirella okay Osa AGTOUpa rns eects tan + he te eck aeeeat nie, eae: ee eteged were ls ees 47 The mitochondrial lineage of Rhinella horribilis........ 0... ccc cece cece ee eee 51 be Ri ellat CRC er OTe S14 eR Ph daha ihe TT Oe LSE LS aN OG Te 51 ThevRhinelazatar ina: Group: 20 fa ek st. «erates owl ohy See ens CRA Ee ote es 54 The Rhinelamaredcitifera Glade. 5 32.0 «4 gM eas ted eee Mee ne baa ieee Me os 56 Rin Hella Sher igstCH ala vc oes a ene te 2 eet Me Arte Le Soy SSRN SLN gh Ph ote PAG Be 57 Ne Ri nellacper aan en sige Toupee 8 Wer: hes thoynn Mae Hace tn ae Mee caen le (ds opera ae | 57 iherRiinellacfestae GtOuye:. aos 5s Bidens wate ayo ghia ae Aenea wc etnane aanee a4 60 he -Riinellavmarcaritifera Groep wr. te str cect nets erage eset haeee en ee oe! 63 Hybridization, deep mitochondrial divergence, and “ghost introgression” in Rhinella...... 69 Comments on the phenotypic evidence considered for Rhinella.................. 000005 fe ‘The fossil record of Rhinella-and*calibration points. + 24% elie cee bets hace a eee ede ee 73 COMEMISIONS A fs foo e esha ard eR ed ahaa RE Ata, ence oe cet aN ee elastin At dctate 75 PECKMO WICC OMPCIS cet rics Ry estes Ra a enepch so RAM cerlonrdn DV eon Ree rhage >. Bale Bicmpcicegct 75 RECT eT CEST eyes) pc be ara -teuetes deer artraned ppb hu tpeness d: muuaneeeh De acarlnalaede dso uchasagert mea inde 2th euch ane eegeet: aka 76 PaW el G/siutaitel =n OO Sa erg IS bela Biv he Whang WAP iy to then Lie istic Re ihc hs ie Os ie in Ms SAR 97 Ex PosEogs6 (31S CCW a0 Im 0] 210) DAE Renee By Ne ckty ARLENE, AUN Rie Nain, aos GA Aen AER ee ROMS An OCA Aex AMET) MUIR ah 155 4 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 AUTHORS Martin O. PEREyRA: Division Herpetologia, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia’-—CONICET, Buenos Aires; and Laboratorio de Genética Evolutiva “Claudio J. Bidau,” Insti- tuto de Biologia Subtropical (IBS, CONICET), Universidad Nacional de Misiones (UNaM), Posadas, Misiones, Argentina. Boris L. BLorro: Divisidn Herpetologia, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia’-CONICET, Buenos Aires; Universidade Estadual Paulista (UNESP), Instituto de Biocién- cias, Departamento de Biodiversidade e Centro de Aquicultura (CAUNESP), Rio Claro, Sao Paulo; and Departamento de Zoologia, Instituto de Biociéncias, Universidade de Sao Paulo, Sao Paulo. DiEGo Bapo: Laboratorio de Genética Evolutiva “Claudio J. Bidau,” Instituto de Biologia Subtropi- cal (IBS, CONICET), Universidad Nacional de Misiones (UNaM), Posadas, Misiones, Argentina. JUAN C. CHAPARRO: Museo de Biodiversidad del Peru, Cusco, Peru; and Museo de Historia Natural de la Universidad Nacional de San Antonio Abad del Cusco, Paraninfo Universitario, Cusco. SANTIAGO R. Ron: Museo de Zoologia, Escuela de Biologia, Pontificia Universidad Catdlica del Ecuador, Quito. AcusTin J. Ex1as-Costa: Divisidn Herpetologia, Museo Argentino de Ciencias Naturales “Ber- nardino Rivadavia’—CONICET, Buenos Aires. Patricia P. Icesias: Laboratorio de Genética Evolutiva “Claudio J. Bidau’, Instituto de Biologia Subtropical (IBS, CONICET), Universidad Nacional de Misiones (UNaM), Posadas, Misiones, Argentina. PABLO J. VENEGAS: Division de Herpetologia-Centro de Ornitologia y Biodiversidad (CORBIDI), Surco, Lima. MarIA TEREZA C. THOME: Universidade Estadual Paulista (UNESP), Instituto de Biociéncias, Departamento de Biodiversidade e Centro de Aquicultura (CAUNESP), Rio Claro, Sao Paulo. JHON JAIRO OsPINA-SARRIA: Departamento de Zoologia, Instituto de Biociéncias, Universidade de Sao Paulo, Sao Paulo, Brazil; and Calima, Fundacion para la Investigacion de la Biodiversidad y Con- servacion en el Trdépico, Cali. NaTaNn M. Maciet: Laboratorio de Herpetologia e Comportamento Animal, Departamento de Ecologia, Instituto de Ciéncias Bioldgicas, Universidade Federal de Goias, Goiania, Brazil. Marco Rapa: Departamento de Zoologia, Instituto de Biociéncias, Universidade de Sao Paulo, Sao Paulo. FRANCISCO KOLENC: Seccién Herpetologia, Museo Nacional de Historia Natural, Montevideo. CLAUDIO BorTEIRO: Seccién Herpetologia, Museo Nacional de Historia Natural, Montevideo. Mauricio RrvERA-CorrEA: Grupo Herpetoldégico de Antioquia, Instituto de Biologia, Universidad de Antioquia, Medellin. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 5 FERNANDO J.M. Rojyas-Runyjaic: Fundacion La Salle de Ciencias Naturales, Museo de Historia Natural La Salle (MHNLS), Venezuela; and Pontificia Universidade Catdlica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil. Jiki Moravec: Department of Zoology, National Museum, Prague, Czech Republic. IGNACIO DE LA Riva: Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid. Warp C. WHEELER: Division of Invertebrate Zoology, American Museum of Natural History, New York. SANTIAGO CASTROVIEJO-FISHER: Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil; and Research Associate, Herpetology, Division of Vertebrate Zoology, American Museum of Natural History, New York. TARAN GRANT: Departamento de Zoologia, Instituto de Biociéncias, Universidade de Sao Paulo, Sao Paulo; and Research Associate, Herpetology, Division of Vertebrate Zoology, American Museum of Natural History, New York. CELIO F.B. HAppAD: Universidade Estadual Paulista (UNESP), Instituto de Biociéncias, Departa- mento de Biodiversidade e Centro de Aquicultura (CAUNESP), Rio Claro, Sao Paulo. JULIAN FaIvovicu: Division Herpetologia, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia’-CONICET, Buenos Aires; Departamento de Biodiversidad y Biologia Experimental, Fac- ultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires; and Research Associate, Herpetology, Division of Vertebrate Zoology, American Museum of Natural History, New York. This publication has been registered in ZooBank (urn:lsid:zoobank.org:pub:56A BAF36-1B0A-4AE3-A3B4-1283545A675F). BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY ABSTRACT True toads of the genus Rhinella are among the most common and diverse group of Neotropical anurans. These toads are widely distributed throughout South America, inhabiting a great diversity of environments and ecoregions. Currently, however, the genus is defined solely on the basis of molecular characters, and it lacks a proper diagnosis. Although some phenetic species groups have traditionally been recognized within Rhinella, the monophyly of some of them have been rejected in previous phylogenetic analyses, and many species remain unassigned to these poorly defined groups. Additionally, the identity and taxonomy of several species are problematic and hinder the specific recognition and description of undescribed taxa. In this work, we first perform phylogenetic analyses of separate mitochondrial and nuclear datasets to test the possible occurrence of hybridiza- tion and/or genetic introgression in the genus. The comparative analysis of both datasets revealed unidirectional mitochondrial introgressions of an unknown parental species into R. horribilis (“ghost introgression’) and of R. dorbignyi into R. bernardoi; therefore, the mitochondrial and nuclear data- sets of these species were considered separately in subsequent analyses. We performed total-evidence phylogenetic analyses that included revised molecular (four mitochondrial and five nuclear genes) and phenotypic (90 characters) datasets for 83 nominal species of Rhinella, plus several undescribed and problematic species and multiple outgroups. Results demonstrate that Rhinella was nonmono- phyletic due to the position of R. ceratophrys, which was recovered as the sister taxon of Rhaebo nasicus with strong support. Among our outgroups, the strongly supported Anaxyrus + Incilius is the sister clade of all other species of Rhinella. Once R. ceratophrys is excluded, the genus Rhinella is monophyletic, well supported, and composed of two major clades. One of these is moderately supported and includes species of the former R. spinulosa Group (including R. gallardoi); the mono- phyletic R. granulosa, R. crucifer, and R. marina Groups; and a clade composed of the mitochondrial sequences of R. horribilis. The other major clade is strongly supported and composed of all the spe- cies from the non-monophyletic R. veraguensis and R. margaritifera Groups, the former R. acrolopha Group, and R. sternosignata. Consistent with these results, we define eight species groups of Rhinella that are mostly diagnosed by phenotypic synapomorphies in addition to a combination of morpho- logical character states. Rhinella sternosignata is the only species that remains unassigned to any group. We also synonymize nine species, treat three former subspecies as full species, and suggest that 15 lineages represent putative undescribed species. Lastly, we discuss the apparently frequent occurrence of hybridization, deep mitochondrial divergence, and “ghost introgression”; the incom- plete phenotypic evidence (including putative character systems that could be used for future phy- logenetic analyses); and the validity of the known fossil record of Rhinella as a source of calibration points for divergence dating analyses. NO. 447 INTRODUCTION GENERAL OVERVIEW True toads of the former genus Bufo are a popular group of anurans distributed nearly worldwide, and widely studied by researchers from different disciplines. The classic book “Evolution in the genus Bufo” (Blair, 1972) synthesized knowledge about the morphology, phylogeny, and biology of the group. Despite having integrated evidence from many sources of characters to elucidate the evolutionary relationships among the species groups of true toads, this work largely revealed the difficul- ties to study their phylogenetic relationships. It was not until the 1990s—2000s that a general picture of these relationships emerged, and the taxonomy of true toads was revised to be con- sistent with phylogenetic hypotheses (Gray- beal, 1997; Pauly et al., 2004; Frost et al., 2006; Pramuk, 2006). Currently, most of the South American true toads of the former genus Bufo are grouped in the large genus Rhinella (Chap- arro et al., 2007). 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) Th Rhinella includes many of the most conspicu- ous and ubiquitous species of the anuran fauna in almost all the major biogeographic areas of the Neotropical region (Duellman, 1999; Frost, 2020; IUCN, 2020). With 92 species, Rhinella is the sec- ond largest genus of Bufonidae, and its species show considerable morphological and biological diversity, including large variation in size, different levels of cranial ossification, integumentary struc- ture, larval morphology, and ecological and repro- ductive diversity characteristics (Trueb, 1971; Cei, 1972a; Toledo and Jared, 1993; Pramuk, 2006; Aguayo et al., 2009; van Bocxlaer et al., 2010; Pereyra et al., 2015; Bandeira et al., 2016; Simon et al., 2016; Hudson et al., 2018). Some common spe- cies of Rhinella (e.g., R. arenarum, R. horribilis, and R. marina) have been employed extensively as model organisms for various biological disciplines, such as biochemistry (e.g., Abel and Macht, 1912; Cei et al., 1968; Rash et al., 2011), developmental biology (e.g., Markovich and Regeer, 1999; Bari- sone et al., 2002; Brown et al., 2002), ecotoxicology (e.g., Lajmanovich et al., 2011), molecular biology (e.g., Estoup et al., 2004, 2010; Rollins et al., 2015; Edwards et al., 2018; Ceschin et al., 2020), and especially physiology (e.g., Houssay and Giusti, 1929; Houssay, 1949; Penhos et al., 1967; Sassone et al., 2015). This genus also contains a highly inva- sive species, R. marina, widely introduced into many countries and islands from different conti- nents (Frost, 2020), where usually it has a highly negative ecological and socioeconomic impact (Jolly et al., 2015; Bacher et al., 2018). SYSTEMATICS OF RHINELLA For decades, all South American true toads were part of the formerly large and poorly defined genus Bufo, which included a heterogeneous group of toads distributed throughout Africa, America, and Eurasia (e.g., Blair, 1972; Graybeal, 1997). Frost et al. (2006) partitioned this polyphy- letic genus into monophyletic units mostly on the basis of the results of their phylogenetic analysis but also on the results of previous studies (e.g., Graybeal, 1997; Pauly et al., 2004). Frost et al. (2006) resurrected Rhinella for the species of the former Bufo margaritifer Group, which they recovered as distantly related to the other species of South American true toads included in their analysis, including Chaunus and Rhaebo (both also resurrected by Frost et al., 2006). Frost et al. (2006) noted that Bufo margaritifer was nested within Chaunus in a previous phylogenetic study (Pauly et al., 2004), a finding that was subse- quently supported by Pramuk (2006) and Chap- arro et al. (2007). Therefore, Rhinella was later redefined to include the species of Chaunus and Rhamphophryne as well (Chaparro et al., 2007). The species groups of the former Bufo now referred to Rhinella were all recognized primarily on the basis of osteological characters and external morphology that were interpreted without quanti- tative phylogenetic analyses (Tihen, 1962; Cei, 1972a; R.E Martin, 1972a, 1972b; Duellman and Schulte, 1992), including the R. crucifer, R. granu- losa, R. margaritifera, R. marina, R. spinulosa, and R. veraguensis Groups. Pramuk (2006) studied the phylogenetic relationships of these toads on the basis of a combined analysis of morphological (mostly osteological) and molecular evidence. She rejected the monophyly of some of these species groups (e.g., the R. veraguensis Group is polyphy- letic with respect to R. ocellata, the R. margaritifera Group, and Rhamphophryne), but did not modify their composition or diagnosis. The subsequent increase in the knowledge of relations within Rhinella was limited to the addi- tion of available sequences of some species in extensive phylogenetic analyses of Bufonidae or Anura (e.g., van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Jetz and Pyron, 2018). Figure 1 summarizes the main results of the more inclusive analyses of Rhinella. For well over a decade, the systematics of Rhi- nella as a whole has languished, although several efforts focusing on the relationships and taxon- omy of parts of the genus have been undertaken. These include phylogenetic analyses of presump- tively monophyletic species groups (i.e., the R. crucifer, R. granulosa, and R. marina Groups; Maciel et al., 2006, 2010; Thomé et al., 2010, 2012; 8 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY Vallinoto et al., 2010; Pereyra et al., 2016a) or frac- tions of the diversity of certain groups (i.e., the R. festae and R. margaritifera Groups; Fouquet et al., 2007a; Moravec et al., 2014; Santos et al., 2015; Cusi et al., 2017; Avila et al., 2018). Most recent studies on Rhinella aimed primarily to resolve species-level taxonomic problems (e.g., Fouquet et al., 2007a; Narvaes and Rodrigues, 2009; Jansen et al., 2011; Grant and Bolivar-G., 2014; Moravec et al., 2014; Cusi et al., 2017). Consequently, more than a decade after Pramuk’s (2006) revision, spe- cies groups remain poorly defined, several species cannot be assigned to any of them, and few addi- tional phenotypic synapomorphies have been pro- posed for Rhinella or its internal clades (Hoogmoed, 1986; 1990; La Marca and Mijares- Urrutia, 1996; Pramuk, 2006; Chaparro et al., 2007; Padial et al., 2009; Blotto et al., 2014; Grant and Bolivar-G., 2014; Pereyra et al., 2016a). Natural hybridization is common in several groups of Bufonidae, including many species of Rhinella (Blair, 1972; Feder, 1979; Haddad et al., 1990; Masta et al., 2002; Azevedo et al., 2003; Green and Parent, 2003; Yamazaki et al., 2008; Fontenot et al., 2011; Guerra et al., 2011), and mitochondrial and nuclear introgression have been corroborated in some of these clades (e.g. Green and Parent, 2003; Yamazaki et al., 2008; Fontenot et al., 2011; Dufresnes et al., 2019). Pereyra et al. (2016a) dem- onstrated the occurrence of hybridization events in the R. granulosa Group and unidirectional mito- chondrial introgression of R. dorbignyi into R. ber- nardoi. A similar situation might exist between R. marina and R. diptycha, although the evidence is not conclusive (Sequeira et al., 2011; Vallinoto et al., 2017). The impact of these phenomena on the inference of phylogenetic relationships (Hennig, 1966; McDade, 1992; Posada and Crandall, 2002) could be mitigated, at least partially, if detected. A detailed evaluation of the discordance between mitochondrial and nuclear genomes together with a critical taxonomic evaluation provide an effective way to detect hybridization/introgression (Pereyra et al., 2016a). In this paper, we present a densely sampled phylogenetic analysis of Rhinella, including 83 of NO. 447 its 92 species, using molecular (four mitochon- drial and five nuclear genes) and phenotypic char- acters (90 characters from multiple character systems). The goals of this study are to (1) per- form a stringent test of the monophyly of Rhinella as well as similar tests on all its species groups, (2) identify phenotypic synapomorphies to diagnose the species groups of Rhinella, and (3) to evaluate the taxonomic status of several taxa. MATERIAL AND METHODS TAXONOMIC SAMPLING For the complete dataset (molecular and phe- notypic), we sampled 83 described species of Rhinella (including all but nine of the currently recognized species), and 36 exemplar species of other bufonid genera as outgroups (see below). The outgroup species were chosen to provide a severe test of the monophyly of Rhinella, whereas the dense sampling within Rhinella allowed us to rigorously test the monophyly of all its species groups. All specimens scored for phenotypic data were associated with the most morphologi- cally similar and/or geographically closest con- specific terminal of the molecular dataset for the total evidence (TE) analysis. Collection and locality data of vouchers for sequences used in this study, including the information of the sources of the sequences (this work or previous studies), are detailed in appendix 1, and GenBank accession numbers are listed in appendix 2. A list of the species, specimens, and bibliography analyzed for char- acter scoring of the phenotypic dataset is given in appendix 3, and the collection and locality data of specimens studied for morphology are provided in appendix 4. OUTGROUPS For outgroup sampling, we considered the results of the most recent phylogenetic analyses (Frost et al., 2006; Pramuk, 2006; van Bocxlaer et 2071 A Pauly et al. (2004) other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (1) R. granulosa Group (1) R. crucifer Group (1) R. marina Group (1) R. margaritifera Group (1) D Chaparro et al. (2007) other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (?) R. spinulosa Group (?) R. granulosa Group (3) R. crucifer Group (1) R. marina Group (6) “R. veraguensis Group” (3) R. margaritifera Group (8) R. acrolopha Group (1) + “R. veraguensis Group” (3) G Pyron (2014) other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (4) R. spinulosa Group (2) R. granulosa Group (2) R. crucifer Group (1) R. marina Group (6) “R. veraguensis Group” (2) R. margaritifera Group (4) R. acrolopha Group (3) “R. veraguensis Group” (3) B Frost et al R R R R R alls _ (2006) other bufonids . acrolopha Group (1) . margaritifera Group (1) “old world” bufonids Anaxyrus Incilius “R. spinulosa Group” (1 “R. spinulosa Group” (1) . granulosa Group (1) ) . marina Group (3) . veraguensis Group (1) E van Bocxlaer et al. (2010) A Pall other bufonids naxyrus Incilius “old word” bufonids R. spinulosa Group (2 R. spinulosa Group (5 R. granulosa Group (4) R. crucifer Group (1) R. marina Group (6) “R. veraguensis Group” (3) R. margaritifera Group (8) R. acrolopha Group (1) + “R. veraguensis Group” (3) ) ) H Pereyra et al. (2016a) Cll other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (2) R. granulosa Group (12) “R. marina Group” (1) “R. marina Group” (2) R. crucifer Group (2) R. sternosignata R. margaritifera Group (4) R. veraguensis Group (1) PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 2 C Pramuk (2006) other bufonids “old world” bufonids Anaxyrus Incilius R. granulosa Group (3) R. crucifer Group (1) R. marina Group (6) R. spinulosa Group (6) “R. veraguensis Group” (3) R. margaritifera Group (8) R. acrolopha Group (1) + “R. veraguensis Group” (2) F Pyron and Wiens (2011) other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (4) R. spinulosa Group (2) R. granulosa Group (2) R. crucifer Group (1) + R. marina Group (6) “R. veraguensis Group” (2) R. margaritifera Group (4) R. acrolopha Group (3) “R. veraguensis Group” (3) | Jetz and Pyron (2018) other bufonids “old world” bufonids Anaxyrus Incilius R. spinulosa Group (2) R. spinulosa Group (4) R. granulosa Group (6) R. crucifer Group (6) + R. marina Group (10) “R. veraguensis Group” (1) R. margaritifera Group (8) R. acrolopha Group (3) + “R. veraguensis Group” (4) FIG. 1. Summarized relationships of Rhinella according to the main published phylogenetic hypotheses of the group. Only the topological sections corresponding to Rhinella, and putative most related outgroups (i.e., Anaxyrus and Incilius) are shown. The number of species sampled within each clade is reported in parentheses. (A) Pauly et al. (2004: fig. 2). (B) Frost et al. (2006: fig. 50). (C) Pramuk (2006: fig. 4). (D) Chaparro et al. (2007: fig. 9). (E) van Bocxlaer et al. (2010: fig. $1). (F) Pyron and Wiens (2011: fig. 2). (G) Pyron (2014: suppl. information “amph_shl.tre”). (H) Pereyra et al. (2016a: fig. 3 and appendix $12). (I) Jetz and Pyron (2018: suppl. information “amph_shl_new.tre”). 10 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Por- tik and Papenfuss, 2015; Jetz and Pyron, 2018). The previous hypotheses disagree about the phyloge- netic placement of Rhinella, recovering it: (1) as closely related to Incilius and Anaxyrus, and deeply nested within an “old world” bufonid clade (Pauly et al., 2004; Frost et al., 2006; Pramuk, 2006; Chap- arro et al., 2007; Pereyra et al., 2016a); (2) as sister taxon of a clade containing all the “old world” bufo- nid genera (van Bocxlaer et al., 2010); or (3) in a clade together with Anaxyrus + Incilius that is, in turn, sister taxon of the “old world” bufonid clade (Pyron and Wiens, 2011; Pyron, 2014; Portik and Papenfuss, 2015; Ron et al., 2015; Jetz and Pyron, 2018). As all alternative hypotheses have low sup- port values for most relevant nodes around Rhi- nella, we preferred to sample a broad diversity of bufonid genera representing most of the phyloge- netic diversity of the family to rigorously test the relationships and monophyly of the genus. Conse- quently, we targeted 36 species of 22 bufonid genera as outgroup taxa for the combined molecular data- set and 21 of these species for the phenotypic data- set. Outgroup sequences were obtained exclusively from GenBank (see appendices 1, 2). Thus, in order to increase the number of included genes for out- group terminals (considering that the number of sampled genes for the ingroup in this work was higher than previous phylogenetic analyses of Bufonidae), we combined sequences from different specimens of the same species to construct several composite outgroup terminals (see justification by Campbell and Lapointe, 2009). These composite terminals (see appendices 1, 2) were constructed only when their uncorrected p-distances (UPDs) in the 16S rRNA gene were less than 0.5%, which is less than the estimated mean divergence observed between sister species of most anurans (Vences et al., 2005a; Fouquet et al., 2007b; Funk et al., 2011). In taxonomy, the exclusive use of pairwise distances and fixed thresholds is questionable (e.g., Will and Rubinoff, 2004; Grant et al., 2006; Meier et al., 2008), but they serve as a useful heuristic for spe- cies identification and, in the present context, reduce the risk of constructing composited termi- nals that could compromise the phylogenetic analy- NO. 447 sis. Moreover, preliminary analyses including all the sequences of both conspecific specimens recov- ered them as monophyletic with high support (par- simony jackknife supports >97%, see below). THE INGROUP: RHINELLA We included 278 terminals representing 83 described species of Rhinella for the combined (molecular + phenotypic) dataset. For practical purposes, the included taxa are presented below in the species groups to which they were assigned by Duellman and Schulte (1992), but considering subsequent modifications to this proposal (details of the assignation of each species to species groups by different authors are given in appendix 5). For the purposes of our analysis, we recognize the following seven species groups within Rhi- nella: the R. acrolopha Group, the R. crucifer Group, the R. granulosa Group, the R. mar- garitifera Group, the R. marina Group, the R. spinulosa Group, and the demonstrably paraphy- letic “R. veraguensis Group.” Moravec et al. (2014) also proposed the Rhinella festae Group to include three species of the former Rham- phophryne and four species of the paraphyletic R. veraguensis Group (see Pramuk, 2006; Chaparro et al., 2007; van Bocxlaer et al., 2010; Pyron and Wiens, 2011), which they recovered as a clade in their molecular phylogenetic analysis. Although this resolves the nonmonophyly of the analyzed species of the R. veraguensis Group, the authors did not diagnose either their R. festae Group or their restricted R. veraguensis Group or address the placement of the remaining species of the former Rhamphophryne. Given that recognizing the R. festae Group left many species of the for- mer Rhamphophryne and R. veraguensis Group s.l. unassigned to any group due to the lack of diagnoses, we exclude the R. festae Group below. Grant and Bolivar-G. (2014) proposed the Rhinella acrolopha Group to include the species previously assigned to Rhamphophryne. Although molecular phylogenetic analyses have consis- tently supported the monophyly of this group (albeit on the basis of a small fraction of its spe- 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 11 cies; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Jetz and Pyron, 2018), its rec- ognition renders the R. veraguensis Group para- phyletic (see Pramuk, 2006; Chaparro et al., 2007; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Jetz and Pyron, 2018). Thus, as discussed by Grant and Bolivar-G. (2014), the composition and diagnosis of the R. festae Group, the R. acrolopha Group, and the R. veraguensis Group are problematic and will be addressed in the context of our results. For the time being, we employ the putatively monophyletic R. acrolopha Group and demonstrably paraphyletic “R. veraguensis Group” only to characterize and pro- vide background on the ingroup. THE RHINELLA ACROLOPHA GROUP This group consists of 10 small to medium- sized species of Rhinella characterized by a pro- jecting snout, small and inconspicuous parotoid macroglands, heavily ossified skull with some degree of co-osification, well-defined cranial crests (at least in some species), tympanic mem- brane and annulus absent (except in R. truebae), m. levator mandibulae externus undivided with trigeminal nerve passing medial (deep) to the muscle, m. adductor longus absent, and large and unpigmented eggs (Trueb, 1971; Lynch and Renjifo, 1990; Grant and Bolivar-G., 2014). These species are distributed from southern Panama to southern Ecuador, and many of them are criti- cally endangered (Rueda-Almonacid et al., 2004). We sampled the following species: Rhinella acrolopha, R. festae, R. lindae, R. macrorhina, R. nicefori, R. paraguas, R. ruizi, and R. tenrec. We also included an undescribed species from Colombia (Rhinella sp. C sensu Machado et al., 2016). Sequences of R. macrorhina and R. rostrata available from GenBank (A. G. Gluesenkamp, unpublished) were not included because our pre- liminary analyses (data not shown) revealed that the sequences of the fragments of 12S and 16S rRNA genes of each specimen appear to be chime- ric and/or contaminated with R. festae, and we cannot determine with certainty which sequences correspond to each taxon (see also Cusi et al., 2017). Tissues samples of R. rostrata were not available for this study. This poorly known species (Noble, 1920) was described from “Santa Rita Creek,’ 23 km N of Mesopotamia town, in the southern part of the departamento de Antioquia, Colombia. There is great uncertainty about this locality, because it has never been possible to locate or document it in the literature a stream with that name near Mesopotomia (today part of the municipality of La Union, Antioquia). Addi- tionally, we could not obtain samples of R. true- bae, a species known only from the holotype and for which the precise locality is unknown (Lynch and Renjifo, 1990; Vélez-Rodriguez, 2004a). THE RHINELLA CRUCIFER GROUP This putatively monophyletic species group is currently composed of six medium-sized species whose distribution is mainly associated with the Atlantic Forest of Argentina, Brazil, and Paraguay (Duellman and Schulte, 1992; Baldissera et al., 2004; Thomé et al., 2010, 2012; Roberto et al., 2014). The following characters have been pro- posed to diagnose this species group: skull heavily ossified with slightly elevated cranial crests, dorsal skin smooth with low, scattered tubercles, lateral row of enlarged tubercles present, pale mid verte- bral line well-defined, and parotoid macroglands elongated, moderate in size (Duellman and Schulte, 1992; Baldissera et al., 2004; Pramuk, 2006). This species group was recognized as distinct from the Rhinella marina Group by R.E. Martin (1972b) and Duellman and Schulte (1992) and all its forms were considered as a single species (Bufo crucifer) for a long time (see Lutz, 1934; Cochran, 1955; Cei, 1980; Duellman and Schulte, 1992). Baldissera et al. (2004) revised the taxonomy of this species group and recognized five species based on morphology and morphometrics: Rhinella abei (Baldissera et al., 2004), R. crucifer (Wied, 1821), R. henseli (Lutz, 1934), R. ornata (Spix, 1824), and R. pombali (Baldissera et al., 2004). Subsequent to the revision of Baldissera et al. (2004), two additional species, Rhinella inopina and R. casconi, were 12 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY described from wet forests within the Cerrado and Caatinga habitats of Brazil, respectively (Vaz-Silva et al., 2012; Roberto et al., 2014). Pramuk (2006) only included one species (R. ornata, as Bufo cruci- fer) of this group in her phylogenetic analysis, and recovered it as the sister taxon of the R. marina Group. Thomé et al. (2010, 2012) corroborated the monophyly of the R. crucifer Group although the outgroup sampling was limited. They also high- lighted problems in the taxonomy proposed by Baldissera et al. (2004), as the recognized species did not fully correspond with genetic structuring in the group. Thomé et al. (2010, 2012) found that samples from specimens identified as R. pombali are nested within R. crucifer and/or R. ornata in the mitochondrial phylogenies and are associated with intermediate nuclear genomes in nonphylogenetic analysis (see factorial correspondence analyses [FCA] in Thomé et al., 2012). In addition to these results, a geographic distribution between that of R. crucifer and R. ornata (Baldissera et al., 2004) is congruent with R. pombali as a hybrid complex between the last two species (Thomé et al., 2010, 2012). Furthermore, samples from R. abei were nested within R. ornata. Thomé et al. (2012) pro- posed to synonymize R. pombali with both parental species and suggested further reassessment of the taxonomic status of R. abei with additional molecu- lar markers. Their results were congruent with 2D geometric morphometrics of the skull performed by Bandeira et al. (2016), who found R. pombali to be morphologically intermediate between R. cruci- fer and R. ornata, and R. abei nested within R. ornata in the multivariate space. Several specimens of the six valid species (Rhinella abei, R. casconi, R. crucifer, R. henseli, R. inopina, and R. ornata) were included in our analyses to test the monophyly of this group and the results of Thomé et al. (2010, 2012). We car- ried out a preliminary analysis (data not shown) including additional nuclear and mitochondrial sequences of two specimens of “R. pombali” and the results supported their findings (see Hybrid- ization and genetic introgression in Rhinella sec- tion), so we did not include specimens of “R. pombali” in our subsequent analyses. NO. 447 THE RHINELLA GRANULOSA GROUP This monophyletic species group is currently composed of 14 medium- to small-sized species of Rhinella (Pramuk, 2006; Pereyra et al., 2016a; Murphy et al., 2017). The following characters have been proposed to diagnose this species group: skull heavily ossified and exostosed with low, granular or elevated cranial crests, dorsal skin with small, keratinous-tipped tubercles, and lateral row of enlarged tubercles absent (Gal- lardo, 1957, 1965; R.F. Martin, 1972a, 1972b; Cei, 1980; Duellman and Schulte, 1992; Pramuk, 2006). All species of the R. granulosa Group are mostly distributed in open areas of South Amer- ica and Panama (Gallardo, 1965; Duellman and Schulte, 1992; Duellman, 1999; Narvaes and Rodrigues, 2009; Sanabria et al., 2010). The taxonomy of this species group was first revised by Gallardo (1965) and more recently by Narvaes and Rodrigues (2009). The latter authors recognized and diagnosed 12 species on the basis of morphological and morpho- metrical analyses. Subsequently, Sanabria et al. (2010) described a new species (R. bernardoi) from San Juan, western Argentina. The phylo- genetic analyses of Pramuk (2006) and Pereyra et al. (2016a), comprising very different samples of species and characters, recovered this species group as monophyletic and discussed several of its phenotypic synapomorphies. Moreover, Pereyra et al. (2016a) documented the occur- rence of hybridization between sympatric spe- cies as well as past mitochondrial introgression and proposed several morphological synapo- morphies for the group. Vera Candioti et al. (2016) proposed some additional synapomor- phies from the embryonic morphology (a very short third pair of gills, type A adhesive glands, the adhesive gland subdivision immediately before the gills reach their maximum develop- ment, and a short dorsal line of hatching glands mostly restricted to the cephalic region). More recently, Murphy et al. (2017) found the pop- ulations of R. humboldti on both sides of the Andes to be phylogenetically distinct, leading 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 13 them to restrict R. humboldti to the western Andean populations and resurrect R. bebeei for the eastern ones. In our phylogenetic analyses, we included most species of this group (Rhinella azarai, R. beebei, R. bergi, R. bernardoi, R. centralis, R. dorbignyi, R. fernandezae, R. granulosa, R. humboldti, R. major, R. merianae, R. mirandaribeiroi, and R. pygmaea) with the exception of R. nattereri, a species known from a restricted area in the border between Bra- zil, Guyana, and Venezuela (Bokermann, 1967; Narvaes and Rodrigues, 2009). THE RHINELLA MARGARITIFERA GROUP The definition of this species group is contro- versial, as diagnoses have been largely based on morphological variation of the Rhinella mar- garitifera species complex (e.g., R.E Martin, 1972b; Hoogmoed, 1986; Pramuk, 2006) or sub- jective notions of similarity without consideration of character polarity (e.g., Cei, 1972a; Hoogmoed, 1990; Duellman and Schulte, 1992). The following characters have been used to diagnose this species group: skull relatively lightly ossified with variable amounts of dermal ornamentation and prominent cranial crests, dorsal skin smooth or with small, scattered tubercles, and a lateral row of enlarged tubercles present (Hoogmoed, 1990; Duellman and Schulte, 1992; Vélez-Rodriguez, 2004b; Pramuk, 2006). Nevertheless, this definition does not accomodate the morphology of species recently included in the group (R. ocellata and R. yunga, the putative sister species to the remaining species of the group, see Moravec et al., 2014). Similarly, the taxonomy of the species of the Rhinella margaritifera Group is also conflicted due to imprecise type localities, extreme sexual dimorphism, and the extensive ontogenetic vari- ation that hinder the specific recognition and description of some putative undescribed species (Hoogmoed, 1977; 1986; 1990; Hass et al., 1995; De la Riva et al., 2000; Vélez-Rodriguez, 2004b; Fouquet et al., 2007a, 2007b, 2007c; Lavilla et al., 2013, 2017). Currently, this group is composed of 20 medium-sized species (see appendix 5) dis- tributed from Panama to southern Brazil, includ- ing the Amazonia and Guiana Shield. We sampled 17 species of this group: Rhinella acutirostris, R. alata, R. castaneotica, R. dapsilis, R. gildae, R. hoogmoedi, R. lescurei, R. magnus- soni, R. margaritifera, R. martyi, R. ocellata, R. paraguayensis, R. proboscidea, R. scitula, R. sclerocephala, R. stanlaii, and R. yunga. This sam- pling also includes numerous specimens of the R. margaritifera species complex throughout its distribution. Additionally, we included two undescribed species of this group, one from Ecuador and another one from Colombia, Peru, and Venezuela. Two species of this group were unsampled: R. roqueana, which occurs along the lowlands east of the Andes in southern Ecuador and adjacent northern Peru (Hoogmoed, 1990), and R. sebbeni, which is known only from a few localities of the riparian and dry seasonal forests in the Cerrado biome (Vaz-Silva et al., 2015). THE RHINELLA MARINA GROUP This species group is currently composed of 11 large species (Duellman and Schulte, 1992; Maciel et al., 2010; Vallinoto et al., 2010; Lavilla and Brus- quetti, 2018). The group is distributed from the southern United States to Argentina, and its species inhabit both open and forested areas (Duellman and Schulte, 1992; Frost, 2020). The following char- acters have been proposed as diagnostic of this spe- cies group: extremely ossified and exostosed skulls, elevated (keratinized or not) cranial crests, dorsal skin with small and large tubercles, and lateral row of enlarged tubercles absent (Duellman and Schulte, 1992; Pramuk, 2006; Maciel et al., 2010). Maciel et al. (2010) and Vallinoto et al. (2010) studied the phylogenetic relationships in this species group. Maciel et al. (2010) included phenotypic (morpho- logical and parotoid-macrogland secretions) and molecular (sequences of three mitochondrial and one nuclear genes) characters and found this group as monophyletic, being the sister taxon of the Rhi- nella crucifer Group. Alternatively, Vallinoto et al. (2010) found the R. crucifer Group nested within the R. marina Group. Sequeira et al. (2011) reported 14 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY the occurrence of extensive unidirectional intro- gression between R. diptycha (as R. schneideri) and some populations of R. marina that could contrib- ute to biased inferences in the phylogenetic rela- tionships. More recently, Vallinoto et al. (2017) reevaluated this hypothesis by including additional samples and molecular markers and found a more complex scenario with no evident pattern of unidi- rectional introgression and a doubtful taxonomic status of some R. marina populations. Finally, based on a phylogenetic analysis using mitochon- drial genes and morphometric data, Acevedo et al. (2016) resurrected R. horribilis for the western Andean populations previously considered R. marina. Recently Bessa-Silva et al. (2020) found evidence of interspecific nuclear differentiation between these species and a marked discordance between mitochondrial and nuclear phylogenetic inferences in the R. marina Group. We included samples of several populations from all the currently recognized species of this group: Rhinella achavali, R. arenarum, R. cerraden- sis, R. diptycha, R. horribilis, R. icterica, R. jimi, R. marina, R. poeppigii, R. rubescens, and R. veredas. For R. arenarum, we also included samples of the populations historically assigned to the subspecies R. arenarum mendocina (see Laurent, 1969). THE RHINELLA SPINULOSA GROUP Nine species are currently assigned to this group, which are distributed in the Andean region from southern Ecuador to southern Argentina and Chile, except for Rhinella achalen- sis, which is endemic to the Sierras Pampeanas Centrales in central Argentina (Cei, 1972b; Pramuk and Kadivar, 2003). The species of this group are medium sized and have a moderately to lightly ossified skull that lacks dermal sculp- turing and exostosis. They also have a marked sexual dimorphism in skin texture and color- ation (Vellard, 1959; Cei, 1972a, 1972b; Duellman and Schulte, 1992). This group was recovered as monophyletic in the combined phylogenetic analysis of Pramuk (2006: fig. 4) but paraphyletic in the separate molecular or morphological anal- NO. 447 yses (Pramuk, 2006: figs. 1-3). Some subspecies have been recognized for the nominal species of this group, which is a putative species complex (Vellard, 1959; Cei, 1972a; Ferraro et al., 2018). We included all recognized species of this group: Rhinella achalensis, R. amabilis, R. arequi- pensis, R. arunco, R. atacamensis, R. limensis, R. rubropunctata, R. spinulosa (including popula- tions historically assigned to the subspecies R. s. papillosa, R. s. spinulosa, and R. s. trifolium), and R. vellardi. We were unable to sample popula- tions assigned to two subspecies of R. spinulosa: R. s. altiperuviana and R. s. flavolineata. THE “RHINELLA VERAGUENSIS GROUP” This nonmonophyletic group is composed of 17 small- to medium-sized species, all of which occur in the cloud forest of the Andes from northern Peru to northern Argentina, excepting Rhinella chrysophora, a species from north-cen- tral Honduras (Cei, 1972a; Duellman and Schulte, 1992; Chaparro et al., 2007; Cusi et al., 2017; McCranie, 2017). Members of this group are morphologically diverse with terrestrial, semiaquatic, or arboreal habits. The following characters have been considered diagnostic for the Rhinella veraguensis Group: skull with weak exostosis, cranial crests absent or weak, dorsal skin bearing small elevated tubercles, and a lateral row of enlarged tubercles in some species (Gallardo, 1961; Cei, 1972a; Duellman and Schulte, 1992; Pramuk, 2006). This group has been consis- tently recovered as nonmonophyletic (Pramuk, 2006; Chaparro et al., 2007; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Moravec et al., 2014; Pyron, 2014; Cusi et al., 2017; Jetz and Pyron, 2018) and its definition and composition are problematic (see Pereyra et al., 2015; and comments regarding the definition of the R. acrolopha and R. festae Groups above). For purposes of description of the ingroup, R. lilyrodriguezae is included in this group (according to the phylogenetic relationships recov- ered by Cusi et al., 2017), although this species was assigned to the R. festae Group in the original 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 15 description. We included samples of most species of this group (R. amboroensis, R. arborescandens, R. chavin, R. fissipes, R. inca, R. justinianoi, R. leptos- celis, R. lilyrodriguezae, R. manu, R. multiverrucosa, R. nesiotes, R. quechua, R. rumbolli, R. tacana, R. veraguensis, and R. yanachaga). ‘The only unsam- pled species was R. chrysophora, which occurs in the Wet Forest in the central and western portions of the Cordillera Nombre de Dios, central-north Honduras. This species has not been observed since 1996 and is thought to be extinct (McCranie, 2017). SPECIES UNASSIGNED TO ANY GROUP Six species of Rhinella are not currently assigned to any group (for a history of previous group assignments see appendix 5). Among them, we included R. ceratophrys, R. gallardoi, and R. sterno- signata in our phylogenetic analyses. The following species were not included in the phylogenetic anal- yses: (1) R. cristinae (Vélez-Rodriguez and Ruiz- Carranza, 2002), a species known only from the type locality (Vereda Tarqui, km 53-54 on road Altamira-Florencia, Municipio de Florencia, Departamento del Caqueta, Colombia) and col- lected in 1990 for the last time; (2) R. gnustae (Gal- lardo, 1967), which is only known from the holotype collected in 1925 from an imprecise local- ity (Rio Grande, Jujuy, Argentina; see Lavilla et al., 2002); and (3) R. iserni (Jiménez de la Espada, 1875), which is also known with certainty only from the holotype and its type locality is imprecise (Andes de Chanchamayo, Peru). MOLECULAR DATA TISSUE SAMPLING The molecular data were the main source of evidence in terms of both number of scored char- acters and sampled terminals. As one of the main goals of this paper was to test the monophyly of all the species groups of Rhinella, we attempted to obtain tissue samples from as many species as possible, with particular emphasis on putative nonmonophyletic species groups. Additionally, we included specimens from multiple populations of species that include recognized subspecies (e.g., R. arenarum and R. spinulosa), species that might represent species complexes (e.g., R. dapsilis, R. margaritifera, and R. proboscidea), and widely dis- tributed taxa (e.g., R. diptycha, R. marina, and R. veraguensis) to evaluate their taxonomy. We included GenBank sequences only in cases where precise voucher number and locality data are pro- vided, for specimens sequenced for at least the 16S rRNA gene. Besides, we made an effort to cor- roborate the identity of most relevant vouchers. A detailed list of all the terminals included in our analyses is given in appendices 1 and 2. LABORATORY PROTOCOLS We extracted total genomic DNA from etha- nol-preserved tissues (liver, muscle, or fingertips) using the Qiagen DNeasy kit. We carried out PCR amplifications in a total volume of 25 ul reactions using 0.2 ul Taq (Fermentas). The PCR protocol consisted of an initial denaturation step of 3 min. at 94° C followed by 35 (for mitochondrial genes) or 45 (for nuclear genes) cycles consisting of 30 seconds at 94° C for denaturation, 40 seconds at 48°-62° C for annealing, and 30-60 seconds at 72° C for extension, and a final extension step of 10-15 minutes at 72° C. We cleaned PCR-ampli- fied products using 10U of Exonuclease plus 1U of alkaline phosphatase per reaction. We sequenced the products with an automatic sequencer ABI 3730XL (Applied Biosystems) in both directions to check for potential errors and nuclear polymorphisms. We processed the chro- matograms using the software Sequencher version 4.5 (Gene Codes, Ann Arbor, MI) and edited the complete sequences with BioEdit (Hall, 1999). Sequences are deposited in GenBank under the accession numbers MW002838-MW003700. GENOTYPIC CHARACTER SAMPLING The mitochondrially encoded loci sampled for the phylogenetic analyses include: (1) the 12S 16 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY rRNA, the tRNA Valine, and the 16S rRNA genes (12S-tRNA“'- 16S; 2469 bp), (2) a fragment com- prising the upstream section of the 16S rRNA gene and the tRNA Leucine, NADH dehydrogenase sub- unit 1, and tRNA Isoleucine genes (16S-tRNA’*“- NDI- tRNA!; 1305 bp), and (3) a fragment of cytochrome b gene (cytb; 700 bp), for a total of up to 4474 bp. The nuclear loci include: (1) the C-X-C motif chemokine receptor 4 gene (cxcr4; 676 bp), (2) the solute carrier family 8 member Al gene (slc8a1; 715 bp), (3) the proopiomelanocortin gene (pomc; 559 bp), (4) two nonoverlapping fragments of the recombination activating 1 gene (ragl-a and rag1-b; 936 and 429 bp respectively), and (5) the rhodopsin gene (rho; 316 bp), for a total of 3631 bp. Primers and their sources are detailed in table 1. For the parsimony total evidence and maxi- mum-likelihood (ML) analyses (see below), the amount of sequence data analyzed per terminal ranged from 447 bp (Rhinella gildae URCA 12651 obtained from GenBank) to 8089 bp (R. henseli CFBH 20117), with a mean of 4378 bp per terminal. All the phylogenetic datasets employed in the analyses are available at https:// doi.org/10.5531/sd.sp.46). PHENOTYPIC DATA The phenotypic dataset consisted of direct observations on specimens and bibliographic information for 90 characters, scored for 106 terminals (84 from the ingroup, 22 from out- groups). The scoring was recorded using Mes- quite version 3.51 (Maddison and Maddison, 2018). The dataset was assembled from the fol- lowing character systems: 33 from adult osteol- ogy, 15 from hand and foot musculature, 3 from the tympanic middle ear, 1 from adult visceral anatomy, 15 from adult external morphology, 9 from larval external morphology, 3 from larval chondrocranium, 4 from embryonic external morphology, 6 from natural history, and 1 from cytogenetics. Phenotypic characters are described below (see List and Description of Characters); the phenotypic matrix is included NO. 447 as supplementary data 1 (available at https:// doi.org/10.5531/sd.sp.46). Cranial and postcranial osteology follows the terminology employed by Trueb (1973, 1993), that of cranial crests follows Mendelson (1997a), and hand and foot myology follows Blotto et al. (2020). Terminology for larval external morphol- ogy follows Altig and McDiarmid (1999) and the characterization of embryonic structures follows Nokhbatolfoghahai and Downie (2005, 2008). Osteology was studied in (1) cleared and double- stained specimens prepared following the tech- niques of Wassersug (1976), (2) dry skeletons, and (3) u-CT scans (available for download at www. morphosource.org, Duke University). Additional information was obtained from detailed osteologi- cal descriptions in the literature (see appendix 3). Visualization and data processing of u-CT images was done in MeshLab (Cignoni et al., 2008). For the study of myology, dissections of the hand and foot musculature were performed to remove superficial layers and observe successively deeper muscles as outlined by Blotto et al. (2020). Topical applications of the iodine/potassium iodide solu- tion of Bock and Shear (1972) were used when necessary to enhance contrast. The remaining characters were scored from the literature, unless specified (see appendix 3). We scored multiple states for uncertainty or ambiguity in the condition of a terminal (among some states, but not all the character states) for some characters (see Pol and Apesteguia, 2005). This way of scoring let us incorporate relevant information (mainly from descriptions obtained from the bibliography) even when descriptions were not detailed enough. For 19 series of trans- formation, we used composite coding (sensu Maddison, 1993), which minimizes the occur- rence of inapplicable or missing entries (Pimen- tel and Riggins, 1987; Maddison, 1993; Strong and Lipscomb, 1999). PHYLOGENETIC ANALYSES The final taxon sample for the phylogenetic analyses was defined by means of a series of 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) i, preliminary analyses that clarified the situation of many problematic terminals. As hybridiza- tion and genetic introgression, both nuclear and mitochondrial, seem to be common in some species of Rhinella (Sequeira et al., 2011; Pereyra et al., 2016a; Vallinoto et al., 2017), we first per- formed exploratory analyses of mitochondrial (MD) and nuclear (ND) datasets independently to detect nuclear-mitochondrial discordance as indicative of putative genetic introgression. Subsequently, we performed a total evidence (TE) analysis (Kluge, 1989, 2004; Nixon and Carpenter, 1996) combining nonintrogressed nuclear and mitochondrial sequences and the phenotypic dataset (see details in appendix 2). The phylogenetic analyses of each separate molecular dataset (nuclear and mitochondrial, see below) and the total evidence analysis were performed in TNT version 1.5 (Goloboff et al., 2008; Goloboff and Catalano, 2016). Gaps were considered as a fifth state in all parsi- mony analyses (nuclear, mitochondrial, and total evidence analyses) and all classes of transformation events were equally weighted. In addition, we performed a total evidence analysis considering gaps as missing data for comparisons with the maximum likelihood analysis (see below). Unless otherwise stated, all results shown refer to parsimony analyses in which gaps were treated as a fifth state. We favoured parsimony as optimality criterion because the cladogram that minimizes trans- formations to explain the observed variation is the simplest one, maximizes evidential con- gruence, and has the greatest explanatory power (Farris, 1983; Goloboff, 2003; Goloboff and Pol, 2005; Kluge and Grant, 2006; Wheeler et al., 2006). Sequences were aligned using the online software MAFFT v7 (Katoh and Toh, 2008; Katoh et al., 2019) under the strategy E-INS-i (for the 12S-tRNA“!-16S fragment) and L-INS-i or G-INS-i (for remaining frag- ments), with default parameters for gap open- ing and extension. These alignments were used for both phylogenetic analyses and clade sup- ports estimations (see details below). SEPARATE PHYLOGENETIC ANALYSES OF NUCLEAR AND MITOCHONDRIAL SEQUENCES Both nuclear and mitochondrial datasets were analyzed in TNT using “New Technology” searches and performing a combination of secto- rial searches, ratchet, and tree fusing (Goloboff, 1999; Nixon, 1999), using the default settings for these strategies. Tree searches were performed until the consensus was stabilized 10 times, with a factor of 75 (see Goloboff, 1999; Giribet, 2005). The strict consensus tree resulting from the analysis of all sampled taxa of the nuclear dataset (= ND) was poorly resolved (data not shown). A poor resolution of the consensus can be due to the effect of just a small number of wildcard or rogue taxa, which are those that assume varying phylogenetic positions in the most parsimonious trees (MPT) (Nixon and Wheeler, 1992; Wilkin- son, 1996; Aberer et al., 2013; Goloboff and Szu- mik, 2015). To avoid including terminals that act as wildcard taxa due to the lack of evidence, we included only terminals with more than three nuclear sequenced fragments (see appendix 2). Although there is an imperfect relationship between missing data and wildcard behavior, we identified three loci as the critical number to obtain an informative and comparable consensus in preliminary analyses. After excluding termi- nals with fewer than three nuclear fragments from the dataset, we reanalyzed this restricted nuclear dataset (rND) to estimate the consensus tree and clade supports (see below). The mito- chondrial dataset was analyzed using the same terminals as the restricted nuclear dataset (i.e., restricted mitochondrial dataset, rMD) and simi- lar parameters of analysis (see above), to allow the comparison. TOTAL EVIDENCE ANALYSIS For the TE analysis, we followed the strategy described above for the separate nuclear and mito- chondrial analyses. In this analysis, we included: (1) all the nuclear sequences from the complete nuclear dataset, (2) all the mitochondrial sequences 18 Genome Mitochondrial Nuclear Gene 12S- 16S ndl cytb cxcr4 pomc ragla raglb rho slc8al BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 TABLE 1 Primers used to amplify and sequence DNA in this study See appendix 2 for gene abbreviations. Primer Direction Primer sequence 5’>3’ Source MVZ59 Forward ATAGCACTGAAAAYGCTDAGATG Graybeal, 1997 Phe2-L Forward AAAGCATAACACTGAAGATGTTAAGATG Wiley et al., 1998 12S F-H Reverse CTTGGCTCGTAGTTCCCTGGCG Goebel et al., 1999 12S A-L Forward AAACTGGGATTAGATACCCCACTAT Goebel et al., 1999 tRNAval-H Reverse GGTGTAAGCGARAGGCTTTKGTTAAG Goebel et al., 1999 12Sm Forward GGCAAGTCGTAACATGGTAAG Pauly et al., 2004 L13 Forward TTAGAAGAGGCAAGTCGTAACATGGTA Feller and Hedges, 1998 Titus I Reverse GGTGGCTGCTTTTAGGCC Titus and Larson, 1996 L2A Forward CCAAACGAGCCTAGTGATAGCTGGTT Hedges, 1994 H10 Reverse TGATTACGCTACCTTTGCACGGT Hedges, 1994 AR Forward CGCCTGTTTATCAAAAACAT Palumbi et al., 1991 Wilkinson2 Reverse GACCTGGATTACTCCGGTCTGA Wilkinson et al., 1996 16S-frog Forward TTACCCTRGGGATAACAGCGCAA Wiens et al., 2005 tMet-frog Reverse TTGGGGTATGGGCCCAAAAGCT Wiens et al., 2005 ND1 Fl Forward AGCCATAATCATCTGAACC Smith et al., 2005 ND1 RI Reverse TCCTCCCTATCAAGGAGGTCC Smith et al., 2005 CytbDen3-L Forward AAYATYTCCRYATGATGRAAYTTYGG an ey cee eu Reverse GCRAANAGRAAGTATCATTCNGGYTTRAT ee ane Ganngtelé CXCR4-C Forward GTCATGGGCTAYCARAAGAA Biju and Bossuyt, 2003 CXCR4-G Reverse AGGCAACAGTGGAARAANGC Biju and Bossuyt, 2003 POMC-1 Forward GAATGTATYAAAGMMTGCAAGATGGWCCT Wiens et al., 2005 POMC-2 Reverse TAYTGRCCCTTYTTGTGGGCRTT Wiens et al., 2005 POMC-2B Reverse GCATTYTTGAAAAGAGTCATTARTGGAGTCTG Pramuk, 2006 MartFll Forward AGCTGCAGYCARTAYCAYAARATGTA Hoegg et al., 2004 AmpRI1 Reverse AACTCAGCTGCATTKCCAATRTCA Hoegg et al., 2004 R1-GFF Forward GAGAAGTCTACAAAAAVGGCAAAG Faivovich et al., 2005 R1-GFR Reverse GAAGCGCCTGAACAGTTTATTAC Faivovich et al., 2005 RAGI1 TGIF Forward CCAGCTGGAAATAGGAGAAGTCTA Grant et al., 2006 RAGI TGIR Reverse CTGAACAGTTTATTACCGGACTCG Grant et al., 2006 Rhod1A Forward ACCATGAACGGAACAGAAGGYCC ey nay RhodIC Reverse +CCAAGGGTAGCGAAGAARCCTTC Resse sae on. itch, 2000 NACAL Forward +TCCAAAGCAGATATTGAAATGGA Seis ane Rossy Roelants and Bossuyt, NACAO Reverse ATACCTGCATGATCATCATCAAA 3005 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 19 from the complete mitochondrial dataset, and (3) the phenotypic dataset. The following criteria were used to treat putatively conspecific sequences as pertaining to the same or different terminals: (1) sequences from the same individual or conspecific individuals placed in well-supported discordant positions in the separate nuclear and mitochon- drial analyses were considered as independent ter- minals, because discordance suggests mitochondrial introgression between different spe- cies (see Pereyra et al., 2016a); and (2) terminals from the phenotypic dataset were combined with the more closely related conspecific terminal of the molecular dataset (mitochondrial + nuclear). When mitochondrial and nuclear sequences of a specimen were included separately, the phenotypic data were combined with the nuclear sequences. Appendix 2 provides a list of all the terminals included and excluded in the TE analysis. RESAMPLING SUPPORT MEASURES Two types of resampling support measures were estimated for the datasets in TNT version 1.5 (Goloboff and Catalano, 2016): (1) parsimony jackknife absolute frequencies (JAF; Farris et al., 1996) and (2) parsimony jackknife frequency dif- ferences (JGC; Goloboff et al., 2003). For estima- tion of both measures, we performed 1,000 replicates using “New Technology” searches con- sisting of a combination of sectorial searches, ratchet, and tree fusing (Goloboff, 1999; Nixon, 1999), reaching minimum length two times (pre- liminary analyses showed that minimum lengths are hit with this search strategy). Goloboff et al. (2003) noted that the resampling support for a clade does not necessarily correlate with the abso- lute frequency itself (ie., the number of times a group is recovered in the resampled matrices), because groups with positive support (= 50%) can have much lower frequencies than groups with no support at all (<50%). To solve this situation, these authors proposed to also consider the value GC (i.e., frequency difference), which indicates the frequency differences between a group and the most frequent contradictory group. Values of this score range between -100% (maximum contradic- tion) and 100% (maximum support). MAXIMUM-LIKELIHOOD ANALYSIS Maximum-likelihood analysis was performed with IQ-TREE v1.6.12 (Nguyen et al., 2015) con- sidering the same dataset (DNA sequences + phe- notypic characters) as the TE analysis under parsimony. ModelFinder (Kalyaanamoorthy et al., 2017), which is implemented in IQ-TREE, was used to select the optimal partition scheme and substitution models for molecular characters. ModelFinder implements a greedy strategy (Lan- fear et al., 2012) that starts with the full partition model and subsequentially merges two genes until the model fit does not increase any further. The best partition scheme included two subsets (see table 2). For morphological data we use the two morphological ML models (see Lewis, 2001) implemented in IQ-TREE (ie. MK and ORDERED, for unordered and ordered characters respectively) considering the ascertainment bias correction (ASC) method. We consider edge- linked-proportional partition model but separate substitution models and rate evolution between partitions (-spp option). The maximum-likeli- hood tree was conducted with 1000 ultrafast boot- strap replicates (Minh et al., 2013; Hoang et al., 2018) using the option -bnni that reduces the risk of overestimating branch supports due to severe model violations. The resulting tree was visualized and edited in FigTree 1.4.3 (Rambaut, 2016). Par- titions and models selected are detailed in table 2. TAXONOMIC EVALUATION We considered the following criteria in assess- ing the taxonomic status of each lineage: (1) the cladogram topology resulting from the phyloge- netic analyses, (2) the uncorrected pairwise dis- tances (UPDs) of a fragment of the 16S rRNA gene (delimited by the primers AR and WILK2; see Vences et al., 2005a, 2005b; Fouquet et al., 2007b) calculated in PAUP* (Swofford, 2002), and (3) the known phenotypic evidence for each 20 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 TABLE 2 Best partition scheme and best-fit models selected by ModelFinder for the molecular data. For phenotypic data, we used morphological models considering the ascertainment bias correction (ASC) method. Subset Data blocks Model ! a ee emine wy Coding mitochondrial sequences 3rd TN+F+1+G4 6: Unordered phenotypic characters MK+ASC 4 Ordered phenotypic characters ORDERED+ASC taxon. The phenotypic criterion was mainly con- sidered in cases where relationships were unre- solved (i.e. occurrence of polytomies) or poorly supported (JGC <50%) within a clade. For estima- tion of UPDs, datasets containing only sequences of the 16S rRNA gene for each species group (as are redefined in the Results section) were aligned in MAFFT under the strategy G-INS-i. LIST AND DESCRIPTION OF CHARACTERS Characters modified from previous phyloge- netic studies are indicated with an asterisk (*). ADULT OSTEOLOGY Most of the osteological characters used here are those of Pramuk (2006), so they are not described in detail except when relevant (e.g., when character states were modified or addi- tional character states were considered). Described characters refer to adult individuals of both sexes unless specified. Skull 0. Preorbital crest (on the maxillary process of nasal), occurrence: (0) absent or indistin- guishable, (1) weak, (2) well developed. Addi- tive. Cranial crests were considered osteological characters, although it could also be scored from whole-preserved specimens. The use of presence/ absence of cranial crests has a long history in bufonid taxonomy, and they were used in a phy- logenetic context by Pramuk (2006: chars. 63-69). However, unlike Pramuk (2006), we dif- ferentiate between weak and well-developed crests. State 1 (weak) refers to cranial crests that are faint or not evident externally in living or intact preserved specimens, but evident in osteo- logical preparations. State 2 (well developed) refers to crests that are evident externally in both intact and osteologically prepared specimens. When osteological preparations were not avail- able to precisely determine the absent or weak state of the crest (since both states are similar in complete specimens) we scored these uncertain- ties as multiple states (i.e., 0/1, see Phenotypic data scoring in Material and methods section). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 29*), Morrison (1994: char. 13*), Mendelson (1997a: char. 6*), Pramuk (2006: char. 65*), Mendelson et al. (2011: char. 6*). 1. Supraorbital crest (on frontoparietals), occurrence: (0) absent or indistinguishable, (1) weak, (2) well developed. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 29*), Morrison (1994: char. 14*), Mendelson (1997a: char. 7*), Pramuk (2006: char. 68*), Mendelson et al. (2011: char. 7*). 2. Pretympanic crest (on the zygomatic ramus of squamosal), occurrence: (0) absent or indistin- guishable, (1) weak, (2) well developed. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 16*), Mendelson (1997a: char. 11*), Pramuk (2006: char. 66%), Mendelson et aliv(2011s-chans11*). 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 21 3. Supratympanic crest (on the otic ramus of squamosal), occurrence in females: (0) supratym- panic crest inconspicuous or developed, but that does not extend beyond the level of the cranial roof dorsally, (1) supratympanic crest hypertrophied extending beyond the level of the cranial roof dor- sally. This character was codified separately for males and females since a dimorphic condition was detected. Large supratympanic crest occurs mainly in adult females of many species of the Rhinella margaritifera Group (Hoogmoed, 1990; Duellman and Schulte, 1992). However, males of some of these species also have large supratympanic crest (Hoogmoed, 1990). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 17*), Mendelson (1997a: char. 10*), Vélez-Rodriguez (2004b: char. 35%), Pramuk (2006: char. 69*), Mendelson et al. (2011: char. 10*). 4, Supratympanic crest (on the otic ramus of squamosal), occurrence in males: (0) supra- tympanic crest inconspicuous or developed, but that does not extend beyond the level of the cra- nial roof dorsally, (1) supratympanic crest hyper- trophied extending beyond the level of the cranial roof dorsally. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 17*), Mendelson (1997a: char. 10*), Vélez-Rodriguez (2004b: char. 35%), Pramuk (2006: char. 69*), Mendelson et al. (2011: char. 10*). 5. Parietal crest (on frontoparietal), occur- rence: (0) absent or indistinguishable, (1) weak, (2) well developed. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 29*), Morrison (1994: char. 15*), Mendelson (1997a: char. 8*), Pramuk (2006: char. 64*), Mendelson et al. (2011: char. 8%). 6. Nasals, shape of anterior margins: (0) relatively blunt, (1) acuminate. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Mendelson (1997a: char. 34*), Scott (2005: char. 64*), Pramuk (2006: char. 4), Nussbaum and Wu (2007: char. 52*), Mendelson et al. (2011: char. 25”). 7. Nasals, medial contact: (0) not in contact medially, (1) in contact medially. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Lynch (1978: char. 2*), Clarke (1981: char. 1*), Ford (1990: char. 1), Morrison (1994: char. 1*), Baez and Basso (1996: char. 2), Mendelson et al. (2000: char. 32), Scott (2005: char. 63), Fabrezi (2006: char. 1), Pramuk (2006: char. 3), Nuss- baum and Wu (2007: char. 51), Ponssa (2008: char, 52*). 8. Contact between nasal and frontoparie- tal: (0) anterior margin of frontoparietal does not articulate with posterior margin of nasal (fig. 2A), (1) articulate only laterally (fig. 2B), (2) articulate along most of its margin but not com- pletely (fig. 2C), (3) articulate along the entire margin (fig. 2D). Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Heyer and Liem (1976: char. 2*), Morrison (1994: char. 6*), Mendelson (1997a: char. 2%), Pugener et al. (2003: char. 12* [adult morpho- logical characters]), Pramuk (2006: char. 8%), Mendelson et al. (2011: char. 2*). 9. Dermal roofing bones, sculpturing: (0) dermal bones of the skull completely smooth, (1) lightly exostosed, (2) heavily ornamented with pits, striations, and rugosities. Additive. Hyperos- sification in anurans involves the level of sculptur- ing and the number and identity of exostosed bones (see revision by Blotto et al., 2021). Although species of Rhinella display a relatively high diversity of hyperossification, for the time being, we scored the variation only in the dermal roofing bones (nasals and frontoparietal), until more detailed analyses of the skull morphology are carried out. PREVIOUS USAGE IN PHYLOGENETIC STUD- IES: Clarke (1981: char. 2*), Ford (1990: char. 4*), Morrison (1994: char. 11), Mendelson et al. (2000: char. 28*), Pugener et al. (2003: char. 10* [adult morphological characters]), Scott (2005: char. 61*), Fabrezi (2006: char. 2%), Pramuk (2006: char. 2), Nussbaum and Wu (2007::char: 62*): 10. Occipital artery pathway, coverage with bone: (0) occipital canal not covered by bone, (1) partially covered, (2) completely covered with bone. Additive. po) BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 FIG. 2. Skulls (dorsal view) showing the different level of contact between nasals and frontoparietals (both bones in gray): A, Nannophryne cophotis KU 218525 (char. 8.0; species not included in this study); B, Rhinella yanachaga MUSM 24509 (char. 8.1); C, R. crucifer KU 93112 (char. 8.2); D, R. marina KU 152914 (char. 8.3). Panels A, C, D redrawn from Pramuk (2006), B redrawn from Lehr et al. (2007). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 7*), Inger (1972: char. 10*), Heyer and Liem (1976: char. 3*), Lynch (1978: char. 4*), Clarke (1981: char. 4*), Morri- son (1994: char. 10*), Mendelson (1997a: char. 4*), Mendelson et al. (2000: char. 38*), Pugener et al. (2003: char. 15* [adult morphological char- acters]), Wiens et al. (2005: char. 16*), Pramuk (2006: char. 9), Mendelson et al. (2011: char. 4*). 11. Squamosal, medial extension of otic ramus: (0) otic ramus of squamosal present, but not enlarged, (1) otic ramus of squamosal slightly enlarged, overlapping with the dorsal surface of the crista parotica, (2) otic ramus enlarged, in contact with posterolateral margin of frontoparietal, form- ing a continuous temporal arcade. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 12*), Lynch (1978: char. 5%), Clarke (1981: char. 5*), Ford (1990: char. 29%), Baez and Basso (1996: char. 16*), Mendelson (1997a: char. 33*), Faivovich (2002: char. 4%), Scott (2005: char. 65*), Wiens et al. (2005: char. 15*), Fabrezi (2006: char. 10*), Pramuk (2006: char. 15), Nussbaum and Wu (2007: char. 85%), Araujo- Vieira et al. (2019: char. 28”). 12. Nasals, extension of anterior margin: (0) anterior margins extend beyond the dorsal mar- gins of the alary processes of the premaxillae (fig. 3A), (1) anterior margins are flush with the dor- sal margins of the alary processes (fig. 3B), (2) anterior margins lie posterior to the dorsal mar- gins of the alary processes (fig. 3C). Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Pramuk (2006: char. 21), Ponssa (2008: char. 57*). 13. Premaxilla, orientation of alary process: (0) angled posteriorly to the anterior margin of the premaxillae (fig. 4A), (1) dorsally projected to the anterior margin of the premaxillae (fig. 4B), (2) angled anteriorly to the anterior margin of the premaxillae (fig. 4C). Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Ford (1990: char. 12), Morrison (1994: char. 42), Mendelson (1997a: char. 23), Scott (2005: char. 78), Pramuk (2006: char. 26), Nussbaum and Wu (2007: char. 68), Ponssa (2008: char. 33), Barrion- uevo (2017: char. 6*), Araujo- Vieira et al. (2019: char. 19*). 14. Septomaxilla, level of development of the anterior end: (0) not developed, (1) very developed and exposed anteriorly to the alary process of the premaxilla. Alcalde (2017) showed that bones previously described as “rostrals” (Pregill, 1981) or “prenasals” (Pramuk, 2000, 2006) in some bufonids are actually part of the 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 2a B th. Ph [> : D>, FIG. 3. Skulls (lateral view of the anterior region) showing the relation between the anterior margin of the nasal (black arrow) and the dorsal margin of the alary process of the premaxilla (gray arrow): A, Rhinella yanachaga MSM 24509 (char. 12.0), B, R. amabilis KU 124587 (char. 12.1), C, Schismaderma carens USNM 153380 (char. 12.2). Panels A and B redrawn from Lehr et al. (2007) and Pramuk (2006), respectively. Black arrows indicate the anterior margin of the nasal, gray arrows indicate the dorsal margin of the alary process. enlarged and exposed anterior ends of the septo- maxillae (and thus char. 42 of Pramuk [2006] refers to this structure instead to prenasals bones). Alcalde (2017) also pointed out the pres- ence of an unpaired bone in the anterior end of the snout in Rhinella dorbignyi (as R. fernan- dezae, from the R. granulosa Group). He stated that it is homolog to the prenasal bone in some Lophyohylini (Hylidae; Trueb, 1970); even if pri- mary homologs, they clearly represent indepen- dent instances of evolution. We observed this element in R. beebei (USNM 566017-8), but we could not determine its occurrence in other spe- cies of the group for which we do not consider this bone as a different character (see comments on the preservation and identification of this structure in Alcalde, 2017). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Pramuk (2006: char. 42*). 15. Squamosal, articulation of zygomatic and ventral rami: (0) the zygomatic ramus of the squa- mosal is free from the ventral ramus, (1) the zygo- matic ramus of the squamosal articulates with the ventral ramus of the squamosal. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Mendelson (1997a: char. 32*), Vélez-Rodriguez (2004b: char. 32*), Pramuk (2006: char. 14*). 16. Jaw articulation: (0) posterior to the fenestra ovalis, (1) opposite to the fenestra ovalis, (2) anterior to the fenestra ovalis. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Pramuk (2006: char. 25), Baez et al. (2012: char. 39). 17. Supraorbital flange on the frontoparie- tals: (0) frontoparietal does not extend laterally beyond the lateral margin of the sphenethmoid, (1) frontoparietal extends laterally beyond the lateral margin of the sphenethmoid. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 4), Mendelson (1997a: char.1), Mendelson et al. (2000: char. 36), Pugener et al. (2003: char. 13 [adult morphologi- cal characters]), Wiens et al. (2005: char. 13), Pramuk (2006: char. 72), Mendelson et al. (2011: char. 1). 18. Sphenethmoid, extent of anterior ossifi- cation: (0) bony sphenethmoid reaches the level of palatines, but not beyond, (1) bony spheneth- moid beyond palatines, but does not reach the level of the premaxillae, (2) bony sphenethmoid reaches the level of the premaxillae anteriorly. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 20), Mendelson (1997a: char. 13*), Vélez-Rodriguez (2004b: char. 21*), Pramuk (2006: char. 34*), Araujo-Vieira et al. (2019: char. 9*). 19. Pterygoid, articulation of the anterior ramus with maxilla: (0) anterior ramus of ptery- goid articulates along the margin of maxilla, but does not contact with the palatine, (1) anterior ramus of pterygoid articulates along the margin of maxilla and contacts the palatine. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Ford (1990: char. 32*), Morrison (1994: char. 52), 24 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY B Ch. 13.1 NO. 447 C Ch. 13.2 ! ! | ! ! ! l ! ! ! I! ek l FIG. 4. Skulls (lateral view of the anterior region) showing the orientation of alary process of the premaxilla in relation to the anterior margin of the premaxilla (premaxilla in gray): A, Nannophryne cophotis KU 218525 (char. 13.0; species not included in this study; B, R. crucifer KU 93112 (char. 13.1); C, R. sp. margaritifera Group (char. 13.2). All the figures redrawn and slightly modified from Pramuk (2006). The voucher number provided for the specimen of the R. sp. margaritifera Group was erroneously stated in Pramuk’s (2006) figures according to the information provided in appendix 1 of that publication and in VertNet database (http://portal.vertnet.org/). Clarke (1981: char. 13*), Mendelson (1997a: char. 28*), Pugener et al. (2003: char. 40* [adult mor- phological characters]), Vélez-Rodriguez (2004b: char. 17*), Ponssa (2008: char. 67), Barrionuevo (2017: char. 29*). 20. Palatine, ventral ridge: (0) absent or indistinguishable, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 18*), Morrison (1994: char. 33*), Mendelson (1997a: char. 15*), Mendelson et al. (2000: char. 10*), Pramuk (2006: char. 38), Mendelson et al. (2011: char. 14”). 21. Pterygoid, contact of medial ramus with ala of parasphenoid: (0) the medial ramus of the pterygoid is not in contact nor fused with the anterolateral margin of the ala of the parasphenoid, (1) the medial ramus of the pterygoid is fused with the anterolateral margin of the parasphenoid, (2) the medial ramus of the pterygoid is fused and extends medially along approximately half the length of the parasphenoid ala. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Lynch (1978: char. 9*), Clarke (1981: char. 14%), Ford (1990: char. 34*), Morrison (1994: char. 54), Baez and Basso (1996: char. 28*), Mendelson (1997a: ch 29*), Vélez-Rodriguez (2004b: char. 19*), Pramuk (2006: char. 19). 22. Pterygoid, suture between the medial ramus and parasphenoid alae: (0) the surface of contact is smooth, (1) jagged or scalloped. This character is not applicable for specimens where the medial ramus of the pterygoid is not in con- tact or not fused with the anterolateral margin of the ala of the parasphenoid (char. 21.0). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grandison (1981: char. 13*), Pramuk (2006: char. 31). 23. Parasphenoid, shape of anterior margin of cultriform process: (0) acute and narrow (fig. 5A), (1) broadly rounded anteriorly (fig. 5B), (2) truncated (fig. 5C), (3) jagged or scalloped (fig. 5D). Nonadditive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Clarke (1981: char. 12*), Ford (1990: char. 45%), Morrison (1994: char. 36*), Mendelson (1997a: char. 20*), Scott (2005: char. 54*), Pramuk (2006: char. 29*), Nussbaum and Wu (2007: char. 98*), Araujo- Vieira et al. (2019: char. 42”). 24. Bony protrusion at the angle of jaws: (0) absent or indistinguishable, (1) weak, (2) devel- oped into a processus. Additive. A bony protru- sion (“or bony knob”) is caused by a variable level of thickening of the ventrolateral margin of the quadratojugal. The level of development of the bony protrusion could also be determined both in living or intact specimens as in osteologi- cal preparations. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Vélez-Rodriguez (2004b: char. 36*). 25. Hyoid, posterior lobe of the anterolat- eral process: (0) absent or indistinguishable (fig. 6A), (1) present (fig. 6B). 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 25 FIG. 5. Skulls (ventral view) showing the anterior margin of cultriform process of the parasphenoid (in gray): A, Rhinella marina KU 152914 (char. 23.0); B, Nannophryne cophotis KU 218525 (char. 23.1); C, R. festae USNM 167168 (char. 23.2); D, R. cristinae ICN 26233 (char. 23.3). Panels redrawn from Pramuk, 2006 (A, B); Trueb, 1971(C) and Vélez-R. and Ruiz-C., 2002 (D). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Vélez-Rodriguez (2004: char. 42). VERTEBRAL COLUMN 26. Presacral vertebrae, level of develop- ment of neural spine: (0) neural spine flat or slightly elevated, (1) neural spine notably ele- vated, protruding externally. The level of devel- opment of the neural spines can be determined both in intact-preserved specimens and in osteo- logical preparations. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Vélez-Rodriguez (2004b: char. 40*). 27. Presacral vertebrae, number: (0) eight, (1) seven. This number refers to the number of vertebrae even if there is some level of fusion between them. The number can be traceable even when there is fusion of centra due to the persistence of the intervertebral foramina (see Trueb, 1973; Cannatella, 1986). PREVIOUS USAGE IN PHYLOGENETIC STUD- 1ES: McDiarmid (1971: char. 23*), Lynch (1973: char. 1*), Grandison (1981: char. 15*), Canna- tella (1986: char. 3*), Morrison (1994: char. 65*), Baez and Basso (1996: char. 30*), Wiens et al. (2005: char. 51*), Fabrezi (2006: char. 34), Pramuk (2006: char. 44*), Nussbaum and Wu (2007: char. 139), Mendelson et al. (2011: char. 43). 28. Presacral vertebrae I and II, fusion: (0) absent, (1) present. The fusion of the centra of both vertebrae into a single element may be identified for the occurrence of transverse pro- cesses and two foramina for vertebral nerves in the anterior presacral element. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 24*), Lynch (1973: char. 2), Heyer and Liem (1976: char. 9), Cannatella (1986: char. 4*), Ford (1990: char. 66), Morrison (1994: char. 66), Wiens et al. (2005: char. 50), Grant et al. (2006: char. 145*), Nussbaum and 26 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY A ‘Ch. 25.0 NO. 447 B & J FIG. 6. Hyoid plate: A, Rhinella cristinae ICN 26233 (char. 25.0), B, R. manu MHNC 4404 (char. 25.1). Arrow- heads indicate the occurrence of posterior lobes of the anterolateral processes in B. Panels redrawn from Vélez-R. and Ruiz-C., 2002 (A); Chaparro et al., 2007 (B). Wu (2007: char. 137), Baez et al. (2012: char. 49*), Barrionuevo (2017: char. 43). 29. Sacrum, shape of sacral diapophyses: (0) the maximum width of the sacral diapophysis is smaller than its maximum length, (1) the maxi- mum width of the sacral diapophysis is equal to, or greater than, its maximum length. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Heyer (1975: char. 34*), Heyer and Liem (1976: char. 12*), Ford (1990: char. 75*), Morrison (1994: char. 70*), Baez and Basso (1996: char. 36*), Faivovich (2002: char. 21*), Pugener et al. (2003: char. 57* [adult morphological characters]), Fabrezi (2006: char. 42*), Grant et al. (2006: char. 143*), Pramuk (2006: char. 51), Araujo- Vieira et al. (2019: char. 95”). 30. Sacrum, orientation of anterior edge of sacral diapophyses: (0) posterior to the midline axis of the vertebral column, (1) perpendicular to the midline axis of the vertebral column, (2) anterior to the midline axis of the vertebral col- umn. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Scott (2005: char. 16), Pramuk (2006: char. 52), Nussbaum and Wu (2007: char. 142). 31. Sacrum and urostyle, fusion: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 25*), Lynch (1973: char. 3), Ford (1990: char. 76), Pugener et al. (2003: char. 58* [adult morphological characters]), Wiens et al. (2005: char. 60*), Nussbaum and Wu (2007: char. 138), Baez et al. (2012: char. 51*). 32. Ilium, dorsal protuberance, level of devel- opment: (0) large and slightly anteriorly or more dorsally directed, (1) small, low, and laterally pro- jected. Gémez and Turazzini (2016) comment on the morphological variation and taxonomic distri- bution of this structure in anurans. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Clarke (1981: char. 21*), Morrison (1994: char. 87*), Scott (2005: char. 12*), Pramuk (2006: char. 54), Baez et al. (2012: char. 65*). ADULT MUSCULATURE Foot (ventral surface) 33. Discrete superficial cutaneous tendons, occurrence: (0) absent, (1) present. Burton (2004: 212, 220) described briefly this group of superficial tendons and Blotto et al. (2020) for- malized this name. We scored if the superficial tendons are discrete or if they are absent or transformed into a sheet of connective tissue or fascia over the plantar side of the foot. Addi- tional studies are needed to determine whether this group of tendons must be considered as a whole (as here) or individual superficial cutane- ous tendons of each digit should be treated as independent characters. See further comments in Blotto et al. (2020). 34. M. interphalangeus proximalis digiti V, medial slip, occurrence: (0) absent, (1) present. See Dunlap (1960), Burton (2001, 2004), and Blotto et al. (2020) for descriptions of the mm. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 27 interphalangei of the foot and comments on its taxonomic distribution in Anura. 35. M. interphalangeus proximalis digiti V, lateral slip, occurrence: (0) absent, (1) present. 36. M. abductor brevis plantaris hallucis, occurrence: (0) absent, (1) present. See Burton (2001, 2004) and Blotto et al. (2017) for charac- terization of this muscle and taxonomic distribu- tion in nonbufonid taxa. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Burton (2004: char. 30*), Faivovich et al. (2005: char. 7), Hoyos et al. (2014: char. 44), Blotto et al. (2017: char. 1). 37. M. flexor digiti II (FDM II), position of the origin with respect to the m. intermetatar- salis 1 (IMT 1): (0) FDM II ventral to the IMT 1, (1) FDM II dorsal to the IMT 1, (2) FDM II ven- tral and dorsal to the IMT 1. Nonadditive. See Dunlap (1960: 42) for an account under the name of m. flexor teres (for the FDM II) and transver- sus metatarsus (for the m. intermetatarsalis). 38. M. interosseus cruris, presence of an additional origin from the tibiale: (0) absent, (1) present. Most species have both an origin from the tibiale and from the fibulare (Gaupp, 1896; Dunlap, 1960; Burton, 2004). Among bufo- nid taxa, state 0 was reported for Atelopus (see Dunlap, 1960: 30), under the name of m. inter- tarsalis. The only species from our sampling that has state 0 is Rhinella paraguas. Foot (dorsal surface) 39. M. extensor digitorum longus (EDL), insertion on metatarsophalangeal joint of digiti IV: (0) absent, (1) present. We scored the inser- tion of the EDL in each digit as an independent character, contra Burton (2004: char. 48), as dis- cussed by Faivovich et al. (2005: 201). We found informative variation for the insertions on digits IV and V (next char.). The insertions on the meta- tarsophalangeal joint of the digits IV and V may be by an independent tendon or through a com- mon tendon with the m. extensor brevis superfi- cialis, m. extensor brevis medius, and/or the m. dorsometatarsalis proximalis, a source of variation not considered in the present study. See Dunlap (1960) and Burton (2004) for descriptions and variation of the insertion of this muscle, under the name m. extensor digitorum communis longus. The intraspecific variation reported by Inger (1972: 103) for the absence/presence of the inser- tion on each digit should be further tested; only Nannophryne variegata from our sampling was studied from more than one specimen to test this potential intraspecific variation. 40. M. extensor digitorum longus, insertion on metatarsophalangeal joint of digit V: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 26”). 41. M. extensor brevis medius hallucis, occurrence: (0) absent, (1) present. See Dunlap (1960: 52-53) for description and variation across Anura. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Hoyos et al. (2014: char. 37). 42. Lateral m. dorsometatarsalis proxima- lis digiti IV, discrete and independent tendon inserting on the proximal interphalangeal joint of digit IV: (0) absent, (1) present. Dunlap (1960: 57) considered the muscles dorsometatarsales proximales and the dorsometatarsales distales (both as mm. extensores breves profundi) as the same muscle (see discussion in Blotto et al., 2020). This fact partially precludes the understanding of the variation and taxonomic distribution described by Dunlap (1960). On the other hand, the extensive study of Hylidae by Burton (2004: char. H) suggests a great intraspecific variation when considering the number of tendons of insertion of the mm. dor- sometatarsales proximales HI-V (as extensores breves profundi). In our sampling, all species have a tendon of the lateral m. dorsometatarsalis proxi- malis digiti IV inserting on the distal interphalan- geal joint of digit IV, while Rhinella crucifer and R. henseli have an additional independent tendon of insertion on the proximal interphalangeal joint. In the light of the variation found in Bufonidae, as well as in other clades of Anura (B.L.B., personal obs.), we decided to tentatively consider each ten- don to each interphalangeal joint as independent transformation series. 28 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY Hand (ventral surface) 43. Medial m. lumbricalis brevis digiti V, slip from distal carpal 3-4-5: (0) absent, (1) present. The medial m. lumbricalis brevis digiti V may have two slips, one from the distal carpals and the other one from the flexor plate/adjacent tendo superficia- lis digiti V; both with a common or independent insertions (Burton, 1998: 59; this study). Neverthe- less, Burton (1998: char. 18) discarded further dis- cussion and comparison of the nature of this muscle given the extreme degree of variation found within his sampling (“Leptodactylidae’” s.1.). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Burton (1998: char.18). Hand (dorsal surface) 44, M. extensor digitorum, insertion on metacarpophalangeal joint of digiti III: (0) absent, (1) present. Some species lack the insertion on the meta- carpophalangeal joint of the digit II. This insertion may be through a common tendon after inserting on the dorsal fascia of other muscles (usually mm. extensores breves super- ficiales) or by an independent tendon (Burton, 1998; this study). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Burton (1998: char. 22*). 45. M. extensor digitorum, insertion on metacarpophalangeal joint of digiti V: (0) absent, (1) present. The slip of the m. extensor digitorum to the digit V may have two inser- tions, one on the metacarpophalangeal joint and a second insertion on the lateral side of the metacarpal V. The presence of both insertions varies independently across Anura (B.L.B., per- sonal obs.), for which we scored their presence as independent transformation series. Within the current sampling of Bufonidae, the lateral inser- tion on metacarpal V is invariably present, and thus variation is restricted to the presence of the insertion on the metacarpophalangeal joint. This insertion may be through a common tendon after insertion on the dorsal fascia of other mus- cles or by an independent tendon (Burton, 1998; Araujo-Vieira et al., 2019; this study). NO. 447 PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Araujo- Vieira et al. (2019: char. 171). 46. M. extensor carpi ulnaris, occurrence of a head from the radioulna: (0) absent, (1) present. This head was not previously reported in the lit- erature. It originates from the distal half or quarter of the radioulna, laterally to the origin of the m. abductor pollicis longus. The head converges with the head from the humerus, which attaches to the ulnare and distal carpal 3-4-5 (fig. 7). 47. M. extensor carpi ulnaris, nature of the origin of the head from the radioulna: (0) fleshy (fig. 7B), (1) via a flat tendon (fig. 7D). This character is not applicable for specimens that lack a supplementary head from the radio- ulna (char. 46.0). TYMPANIC MIDDLE EAR COMPLEX Pereyra et al. (2016b) reported the range of variation in structures of the tympanic middle ear (i.e., columella, annulus tympanicus, and tym- panic membrane) in Bufonidae and demonstrated its unique evolutionary pattern within Anura. 48. Columella, occurrence: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grandison (1981: char. 1*), Cannatella (1986: char. 6*), Ford (1990: char. 11), Morrison (1994: char. 27), Mendelson (1997a: char. 38”), Pugener et al. (2003: char. 47* [adult morphological char- acters]), Scott (2005: char. 81), Pramuk (2006: char. 17), Nussbaum and Wu (2007: char. 67*), Mendelson et al. (2011: char. 27). 49. Annulus tympanicus, occurrence: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 28*), Drewes (1984: char. 23%), Cannatella (1986: char. 8*), Scott (2005: char. 80*), Wiens et al. (2005: char. 35*), Nussbaum and Wu (2007: char. 66). 50. Tympanic membrane: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Inger (1972: char. 28*), Heyer (1975: char. 2*), Drewes (1984: char. 23*), Cannatella (1986: char. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 29 FIG. 7. Musculature of the dorsal surface of the hand and forearm: A, B. Rhinella paraguas CD 870. C, D. R. dorbignyi MACN 39350. A. First muscular layer: m. extensor digitorum; the m. extensor carpi ulnaris is also shown. Elements figured: 1, m. extensor digitorum and the m. extensor carpi ulnaris (head from humerus): common tendon of origin; 2, m. extensor digitorum; 3, m. extensor digitorum: slip to the dorsal surface of the m. extensor brevis superficialis digiti [V (both muscles attaches to the metacarpophalangeal joint of digit IV via a common tendon); 4, m. extensor digitorum: slip to metacarpal V; 5, m. extensor carpi ulnaris: head from humerus; 6, m. extensor carpi ulnaris (head from humerus): tendon of insertion. B. Second muscular layer: m. abductor pollicis longus and mm. extensores breves superficiales; the head from radioulna of the m. extensor carpi ulnaris is also shown (the head from humerus was removed). Elements figured: 1, m. extensor carpi ulnaris (head from radioulna): fleshy origin; 2, m. extensor carpi ulnaris: head from radioula; 3, m. extensor carpi ulnaris (heads from humerus and radioulna): common tendon of insertion on distal carpal 3-4-5; 4, m. abductor pollicis longus; 5, m. extensor indicis brevis superficialis: slip from radiale; 6, m. exten- sor indicis brevis superficialis: slip from ulnare; 7, m. extensor brevis superficialis digiti III; 8, m. extensor brevis superficialis digiti IV: slips from ulnare and distal carpal 3-4-5; 9, m. extensor brevis superficialis digiti V. C. First muscular layer: m. extensor digitorum; the m. extensor carpi ulnaris is also shown. Elements fig- ured: 1, m. extensor digitorum and m. extensor carpi ulnaris (head from humerus): common tendon of origin; 2, m. extensor digitorum; 3, m. extensor digitorum: slip to the dorsal surface of the m. extensor brevis super- ficialis digiti [V (both muscles attaches to the metacarpophalangeal joint of digit IV via a common tendon); 4, m. extensor digitorum: slip to metacarpal V; 5, m. extensor carpi ulnaris: head from humerus; 6, m. exten- sor carpi ulnaris: tendon of insertion. D. Second muscular layer: m. abductor pollicis longus and mm. exten- sores breves superficiales; the head from radioulna of the m. extensor carpi ulnaris is also shown (the head from humerus was removed). Elements figured: 1, m. extensor carpi ulnaris (head from radioulna): tendon of origin; 2, m. extensor carpi ulnaris: head from radioulna; 3, m. extensor carpi ulnaris (heads from humerus and radioulna): common tendon of insertion on distal carpal 3-4-5; 4, m. abductor pollicis longus; 5, m. extensor indicis brevis superficialis: slip from ulnare inserting on metacarpal II (in common with the m. abductor pollicis longus); 6, m. extensor indicis brevis superficialis: slip from ulnare inserting on metacarpo- phalangeal joint; 7, m. extensor brevis superficialis digiti HII: slips from ulnare and distal carpal 3-4-5; 8, m. extensor brevis superficialis digiti IV: slip from distal carpal 3-4-5; 9, m. extensor brevis superficialis digiti V. Characters figured: char. 46.1, presence of the head from radioulna of the m. extensor carpi ulnaris; char. 47.0, fleshy origin of the head from radioulna of the m. extensor carpi ulnaris; char. 47.1, origin via a flat tendon of the head from radioulna of the m. extensor carpi ulnaris. Scale bars = 1 mm. 30 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY 8*), Morrison (1994: char. 96*), Scott (2005: char. 144*), Wiens et al. (2005: char. 108*), Ohler and Dubois (2006: char. 4*), Nussbaum and Wu (2007: char. 4), Barrionuevo (2017: char. 60). ADULT VISCERAL ANATOMY 51. Inguinal fat bodies, occurrence: (0) absent, (1) present. Boulenger (1910) first reported the occurrence of elongated bodies associated to the muscles of the inguinal region in several species of Bufonidae. Later, Plytycz and Szarski (1987) and da Silva and Mendelson (1999) corroborated the occurrence of these inguinal fat bodies in many other species of sev- eral bufonid genera. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Mendelson (1997a: char. 45), Pramuk (2006: char. 79), Mendelson et al. (2011: char. 34). ADULT EXTERNAL MORPHOLOGY All described characters of external morphol- ogy refer to adult individuals of both sexes, except when sexually dimorphic characters are considered (i.e., chars. 53-54 and 58-59). 52. Dorsal skin, macroscopic glandular struc- tures in females: (0) indistinct; (1) small and smooth; (2) greatly enlarged and smooth; (3) with small tubercles, without cornified tip; (4) conical with a single cornified tip; (5) hemispherical with multiple cornified tips. Nonadditive. The skin of bufonids varies from completely smooth to highly tuberculated and warty due to modifications of dermal and/or epidermal components (Elias and Shapiro, 1959). It differs between sexes and its structure is affected (at least in males) by the repro- ductive condition of the specimens (see Cei, 1980; Duellman and Trueb, 1986). Therefore, when scor- ing skin diversity we considered: (1) as indepen- dent character structures present in both sexes (chars. 53, 54); (2) the maximum level of develop- ment reported for the dorsal structures of skin within the studied specimens; and (3) the most common structures present in the dorsal skin. Although some character states seem to be com- NO. 447 posed of a progressive series of transformation of glandular structures, detailed histological studies are required to understand the various components that are differentiated in each structure. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 39*), Morrison (1994: char. 99*), Grant et al. (2006: char. 0*). 53. Dorsal skin, macroscopic glandular structures in males: (0) indistinct; (1) small and smooth; (2) greatly enlarged and smooth; (3) with small tubercles, without a cornified point; (4) conical with a single cornified point; (5) hemispherical with multiple cornified points. Nonadditive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 39*), Morrison (1994: char. 99*), Grant et al. (2006: char. 0*). 54. Vertebral line, occurrence: (0) absent, (1) present. Boulenger (1897) first noted the distinc- tiveness and independence between a filiform line or raphe and the vertebral line. The former is a line of thin skin extending along the middle of the back from the snout to the vent. This raphe is very common in bufonids and gives rise to a light vertebral line. As pointed out by Boulenger (1897), the independence of these structures is evident in cases of deviation of the former (see Boulenger, 1897: fig. 9). We consider the occur- rence of a distinctive light vertebral line only. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Heyer (1978: char. 1*), Ohler and Dubois (2006: char. 13*), Ponssa (2008: char. 1*). 55. Parotoid gland, occurrence: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Cannatella (1986: char. 11), Morrison (1994: char. 95*), Mendelson (1997a: char. 43*), Wiens et al. (2005: char. 109), Fabrezi (2006: char. 72), Pramuk (2006: char. 73*). 56. Parotoid gland, shape: (0) approximately ellipsoid, longer than wide (fig. 8A); (1) subtri- angular (fig. 8B); (2) round to ovoid mostly sym- metrical (fig. 8C); (3) triangular and bulky (fig. 8D). Nonadditive. This character is not appli- cable for specimens that lack a parotoid gland (char. 55.0). 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 31 C hd ee pt Ne ae oy A) Saree NS) CS LO FIG. 8. Head (lateral and dorsal views) showing the shape of the parotoid gland (in gray): A, Rhinella aff. cerradensis (char. 56.0); B, R. acutirostris (char. 56.1); C, R. arunco (char. 56.2); D, R. marina (char. 56.3). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 95*), Pramuk (2006: char. 73*), Mendelson et al. (2011: char. 32”). 57. Skin, occurrence of row of dorsolateral tubercles: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Morrison (1994: char. 97), Mendelson (1997a: char. 44*), Pramuk (2006: char. 81), Mendelson et al. (2011: char. 33). 58. Vocal sac, occurrence in adult males: (0) absent, (1) present. The vocal sac develops as ven- tral diverticula of the mouth floor into spaces among submandibular muscles (Noble, 1931; Tyler, 1971). This second cavity communicates with the buccal cavity via single or paired aper- tures, the vocal slits. In this way, the presence of a vocal sac automatically implies the presence of at least one vocal slit (and vice versa). Modifications of the gular skin (i.e. “internal” or “external” vocal sacs sensu Gunther, 1858a) can be absent or pres- ent without affecting the codification of this char- acter. Vocal sacs are either absent or present in adult males of the majority of species, with few exceptions where both states cooccur in different specimens (Liu, 1935; Inger and Greenberg, 1956, Hayes and Krempels, 1986; Mendelson, 1997b). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Liem (1970: char. 36*), Drewes (1984: char. 20*), Cannatella (1986: char. 10*), Hillis and de Sa (1988: char. 6), Mendelson et al. (2000: char. 51*), Grant et al. (2006: char. 76*), Pramuk (2006: char. 75*), Ohler and Dubois (2006: char. 14), Mendelson et al. (2011: char. 31*). 59. Vocal slits, number: (0) unilateral, (1) bilateral. Several authors (e.g., Boulenger, 1897; Liu, 1935; Inger and Greenberg, 1956) reported the occurrence of specimens with a single vocal slit. This condition was observed in some species of Bufonidae and has not been reported in other anuran families. The single vocal slit can either be on the left or the right side of the tongue in different specimens of the same species. Further- more, there are species where one (on either side) or two vocal slits can occur. This character is scored as not applicable for taxa lacking vocal sacs (see char. 58.0). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Drewes (1984: char. 20*), Cannatella (1986: char. 10*), Mendelson (1997a: char. 42*), Mendelson et al. (2000: char. 51*), Pramuk (2006: char. 75*), Mendelson et al. (2011: char. 31*). 60. Vocal sac, shape when fully inflated: (0) spherical or subspherical, (1) projected anteriorly. Simple subgular vocal sacs are often spherical or subspherical. Nevertheless, in a few species, they project anteriorly deviating from a spherical shape. The degree of projection ranges from a slight deformation to a large, vertically oriented lobe. McAllister (1961) reported on this variation in North American bufonids and their putative oz BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY relationship with vocalization, but this character has not been used in phylogenetic studies. This character is not applicable for specimens that lack a vocal sac (char. 58.0) 61. Nuptial pads, occurrence in males: (0) absent, (1) present. Nuptial pads are sexually dimorphic structures that can be present in the fin- gers of males; their structure and diversity were recently studied (Luna et al., 2018). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Liem. (1970; char. 35), Heyer (1975: char.3*), Scott (2005: char. 132*), Wiens et al. (2005: char. 100), Grant et al. (2006: char. 23), Ohler and Dubois (2006: chars. 16-18*), Ponssa (2008: char. 24*), Barrionuevo (2017: char. 69). 62. Nuptial pads, coloration: (0) light col- ored, (1) dark colored. Following Luna et al. (2018) we distinguished between dark- and light- colored nuptial pads, where “dark-colored” includes all tones of brown and black and “light- colored” includes beige/uncolored pads. These differences in coloration result from minor changes in the stratum corneum of the epidermis and are independent of the number of layers of this stratum (Luna et al., 2008). This character is not applicable for specimens that lack nuptial pads (char. 61.0). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Ohler and Dubois (2006: char. 24”). 63. Manus, occurrence of webbing between fingers: (0) absent or poorly developed, (1) pres- ent, well developed. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Wiens et al. (2005: char. 99), Pramuk (2006: char. 77*), Nussbaum and Wu (2007: char. 12*). 64. Pes, edge of foot webbing: (0) smooth, (1) serrated. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Vélez-Rodriguez (2004b: char. 12*). 65. Tarsus, occurrence of tarsal fold: (0) absent, (1) present. A tarsal fold is a dermal fold on the medial-ventral surface of the foot, extending proxi- mally from the inner metatarsal tubercle. PREVIOUS USAGE IN PHYLOGENETIC STUD- 1ES: Inger (1972: char. 31*), Heyer (1975: char. 6*), Scott (2005: char. 156*), Grant et al. (2006: NO. 447 char. 28), Ohler and Dubois (2006: char. 11”), Ponssa (2008: char. 19), Barrionuevo (2017: chart 77). 66. Relative size of adult females and males: (0) adult females similar in size or larger than adult males, (1) adult males much larger than adult females. As a first approxi- mation, we consider only two states due to the occurrence of a more evident gap in size according to published data. However, a more detailed study of sexual dimorphism in Rhi- nella could help to partition these into more additional states. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Scott (2005: char. 139), Fabrezi (2006: char. 78*), Ponssa (2008: char. 110*). LARVAL EXTERNAL MORPHOLOGY 67. Body, morphology of the peribranchial and abdominal regions: (0) absence of external modifications, (1) presence of bulging regions lat- eral to the oral disc, (2) occurrence of an abdomi- nal sucker. Additive. Most species of Rhinella have lentic larvae that lack external modifications in the peribranchial and abdominal regions (state 0). Modifications in these regions are typical of some lotic forms (McDiarmid and Altig, 1999; Hoff et al., 1999) and two different states occur within Rhinella. Larvae of Rhinella rumbolli have a central depression delimited by bulbous lateral regions in the peribranchial zone (state 1). More- over, some other species of the R. veraguensis Group have a well-developed abdominal sucker that is bounded anteriorly by the oral disc, and the lateral and posterior edges are free from the body (state 2). We consider the character states to represent an ordered series of transformation for which the states are considered as additive. 68. Body, dorsal coloration: (0) light brown, (1) dark brown, (2) sharply defined dark mark- ings on pale ground. Nonadditive. 69. Caudal musculature, ocurrence of an unpigmented longitudinal stripe along the infe- rior edge in the caudal musculature: (0) absent, (1) present. An unpigmented longitudinal stripe 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 33 along the inferior edge of the caudal musculature sometimes occur in the caudal musculature of lar- vae having a dark coloration of the tail. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Mendelson et al. (2011: char. 38”). 70. Caudal musculature, occurrence of irregular transverse whitish stripes: (0) absent, (1) present. In some species of the Rhinella gran- ulosa and R. veraguensis Groups there are irregu- lar transverse whitish stripes of variable extension due to the absence of melanocytes contrasting with the general dark coloration of the dorsal musculature (see Blotto et al., 2014, for taxo- nomic distribution in Rhinella). 71. Oral disc, occurrence of submarginal papillae: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grant et al. (2006: char. 91*), Barrionuevo (2017: char. 86*), Araujo-Vieira et al., (2019: char. 135). 72. Oral disc, number of posterior labial tooth rows: (0) two, (1) three. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Hillis and de Sa (1988: char. 2*), Wiens et al. (2005: char. 122*), Grant et al. (2006: char. 94*), Ohler and Dubois (2006: char. 31*), Barrionuevo (2017: char. 90*), Araujo- Vieira et al. (2019: char. 141*). 73. Oral disc, condition of the labial tooth row A2: (0) complete, (1) divided. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Mendelson et al. (2011: char. 37*). 74. Oral disc, condition of the labial tooth row P1: (0) complete, (1) divided. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Wiens et al. (2005: char. 124), Araujo-Vieira et al. (2019: char. 142) 75. Vent tube, opening: (0) medial, (1) dextral. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grant et al. (2006: char. 96*), Barrionuevo (2017: char. 93), Araujo- Vieira et al., 2019 (char. 145). LARVAL CHONDROCRANIUM Oliveira et al. (2014) studied the chondrocra- nium of some species of Rhinella and reviewed the information available for other bufonids. 76. Otic capsule, larval crista parotica, occurrence of processus anterolateralis: (0) absent or indistinguishable, (1) poorly developed with a rounded aspect, (2) well developed with an acute appearance. Additive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Larson and de Sa (1998: char. j*), Haas (2003: char. 66*); Miranda et al. (2015: char. 61*). 77. Procesus ascendens, angle of attaching to the braincase: (0) obliquely attached, (1) per- pendicularly attached. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Larson and de Sa (1998: char. o*), Miranda et al. (2015: char. 65*). 78. Copula anterior, occurrence: (0) absent, (1) present. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Haas (2003: char. 105), Pugener et al. (2003: char. 35 [larval characters]), Miranda et al. (2015: char. 73). EMBRYONIC MORPHOLOGY Vera Candioti et al. (2016) studied the early ontogeny and described the informative varia- tion found in several species of Bufonidae. All the characters considered on embryonic mor- phology were described in detail in that publication. 79. Third pair of external gills, condition: (0) absent or indistinguishable, (1) short, (2) long. Additive. 80. Dorsal line of hatching glands: (0) short (cephalic region only), (1) long (beyond cephalic region). 81. Type of adhesive gland: (0) A, (1) B. 82. Time of division of adhesive gland: (0) slightly after the second-gill pair branches off before operculum at the gill base, (1) immedi- ately before the gills reach their maximum devel- opment, (2) immediately after opercular fusion. Additive. NATURAL HISTORY 83. Diel activity of adults: (0) diurnal, (1) nocturnal. 34 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grant et al. (2006: char. 115*). 84. Habits: (0) terrestrial, (1) arboreal, (2) aquatic. Nonadditive. Some species of the Rhi- nella veraguensis Group are completely arboreal. We do not consider as arboreal the mostly ter- restrial species that have the ability to climb up the vegetation to rest during the night (de Noronha et al., 2013). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Grant et al. (2006: char. 114”). 85. Oviposition site: (0) aquatic, (1) terres- trial, (2) phytotelmata. Nonadditive. Following van Bocxlaer et al. (2010), terrestrial oviposition refers to eggs that are placed on the ground, in leaf litter, or under stones, and are exposed to little or no free water at the time of oviposition. Phytotelmata refers to any chambers in a plant that is used as oviposition site (e.g., water-filled nut, tree holes, leaf axils; see Lehtinen et al., 2004; Grant et al., 2006). PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Faivovich (2002: char. 83*), Grant et al. (2006: char. 107*), Araujo-Vieira et al. (2019: char. 191%). 86. Structure of the spawn: (0) strings, (1) open clump, (2) mass, (3) strands. Nonadditive. Altig and McDiarmid (2007) reviewed in detail the terminology and diversity of arrangement of deposited eggs in Amphibia. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Haas (2003: char. 141*). 87. Egg disposition in strings: (0) uniserial, (1) biserial, (2) multiserial. Nonadditive. Mature oocytes are surrounded by jelly layers as they are displaced through the different regions of the oviduct (Salthe, 1963; Altig and McDiarmid, 2007). The number and type of jelly layers are not well characterized in Rhinella (Pereyra et al., 2015), and there is no information about a direct relation between the diversity of strings and the eggs disposition within the string. Thus, we can- not infer a series of transformation and we con- sider this character as nonadditive. 88. Ovum pigmentation: (0) unpigmented, (1) animal pole pigmented. NO. 447 PREVIOUS USAGE IN PHYLOGENETIC STUDIES: McDiarmid (1971: char. 42*), Grandison (1981: char. 4*), Cannatella (1986: char. 14*), Grant et al. (2006: char. 68*), Ohler and Dubois (2006: char. 29), Mendelson et al. (2011: char. 40*). CYTOGENETICS 89. Nucleolar Organizer Regions, loca- tion: (0) terminal position of the short arms of the chromosome pair 1, (1) pericentromeric position of the long arms of the chromosome pair 1, (2) terminal position of the long arms of the chromosome pair 5, (3) terminal posi- tion of the long arms of the chromosome pair 6, (4) interstitial position of the short arms of the chromosome pair 7, (5) interstitial posi- tion of the long arms of the chromosome pair 10, (6) terminal position of the long arms of the chromosome pair 10, (7) interstitial posi- tion of the short arms of the chromosome pair 11. Nonadditive. PREVIOUS USAGE IN PHYLOGENETIC STUDIES: Faivovich (2002: char. 82*). RESULTS SEPARATE ANALYSES OF RESTRICTED NUCLEAR (RND) AND MITOCHONDRIAL (RMD) DATASETS The parsimony analyses, reaching a stable consensus 10 times, retained 706 unique MPTs of length 1757 for the rND and one MP's of length 11,436 for the rMD. Within the ingroup (i.e., Rhi- nella), the main incongruence between the rND and rMD analyses involved the position of the specimens of R. horribilis, which are deeply nested within the R. marina Group in the rND analysis, but were recovered as the sister clade of the R. marina + R. crucifer Groups in the rMD analysis (fig. 9). Based on these observations and previ- ous published results (Pereyra et al., 2016a), we included the mitochondrial and nuclear genomes of R. bernardoi and R. horribilis as independent terminals in the TE analysis (see Discussion sec- tion for comments on the putative mitochon- drial or nuclear introgression in these terminals 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 35 and the rationale for the considerations of both genomes as independent terminals). Mitochon- drial introgression and hybridization between R. diptycha and R. marina might have occured in the area south of the Amazon River (see Sequeira et al., 2011), but the evidence is not conclusive (see Vallinoto et al., 2017). For this reason, we did not include sequences of these species from this complex area. In appendix 2, we list the terminals considered in the TE analysis. TOTAL EVIDENCE ANALYSIS Molecular data were included for all 320 termi- nals of 124 species, whereas phenotypic data were restricted to 106 specimens of 102 species (90 characters; ~50 scores/terminal). The TE analysis using parsimony, reaching a stable consensus 10 times, retained 657 unique MPTs (length 25,399). One of the optimal topologies is shown in figures 10-14 (fig. 10 for outgroup relationships, figs. 11-14 for Rhinella relationships). A summary tree of Rhinella relationships to species level is shown in the supplementary data 2 (available at https:// doi.org/10.5531/sd.sp.46). In depicting all unre- futed clades, we employ the strict consensus of the optimal phylogenetic hypotheses resulting from this TE analysis treating gaps as fifth state as the basis of our discussion of taxonomy. The results of the TE analysis considering gaps as missing data (see supplementary data 3.1-3.5, available at https://doi.org/10.5531/sd.sp.46) and the ML analysis (see supplementary data 4.1-4.5, available at https://doi.org/10.5531/sd.sp.46) were highly congruent with the TE analysis considering gaps as fifth state. The few differences between these hypotheses are discussed when relevant. The MPTs resulting from the TE analysis recovered Rhinella as nonmonophyletic due to the position of R. ceratophrys that is the sister taxon of Rhaebo nasicus with strong support (JGC and JAF = 100%; see fig. 10). Among outgroups the strongly supported Anaxyrus + Incilius (JGC = 96%, JAF = 97%) is the sister clade of all the other species of Rhinella. The monophyly of the clade composed of these three genera is poorly sup- ported (JGC = 63%, JAF = 73%). The species of Rhinella (excluding R. ceratophrys) are monophy- letic, well supported (JGC and JAF = 98%), and grouped in two major clades. One of these is moderately supported (JGC = 88%, JAF = 92%) and includes the species of the former R. spinulosa Group (including R. gallardoi; see Discussion) and those of the R. granulosa, R. crucifer, and R. marina Groups (figs. 11, 12). The other is strongly supported (JGC and JAF = 99%) and composed of all the species from the nonmonophyletic R. veraguensis and R. margaritifera Groups, the for- mer R. acrolopha Group (see Discussion section), and R. sternosignata (figs. 13, 14). UNCORRECTED P-DISTANCES The patterns of UPDs found within each spe- cies group vary largely (see below), so we did not consider a single value as a threshold to delimit species, but each particular situation was consid- ered in the context of the genetic distances found within each species group. Interspecific distances among all the species addressed by the taxo- nomic revision are presented in the Discussion section of each species group. Throughout the text the UPDs are expressed as percentage. DISCUSSION SYSTEMATICS AND TAXONOMY RELATIONSHIPS AMONG OUTGROUPS AND RHINELLA Our outgroup sample was designed exclusively to provide a rigorous test of the monophyly of Rhi- nella and does not constitute a critical test of previ- ously hypothesized relationships among other clades of Bufonidae (e.g., Frost et al., 2006; Pramuk, 2006; Pramuk et al., 2008; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Portik and Papenfuss, 2015; Jetz and Pyron, 2018). Indeed, most of the basal relationships of Bufonidae are unresolved or poorly supported in the TE analysis (fig. 10). Nevertheless, we found Anaxyrus + Incilius to be the sister clade of Rhinella with low support 36 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 rND OUTGROUPS “R. SPINULOSA” GROUP a “R. SPINULOSA” GROUP Gi R. GRANULOSA GROUP R. henseli MNRJ Q 3 o ie) dnows) Yay/DNYO “YY inopina © inopina | inopina \WiZUS icterica C\ icterica C aff. cerradensis aff. cerradensis cf. icterica Wi rubescens veredas CFB! poeppigii US poeppigii MH poeppigii marina | jimi CF ¢ cf. diptycha diptycha « horribilis horribilis Q horribilis © sternosignata R. FESTAE + “R. VERAGUENSIS” GROUPS a> R. MARGARITIFERA GROUP “R. VERAGUENSIS” GROUP “R. MARINA GROUP” ANOS VNINVW ‘YY, R. CRUCIFER GROUP DAADADADAANAAANNNAANNANNAAAANADAAAAA: rMD OUTGROUPS R. SPINULOSA GROUP ps R. GRANULOSA GROUP > R. horribilis R. horribilis R. horribilis R. rubescens R. icterica ( R. cf. icterica R. icterica R. aff.cerradensis | R. aff.cerradensis | R. poeppigii R. poeppigii | R. poeppigii | R. veredas R. marina | R. jimi CFBH R. cf. diptycha R. diptycha R. henseli \ R. henseli R. henseli R. casconi R. casconi R. crucifer R. inopina Cc R. inopina MZUSP R. inopina MZUSP R. ornata Z| R. ornata R. abei ci R. abef MZUSP R. ornata R. ornata LG! R. sternosignata R. FESTAE + “R. VERAGUENSIS” GROUPS R. MARGARITIFERA GROUP a “R. VERAGUENSIS” GROUP Jackknife support values (JGC and JAF) @=298% ©90%-97% ©70%-89% @<69% | FIG. 9. Comparison between the strict consensuses resulting from the analyses of the restricted nuclear dataset (rND) and restricted mitochondrial dataset (rMD), showing the alternative positions of Rhinella horribilis in both analyses. Circles on nodes indicate parsimony jackknife frequencies (frequency differences value [above]/absolute [below]). Nodes lacking circles have <25% frequency difference values or < 50% jackknife absolute frequencies. (JGC = 63%, JAF = 73%). The clade composed of these three genera, in turn, is the sister taxon of a large and poorly supported clade (JGC <25%, JAF <50%) of African and Eurasian bufonids. An almost identical relationship was recovered in the ML analysis (see supplementary data 4.1). The sis- ter-group relationship between Rhinella and Anaxyrus + Incilius is consistent with the results of most previous phylogenetic analyses (e.g., Pramuk, 2006: fig. 4; Pramuk et al., 2008; Pyron and Wiens, 2011; Pyron, 2014: suppl. information “amph_shl. tre”; Portik and Papenfuss, 2015; Jetz and Pyron, 2018: suppl. information “amph_shl_new.tre” Alternatively, van Bocxlaer et al. (2010: fig. $1) recovered Rhinella as the sister taxon of a clade comprising all African and Eurasian bufonids. Although the vast majority of species of Rhi- nella form an exclusive clade, it is polyphyletic because R. ceratophrys was recovered as the sister taxon of Rhaebo nasicus with strong support (JGC and JAF = 100%). This relationship is not surpris- ing, given that the morphological resemblance between both species was pointed out previously (e.g., Hoogmoed, 1977; Fenolio et al., 2012). 2021 Melanophryniscus stelzneri Amazophrynella minuta Nannophryne variegata * 98 99 Rhaebo guttatus Rhaebo nasicus xp Rhinella ceratophrys JMP 2284 - Colombia:# Amazonas xl Rhinella ceratophrys QCAZ 40240 - Ecuador: Sucumbios Peltophryne lemur Peltophryne empusa 98 98 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) Rhaebo ecuadorensis 7. “RHAEBO” Ingerophrynus galeatus 90 92 76 82 61 69|__46 29 66 52) * 88 92 61 73 * 71 78) 57 76 * * * * * 96 97 * 63 73 * * * 98) 98 Anaxyrus americanus Anaxyrus woodhousii Incilius valliceps Incilius nebulifer R. MARINA CLADE (figs. 11, 12) Ansonia longidigita Pelophryne misera Bufotes luristanicus Duttaphrynus melanostictus Nectophrynoides tornieri Bufo bufo Bufo gargarizans Phrynoidis juxtaspera Rentapia hosii Leptophryne borbonica Wolterstorffina parvipalmata Vandijkophrynus robinsoni Schismaderma carens Mertensophryne loveridgei Sclerophrys mauritanica Sclerophrys regularis Sclerophrys garmani Sclerophrys brauni Sclerophrys steindachneri Anaxyrus boreas Pe WE ie Anaxyrus quercicus cq Incilius alvarius Incilius coniferus ” RHINELLA = R. MARGARITIFERA CLADE (figs. 13, 14) FIG. 10. Phylogenetic relationships of Rhinella and outgroups recovered in one of the most parsimonious trees from the total evidence analysis with TNT considering gaps as a fifth state (length 25,399 steps). Values around nodes are parsimony jackknife frequencies (frequency differences value [above]/absolute [below]). An asterisk (*) indicates 100% jackknife support. Clades lacking references have <25% frequency difference values or <50% jackknife absolute frequencies. Lower left inset shows the entire cladogram with present view marked in white. ferring R. ceratophrys to Rhaebo) and well supported (JGC and JAF = 98%). The monophyly of Rhinella was previously recovered by several phylogenetic studies that used fewer taxa (e.g., Although Rhaebo was paraphyletic in our TE analysis (fig. 10; but see results of the ML analysis in supplementary data 4.1), our taxon sampling was not designed to test its monophyly. Thus, we transfer Rhinella ceratophrys to Rhaebo as Rhaebo ceratophrys (Boulenger, 1882), new combination. RHINELLA AND ITS INTERNAL RELATIONSHIPS In the parsimony total evidence analysis, Rhi- nella was recovered as monophyletic (after trans- Pauly et al., 2004: fig. 4; Pramuk, 2006; Pyron and Wiens, 2011; Pyron, 2014: suppl. informa- tion “amph_shl.tre”; Portik and Papenfuss, 2015; Jetz and Pyron, 2018: suppl. information “amph_ shl_new.tre”). In contrast to all previous studies, we found that Rhinella is composed of two major, well-supported clades (figs. 11-14; see below). 38 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 25 R. arunco KU 217369 - Chile: Santiago * R. rubropunctata MACN 52275 - Argentina: Chubut R. ARUNCO *[4-R. atacamensis KU 217352 - Chile: Coquimbo GROUP *R. atacamensis AMNH 168401 - nd 95 R. limensis RGP 4719 - Peru: Arequipa oer FR. limensis nv - Peru: Lima 68 R. amabilis QCAZ 68471 - Peru: Cajamarca |R. cf. amabilis 82 be R. limensis KU 215587 - Peru: Ancash R. vellardi KU 211765 - Peru: Cajamarca atR. vellardi MUBI 14291 - Peru: La Libertad ge ‘ir R. vellardi CORBIDI 7626 - Peru: La Libertad R. SPINULOSA * 98 ___ 8° ER. vellardi MUBI 14281 - Peru: La Libertad GROUP 98\yR. S. trifolium nv - Peru: Junin vase R. s. trifolium CORBID| 5530- Peru: Lima |. trifolium al R. arequipensis LGE 2516 - Peru: Arequipa * Rs. spinulosa MUBI 10737 - Peru: Cusco * R. s. spinulosa MUBI 10770 - Peru: Cusco R. s. spinulosa nv - Peru: Puno R. arequipensis KU 214792 - Peru: Arequipa Rs. spinulosa IDLR 3837 - Bolivia: La Paz R. Ss. spinulosa MACN 49701 - Argentina: Jujuy Rs. spinulosa MNCN 419889 - Bolivia: La Paz xR. gallardoi LGE 4546 - Argentina: Jujuy *IR_ gallardoi LGE 4735 - Argentina: Jujuy R. achalensis MACN 52406 - Argentina: San Luis 5677 R. S. papillosa BB 983 - Argentina: Neuquén 66 R. s. papillosa NB 96-23 - Argentina: San Luis R. papillosa R. s. papillosa MACN 49782 - Argentina: Chubut R. s. papillosa BB 1032 - Argentina: Rio Negro ap NuG R. bernardoi UNSJ 5046 - Argentina: San Juan *LNuG R. bernardoi FML 23921 - Argentina: San Juan R. cf. dorbignyi CFBH 14062 - Brazil: Rio Grande do Sul *J99\-R. fernandezae GE 8718 - Argentina: Santa Fe *L| “R. fermandezae LGE 8717 - Argentina: Corrientes R. dorbignyi MNHN-Uy 9492 - Uruguay: Treinta y tres R. dorbignyi MACN 43695 - Argentina: Buenos Aires MtG R. bemardoi FML 23921 - Argentina: San Juan * 98/MtG R. bernardoi UNSJ 5046 - Argentina: San Juan JR. Aorbignyi Pp a7-R. pygmaea CFBH 2894 - Brazil: Rio de Janeiro *-R. pygmaea CFBH-T 15163 - Brazil: Rio de Janeiro R. GRANULOSA ay—R. major MNCN-ADN 6232 - Bolivia: Cochabamba *-L R. major LGE 8720 - Argentina: Salta GROUP 66 “8 = R. azarai LGE 8710 - Argentina: Misiones 74 * *L—R. azaral LGE 8711 - Argentina: Misiones 4 «pR. bergi MACN 46555 - Argentina: Chaco *LR. bergi LGE 8723 - Argentina: Formosa ay-R. granulosa CFBH 18706 - Brazil: Espirito Santo *L—R_ granulosa CFBH 7341 - Brazil: Alagoas 99 x R. mirandaribeiroi CFBH 10254 - Brazil: Tocantins 99 = j *L—R. mirandaribeiroi CFBH 13849 - Brazil: Maranhao ap R. merianae CFBH 16641 - Brazil: Amazonas 80 *L.R. merianae MTR 20517 - Brazil: Roraima R. humboldti CZUT1717 - Colombia: Tolima J« ay FR. humboldti AJC 3533 - Colombia: Santander R. humbolati SOF R. centralis CH 9383 - Panama: Coclé R. centralis MVUP 2305 - Panama: Coclé R. beebei UWIZM 2012.27.72.3 - Trinidad and Tobago: Trinidad 94 7A. beebei CBA5732 - Venezuela: Bolivar 94 WR. beebeil nv - Venezuela: Amazonas 88/1. beebel |CN 55784 - Colombia: Casanare 88 "R. beebei ICN 55776 - Colombia: Casanare oO R. spinulosa R. altiperuviana 88 92 BR ~ Leb 287 87 i ‘ © a Be 58 66 29 Figure 12 FIG. 11. Phylogenetic relationships of Rhinella recovered in one of the most parsimonious trees from the total evidence analysis with TNT considering gaps as a fifth state (length 25,399 steps). The clades and species groups shown are those recognized in this study. Part 1 of 4. The R. marina Clade (1): the R. arunco, R. spi- nulosa, and R. granulosa Groups. Black circles indicate nodes that collapse in the strict consensus. Values around nodes are parsimony jackknife frequencies (frequency differences value [above]/absolute [below]). An asterisk (*) indicates 100% jackknife support. Clades lacking references have <25% frequency difference values or <50% jackknife absolute frequencies. Lower left inset shows the entire cladogram with present view marked in white. Abbreviations: MtG, mitochondrial genome; NuG, nuclear genome. 2021 29,MtG R- horribilis KRL744 - 99 MtG R. horribilis MAR 2057 - Colombia: Valle del Cauca MtG R. horribilis KU 289750 - El Salvador: Ahuachapan 99 55) MtG R- horribilis UTA 54 71 * 61 * 11 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) Panama: Coclé “GHOST INTROGRESSED MITOCHONDRION” 882 - Mexico: Veracruz MtG R. horribilis KU 217482 - Ecuador: Loja MtG R. horribilis QCAZ 47444 - Ecuador: Loja 401997 MtG R. horribilis «KU 202274 - Ecuador: Pichincha 99 MtG R. horribilis QCAZ 50698 - Ecuador: Manabi mtG Rhinella sp. 1 R. henseli MNRJ 33006 - Brazil: Rio Grande do Sul R. henseli CFBH 20117 - Brazil: Rio Grande do Sul R. henseli UFRGS 3569 - Brazil: Rio Grande do Sul «)R. casconi CFBH 22863 - Brazil: Ceara *!R. casconi CFBH 22865 - Brazil: Ceara R. crucifer CFBH 24630 - Brazil: Bahia R. crucifer CFBH 2867 - Brazil: Espirito Santo R. inopina CHUNB 51110 - Brazil: Bahia 99L TR. inopina MZUSP 142094 - Brazil: Sao Paulo 98 38 R. inopina MZUSP 142356 - Brazil: Sao Paulo 98]/— R. ornata ZUECDCC 3392 - Brazil: Rio de Janeiro R. ornata CFBH 18815 - Brazil: Rio de Janeiro R. ornata CFBH 38375 - Brazil: Rio de Janeiro R. abei CFBH 18141 - Brazil: Parana R. abei MACN 46672 - Brazil: Santa Catarina R. abei MZUSP 128425 - Brazil: Parana R. omata USNM 303015 - Brazil: Sdo Paulo R. omata LGE 19020 - Argentina: Misiones R. omata_ LGE 19027 - Argentina: Misiones R. omata LGE 6503 - Argentina: Misiones R. omata LGE 8729 - Argentina: Misiones R. poeppigii USNM 268824 - Peru: Madre de Dios R. poeppigii MUBI 6863 - Peru: Cusco R. poeppigii MUBI 6864 - Peru: Cusco 98>— R. veredas CHUNB 44609 - Brazil: Minas Gerais 98 R. veredas CFBH 20516 - Brazil: Bahia R. marina’ MAR 1982 - Colombia: Caqueta R. marina \VUB 1965 - Suriname R. marina SBH 190696 - Jamaica R. diptycha MACN 51118 - Argentina: Santiago del Estero R. diptycha MNCN-ADN 6044 - Bolivia: La Paz R. cf. diptycha LGE 9867 - Argentina: Misiones R. diptycha KU 289057 - Paraguay: Concepcién R. jimi CFBH 19523 - Brazil: Bahia R. jimi CFBH 19335 - Brazil: Bahia NuG R. horribilis §QCAZ 50698 - Ecuador: Manabi NuG R. horribilis QCAZ 47444 - Ecuador: Loja NuG R. horribilis KU 217482 - Ecuador: Loja B- NuG R. horribilis MAR 2057 - Colombia: Valle del Cauca NuG R. horribilis «KU 289750 - El Salvador: Anuachapan R. a. arenarum MNHN-Uy 9935 - Uruguay: Cerro Largo R. a. arenarum MNCN-ADN 5972 - Bolivia: Tarija R. arenarum AR 305 - Argentina R. a. arenarum MACN 38639 - Argentina: San Luis R. a. mendocina MACN 49141 - Argentina: Mendoza R. aff. cerradensis MNHN-Uy 9514 - Uruguay: Rivera ger R. aff. cerradensis GE 19096 - Argentina: Misiones 88L_R. aff. cerradensis GE 19103 - Argentina: Misiones g9-— R. rubescens CFBH 5836 - Brazil: Minas Gerais gg] 99 R. rubescens CFBH 7696 - Brazil: Goias 99.7 R. cerradensis CFBH 20517 - Brazil: Bahia R. cerradensis CHUNB 38671 - Brazil: Distrito Federal { at 35 R. cerradensis CHUNB 39953 - Brazil: Distrito Federal f R. icterica CFBH 13965 - Brazil: Rio de Janeiro R. icterica CFBH 38392 - Brazil: Minas Gerais R. Icterica| CFBH 11027 - Brazil: Santa Catarina R. Icterica CFBH 27410 - Brazil: Rio de Janeiro R. cf. icterica MACN 43789 - Argentina: Misiones R. sp. gr. marina LGE 19195 - Argentina: Misiones R. achavali MNHN-Uy 9301 - Uruguay: Treinta y tres R. achavali ZVC 3801 - Uruguay: Treinta y tres * * * 91 94) 99) R. CRUCIFER GROUP 56, 58h 56 72 SARS ole 74 77 40 62 52 78 38 54 R. diptycha R. MARINA GROUP 79 NuG Rhinella_ sp. 1 R. arenarum 85 99 R. icterica 1p 57 49 52 80.0 FIG. 12. Phylogenetic relationships of Rhinella recovered in one of the most parsimonious trees from the total evidence analysis with TNT considering gaps as a fifth state (length 25,399 steps). The clades and species groups shown are those recognized in this study. Part 2 of 4. The R. marina Clade (2): the ghost introgressed mito- chondrion and the R. crucifer and R. marina Groups. Black circles indicate nodes that collapse in the strict consensus. Values around nodes are parsimony jackknife frequencies (frequency differences value [above]/ absolute [below]). An asterisk (*) indicates 100% jackknife support. Clades lacking references have <25% fre- quency difference values or <50% jackknife absolute frequencies. Lower left inset shows the entire cladogram with present view marked in white. Abbreviations: MtG, mitochondrial genome; NuG, nuclear genome. 40 R. r 97 VERAGUENSIS +1 99 R. amboroensis MNK 5302 - Bolivia: Santa Cruz os taeaie GROUP x R. quechua MNCN-ADN 3927 - Bolivia: Cochabamba a 99) 99) 49 80.0 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 R. sternosignata nv - Venezuela: Barinas R. fissippes MNCN-ADN 6310 - Bolivia: Beni-Cochabamba 67 *f R. rumbolli MACN 53782 - Argentina: Salta 761 96 *" R. rumbolli MACN 43719 - Argentina: Salta 99 R. justinianoi MNCN-ADN 6065 - Bolivia: Santa Cruz R. veraguensis MNCN-ADN 5808 - Bolivia: Cochabamba R. veraguensis \UBI 5946 - Peru: Puno 7 R. veraguensis USNM 346048 - Peru: Cusco 16 R. veraguensis IDLR 3820 - Bolivia: La Paz 76\| R. leptoscelis MUBI 14523 - Peru: Oxapampa R. leptoscelis CORBID! 7266 - Peru: Oxapampa | Rhinella sp. 2 R. leptoscelis MUSM 31150 - Peru: Oxapampa R. leptoscelis NMP6V 74749 - Peru: Oxapampa +f R. leptoscelis MUBI 5989 - Peru: Puno R. leptoscelis MUBI 5976 - Peru: Puno ggf__ FR. inca MNCN 44406 - Peru: Cusco go" R. inca LGE 2554 - Peru: Cusco 88 R. inca MNCN 44405 - Peru: Cusco R. inca CORBIDI 6920 - Peru: Ayacucho x1 R. manu MUBI 11372 - Peru: Cusco $8 & *" R. manu MNCN-ADN 20672 - Peru: Cusco R. manu CORBIDI 5152 - Peru: Madre de Dios | ; »| 99 R. manu MUBI 10487 - Peru: Cusco Rhinella sp. 3 *| Qf R. nesiotes CORBIDI 13953 - Peru: Huanuco *| PR. nesiotes CORBIDI 8122 - Peru: Cusco 2 R. tacana MUBI 7007 - Peru: Cusco R. FESTAE 80 R. tacana UTA 53310 - Bolivia: La Paz GROUP 85 85, R. tacana MNK 7187 Bolivia: La Paz 86 g5/4> R. tacana MUBI 6950 - Peru: Cusco 73 *’ R. tacana MUBI 7409 - Peru: Cusco R. lilyrodriguezae MUSM 32205 - Peru: San Martin +!" R. lilyrodriguezae CORBID! 8839 - Peru: San Martin _ | R. lilyrodriguezae CORBIDI 6778 - Peru: San Martin 9% R. lilyrodriguezae CORBID! 6780 - Peru: San Martin 97 * - R. chavin MTD 43789 - Peru: Huanuco R. multiverrucosa MUB! 11455 - Peru: Huanuco] R. cf. multiverrucosa R. yanachaga CORBID! 7269 - Peru: Pasco 21 R. yanachaga MUBI 7119 - Peru: Pasco 75 R. yanachaga MUBI 7121 - Peru: Pasco 83 * R. arborescandens CORBID! 2020 - Peru: Amazonas *lga R. arborescandens MUB! 14076 - Peru: Amazonas R. arborescandens MUB! 14082 - Peru: Amazonas «| R. festae CORBIDI 7505 - Peru: Loreto 90) * R. festae QCAZ 41490 - Ecuador: Zamora 93 R. festae QCAZ 46457 - Ecuador: Morona 98/lz] R. festae KU 217501 - Ecuador: Pastaza 99 *" R. festae QCAZ 18203 - Ecuador: Napo 7 68 #[- R. nicefori MHUA 4793 - Colombia: Antioquia | R. ef. nicefori 89 79 *L)R. ruizi AML 39 - Colombia: Antioquia 99 PR. ruizi AML 40 - Colombia: Antioquia 3 35 28 R. sp. gr. acrolopha 1G 2115 - Colombia: Caldas | Budell 61 R. paraguas TG 1480 - Colombia: Valle del Cauca p. Ed bw coke Oko Ed ba ++ +1 + cd kal Oho +] + * i. R. paraguas TG 1415 - Colombia: Valle del Cauca «| R. lindaeé MAR 3431 - Colombia: Antioquia *1 R. lindae MAR 3432 - Colombia: Antioquia - 99 R. lindaé MAR 3330 - Colombia: Antioquia 99 *{~ R.acrolopha MAR 1426 - Colombia: Chocé R. acrolopha MAR 1425 - Colombia: Choco 6 x!) R. tenrec MAR 3584 - Colombia: Antioquia 99 *" R. tenrec MAR 3585 - Colombia: Antioquia 9\y R. macrorhina MHUA 8319 - Colombia: Antioquia = R. macrorhina MHUA 10262 - Colombia: Antioquia gly R. macrorhina MAR 2867 - Colombia: Caldas } 99 ©. macrorhina MAR 2903 - Colombia: Caldas Figure 14 FIG. 13. Phylogenetic relationships of Rhinella recovered in one of the most parsimonious trees from the total evidence analysis with TNT considering gaps as a fifth state (length 25,399 steps). The clades and species groups shown are those recognized in this study. Part 3 of 4. The R. margaritifera Clade (1): R. sternosignata and the R. veraguensis and R. festae Groups. Black circles indicate nodes that collapse in the strict consensus. Values around nodes are parsimony jackknife frequencies (frequency differences value [above]/absolute [below]). An asterisk (*) indicates 100% jackknife support. Clades lacking references have <25% frequency difference values or <50% jackknife absolute frequencies. Lower left inset shows the entire cladogram with present view marked in white. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 4] R. sp. gt marganitifera QCAZ 53072 - Ecuador: Pastaza , R_sp. gr.marganitifera QCAZ 53142 - Ecuador: Pastaza | Rhinella sp. 5 R. ocellata ZUFG 8519 - Brazil: Goids * R. ocellata CFBH 26592 - Brazil: Maranhao 66 R. ocellata LAJ 210 - Brazil: Tocantins se 67+ R. ocellata MZUSP 103261 - Brazil: Tocantins 60] =o R. yunga NMP6V 75552 - Peru: Junin * +] R. yunga MUSM 31950 - Peru: Junin Bp F ; G 79 4 R. yunga MUSM 31096 - Peru: Cusco |/*- ‘S@/M! 75 | R. yunga MUSM 31097 - Peru: Cusco 95 R. magnussoni_ APL 20530 - Brazil: Para 93 96 = . Cf. margaritifera_ANDES 1723 - Colombia: Amazonas | Rhinella sp. 6 94|'- R. cf. margaritifera PD 16 - Brazil: Amazonas |Rhinella Sp. 7 ; 7 = R. proboscidea QCAZ 28573 - Ecuador: Sucumbios | Rhinella sp. 8 28 78 R. proboscidea CORBID!I 102 - Peru: Loreto 58 72/ ofe4— R. proboscidea CORBIDI 5835 - Peru: Loreto | Rhinella sp. 9 (7 gp 4— R. proboscidea MNCN-ADN 26559 - Peru: Loreto 33 R. sclerocephala MHNLS 7495 - Venezuela: Cojedes for) R. alata QCAZ 11597 - Ecuador: Esmeralda 26/"- R. alata QCAZ 13896 - Ecuador: Cafiar Hf - 97}ee- R. alata MHUA 8415 - Colombia: Antioquia |“R. alata 58 R. alata CH 9192 - Panama: Colén FR. alata_ MAR 2574 - Colombia: Tolima f 99 3483 R. cf. margaritifera QCAZ 42269 -Ecuador: Napo | Rhinella sp. 10 99 80 R. cf. margaritifera CHUNB 32342 - Brazil: Amazonas | Rhinella sp. 11 2 [ir R. cf. margaritifera ROM 40103 - Peru: Madre de Dios . u R. cf. margaritifera NMP6V 74260 - Bolivia: Pando Rhinella sp. 12 R. cf. margaritifera USNM 268828 - Peru: Madre de Dios 99] g4xy R. acutirostris MTR 36593 - Brazil: Amazonas 99/79 |" R. acutirostris MTR 366864 - Brazil: Amazonas ih J R. acutirostris CORBIDI 4635 - Peru: Loreto R. acutirostris QCAZ 10601 - Ecuador: Orellana ah RR. acutirostris QCAZ 28379 - Ecuador: Sucumbios 89 29r R. sp. gr. margaritifera MHNLS 21837 - Venezuela: Miranda Ge gol Fct. sternosignata MAR 1314 - Colombia: Boyaca Rhinella 97 coy cf. margaritifera. CORBID! 5840 - Peru: Loreto 98 4o7t- R. cf. marganitifera CORBIDI 5848 - Peru: Loreto sp. 13 R. cf. margaritifera \MUBI 6374 - Peru: Loreto 52 38% R. cf. sternosignata MAR 1955 - Colombia: Caqueta eto Com ages NMP 6V74261 - Bolivia: Pando . castaneotica LSUMZ 17429 - Brazil: Para iB en, 96 R. cf. castaneotica ZUFG 8171 - Brazil: Acre R. castaneotica 96 R._ cf. castaneotica BM 131 - French Guiana: Mataroni 8 R. proboscidea AMNHFS 20085 - Brazil: Roraima : 8678 R. proboscidea CTGA-UF AM 5602 - Brazil: Amazonas | R. proboscidea 8251+ R. cf. castaneotica MTR 10003 - Brazil: Amazonas 55 54 »f PR. lescurei MNHN-Fr 2006.2611 - French Guiana 73 *Lly R. lescurej MC 5 - French Guiana: Cisame g6/' R. lescurei AF 1613 - French Guiana: St Laurent 96 R. hoogmoedi CFBH 13286 - Brazil: Bahia *F R. hoogmoedi MTR 16199 - Brazil: Bahia *[98) R. hoogmoedi_ CFBH 15962 - Brazil: Sao Paulo 97 98! R. hoogmoedi ZUECDCC 3393 - Brazil: Rio de Janeiro 98 7 R. cf. margaritifera \MNCN-ADN 20639 - Peru: Puno o7Ly R. cf. margaritifera KU 215146 - Peru: Madre de Dios 86lo7/*_R. cf. margaritifera KU 215145 - Peru: Madre de Dios Bq) 977 R. cf. margaritifera CORBIDI 5468 - Peru: Cusco : 96 95] R_cf. margaritifera NMP6V 74915 - Peru: Ucayali , 7 99] %lg R. cf. margaritifera MUBI 14775 - Peru: Ucayali Rhinella sp. 14 99 R. cf. margaritifera MUBI 14776 - Peru: Ucayali R. aff. stanlaii, MNCN-ADN 4159 - Bolivia: La Paz | Rhinella sp. 15 98] ay R. scitula IIBP 849 - Paraguay: Concepcion ; 98] [74r R. paraguayensis UFMT 1876 - Brazil: Mato Grosso do Sul [R_ scitula go~ R. Cf. scitula CFBH 42359 - Brazil: Mato Grosso do Sul 73/64" R. stanlaii_ MNCN-ADN 6274 - Bolivia: Cochabamba 76 [sf R. stanlaii MNCN-ADN 4160 - Bolivia: Santa Cruz 56} R_cf. paraguayensis SMF 88237 - Bolivia: Santa Cruz R. stanlaii ZUFG 6456 - Brazil: Minas Gerais QI Nia oO R. stanlaii gg) R. ee MNHN-Fr 2006.2602 - Suriname i 75 fog] &. martyi MC 156 - French Guiana: Trijonction |r margaritifera 80 R. martyi MW 1006 - Guyana 98I\ - R_cf. dapsilis CORBIDI 1969 - Peru: Amazonas al R. cf. margaritifera |\WU 334 - Peru: Junin 99 4! R. cf. margaritifera MUSM 32715 - Peru: Junin 99 » R. gildae URCA 12651 - Brazil: Maranhao R. gildae CFBH 11400 - Brazil: Tocantins R. MARGARITIFERA R. cf. dapsilis MZUSP 139598 - Brazil: Para GROUP R. cf. dapsilis MTR 6313 - Brazil: Para R. gildae ESTR 173 - Brazil: Maranhao R.sp. gt margaritifera MC 204 - French Guiana: Saul R.sp. gt margaritifera PG 144 - French Guiana: Patawa 98- R. dapsilis QCAZ 38892 - Ecuador: Pastaza 98! R. cf. dapsilis QCAZ 39474 - Ecuador: Orellana i R. dapsilis QCAZ 43967 - Ecuador: Orellana 80.0 as R. dapsilis QCAZ 17719 - Ecuador: Napo 77 ea ion R. cf. dapsilis QCAZ 38621 - Ecuador: Pastaza R. dapsilis FIG. 14. Phylogenetic relationships of Rhinella recovered in one of the most parsimonious trees from the total evidence analysis with TNT considering gaps as a fifth state (length 25,399 steps). The clades and species groups shown are those recognized in this study. Part 4 of 4. The R. margaritifera Clade (2): the R. mar- garitifera Group. Black circles indicate nodes that collapse in the strict consensus. Values around nodes are parsimony jackknife frequencies (frequency differences value [above]/ absolute [below]). An asterisk (*) indi- cates 100% jackknife support. Clades lacking references have <25% frequency difference values or <50% jackknife absolute frequencies. Lower left inset shows the entire cladogram with present view marked in white. 42 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY Our results support the R. crucifer, R. granulosa, and R. marina Groups as monophyletic. Other- wise, the R. spinulosa Group is recovered para- phyletic due to the nested position of R. gallardoi (a species unassigned to any group). The R. mar- garitifera Group is polyphyletic due to the posi- tion of the former R. ceratophrys nested in Rhaebo. The R. veraguensis Group is polyphyletic due to the position of several taxa (i.e., R. arbor- escandens, R. chavin, R. lilyrodriguezae, R. manu, R. multiverrucosa, R. nesiotes, R. tacana, and R. yanachaga) more closely related to the R. mar- garitifera Group, and with the monophyletic R. acrolopha Group nested within them. The ML analysis of the molecular + phenotypic datasets supported most of these results (supplementary data 4.2-4.5), and we only discuss the relevant differences between analyses. Below, we provide a revised account and comments for Rhinella and its main clades and species groups on the basis of these results. RHINELLA Diacnosis: The long third pair of external gills (char. 79.2) optimizes as the only phenotypic syn- apomorphy of Rhinella in all the MPTs, which reverts to short third pair of external gills, the ple- siomorphic bufonid condition, in the R. granulosa Group. An unequivocal diagnosis of this genus is obscured by the large phenotypic variation within Rhinella that overlaps with the diversity of many of the related bufonid genera. Nevertheless, this genus can be diagnosed from most of the related bufonids by the combination of the following phe- notypic characters: (1) nasals and frontoparietal heavily ornamented with pits, striations, and rugosities (char. 9.2); (2) presence of a row of dor- solateral tubercles on skin (char. 57.1); and (3) nucleolar organizer regions (NORs) located on interstitial position of the short arms of the chro- mosome pair 7 (char. 89.4). SISTER CLADE: The well-supported clade composed of Anaxyrus + Incilius JGC = 96%, JAF = 97%). DISTRIBUTION: Mostly Neotropical, ranging from the southern United States to southern NO. 447 South America. Rhinella marina is a highly inva- sive species introduced in many countries and islands outside its native distribution (e.g., Antil- les, Australia, Hawaii, Philippines, Taiwan, etc.; see Frost, 2020; IUCN, 2020). CoMMENTs: The exclusion of the former Bufo ceratophrys renders Rhinella monophyletic. The two large clades of Rhinella were not recovered in previous phylogenetic analyses (e.g., Pramuk, 2006: fig. 4; Chaparro et al., 2007; Pramuk et al., 2008; van Bocxlaer et al., 2010: suppl. informa- tion S1; Pyron and Wiens, 2011; Pyron, 2014: supp. information “amph_shl.tre”; Portik and Papenfuss, 2015; Pereyra et al., 2016a; Jetz and Pyron, 2018: suppl. information “amph_shl_new. tre’). Vera Candioti et al. (2016) proposed the long third pair of external gills as a putative syn- apomorphy of Rhinella in the context of a review of embryonic morphology of Bufonidae. Our TE analysis supports this character state as synapo- morphy of the genus, although the embryonic morphology of many genera of Bufonidae and species of the R. margaritifera Clade (see below) is unknown. This synapomorphy of Rhinella reverts to the plesiomorphic state (short third pair of external gills) in the R. granulosa Group. As a result of our TE analysis (also see ML result), we define two major clades, the Rhinella marina Clade and the R. margaritifera Clade, composed of eight species groups within Rhi- nella. The R. marina Clade includes (1) the R. arunco Group (new species group); (2) the R. crucifer Group; (3) the R. granulosa Group; (4) the R. marina Group; and (5) the R. spinulosa Group as redefined here. The second clade, the R. margaritifera Clade, is composed of (1) R. sternosignata, a species unassigned to any group; (2) the R. festae Group as redefined here; (3) the R. margaritifera Group as redefined here; and (4) the R. veraguensis Group as redefined here. Below, we provide diagnoses, content, and com- ments on the distribution and systematics of each of the newly defined major clades and all species groups of Rhinella. The clades and species group are presented in the order described above and correspond to the sequence in which they 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 43 appear in the TE tree (figs. 10-14) from base to tip and top to bottom. THE RHINELLA MARINA CLADE (figs. 11, 12) Diacnosis: This clade is moderately sup- ported (JGC = 88%, JAF = 92%) and diagnosed by a phenotypic synapomorphy: larval otic cap- sule with poorly developed processus anterolate- ralis with a rounded aspect (char. 76.1), with one instance of homoplasy in Sclerophrys regularis. SISTER CLADE: The Rhinella margaritifera Clade (figs. 13, 14). ConTENts: The Rhinella marina Clade is composed of the R. crucifer, R. granulosa, and R. marina Groups, the R. spinulosa Group as rede- fined here, and the R. arunco Group, a new group defined here (see below). Moreover, we found a divergent mitochondrial lineage introgressed into R. horribilis (hereafter referred to as GIM [ghost introgressed mitochondrion], see below and discussion) that does not seem to belong to any recognized extant species of Rhinella and was recovered as sister clade of the R. marina + R. crucifer Groups (see fig. 12), although with poor support (see below). DISTRIBUTION: The species of this clade natu- rally occur in all main biogeographic regions of the Neotropics. ComMENts: The Rhinella marina Clade is composed of two subclades. One is poorly sup- ported (JGC = 68%, JAF = 82%) and includes the R. arunco + R. spinulosa Groups (fig. 11). It is diagnosed by four phenotypic synapomorphies: (1) the supraorbital flange on frontoparietal does not extend laterally beyond the lateral margin of the sphenethmoid (char. 17.0, with instances of homoplasy in R. quechua and some outgroups); (2) the m. extensor digitorum on the metacar- pophalangeal joint of digiti III (char. 44.1, with instances of homoplasy in Anaxyrus woodhousii [polymorphic], Rhinella hoogmoedi, R. jimi, and R. rumbolli); (3) parotoid gland round to ovoid, mostly symmetrical (char. 56.2, with instances of homoplasy in R. bergi and several species of the R. margaritifera Clade); and (4) vocal sac absent in adult males (char. 58.0, with instances of homoplasy within Rhinella and outgroups). The other subclade is well supported (JGC and JAF = 99%) and includes the R. crucifer, R. granu- losa, and R. marina Groups, and the GIM (figs. 11, 12). Three phenotypic synapomorphies are recovered for this subclade: (1) occurrence of a well-developed supraorbital crest (char. 1.2, with instances of homoplasy in several bufonids); (2) occipital artery pathway completely covered with bone (char. 10.2, with instances of homoplasy in bufonids); and (3) general pattern of coloration of caudal musculature of larvae uniformly dark except an unpigmented longitudinal stripe along the inferior edge (char. 69.1, with instances of homoplasy in R. quechua, R. veraguensis, and some outgroups). Previous phylogenetic studies including less complete sampling of Rhinella (Pramuk, 2006; Pramuk et al., 2008; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Portik and Papenfuss, 2015; Pereyra et al., 2016a; Jetz and Pyron, 2018) never found a sister relation between the clade composed of the R. arunco + R. spinulosa Groups and the clade composed of the R. granulosa + (R. crucifer + R. marina) Groups. Instead, these studies found the R. arunco and R. spinulosa Groups as: (1) the sister clade of the species of the R. margaritifera Clade as defined here (Pramuk, 2006), (2) as sister clade of the remaining species of Rhinella (Pramuk et al., 2008; Pereyra et al., 2016a), or (3) as successive sister clades of the remaining spe- cies of Rhinella (van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Portik and Papen- fuss, 2015; Jetz and Pyron, 2018). In contrast to all these analyses, our ML analysis recovers the R. arunco Group as the sister clade of the remain- der of the R. marina Clade, whereas the R. spinu- losa Group is the sister taxon of the clade composed of R. granulosa + (R. crucifer + R. marina) Groups. This last clade has always been recovered as monophyletic in previous phyloge- netic analyses (Pramuk, 2006; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Por- tik and Papenfuss, 2015; Pereyra et al., 2016a; Jetz and Pyron, 2018). 44 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY THE RHINELLA ARUNCO GROUP D1aGnosis: Two phenotypic synapomor- phies diagnose this strongly supported (JGC and JAF = 100%) species group: (1) jaw articu- lation opposite to the fenestra ovalis (char. 16.1, with instances of homoplasy in some species of the Rhinella granulosa Group, the R. mar- garitifera Clade, and in Nannophryne variegata); and (2) anterior edge of sacral diapophyses per- pendicular to the midline axis of the vertebral column (char. 30.1, with instances of homo- plasy in R. crucifer, R. quechua, R. rubescens, R. spinulosa, and R. vellardi). The presence of an insertion of the m. extensor digitorum longus on metatarsophalangeal joint of digit V (char. 40.1) and the presence of an insertion of the m. extensor digitorum on metacarpophalangeal joint of digiti V (char. 45.1) could represent two additional synapomorphies of this group or an internal clade. Moreover, species of the R. arunco Group can be distinguished from mem- bers of the other species groups of Rhinella by the following combination of character states: (1) preorbital crest weak (char. 0.1), (2) occipi- tal artery pathway uncovered with bone (char. 10.0), (3) frontoparietal that does not extend laterally beyond the lateral margin of the sphen- ethmoid (char. 17.0), (4) medial ramus of the pterygoid fused with the anterolateral margin of the parasphenoid (char. 21.1), (5) m. exten- sor digitorum longus with an insertion on the metatarsophalangeal joint of the digit IV (char. 39.1), (6) m. extensor digitorum with an inser- tion on the metacarpophalangeal joint of digiti III (char. 44.1), (7) inguinal fat bodies present (char. 51.1), (8) row of dorsolateral tubercles absent (char. 57.0), (9) vocal sac absent in adult males (char. 58.0), and (10) eggs biserially dis- posed in strings (char. 87.1). SISTER CLADE: The Rhinella spinulosa Group. CONTENTS (3 SPECIES): Rhinella arunco (Molina, 1782), R. atacamensis (Cei, 1962), and R. rubropunctata (Guichenot, 1848). DISTRIBUTION: Species of the Rhinella arunco Group are distributed in Argentina and Chile: Rhinella arunco and R. atacamensis in the Atac- NO. 447 ama Desert region, R. rubropunctata in the Aus- tral Temperate Forest region (Cei, 1962, 1980; Correa et al., 2013). See map 1 (available at https://doi.org/10.5531/sd.sp.46) for type locali- ties and sampled localities. COMMENTS: Pramuk (2006) found the Rhi- nella spinulosa Group (sensu Duellman and Schulte, 1992) as nonmonophyletic and excluded the species now placed in Nannophryne (i.e., N. apolobambica, N. cophotis, N. corynetes, and N. variegata; see Frost et al., 2006; Frost, 2020). The remaining species constituted a well-supported clade in her combined (morphological and molecular) analysis, being the sister taxon to all the remaining species of Rhinella. A subsequent molecular phylogeny (Pramuk et al., 2008) con- sidering a similar taxon sampling and mitochon- drial genes, but several different nuclear genes with respect to Pramuk (2006; cxcr4 and ragl-a vs pomc and ragl-a), recovered this redelimited group as monophyletic with poor support. Previ- ous and subsequent analyses with slightly increased taxon and gene sampling, however, found this group as paraphyletic with respect to all remaining species groups of Rhinella (Frost et al., 2006; van Bocxlaer et al., 2010; Pyron and Wiens, 2011; Pyron, 2014; Portik and Papenfuss, 2015; Jetz and Pyron, 2018), or as the (poorly supported) sister taxon of all other species of Rhinella (Pereyra et al., 2016a). In our TE analy- sis, the former R. spinulosa Group (including R. gallardoi, see below) was recovered as monophy- letic but poorly supported (JGC = 68%, JAF = 82%). Moreover, the individual monophyly of its sister subclades is strongly supported (both with JGC and JAF = 100%) and can be diagnosed by phenotypic synapomorphies (see Diagnosis of both groups). Our ML analysis found the former R. spinulosa Group paraphyletic with respect to the remaining species groups of the R. marina Clade (supplementary data 4.2). Based on these observations, we restrict the R. spinulosa Group to the strongly supported clade containing most species of the former R. spinulosa Group (and including R. gallardoi), and exclude the extra- Andean species R. arunco, R. atacamensis, and R. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 45 rubropunctata that constitute another well-sup- ported clade, herein recognized as the R. arunco Group. The southernmost distributed species R. arunco and R. rubropunctata are recovered as sis- ter taxa, although with poor support (JGC = 25, JAF < 50%). The three species of this group show a high genetic differentiation in comparison to other species groups of the R. marina Clade (see tables 3-6). Natural hybridization between R. arunco and R. atacamensis was reported by Cor- rea et al. (2012, 2013), but they did not find mitochondrial and nuclear introgression outside a narrow hybrid zone. THE RHINELLA SPINULOSA GROUP Diacnosis: The following character states optimize as phenotypic synapomorphies of this strongly supported group (JGC and JAF = 100%) in our TE analysis: (1) pretympanic crest absent or indistinguible (char. 2.0, with instances of homoplasy in Rhinella arunco, R. castaneotica, R. festae, and some outgroups); (2) nasal and fron- toparietal bones articulating only laterally (char. 8.1, homoplastic in R. quechua, R. rubropunctata, R. veraguensis, R. yanachaga, Rhinella sp. 14, and some outgroups); (3) lightly exostosed dermal roofing bones (char. 9.1, homoplastic in the R. festae Group, in several species of the R. marina Group, and outgroups); and (4) slightly enlarged otic ramus of squamosal, overlapping with the dorsal surface of the crista parotica (char. 11.1). In addition, species of the R. spinulosa Group can be distinguished from members of the other species groups of Rhinella by the following com- bination of character states: (1) occipital artery pathway not covered by bone (char. 10.0), (2) frontoparietal that does not extend laterally beyond the lateral margin of the sphenethmoid (char. 17.0), (3) medial ramus of the pterygoid fused with the anterolateral margin of the paras- phenoid (char. 21.1), (4) m. extensor digitorum longus with an insertion on metatarsophalangeal joint of digiti IV (char. 39.1), (5) m. extensor digitorum with an insertion on the metacarpo- phalangeal joint of digiti III (char. 44.1), (6) inguinal fat bodies present (char. 51.1), (7) mul- tiserial configuration of eggs in the jelly string (char. 87.2), (8) tarsal fold present (char. 65.1), and (9) adhesive gland divided after fusion of the operculum in embryo (char. 82.2). SISTER CLADE: The Rhinella arunco Group. CONTENTS (9 spEcrEs): Rhinella achalensis (Cei, 1972b), R. altiperuviana (Gallardo, 1961) new status, R. amabilis (Pramuk and Kadivar, 2003), R. gallardoi (Carrizo, 1992), R. limensis (Werner, 1901), R. papillosa (Philippi, 1902), new status, R. spinulosa (Wiegmann, 1834) [including R. arequipensis (Vellard, 1959), new synonymy, see below], R. trifolium (Tschudi, 1845) new status, and R. vellardi (Leviton and Duellman, 1978). DISTRIBUTION: This species group is mostly distributed in arid regions along the Andes of Argentina, Bolivia, Ecuador, Chile, and Peru, except Rhinella gallardoi that inhabits the humid subandean forest of Argentina (Vellard, 1959; Cordova, 1999; Pramuk and Kadivar, 2003; Lavilla and Cei, 2001). Rhinella achalensis and R. limensis are the only species of this group with an extra-Andean distribution in the Sierras Pam- peanas Centrales in the Pampas region of Argen- tina and Atacama Desert of Peru respectively (Vellard, 1959; Cei, 1972b). See map 2 (available at https://doi.org/10.5531/sd.sp.46) for type localities and sampled localities. ComMENts: The Rhinella spinulosa Group as redelimited here is composed of some taxa with controversial taxonomies that are discussed in the context of our results. The widespread, poly- typic, and poorly defined species R. spinulosa is recovered as nonmonophyletic, with R. achalen- sis, R. gallardoi, and R. arequipensis nested within it (fig. 11). Based on our results and considering that “Peru” is the type locality of R. spinulosa (and putatively confined to southern Peru, see Vellard, 1959), we restrict the species R. spinu- losa s.s. to the well-supported lineage (JGC = 95%, JAF = 97%), composed of the populations from southern Peru and northern Bolivia. The lineage containing these populations of R. spinu- losa also includes the sampled specimen of R. arequipensis from Departamento Arequipa, Peru. Rhinella arequipensis was originally described as 46 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY a subspecies of R. spinulosa based only on differ- ences in coloration and density of granular for- mations in the dorsal tegument (Vellard, 1959). Morrison (1992, 1994), Cérdova (1999), and Aguilar and Gamarra (2004) did not find mor- phological, osteological, karyological, or larval differences that could discriminate between R. spinulosa and R. arequipensis. According to these observations and our results, we consider Bufo spinulosus arequipensis Vellard, 1959, a junior synonym of Rhinella spinulosa (Wiegmann, 1834). Thus, the species R. spinulosa is restricted to the populations distributed mainly along the Andean Puna of Peru and adjacent Bolivia. Populations of Rhinella spinulosa that had been considered as R. s. trifolium were recovered as a distinct and strongly supported lineage (JGC and JAF = 98%) sister to a poorly supported clade (JGC <25%, JAF <50%) containing R. spi- nulosa s.s. and several other species of the group (see below). There are several morphological dif- ferences between R. s. trifolium and R. spinulosa s.s. Vellard (1959) pointed out the disposition of the dorsal glands (longitudinal rows in R. s. tri- folium and a uniform distribution in R. s. spinu- losa) and the occurrence of a middorsal vertebral line in R. s. trifolium, as the main distinguishing characters. Morrison (1992, 1994), Sinsch (1986), Haas (2002), and Pramuk and Kadivar (2003) considered R. spinulosa s.s. and R. s. trifolium (and also R. s. flavolineata) as variations of a single species (see below), although all but Haas failed to provide detailed justification. The mor- phological comparisons were some superficial and a detailed reevaluation of the specimens and comparisons with topotypes is needed. Cordova (1999) and Aguilar and Gamarra (2004) did not find karyological or larval differences between R. s. spinulosa and R. s. trifolium; however, these character systems are conserved in related spe- cies of Rhinella (see Tolledo and Toledo, 2010; Kolenc et al., 2013; Blotto et al., 2014). The UPDs between the specimens of R. s. trifolium and R. spinulosa s.s. are relatively high for this species group (1.11%-1.30%, see table 4). Consequently, the differences in adult morphology proposed by NO. 447 Vellard (1959) and their genetic divergence sup- port the recognition of Rhinella trifolium (Tschudi, 1845) as a distinct species. Some populations currently assigned to Rhi- nella spinulosa s.\. from Jujuy (Argentina) and La Paz (Bolivia) were recovered as another distinct and strongly supported lineage (JGC and JAF = 100%; see fig. 4) with a low UPD between them (0.18%). In the intermediate area of Puna between these localities (~ 800 km) lays the type locality of R. s. altiperuviana (Challapata, Oruro, Bolivia). Gallardo (1961) described this subspe- cies from two adult females; the characters used to differentiate it from R. spinulosa s.l. (i.e., tubercles structure, head shape, tarsal fringe development) show considerable variation, at least, in the studied female specimens from northwestern Argentina. Thus, we tentatively assign these populations to R. s. altiperuviana. In addition to the phylogenetic position, these spec- imens differ in UPDs (see table 4) and adult and larval external morphology (B.L.B., D.B., M.O.P., personal obs.) from other species of the group. For these reasons, these populations should be considered as a distinctive species, R. altiperuvi- ana (Gallardo, 1961) from the Andean Puna of Argentina and Bolivia. A detailed taxonomic revision is beyond the scope of this work but will be discussed in a subsequent contribution (B.L.B. and M.O.P., in prep.). Populations of Rhinella spinulosa that had been considered as R. s. papillosa are recovered as a strongly supported lineage (JGC and JAF = 100%), sister taxon of R. achalensis. Both taxa differ in UPDs (1.10 to 1.47%, see table 4), and are morphologically differentiable from R. spi- nulosa s.s. (B.L.B. and M.O.P., in prep.). Thus, we consider R. papillosa (Philippi, 1902), a valid species from the austral Andes of Argentina and Chile. Rhinella gallardoi is deeply nested within the R. spinulosa Group. In the original description, Carrizo (1992) highlighted the “broad skull” of this species over the general morphological simi- larity with the species of the R. spinulosa Group and assigned it to the “Bufo veraguensis-typho- 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 47 TABLE 3 Percentage of uncorrected p-distances between 16S sequences among species of the Rhinella arunco Group Values reported are mean (range). 1 2 3 1 R. arunco _ (N = 1) R. rubropunctata 2. 2. — iN = 87 3 R. atacamensis 2.80 2.41 0.42 (N = 2) (2.67-2.93) (2.28-2.55) , nius” complex. Moreover, R. gallardoi is the only species of the R. spinulosa Group inhabiting exclusively the Yungas of the Andes in north- western Argentina. The specimens of Rhinella amabilis, R. limensis, and R. vellardi are recovered as a strongly sup- ported clade (JCG and JAF and = 100%), which is the sister taxon of the remaining species of the R. spinulosa Group as redefined here. Within this clade, the specimen of R. amabilis collapses into a basal polytomy with the specimens of R. limensis (the UPDs within the clade composed of these specimens are low [0.19%-0.44%]). Rhinella ama- bilis was differentiated from R. limensis by a few characters (development of cranial crests, pres- ence of vocal slits, and shape of the parotoid gland). Rhinella amabilis is the only species of the R. spinulosa Group distributed north of the Huan- cabamba depression (Loja, Ecuador), and we could not obtain tissues from this area. The only specimen sampled of this species comes from a locality in the Huancabamba depression region but does not fully correspond with the morpho- logical description of the species. An extensive revision of both species, including topotypical material and comparison with the holotypes is necessary to test the validity of R. amabilis. The currently recognized subspecies Rhinella spinulosa flavolineata was not included in our analyses. This subspecies differs from R. trifo- lium only in the conspicuity and time of emer- gence of the vertebral line. Haas (2002) studied the development of specimens he assigned to the subspecies R. s. spinulosa, R. s. trifolium, and R. s. flavolineata from the same locality (Man- taro valley, between Concepcion and Huancayo, Junin department, Peru). This author reported that juveniles with variable development (or even absence) of this vertebral line could be obtained from a single clutch, hence, this char- acter seems not to be relevant in differentiating these taxa. The occurrence of R. spinulosa s.s. in that locality is debatable (see Vellard, 1959, for comments on the distributions of these taxa) and it is possible that Haas (2002) assigned spec- imens of R. trifolium with poorly defined verte- bral line to R. spinulosa s.s. (see Haas, 2002: fig. 1). In any case, the results of that study demon- strate that the tempo and level of development of the vertebral line are highly variable. Consid- ering that the different morphs found by this author correspond to intraspecific variation within R. trifolium, we consider Bufo spinulosus flavolineatus Vellard, 1959, a junior synonym of Rhinella trifolium (Tschudi, 1845). Rhinella trifo- lium is considered to inhabit the Central Andean Wet Puna (Vellard, 1959), but additional studies are necessary to determine the precise limits of its geographic distribution and variation with respect to R. spinulosa s.s. THE RHINELLA GRANULOSA GROUP Diacnosis: This species group is recovered as monophyletic with strong support (JGC and JAF = 100%) as in previous analyses (Pramuk, 2006; Pereyra et al., 2016a). Four phenotypic NO. 447 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY 48 (8¢€°0-00'0) €1'0 (€€1-FI'T) ETT 00°0 dnosry vsojnuids vyjauryy ayy JO saisods Suoure saduenbas Sg] U2I9MJ0q sadUL}sSIp-d po}daII09UN Jo asevJUIIIIg (€€'1-S6'0) rg | (ZS°0-8€'0) 8r'0 61°0 (ZS I-F1'T) 87'T (PE I-FI'T) 6L'l (S6°0-ZS'0) 1Z'0 (61°0-00'0) O1'0 (ZS I-€€'1) (aan (96°0-S6'0) S60 (ZS°0-8€'0) LV'0 (9Z°0-ZS'0) z9'0 (€€1-FI'T) EZ] (ZL°0-9Z2'0) 940 (8¢€°0-61'0) 670 (S6°0-9Z'0) 180 (610) 61'0 00°0 PT (67'Z-1¢'9) 69°9 (8Z°9-¥S'9) 799 (85°9-7¢'9) eV9 (8S°9-91'9) ve9 (61°9-Z1'9) v9 (619-719) v9 (00°0) 00°0 ‘(adue1) Uva ore poysodal sanje, V HTaVL (FI'8-80°Z) SEL (80°Z-€2'9) 169 (80°Z-69'9) 68°9 (LZ7'L-€L£'9) 669 (68°9-02'9) 6L'9 (88°9-69'9) 6L9 (911-960) 90°T 610 (Z1'6-¥0'8) 978 (18°Z-S9'Z) ELL (78°Z-79'L) Cid (S9°Z-97'L) WL S9'L (Z9°L) ZO'L (SO°I-€0'T) €0'T (€F'0) er'0 (9 =N) vsojnuids U (7 =N) winyofiay puvlansadiyjpo vsopjidvd sIsua]DYIV 1oplvyvs Ipavyaa sIsuaUl] syiquuuy ‘J UY (7 =N) Y (vy =N) Y (IT =N) Y (7 =N) UY (y =N) Y (7 =N) UY (I =N) Y 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 49 synapomorphies are recovered for this group: (1) anterior end of the septomaxilla devel- oped (previously considered to be the prenasal bones; see discussion of this character in List and Description of Characters) (char. 14.1); (2) sacral diapophyses with the maximum width greater than its maximum length (char. 29.1), with several instances of homoplasy in Rhi- nella and outgroups; (3) submarginal papillae in the larval oral disc absent (char. 71.0), with instances of homoplasy in several bufonids; and (4) two posterior labial tooth rows in the larval oral disc (char. 72.0), that revert in an internal clade of this group. Moreover, nine additional characters might represent synapomorphies of this group or an internal clade depending on their occurrence in R. bernardoi and R. dorbig- nyi, where they are still unknown: (1) anteriorly oriented alary process of the premaxilla (char. 13.2), which also optimizes as a synapomorphy of the R. margaritifera Clade and is homoplastic in Incilius coniferus, Schismaderma carens, and some species of the R. marina Clade; (2) articu- lation of the zygomatic ramus of the squamosal with the maxilla (char. 15.1), homoplastic in Pel- tophryne lemur and R. sternosignata; (3) articu- lation of the jaw anterior to the fenestra ovalis (char. 16.2), homoplastic in Melanophryniscus gr. stelzneri and Peltophryne lemur; (4) bony sphenethmoid reaching the level of the pre- maxillae anteriorly (char. 18.2); (5) posterior lobe in the anterolateral process of hyoid absent (char. 25.0), homoplastic in Rhaebo ceratophrys, Rhinella acrolopha, and in the R. margaritifera Group; (6) vocal sac projected anteriorly when fully inflated (char. 60.1), homoplastic in some species of Anaxyrus; (7) short third pair of gills in the embryos (char. 79.1), homoplastic in Melanophryniscus gr. stelzneri and Schisma- derma carens; (8) short dorsal line of hatch- ing glands in the embryos (char. 80.0), with an instance of homoplasy in R. marina; and (9) type-A adhesive glands in the embryos (char. 81.0). The species of the Rhinella granulosa Group can be distinguished from members of the other groups of Rhinella by the following combination of charac- ter states: (1) preorbital crest well developed (char. 0.2), (2) supraorbital crest well developed (char. 1.2), (3) pretympanic crest well developed (char. 2.2), (4) nasal and frontoparietal articulate along the entire margin (char. 8.3), (5) occipital artery pathway completely covered with bone (char. 10.2), (6) medial ramus of the pterygoid fused and extending medially along approximately half the length of the parasphenoid alae (char. 21.2), (7) anterior edge of sacral diapophyses perpendicular to the midline axis of the vertebral column (char. 30.1), (8) inguinal fat bodies present (char. 51.1), (9) tarsal fold absent (char. 65.0), (10) caudal mus- culature of larvae uniformly dark except an unpig- mented longitudinal stripe along the inferior edge (char. 70.1), (11) occurrence of irregular transverse whitish stripes in the caudal musculature of larvae (char. 70.1), (12) short third gill pair in the embryo (char. 79.1), and (13) adhesive gland divides imme- diately before the gills reach their maximum devel- opment (char. 82.1). SISTER CLADE: The clade composed of the GIM (see below) and the Rhinella crucifer and R. marina Groups. CONTENTS (13 SPECIES): Rhinella azarai (Gal- lardo, 1965); R. beebei (Gallardo, 1965); R. bergi (Céspedez, 2000); R. bernardoi Sanabria et al., 2010; R. centralis Narvaes and Rodrigues, 2009; R. dorbignyi (Duméril and Bibron, 1841) [includ- ing R. fernandezae (Gallardo, 1957) new synon- ymy, see below]; R. granulosa (Spix, 1824); R. humboldti (Gallardo, 1965); R. major (Miller and Hellmich, 1936); R. merianae (Gallardo, 1965); R. mirandaribeiroi (Gallardo, 1965); R. nattereri (Bokermann, 1967); and R. pygmaea (Myers and Carvalho, 1952). DISTRIBUTION: Species of this group are widely distributed in open areas of Amazonia, Atlantic Forest, Caatinga, Cerrado, Chaco/ Pantanal, Chocd, and Pampas regions and in Panama (Narvaes and Rodrigues, 2009; Sanabria et al., 2010; Pereyra et al., 2016a; Murphy et al., 2017). See map 3 (available at https://doi.org/10.5531/sd.sp.46) for type localities and sampled localities. 50 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY ComMENTs: In a previous molecular phyloge- netic analysis of the Rhinella granulosa Group, Pereyra et al. (2016a) recognized 12 putative phenotypic synapomorphies for the group (three of these were first proposed by Pramuk, 2006, and one by Blotto et al., 2014). Eight of these character states were included as part of homol- ogy hypotheses (characters) in our TE analysis and only three were corroborated as synapomor- phies of the R. granulosa Group in all the MPTs (all the remaining were recovered as synapomor- phies in some MPTs). The remaining four char- acter states (i.e., the presence of an expanded flag-shaped dorsal crest of the ilium in lateral view; nasal bone articulates with the dorsal mar- gin of the pars facialis of the maxilla from the preorbital process to the posterior margin of the narial opening; occipital condyles widely sepa- rated; and ability to build and inhabit holes in the ground) were not included due to the lack of detailed descriptions or preparations for many species of Rhinella. However, these character states are unique of the R. granulosa Group among the most closely related groups and are consequently considered putative synapomor- phies of this group. Taxonomic, genetic, and biological aspects of the Rhinella granulosa Group were addressed in detail by Pereyra et al. (2016a), but some differ- ences need to be stressed. First, we found varia- tions in the inferred relationships among the earlier diverging clades/species of this group. Our TE analysis recovered a basal polytomy that com- prises: (1) R. bernardoi, (2) R. dorbignyi (including R. fernandezae, see below), and (3) a poorly sup- ported clade (JGC = 66%, JAF = 74%) composed of the remaining species of the group. Pereyra et al. (2016a) found R. major to be the sister species of a poorly supported clade (JAF <50, no JGC value reported) comprising all the remaining spe- cies of the group. Although in both analyses the interspecific relationships are poorly supported in general, we presume that these differences are due to the inclusion of phenotypic characters, the inclusion of sequences of R. humboldti, the denser outgroup sampling in this study, and the inclusion NO. 447 of a contaminated fragment of cytochrome D (KP684992; contaminated with R. icterica) in the dataset of Pereyra et al. (2016a). Pereyra et al. (2016a) retained Rhinella dorbig- nyi and R. fernandezae as different species, although they noted the absence of reciprocal monophyly between both taxa and the very low genetic distances among the sampled specimens. Although we did not add additional specimens or sequences to our analyses (but a set of phenotypic characters was added in the TE analysis) and we recovered the same topology as Pereyra et al. (2016a) for the clade containing both taxa, we consider Bufo granulosus fernandezae Gallardo, 1957, a junior synonym of Rhinella dorbignyi (Duméril and Bibron, 1841). This decision is con- sistent with the criteria followed to synonymize other taxa of Rhinella (i.e., absence of reciprocal monophyly, absence of genetic differentiation, and absence of conspicuous differential morphological characters). Different populations of R. dorbignyi s.s. vary only in the level of development of the cranial crest, but not in other phenotypic or molecular characters. We hypothesize that local environmental factors through the area of distri- bution (ie., Espinal, Humid Chaco, Humid Pampa, and Uruguayan Savanna) could affect the levels of ossification in the skull, resulting in dif- ferential development of cranial crests. The genetic and environmental causes of hyperossification are still not well understood in anurans (Paluh et al., 2020; Blotto et al., 2021). The differential patterns of bone deposition on the skull of R. dorbignyi are drastic and generate large morphological differ- ences, making this species an excellent candidate to explore the role and impact of environmental factors on hyperossification. We recovered Rhinella humboldti as distinct from R. beebei, as obtained by Murphy et al. (2017). However, both specimens of R. humboldti collapse in a polytomy together with the well sup- ported R. centralis (JGC and JAF = 99%). Both taxa seem to differ in several morphological char- acters (Narvaes and Rodrigues, 2009; although these authors considered R. beebei and R. hum- boldti as a single taxon) and the UPDs between 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 51 the specimens of both species are 1.04%-1.37% (see table 5). The poor internal resolution of this clade could be due to the reduced gene sampling for both specimens of R. humboldti (see appendix 2). However, a thorough analysis including addi- tional molecular markers and morphological comparisons with R. humboldti s.s. is necessary to test the validity of R. centralis. THE MITOCHONDRIAL LINEAGE OF RHINELLA HORRIBILIS The included mitochondrial sequences of Rhi- nella horribilis together with the R. crucifer + R. marina Groups constitute a strongly supported clade (JGC and JAF = 100%) in the TE analysis. Within this clade, they are recovered as sister taxon of a poorly suported clade (JGC = 56%, JAF = 72%) formed by the two aforementioned groups. Alternatively, this lineage is recovered in the ML analysis as the sister of the R. crucifer Group, with low support (44% ultrafast boot- strap support value). This clade is, in turn, sister to the R. marina Group (supplementary data 4.3). As we discuss below (see “Hybridization and genetic introgression in Rhinella”), the strong phylogenetic incongruence between mito- chondrial and nuclear sequences of all the sam- pled specimens of R. horribilis is the result of a past hybridization with introgression event in which R. horribilis incorporated this mitochon- drial lineage and completely replaced the origi- nal mtDNA of this species. We hypothesize that this mitochondrial lineage corresponds to a still unknown, or perhaps even extinct species of Rhi- nella, as we could not associate it to any of the 92 included species. In addition, two well-supported lineages are genetically differentiated within this mitochondrial clade according to the tree topol- ogy and proportionately large genetic distances (mean UPD = 4.19%, table 6): one includes most populations of R. horribilis from Colombia and Central America, which we associate to R. hor- ribilis s.s., whereas the second lineage includes populations of R. horribilis from Ecuador that represent an undescribed species (Rhinella sp. 1). This structure is not recovered by the nuclear sequences of R. horribilis because they collapse in polytomy. THE RHINELLA CRUCIFER GROUP DiaGnosis: This species group was recov- ered as monophyletic and well supported (JGC and JAF = 100%), as in previous studies (Maciel et al., 2006; Thomé et al., 2010, 2012). Three phenotypic characters states optimize as syn- apomorphies of the Rhinella crucifer Group: (1) insertion of the m. extensor digitorum longus on the metatarsophalangeal joint of digiti IV absent (char. 39.0), which is homoplastic in a subclade of the R. granulosa Group, in the R. margaritifera Clade, and in some of the earlier- diverging bufonids; (2) lateral m. dorsometatar- salis proximalis digiti IV with a discrete tendon inserting on the proximal interphalangeal joint of digiti IV (char. 42.1), with an instance of homoplasy in Nannophryne variegata; and (3) the occurrence of a vertebral line (char. 54.1), with several instances of homoplasy within Rhi- nella. Other additional character states that could optimize as a synapomorphy of this group or an internal clade, depending on their occurence in R. casconi and R. henseli, that are still unknown: (1) dorsal protuberance of the illium small, low, and laterally projected (char. 32.1; condition within the group known only in R. crucifer); and (2) inguinal fat bodies absent (char. 51.0), with instances of homoplasy in R. achavali, R. rumbolli, in a subclade of the R. fes- tae Group, in the R. margaritifera Group, and in several sampled outgroups. Species of the Rhinella crucifer Group can be distinguished from members of the other groups of Rhinella by the following combination of char- acter states: (1) supraorbital crest well developed (char. 1.2), (2) pretympanic crest weak (char. 2.1), (3) nasal and frontoparietal articulate along most of its margin but not completely (char. 8.2), (4) occipital artery pathway completely covered with bone (char. 10.2), (5) medial ramus of the pterygoid fused medially along approximately half the length of the parasphenoid ala (char. 21.2), (6) head of the m. extensor carpi ulnaris NO. 447 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY 52 (SZ 1-00°0) 7Z'0 Il (ILZ7-67°7%) (677-LO'%) (FI'E-89°7) (€9°S-LS'7) GS:% 61°0 OI 9T'7 167 LVS (€€1-SO'L) (SO'€-98'Z) (S6'h-07'F) 6I'T 96'7 LS‘ 120 (ZTL'7-89'7) (¥S'P-10'F) OL'7 8T'P ; (1S°S-6S'F) 00'0 nee 1Z0 6 8 L (9L'F-10'F) SVP (00°P-E€F'€) IZLE (I1'€-Z9°7Z) 6L'7 (ZO'P-¥9'€) €8'e (OF'S-8S'F) L6'V 8¢'0 9 ‘(aduer1) UvsuT ore poyiodai sanje,, dnoiry vsojnuvas vyjau1yy ayy JO satdads Suoure saduanbas Sg] U90MJ0q SadUL}sSIP-d pa}da1109UN Jo a8vJUII19g (€7°S-€9'€) eVy (SS‘P-I8'€) LUP (88°€-€F'¢) 99°¢ (6S5°S-09'F) 60°S (9L°S-LL'P) 9€'S (LS'P-78'€) 61 00°0 S TIadVL (S6°€-90'€) 6r'€ (br €-S8'7Z) STs (76'T-8F'7) 697 (79'¢) rO'E (PL P-10'F) CT (p9"€-S7'€) we (Lr 1-760) SIT LL'0 a4 (SI'P-S0'€) 6r'€ (Z9°€-S0'€) cere (ZL'€-L9°7) 88°7 (10°F-€9'€) Z8"€ (€1°9-S6'F) ISS (07 F-PF'E) ZS'€ (9€°€-67'7Z) 08°7 (6'7-ZL'1) Ol'Z (8¢°0) 80 (S6°€-S0'€) Ore (Zh €-H0'€) ETE (76°C-L¥'7) 69'7 (10°F-Z8'€) 76'€ (16'S-96'F) avs (18°€-€F'€) Z9'E (S9°7-06'1) LTT (16'T-I1Z'T) IST (16° I-€€'1) Z9'T (610) 61'0 (9¢°F-£9'€) 96°¢ (Fr'€-S0'€) LZ (16°7-L9°7Z) SFC (€O'P-€8'¢) 68'€ (8r'S-0F'P) 88'P (10°7-SZ'€) poe (9€°€-89'7) L6'7 (0€°7-OT'7Z) 9T'7Z (97'€-89'7Z) €6°7 (L9°7-67'7Z) v7 (8¢€°0-00'0) 70 I (¢ =N) Y 19q20q (7 =N) SUDAJUII U (7 =N) pjoquny “y,, (7 =N) aDUvIAaU U (7 =N) loMaglivpuvsiwl U (7 =N) vsojnuvls U (7 = N) 18.19 U (7 =N) IDAVZD ae (7 =N) Jofou U (7 =N) vavusdid U (ZL =N) 1Ausiq.op U IT OI 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) a3 TABLE 6 Percentage of uncorrected p-distances between 16S sequences among terminals of the ghost introgressed mitochondrion Values reported are mean (range). R. horribilis (N = 4) Rhinella sp. 1 (N = 4) from the radioulna with an origin via a flat ten- don (char. 47.1), (7) parotoid gland approxi- mately ellipsoid (char. 56.0), (8) tarsal fold present (char. 65.1), (9) caudal musculature of larvae uniformly dark except an unpigmented longitudinal stripe along the inferior edge (char. 69.1), and (10) adhesive gland of the embryo divides after opercular fusion (char. 82.2). SISTER CLADE: The Rhinella marina Group. CONTENTS (5 SPECIES): Rhinella casconi Roberto et al., 2014; R. crucifer (Wied, 1821); R. henseli (Lutz, 1934); R. inopina Vaz-Silva et al., 2012; and R. ornata (Spix, 1824) [including R. abei (Baldissera et al., 2004), new synonymy, see below]. DISTRIBUTION: These species are distributed mainly along the Atlantic Forest region, except R. inopina, which inhabits the Cerrado region (Baldissera et al., 2004; Thomé et al., 2010; Arruda et al., 2014; Roberto et al., 2014). See map 4 (available at https://doi.org/10.5531/sd. sp.46) for type localities and sampled localities. ComMENTSs: The general internal relationships among the species are similar to those reported by Thome et al. (2010, 2012). Previously, Maciel et al. (2006) recovered this group as monophyletic, but the internal relationships among the species were poorly supported. Based on our results and those of previous analyses, we discuss below several rel- evant taxonomic issues of this group. Based on external morphology and morpho- metric analyses, Baldissera et al. (2004) reviewed the taxonomy of Rhinella crucifer. These authors resurrected two species (R. henseli and R. ornata) and recognized two new species (R. abei and R. pombali) for several populations previously con- 1 2 0.89 (0.19-1.34) 4.23 0.49 (3.24-5.98) (0.00-0.86) sidered within R. crucifer. More recently, two additional species were described, R. casconi and R. inopina (Vaz-Silva et al., 2012; Roberto et al., 2014). Three of these species (i.e., R. casconi, R. crucifer, and R. henseli) were recovered as strongly supported lineages (JAF and JGC = 100%), and they have moderate UPDs with respect to other species (>0.98% see table 7). Thomé et al. (2010, 2012) found Rhinella abei nested in R. ornata and stressed the need for including additional molecular markers before taking a taxonomic decision on this species. Our analyses, considering additional genes, recovered R. abei as nonmonophyletic and nested within R. ornata. Moreover, vouchers from multiple locali- ties show no consistent differences in the morpho- logical characters employed by Baldissera et al. (2004) to distinguish these species (e.g., color in preserved specimens, subocular band distinctive- ness, head width, and forearm development; M.O.P. and D.B., personal obs.). Thus, we found no evidence to support the distinctiveness of R. abei, and consider Bufo abei Baldissera et al., 2004, a junior synonym of Rhinella ornata (Spix, 1824). Rhinella ornata (including R. abei) is mono- phyletic, but poorly supported (JGC = 56%, JAF = 58%). Its sister taxon is R. inopina, a putatively independent lineage (see FCA analysis in Thomeé et al., 2012) recovered with strong support (JGC and JAF = 99%) in the TE analysis. The genetic distances between R. ornata and R. inopina are very low for the R. crucifer Group (0.2%-0.7%) and cannot be attributable to evident mitochon- drial introgression (see Thomé et al., 2012; fig. 9); some morphological characters (e.g., adult size, 54 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY the coloration of marks on flanks, cloacae, and the posterior surface of thighs, and the disposition of parotoid macroglands) were proposed to differen- tiate both species. Considering the exceptionally low UPDs between R. ornata and R. inopina and the considerably wide range of R. ornata, further comparative studies accounting for geographical variation in these characters are necessary to defi- nitely support or reject the status of R. inopina as a distinct species. THE RHINELLA MARINA GROUP Dracnosis: Our TE analysis recovered a poorly supported Rhinella marina Group (JGC = 63%, JAF = 79%) as in previous studies with less dense taxon sampling (e.g., Maciel et al., 2010; van Bocxlaer et al., 2010; Pyron, 2014). Two phe- notypic synapomorphies support this species group: (1) the jagged or scalloped articulation between the medial ramus of pterygoid and parasphenoid alae (char. 22.1), with instances of homoplasy in R. atacamensis, R. achalensis, R. sternosignata, in a subclade of the R. festae Group, and in some species of the R. mar- garitifera Group, and (2) the sacral diapophyses with the anterior edge angled posteriorly to the midline axis of the vertebral column (char. 30.0), with instances of homoplasy in Rentapia hosii and Schismaderma carens. Species of the Rhinella marina Group can be distinguished from members of the other species groups of Rhinella by the following combination of character states: (1) preorbital crest well developed (char. 0.2), (2) supraorbital crest well developed (char. 1.2), (3) pretympanic crest well developed (char. 2.2), (4) nasal and frontoparietal articulate along the entire margin (char. 8.3), (5) occipital artery pathway completely covered with bone (char. 10.2), (6) medial ramus of the pterygoid fused and extending medially along approximately half the length of the parasphenoid ala (char. 21.2), (7) m. extensor digitorum longus with an insertion on the metatarsophalangeal joint of digiti IV (char. 39.1), (8) inguinal fat bodies present (char. 51.1), (9) parotoid gland approximately ellipsoid, longer than wide or triangular and bulky (char. 56.0 or 56.3), NO. 447 (10) tarsal fold present (char. 65.1), (11) adhesive gland division after operculum fusion in embryo (char. 82.2), and (12) eggs biserially disposed in strings (char. 87.1). SISTER CLADE: The Rhinella crucifer Group. CONTENTS (10 spEciEs): Rhinella achavali (Maneyro et al., 2004); R. arenarum (Hensel, 1867); R. cerradensis Maciel et al., 2007; R. dip- tycha (Cope, 1862) [including R. jimi (Stevaux, 2002), new synonymy, see below]; R. horribilis (Wiegmann, 1833); R. icterica (Spix, 1824); R. marina (Linnaeus, 1758); R. poeppigii (Tschudi, 1845); R. rubescens (Lutz, 1925); and R. veredas (Brandao et al., 2007). DISTRIBUTION: These species are naturally dis- tributed throughout all the main regions of the Neotropics, except in arid Andean areas and the Austral Temperate Forest region (Cei, 1980; De la Riva, 2002; Stevaux, 2002; Kwet et al., 2006; Brandao et al., 2007; Maneyro and Kwet, 2008; Santana et al., 2010; Acevedo et al., 2016; Saito et al., 2016; Venan- cio et al., 2017). See map 5 (available at https://doi. org/10.5531/sd.sp.46) for type localities and sam- pled localities. CoMMENTSs: Both recovered phenotypic syn- apomorphies were suggested as distinctive char- acter states of this group by Pramuk (2006). Moreover, Maciel et al. (2010) proposed four osteological synapomorphies for the Rhinella marina Group (ventral ramus of the squamosal ventrolateral in posterior view; anterior exten- sion of the cultriform process extends beyond the orbitonasal foramina; sphenethmoid lightly ossified; medial ramus of the pterygoid relatively narrow) and one skin-secretion compound (occurrence of a specific indolealkylamine). These characters were not considered in our TE analysis and should be reevaluated considering a denser sample of outgroups than the one employed by Maciel et al. (2010). The finding of a moderately supported Rhi- nella marina Group contrasts with previous studies that recovered it well supported (e.g., Maciel et al., 2010; van Bocxlaer et al., 2010; Pyron, 2014; Jetz and Pyron, 2018). Two distinc- tive moderately supported clades are evident in 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 55 TABLE 7 Percentage of uncorrected p-distances between 16S sequences among species of the Rhinella crucifer Group Values reported are mean (range). 1 2 i R. henseli 0.25 (N = 3) (0.00-0.38) > R. casconi 3.62 0.00 (N = 2) (3.62) (0.00) 3 R. crucifer 3.14 1.43 (N = 2) (3.04—3.23) (1.33-1.52) 4 R. inopina 3.42 1.33 (N = 3) (3.42) (1.33) 5 R. ornata 3.25 1.30 (N = 11) (3.04-3.61) (1.14-1.52) this genetically and taxonomically complex spe- cies group. The first roughly corresponds to the North-Central Clade of Maciel et al. (2010) and is composed of R. diptycha (including R. jimi), R. horribilis, R. marina, R. poeppigii, and R. veredas, but does not include R. cerradensis (although see MP tree in Maciel et al., 2010: fig. 3). Rhinella poeppigii, R. veredas, and R. marina s.s. (see Acevedo et al., 2016) are successive sis- ter taxa of the remaining species of the North- Central Clade but their positions are poorly supported (JGC <38%, JAF <54%). Except for R. veredas, the monophyly of these species are poorly supported (JGC = 74%, JAF = 77 for R. poeppigii; JGC <25%, JAF <50% for R. marina). The relationships among R. diptycha, R. jimi, and the nuclear sequences of R. horribilis and Rhinella sp. 1 are poorly resolved (see fig. 12). The lack of resolution and support for the inter- nal relations of the North-Central Clade could be due, at least in part, to (1) the nuclear mark- ers employed that do not provide sufficiently informative variation to resolve the relation- ships in the absence of mitochondrial informa- tion for some terminals, and (2) the occurrence of putative past and present hybridization that could not be detected with the available molec- ular evidence. The occurrence of a deep mitochondrial diver- gence in Rhinella horribilis and Rhinella sp. 1 3 4 5 0.19 (0.19) 0.86 0.25 (0.76-0.95) (0.00-0.38) 0.82 0.35 0,29 (0.57-1.14) (0.19-0.57) (0.00-0.76) precluded the combination of the mitochondrial and nuclear sequences into single terminals. This results in an unstable and poorly supported phy- logenetic position for nuclear sequences of both species in the TE analysis (in a polytomy with R. jimi specimens). Because the nuclear sequences employed provided relatively few informative characters, their relationships should be better explored considering additional evidence. More- over, R. horribilis diverges in several morphologi- cal characters (adult morphology, osteology, and larval morphology; see Savage, 2002; Stevaux, 2002; Kwet et al., 2006; Tolledo and Toledo, 2010; Acevedo et al., 2016) from the largely allo- patric R. jimi. Otherwise, the position of R. jimi in a polytomy together with the specimens of R. diptycha and the very low UPDs (0%-0.19%) among the specimens of these species indicate that the taxa are conspecific. Rhinella diptycha and R. jimi are two morphologically similar spe- cies with large parotoid and tibial macroglands. Remarkably, R. jimi has distinctive glands on its forearms and on both sides of the cloaca that were considered as the only distinctive charac- ters from R. diptycha (Stevaux, 2002; Kwet et al., 2006). Mailho-Fontana et al. (2018) found that both species have similar types and distribution of skin glands, although in different proportions. These authors proposed that this differential development could be related to the occupancy 56 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY of xeric environments by R. jimi. We also found a greater glandular development in the forearms and both sides of the cloaca in some specimens of R. diptycha from different localities of the dry Chaco in Argentina (M.O.P. and D.B., personal obs.). Based on these observations, we consider Bufo jimi Stevaux, 2002, a junior synonym of Rhi- nella diptycha (Cope, 1862). More physiological and histological studies, investigating different populations from different environments, could help to understand the patterns of variation in the development of macroglands in this species. The other clade of the Rhinella marina Group is composed of R. achavali, R. arenarum, R. cer- radensis, R. icterica, and R. rubescens (fig. 12), and roughly corresponds to the South-Central Clade of Maciel et al. (2010). Within this clade, R. are- narum is supported as sister taxon of the remain- ing species of the clade with strong support (JGC and JAF = 100%). The sampled specimen from the populations that had been considered as R. arena- rum mendocina is nested within the remaining specimens of R. arenarum. The sister clade of Rhinella arenarum is well supported but it is internally poorly resolved. This includes R. achavali, R. cerradensis, R. icterica, and R. rubescens. Rhinella cerradensis and R. rubescens are reciprocally monophyletic, their UPDs are low (0.19%-0.74%, see table 8), and constitute a strongly supported clade (JGC and JAF = 99%) that collapses in a basal poly- tomy within the clade. Rhinella achavali was recovered nested in a poorly supported clade (JGC <25%, JAF <50%) composed of some pop- ulations of R. icterica and the UPDs within this clade are low (0.37%-0.76%, see table 8). Although R. icterica is quite variable morpho- logically (M.O.P. and D.B., personal obs.) and this species includes several synonymized forms (e.g., Bufo missionum; Faivovich and Carrizo, 1997), this taxon is divergent morphologically from R. achavali (see Maneyro et al., 2004; Kwet et al., 2006; M.O.P., EK., and C.B., personal. obs.). Finally, some specimens tentatively assigned to R. cerradensis (R. aff. cerradensis) collapse into a basal polytomy within the sister NO. 447 clade of R. arenarum. We refrain from taking any decision regarding the taxonomy of these species pending more studies, particularly with respect to understanding the effect of genetic (e.g., nuclear and/or mitochondrial introgres- sions) and environmental (e.g., phenotypic plasticity) factors on their morphological variation. THE RHINELLA MARGARITIFERA CLADE DiaGnosis: This well-supported clade (JGC and JAF = 99%) is diagnosed by two phenotypic synapomorphies: (1) alary process of the pre- maxillae angled anteriorly to the anterior margin of the pars dentalis of premaxillae (char. 13.2), with instances of homoplasy in Incilius coniferus, Rhinella achalensis, R. ornata, R. poeppigii, and Schismaderma carens; and (2) skin of dorsum of females with small tubercles lacking cornified tips (char. 52.3). SISTER CLADE: The Rhinella marina Clade. ConTENTs: Rhinella sternosignata and the R. festae, R. margaritifera, and R. veraguensis Groups. DISTRIBUTION: The species of this clade are mainly distributed throughout Amazonia and montane humid forest of the Andes. Some spe- cies of this clade are also found in the Atlantic Forest, Caatinga, Cerrado, Chaco/Pantanal, and Chocéd regions, and in Central America (Duellman, 1999). ComMENTs: Within this clade, Rhinella ster- nosignata is recovered as the sister taxon of a large, poorly supported clade (JGC = 49%, JAF = 71%). This last clade is supported by a sin- gle phenotypic synapomorphy (ventral ridges on the palatine absent; char. 20.0), which is homoplastic in several species of the R. marina Group and outgroups. The clade is composed of three strongly supported species groups (JGC and JAF = 100%): (1) the redefined R. veraguen- sis Group, (2) the redefined R. festae Group, and (3) the redefined R. margaritifera Group. The R. festae and R. margaritifera Groups were recovered as sister clades with moderate sup- port (JGC = 81%, JAF = 89%) and five character states optimize as phenotypic synapomorphies 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 57 of this clade: (1) discrete superficial cutane- ous tendons absent (char. 33.0); (2) lateral slip of the m. interphalangeus proximalis digiti V (foot) absent, with instances of homoplasy in R. major and R. papillosa (char. 35.0); (3) m. abductor brevis plantaris hallucis absent (char. 36.0), with instances of homoplasy in Anaxyrus woodhousii, Peltophryne empusa (polymorphic), and R. mirandaribeiroi; (4) slip of the medial m. lumbricalis brevis digiti V originating from the distal carpal 3-4-5 absent (char. 43.0) with an instance of homoplasy in Nannophryne var- iegata (polymorphic); and (5) head of the m. extensor carpi ulnaris from the radioulna with a fleshy origin (char. 47.0), with an instance of homoplasy in P. empusa. A similar topology for the main internal clades of the R. margaritifera Clade was recovered in the ML analysis (sup- plementary data 4.4-4.5). RHINELLA STERNOSIGNATA Diacnosis: Rhinella sternosignata (Gunther, 1858b) was recovered as the sister taxon of all other species of the R. margaritifera Clade, with poor support (JGC = 49%, JAC = 71%). Pheno- typic autapomorphies are: (1) acuminate anterior margins of nasals (char. 6.1), with instances of homoplasy in Incilius coniferus and the R. mar- garitifera Group; (2) articulation of the zygo- matic ramus of the squamosal with the maxilla (char. 15.1), with instances of homoplasy in the R. granulosa Group and Peltophryne lemur; (3) articulation between the medial ramus of the pterygoid and parasphenoid alae with a jagged suture (char. 22.1) with instances of homoplasy in R. achalensis, R. atacamensis, some species of the R. festae and R. margaritifera Groups, and in the R. marina Group; (4) parotoid gland round to ovoid mostly symmetrical (char. 56.2); (5) large size of adult males with respect to adult females (char. 66.1), with instances of homoplasy in R. yanachaga, and in several species of the R. marina Clade; and (6) unpigmented eggs (char. 88.0), with instances of homoplasy in Ansonia longidigita, Rhinella justinianoi, R. stanlaii, and in the R. festae Group. DISTRIBUTION: This species inhabits montane forests of the Cordillera de la Costa and the Andean Cordillera de Mérida of Venezuela (La Marca and Mijares-Urrutia, 1996; Barrio- Amoros et al., 2019). See map 5 (available at https://doi.org/10.5531/sd.sp.46) for type and sampled localities. SISTER CLADE: The clade composed of the Rhinella festae, R. margaritifera, and R. veraguen- sis Groups. ComMENts: This species was tentatively asso- ciated with the Rhinella margaritifera (Cei, 1972a; Hoogmoed, 1990; Duellman and Schulte, 1992) or R. granulosa Groups (Gallardo, 1962). Pereyra et al. (2016a) rejected the inclusion of this species in any of these groups, but they could not determine its relationships rigorously due to the poor sam- pling of Rhinella. This species was wrongly reported for many localities outside the Cordillera de la Costa montane forests region in Venezuela as discussed by La Marca and Mijares-Urrutia (1996). Vélez-Rodriguez (1999) recorded this spe- cies in error for Colombia (see Vélez-Rodriguez, 2004b, 2005). Additionally, there are a large num- ber of recent reports of R. sternosignata for Colombia (Acosta-Galvis et al., 2006; Romero et al., 2008; Acosta-Galvis, 2012a, 2012b). Analyzed specimens tentatively assigned to this species from the eastern slope of the Cordillera Oriental in Colombia (MAR 1314, Boyaca and MAR 1955, Caqueta) were unrelated to the specimen of R. sternosignata from Venezuela in the phylogenetic analyses, and instead, they represent an unde- scribed species along with other specimens of the R. margaritifera Group from Loreto, Peru, and Miranda, Venezuela (Rhinella sp. 13, see below). These results, and the absence of comprehensive comparative studies considering topotypical mate- rial of R. sternosignata, indicate that there is no evidence to consider its occurrence in Colombia. THE RHINELLA VERAGUENSIS GROUP DiacGnosis: No phenotypic synapomorphies were recovered for this strongly supported group (JGC and JAF = 100%). This is mainly due to the lack of detailed information for one of its two con- NO. 447 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY 58 00°0 OI (8Z°0-8€'0) 8S'0 (96'0-8€'0) 020 (ZI I-9Z'0) 96'0 (ZS I-ZS'0) 10'T (ZS°0-61'0) 80 ‘sutAuOUAS JU9III IPNOUI YSTI9}se Ue YIM satdads *(a8ueI) UROUT ore poysodal sonjeA, dnory vuiiwu vyjau1yy 24) Jo satdads Suoure saduanbas Sg] Us9Mj9q SaduUL\sIp-d po}d01109UN JO 98e}UII19g (8Z°0-ZS'0) £90 (FI'I-8€'0) €Z0 (9Z°0-61'0) 870 610 L (8Z°0-8€'0) 790 (9Z'0-6T'0) 19°0 (FI'I-8€0) Z8'0 (9Z'0-61'0) rS'0 (8¢'0-00'0) S70 g (ZI I-9Z'0) S30 (ZS I-9Z'0) €0O'T (06° I-¥I'T) Iv'l (ZS 1-S6'0) ZIT (ZS I-9Z'0) 60'°T (8€°0-00'0) ST‘0 S (vE7-06'T) STZ (99°7-06'T) I€'7Z (99°7-06'T) I€7Z (877-121) €0'7 (99°7-06'1) SET (S8°7-87'Z) vS'7 (8¢€°0-00'0) 61'0 74 8 HIaVL (SS°7-877) SV'7 (€6°7-60'7) 6S°7 (767-877) 797 (¥S°7-60°Z) VET (98°7-87'7) 09°7 (€1'€-L9°7Z) 98°7 (6S°0-61'0) 70 (6€°0-61'0) 970 (SLI-IZ'1) ELT (60°7-IZ'1) 06'T (60°-TZ'T) 06'T (IZ 1-ZS'T) Z9'T (60°7-IZ'1) 96'T (87'7-60°7) eZ (ZS°0-61'0) Iv'0 (Z6°0-9Z'0) 680 00°0 Cc (F0°€-99'7) 9L7 (tr €-L¥'Z) 76'7 (Zh €-99°7Z) 86'7 (v0°€-L¥'Z) OL'7 (Zh'€-99'Z) vO'E (08°€-¥0'¢) STE (99°7-06'T) ra (L¥'7-60°7) 977 (60°7-IZ'1) rs'T (8¢°0-00'0) S70 I (7 =N) Y (Il =N) _0NAII “Y yvavyov (€ =N) UY (t=N) UY (€ =N) UY (€ =N) UY (L=N) UY (€ = N) UY (7 =N) UY SISUIPDALII SUdISAGNA sisuapvssad “Tye WNnADUIAAD vyodjdip DULIDU sppasaa » (€ =N) 1181ddaod “y OI 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 59 stituent clades (composed of Rhinella sp. 2 [see below], R. inca and R. leptoscelis). Nevertheless, some character states might represent synapomor- phies for this group or a subclade: (1) the articula- tion of jaw opposite to the fenestra ovalis (char. 16.1), with instances of homoplasy in Nan- nophryne variegata, Rhinella beebei, R. merianae, R. yanachaga, and the R. arunco Group; (2) light- colored nuptial pads (char. 62.0); (3) larval peri- branchial region with bulging regions lateral to the oral disc (char. 67.1); (4) larval oral disc with complete A2 labial tooth row (char. 73.0), with instances of homoplasy in Amazophrynella aff. minuta, Ansonia longidigita, Melanophryniscus gr. stelzneri, Phrynoidis juxtaspera, and Schisma- derma carens; (5) the dextral opening of the vent tube (char. 75.1); and (6) eggs laid in open clumps (char. 86.1; structure of the spawn only known in R. rumbolli within the R. veraguensis Group). The species of the Rhinella veraguensis Group can be distinguished from members of the other species groups of Rhinella by the following combination of character states: (1) preorbital crest weak (char. 0.1), (2) supraorbital crest weak (char. 1.1), (3) pretympanic crest weak (char. 2.1), (4) medial ramus of the pterygoid fused and extending medially along approxi- mately half the length of the parasphenoid ala (char. 21.2), (5) m. extensor digitorum longus without an insertion on the metatarsophalan- geal joint of digiti IV (char. 39.0), and (6) tarsal fold absent (char. 65.0). SISTER CLADE: The clade composed of the Rhinella festae and R. margaritifera Groups. CONTENT (9 SPECIES): Rhinella chrysophora (McCranie et al., 1989); R. fissipes (Boulenger, 1903); R. gnustae (Gallardo, 1967); R. inca (Stej- neger, 1913); R. justinianoi (Harvey and Smith, 1994); R. leptoscelis (Boulenger, 1912); R. que- chua (Gallardo, 1961) [including R. amboroensis (Harvey and Smith, 1993), new synonymy, see below]; R. rumbolli (Carrizo, 1992); and R. veraguensis (Schmidt, 1857). DISTRIBUTION: All species of the Rhinella veraguensis Group are distributed in Andean humid forests of Argentina, Bolivia, and Peru, except R. chrysophora, which inhabits the Cen- tral American Atlantic moist forests in Honduras (Rodriguez et al., 1993; De la Riva et al., 2000; Kohler, 2000; Lavilla and Cei, 2001; Padial et al., 2009; McCranie, 2017). See map 6 (available at https://doi.org/10.5531/sd.sp.46) for type locali- ties and sampled localities. ComMMENTs: The former Rhinella veraguensis Group (see appendix 5) is recovered as polyphy- letic. To remedy this, we restrict its content to the clade of species most closely related to R. veraguensis. This group also comprises two spe- cies not included in the phylogenetic analyses that share several putative synapomorphies with the species sampled here (see below). Two clades were recovered within this redefined R. veraguen- sis Group. One clade is poorly supported (JGC = 67%, JAF = 76%) and comprises the southern- most distributed species of the group. In the sec- ond clade, we were unable to examine the voucher of R. amboroensis (MNK 5302), but this specimen was collected near the type locality of the species. The specimen was recovered as the sister taxon of R. quechua and the genetic dis- tance between the specimens is 0% (see table 9). Both species are very similar morphologically and only a few morphological characters were proposed to differentiate the taxa (i.e., the exten- sion of the foot webbing, ventral skin texture, and finger length). However, these difference are not consistently observed in specimens collected in the type locality of R. amboroensis (1.D.L.R., personal obs.) and they could simply represent variations within R. quechua. For these reasons, we consider Bufo amboroensis Harvey and Smith, 1993, a junior synonym of Rhinella quechua (Gallardo, 1961). The other clade in this group is strongly sup- ported (JGC and JAF = 100%) and includes the northernmost distributed species, R. inca, R. lep- toscelis, and an undescribed species from Oxa- pampa, Peru (Rhinella sp. 2). The UPDs among these three species are relatively low (1.16%- 1.90%; see table 9). Rhinella chrysophora and R. gnustae, two spe- cies not included in the phylogenetic analysis, 60 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY are considered to belong to this species group. Rhinella chrysophora is known only from two localities in northern Honduras and is suppos- edly extinct, not collected since 1996 (McCranie and Castarieda, 2005; McCranie, 2017). This spe- cies was originally described as belonging to a distinct genus (Atelophryniscus; McCranie et al., 1989) of no evident relationships within Bufoni- dae. Pramuk and Lehr (2005), based on a mor- phological phylogenetic analysis, demonstrated that it is related to the species of the R. veraguen- sis Group s.l. Unfortunately, the character scores for R. chrysophora are not available and the con- dition of the double-stained specimen used in that study is very poor (J.J.0.-S., personal obs.). However, morphological evidence indicates that R. chrysophora belongs to the R. veraguensis Group, as it posses all its known putative synapo- morphies (except for oviposition mode, which is unknown; McCranie et al., 1989; Lavilla and de Sa, 2001; Pramuk and Lehr, 2005). Rhinella gnustae (Gallardo, 1967) was described based on a single subadult specimen from an imprecise locality of Jujuy Province (Argentina) (Gallardo, 1967; Cei, 1980; Lavilla and Cei, 2001; Lavilla et al., 2002). We tentatively assign this species to the R. veraguensis Group based on a combination of characters (although none of them synapomorphic) that occur in mul- tiple species of this group: row of dorsolateral tubercles in the skin absent, tarsal fold absent, and small tubercles without a cornified tip. THE RHINELLA FESTAE GROUP Diacnosis: This well-supported group (JGC and JAF = 100%) is diagnosed by the following five phenotypic synapomorphies: (1) skull lightly exostosed (char. 9.1), with instances of homoplasy in Rhinella achavali, R. rubescens, the R. spinulosa Group, and in several outgroups; (2) fusion of medial ramus of pterygoid with anterolateral mar- gin of the parasphenoid ala (char. 21.1), with instances of homoplasy in Rhinella sp. 12, in the R. arunco Group, in some species of the R. spinu- losa Group, and in several outgroups; (3) anterior margin of cultriform process of parasphenoid NO. 447 truncated (char. 23.2); (4) arboreal habits (char. 84.1) that revert in an internal clade of this group, and with instances of homoplasy in Incilius coniferus and Rentapia hosii; and (5) unpigmented eggs (char. 88.0), with instances of homoplasy in Ansonia longidigita, Rhinella justinianoi, R. stan- laii, and R. sternosignata. Other putative synapo- morphies of this group or an internal clade are: (1) additional origin of the m. interosseus cruris from the tibiale absent (char. 38.0; known within the group only for R. paraguas); (2) m. extensor brevis medius hallucis absent (char. 41.0; known within the group only for R. paraguas); and (3) the ter- restrial oviposition (char. 85.1; known within the group only in R. tacana). Moreover, species of the R. festae Group can be distinguished from mem- bers of the other species groups of Rhinella by the following combination of character states: (1) pre- orbital crest absent or indistinguible (char. 0.0), (2) supraorbital crest weak (char. 1.1), (3) discrete superficial cutaneous tendons absent (char. 33.0), (4) lateral slip of the m. interphalangeus proxima- lis digiti V absent (char. 35.0), (5) m. abductor brevis plantaris hallucis absent (char. 36.0), (6) m. extensor digitorum longus without an insertion on the metatarsophalangeal joint of digit IV (char. 39.0), (7) slip of the medial m. lumbricalis brevis digiti V originating from the distal carpal 3-4-5 absent (char. 43.0), (8) head from the radioulna of the m. extensor carpi ulnaris with a fleshy origin (char. 47.0), (9) nuptial pads dark colored (char. 62.1), and (10) tarsal fold absent (char. 65.0). SISTER CLADE: The Rhinella margaritifera Group. CONTENTS (18 Species): Rhinella acrolopha (Trueb, 1971); R. arborescandens (Duellman and Schulte, 1992); R. chavin (Lehr et al., 2001); R. festae (Peracca, 1904); R. lilyrodriguezae Cusi et al., 2017; R. lindae (Rivero and Castafio, 1990); R. macrorhina (‘Trueb, 1971); R. manu Chaparro et al., 2007; R. multiverrucosa (Lehr et al., 2005); R. nesiotes (Duellman and Toft, 1979); R. nicefori (Cochran and Goin, 1970); R. paraguas Grant and Bolivar-G., 2014; R. rostrata (Noble, 1920); R. ruizi (Grant, 2000); R. tacana (Padial et al., 2006); R. tenrec (Lynch and Renjifo, 1990); R. truebae 61 BUFONIDAE) EVOLUTION IN RHINELLA (ANURA: . PEREYRA ET AL. 2021 00°0 (€6°I-16'1) 761 00°0 (91° 1-96'0) 101 (€6°I-ZZ'1) SLT (61°0-00'0) O10 (SI'Z-67'9) IZ9 (06°9-€6'S) ZE9 (€€'L-67'9) LL9 (87°7-S6'0) 7ST (00°9-¥6'S) 96'S (ZL°S-9Z'S) OLS (81'9-¥6'S) 109 (O1'Z-S6'0) LVI 00°0 574 (8¢°9-ZE'9) ve9 (S6°S) So's (Z¢°9-€€'9) seo (8€°P-€7'€) Sse (SO'¢) SO'E ‘(aduer1) Ueda ore poytodas sane, dnory sisuansv.aa vyjauiyy 24} Jo saidads Suowve saduanbas gg] UseMjaq SaduL}sIp-d pa}da1109UN JO aSeJUIIIIqg 6 HIdVL (S6°9-06°9) 1Z9 (SZ°S-9¢'S) co"g (ZZ°9-T€'9) €S°9 (60°9-08'¢) SSP (8€'F-66'¢) 61'F (S6'P-9ZL'P) 98°F 80 (87°Z-ST'L) VOL (87'Z) STL (1L°L-6F'L) 6S°L (ZS°S-86'€) OLY (18°¢) Ig'€ Ze (SLY-LE'F) 9S'P (vy =N) voul U (7 =N) syjaosojday U (vy =N) Z ‘ds vyauiyy (vy =N) SISUaNSDAIA U (7 =N) onysanb Rel (IT =N) 1ouviuysnt U (7 =N) yjoquind a (I=N) sadissyf “Y 62 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY (Lynch and Renjifo, 1990); and R. yanachaga Lehr et al., 2007. DISTRIBUTION: Mainly distributed in Andean humid forests of Bolivia, Colombia, Ecuador, and Peru (Trueb, 1971; Duellman and Lynch, 1988; Lynch and Renjifo, 1990; Duellman and Schulte, 1992; Ruiz-Carranza et al., 1996; Lehr et al., 2001, 2005, 2007; Rueda-Almonacid et al., 2004; Chavez et al., 2013; Grant and Bolivar-G., 2014; Cusi et al., 2017). The only species distrib- uted outside this region is Rhinella acrolopha, which inhabits the Choco region (Darién, Pan- ama; Trueb, 1971). See maps 7 and 8 (available at https://doi.org/10.5531/sd.sp.46) for type locali- ties and sampled localities. ComMENtTs: Analyses of our expanded sample of the Rhinella acrolopha Group corroborated the monophyly of that group; however, they also cor- roborated its placement among a subset of the species referred to the R. veraguensis Group s.l. Insofar as those species are paraphyletic with respect to the R. acrolopha Group, the only means of maintaining the current composition of the R. acrolopha Group would be to recognize two or more groups within the larger clade, which we believe to be unwarranted at this time. Consequently, we redefined the R. festae Group as was originally proposed by Moravec et al. (2014) to also include all the species previously referred to the R. acrolopha Group and three spe- cies (R. arborescandens, R. multiverrucosa, and R. tacana) of the former R. veraguensis Group. Pramuk (2006) proposed a close phyloge- netic relationship between Rhamphophryne and some species of the Rhinella veraguensis Group as formerly defined, but she did not provide a diagnosis for the inclusive clade. As defined in this study, the R. festae Group is a morphologi- cally and ecologically diverse clade of toads; the lack of information for many aspects of these species is remarkable (e.g., adult osteology, adult musculature, larval morphology, natural history; see below). Species of this group have notably high inter- specific UPDs compared to species of other groups of Rhinella (see tables 3-11) except NO. 447 between the pairs R. chavin/R. multiverrucosa and R nicefori/R. ruizi. Sequences of the included specimen assigned to R. multiverrucosa (MUBI 11455) are identical (UPD = 0%) to the topotype of R. chavin (sequence DQ158441 from Pramuk, 2006). Although morphologically most similar to R. multiverrucosa, the specimen MUBI 11455 was not collected near the type locality of this species and was actually collected closer to the type locality of R. chavin (see map 8; available at https://doi.org/10.5531/sd.sp.46). Most of the characters that distinguish these two species involve differences in glandular development. Our results should be tested considering the existing morphological variation within R. chavin and including topotypes of R. multiver- rucosa in a future revision of these species. Similarly, the UPDs between the included specimen of Rhinella nicefori and topotypic spec- imens of R. ruizi is low (UPDs = 0.19%; see table 10). The two species were not explicitly differen- tiated in the original description of R. ruizi (Grant, 2000), but they differ in some characters (degree of cranial ornamentation, the occurrence of vocal slits in adult males, adult size). As we did not sample topotypical material of R. nicefori and cannot discard the occurrence of some addi- tional variation in the diagnostic characters that differentiate the two species, the identity of the included specimen MHUA 4793 should be reevaluated. For this reason, we refrain from tak- ing a taxonomic action, pending a detailed taxo- nomic evaluation of both species, considering topotypical material of R. nicefori and compari- son with type specimens. Two undescribed species within this species group are recovered in our TE analysis. Firstly, some specimens tentatively assigned to Rhinella manu from Madre de Dios and Cusco display high UPDs (3.37%) with respect to specimens of R. manu s.s., suggesting they might represent an undescribed species (Rhinella sp. 3). Second, the specimen of R. sp. “gr. acrolopha” (referred to Rhinella sp. C. by Machado et al., 2016) from Caldas (Colombia) is recovered as sister species of R. paraguas, and the genetic distance between 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 63 them (UPDs = 5.73%-6.11%) is consistent with the hypothesis that it is an undescribed species (Rhinella sp. 4). We could not include Rhinella rostrata and R. truebae in our analyses. Nevertheless, these spe- cies can be placed in the R. festae Group on the basis of several character states that are synapo- morphies of this group or its internal clades: (1) skull lightly exostosed (char. 9.1); (2-4) colu- mella, annulus tympanicus, and tympanic mem- brane absent (chars. 48.0, 49.0, and 50.0) in R. rostrata (present in R. truebae); and (5) finger webbing present (char. 63.1). THE RHINELLA MARGARITIFERA GROUP Dracnosis: No phenotypic synapomorphies were recovered in our TE analysis for this well- supported species group (JGC and JAF = 100%). However, given the lack of information (see Comments on the phenotypic evidence consid- ered for Rhinella section) for its earlier diverg- ing species (e.g., R. ocellata, R. yunga, and Rhinella sp. 5) or closely related clades (i.e., R. sternosignata, the R. festae and R. veraguensis Groups), the inclusion of additional observa- tions in the phenotypic dataset could provide diagnostic synapomorphies for this clade. A putative synapomorphy for this species group (unknown condition in Rhinella sp. 5) is the acuminate anterior margins of nasals (char. 6.1), with instances of homoplasy in Incilius coniferus and R. sternosignata. Moreover, spe- cies of the R. margaritifera Group can be distin- guished from members of the other species groups of Rhinella by the following combina- tion of character states: (1) preorbital crest weak (char. 0.1), (2) medial ramus of the pterygoid fused and extending medially along approxi- mately half the length of the parasphenoid ala (char. 21.2), (3) posterior lobe of the anterolat- eral process of hyoid absent (char. 25.0), (4) discrete superficial cutaneous tendons absent (char. 33.0), (5) lateral slip of the m. interpha- langeus proximalis digiti V absent (char. 35.0), (6) m. abductor brevis plantaris hallucis absent (char. 36.0), (7) m. extensor digitorum longus without an insertion on the metatarsophalan- geal joint of digiti IV (char. 39.0), (8) slip of the medial m. lumbricalis brevis digiti V originat- ing from the distal carpal 3-4-5 absent (char. 43.0), (9) head of the m. extensor carpi ulnaris from the radioulna with a fleshy origin (char. 47.0), (10) inguinal fat bodies absent (char. 51.0), (11) tarsal fold absent (char. 65.0), and (12) submarginal papillae in the oral disc of lar- vae absent (char. 71.0). SISTER CLADE: The Rhinella festae Group. CONTENTS (17 SPECIES): Rhinella acutirostris (Spix, 1824); R. alata (Thominot, 1884); R. cas- taneotica (Caldwell, 1991); R. cristinae (Vélez- Rodriguez and Ruiz-Carranza, 2002); R. dapsilis (Myers and Carvalho, 1945) [including R. gildae Vaz-Silva et al., 2015, new synonymy, see below]; R. hoogmoedi Caramaschi and Pombal, 2006; R. iserni (Jiménez de la Espada, 1875) [including R. yunga Moravec et al., 2014 new synonymy, see below]; R. lescurei Fouquet et al., 2007a; R. magnussoni Lima et al., 2007; R. mar- garitifera (Laurenti, 1768) [including R. martyi Fouquet et al., 2007a, new synonymy, see below]; R. ocellata (Gunther, 1858b); R. probos- cidea (Spix, 1824); R. roqueana (Melin, 1941); R. scitula (Caramaschi and Niemeyer, 2003) [including R. paraguayensis Avila et al., 2010, new synonymy, see below]; R. sclerocephala (Mijares-Urrutia and Arends, 2001); R. sebbeni Vaz-Silva et al., 2015; and R. stanlaii (Lotters and Kohler, 2000). DISTRIBUTION: Mainly distributed in Amazo- nia, but a few species also occur in the Andes, Atlantic Forest, Caatinga, Cerrado, Chocé, Chaco/ Pantanal, and in Central America (Hoogmoed, 1986, 1990; Ruiz-Carranza et al., 1996; Caramas- chi and Pombal, 2006; Kohler et al., 2006; Fouquet et al., 2007a; Moravec et al., 2014; Sugai et al., 2014; Santos et al., 2015; Avila et al., 2018; Freitas et al., 2018; Silva et al., 2018). See maps 9 and 10 (available at https://doi.org/10.5531/sd.sp.46) for type localities and sampled localities. ComMMENTs: This species group is particularly controversial regarding its diagnosis, content, and taxonomy of its species. 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OL ATAVL 66 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY dealing with this group (e.g., Hoogmoed, 1986, 1990; Duellman and Schulte, 1992; Vélez-Rodri- guez, 2004b; Pramuk, 2006; Fouquet et al., 2007a) disagreed with respect to the inclusion of mullti- ple species (e.g., Rhinella cristinae, R. iserni, R. ocellata; see appendix 5). Vélez-Rodriguez (2004b) performed a phylogenetic analysis of the group based on morphological characters and proposed the restriction of its content to a clade diagnosed by two synapomorphies: (1) m. depressor mandibulae composed of two slips with independent origins, on the posterior por- tion of the otic ramus of the squamosal and the anterior portion of the otic ramus of the squamo- sal and tympanic annulus; and (2) thickening of the ventral margin of the quadratojugal (our char. 24.2). This redefinition of the R. margaritifera Group was not supported by the combined (i.e., molecular + morphological characters) phyloge- netic analysis of Pramuk (2006). Pramuk (2006) recovered two synapomorphies for the few exem- plar species of this group that she included: (1) the expansion of the posterior ramus of the pter- ygoid and (2) the occurrence of a lateral articula- tion between the nasals and the preorbital processes of the maxillae (homoplastic). She also found R. ocellata to be the sister species of the R. margaritifera Group. Subsequent studies dealing with the taxon- omy of this group (e.g., Fouquet et al., 2007a; Avila-Pires et al., 2010; Lavilla et al., 2013; Vaz-Silva et al., 2015; Avila et al., 2018) did not explicitly follow a definition based on synapo- morphies and thus its composition varied. Based on our results, we redefine the Rhinella margaritifera Group to include the species listed above that are now grouped exclusively on molecular evidence (although some charac- ter states may result in putative synapomor- phies, see Diagnosis above). Also, the characters proposed by Vélez-Rodriguez (2004) and Pramuk (2006) should be reevalu- ated considering relevant species not included in these studies (e.g., R. sternosignata, R. yunga, and Rhinella sp. 5) to understand their polarity in the context of our results. NO. 447 The lack of a precise type locality and refer- ence specimens, the large intraspecific (including sexual dimorphism) and interspecific variation in adult size and cranial crest shape and develop- ment, and the occurrence of sympatry among some species of the Rhinella margaritifera Group turned its taxonomy chaotic and confusing (Hoogmoed, 1989; 1990; Lavilla et al., 2013). A detailed revision of this complex species group is beyond the scope of the present study. As a result, we have been cautious to take taxonomic actions only when evidence is decisive. The nominal species of the group was described by Laurenti (1768) based on illustra- tions of Seba (1734) of a specimen from “Brasilia” as the type locality. The identity of this taxon remains unclear after more than two and a half centuries (see discussions in Hoog- moed, 1989; Vélez-Rodriguez, 2004; Fouquet et al., 2007a; Avila-Pires et al., 2010; Lavilla et al., 2013, 2017). Avila-Pires et al. (2010) desig- nated the specimen depicted in Seba (1734: pl. 71, figs. 6, 7) as the lectotype of Rana mar- garitifera Laurenti, 1768, and considered the species to be conspecific with Rhinella martyi Fouquet et al., 2007a. Subsequently, Lavilla et al. (2013) invalidated the lectotype designation by Avila-Pires et al. (2010) and, assuming that the type specimen of R. margaritifera was lost, designated and described a neotype for this species. More recently, Lavilla et al. (2017) noted that a previous publication (Milto and Barabanov, 2011) had reported the existence of the type of R. margaritifera, invalidating the neotype. Milto and Barabanov (2011) mentioned two specimens (ZISP 257.1 and 257.2) within the type series of R. margaritifera without additional comments. Photographs of both specimens are inadequate to determine which one was used in the illustration of Seba (1734; or if both were used) because both are adult females that fully agree with the description and illustrations. Con- sequently, it is reasonable to consider the speci- mens found by Milto and Baravanov (2011) to indeed be those used by Seba (1734) and to arbi- 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 67 trarily designate the specimen ZISP 257.1 as lec- totype of Rana margaritifera Laurenti, 1768. Additionally, we follow Avila-Pires et al. (2010) regarding the conspecificity of Rhinella margaritifera and R. martyi because the lectotype and paralectotype of R. margaritifera match almost all the characters used by Fouquet et al. (2007a) to differentiate R. martyi from other spe- cies of the group (heel extension with hind limbs adpressed and iris coloration unknown in the types of R. margaritifera). Thus, we consider Rhi- nella martyi Fouquet et al., 2007a, to be a junior synonym of R. margaritifera (Laurenti, 1768). Having established the identity of Rhinella margaritifera, we now introduce our results regarding this species group. An undescribed species from Pastaza (Ecuador), Rhinella sp. 5, is recovered with low support (JGC = 28%, JAF = 58%) as the sister taxon of all other species of the clade. The sister group of next most inclu- sive clade is poorly supported (JGC = 32%, JAF = 60%) and composed of two morphologically and geographically divergent species, R. yunga and R. ocellata. Rhinella yunga was recently described from the montane forest of the Selva Central, Peru. Distinctive characters used to diagnose this species in the original description are also present in the poorly known R. iserni (skin of dorsum mostly smooth, degree of development of cranial crest, and especially the absence of all the structures of the tympanic middle ear; Jiménez de la Espada, 1875; Moravec et al., 2014; Hoogmoed, personal com- mun.; J.M. and M.O.P., personal obs.). In addi- tion to their morphological resemblance, both species were described from nearby type locali- ties from the Peruvian Yungas region. Thus, we consider Rhinella yunga Moravec et al., 2014, to be a junior synonym of Rhinella iserni (Jiménez de la Espada, 1875). Rhinella magnussoni, R. cf. margaritifera from Amazonas (Colombia), specimens of “R. probos- cidea” from Ecuador and Peru, and an undeter- mined specimen of the R. margaritifera Group from Sao Pedro (Amazonas, Brazil) compose a well-supported clade (JGC = 93%, JAF = 94%). Rhinella magnussoni and R. cf. margaritifera from Amazonas (Colombia) have a relatively high UPD (2.10%), which seem to support the specific distinctiveness of the latter (Rhinella sp. 6). The results of the phylogenetic analysis (see fig. 14) and UPDs among clades (2.60%-10.27%; see table 11) strongly suggest that the specimen from Sao Pedro and both populations of “R. pro- boscidea” from Ecuador (Sucumbios) and Peru (Loreto) correspond to three undescribed species (Rhinella sp. 7-9). The similarity of these unde- scribed taxa with the phylogenetically distantly R. proboscidea s.s. and R. castaneotica (see above) indicates the need for a thorough revision of the “R. proboscidea” complex. Another well-supported clade (JGC and JAF = 99%) includes Rhinella acutirostris, R. alata, R. sclerocephala, R. sternosignata from Colombia, and several apparently undescribed species (see below). A nonmonophyletic Rhinella alata (sensu Santos et al., 2015) and R. sclerocephala constitute a well- supported subclade within this clade (JGC = 96%, JAF = 97%). The latter species was recovered as sister taxon of the Ecuadorian populations of R. alata with poor support (JGC = 61% JAF = 62%), and their UPDs are 1.31%-1.52%. Otherwise, the populations of Panama and Colombia were recoy- ered as a poorly supported lineage (JGC = 55%, JAF = 58%) with UPDs of 0.56%-1.52% with respect to its sister clade. Santos et al. (2015) did not find any morphological character or evident genetic differentiation between the populations from Panama and those from Ecuador as to con- sider them as different taxa. Otherwise, R. sclero- cephala was differentiated from R. alata by several characters such as its larger size, presence of well- developed cranial crests, vertebral apophysis, and bony knob (Mijares-Urrutia and Arends, 2001; Santos et al., 2015), although these characters vary extensively within species of the R. margaritifera Group and might be associated with particular environmental conditions over their areas of dis- tribution (see Kutrup et al., 2006; Bandeira et al., 2016). The absence of evident differences between specimens of both clades of R. alata, the nested position of R. sclerocephala within this clade, and 68 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY the low UPDs seem to support their conspecificity, but we refrain from synonymyzing both taxa due to the low support for the nested position of the specimen of R. sclerocephala and the several mor- phological differences. Nevertheless, it is evident that a detailed taxonomic revision of both taxa considering additional specimens and molecular evidence is required to better resolve the taxonomy within this clade. The other subclade includes Rhinella acutiros- tris and four undescribed species (see also ML topology in supplementary data 4.5): Rhinella sp. 10 from Napo (Ecuador), Rhinella sp. 11 from Amazonas (Brazil), Rhinella sp. 12 from Pando (Bolivia) and Madre de Dios (Peru), and Rhinella sp. 13 from some localities of Colombia (Boyaca and Caqueta), Peru (Loreto), and Venezuela (Miranda). This latter new species was previously recorded from Colombia as R. sternosignata (M.R., personal obs.; see comments for R. sternosignata). The nonmonophyletic Rhinella castaneotica and R. proboscidea compose a well-supported clade (JGC and JAF = 96%). The internal relation- ships among the included specimens are poorly resolved and the UPDs are 0.19%-2.75%. Within this clade, we could not obtain samples from the type locality of R. castaneotica (“near Cachoeira Jarua, Rio Xingu, Para, Brazil”), but we included sequences from a relatively close locality (300 km airline distance) that could represent R. castaneo- tica s.s. (see Fouquet et al., 2012a: fig. S6). We also included sequences of specimens from Manaus (Amazonas, Brazil) and Floresta (Roraima, Brazil) that could be morphologically assigned to R. pro- boscidea. However, the imprecise type locality of this species is “flumen Solimoens (= Rio Solim6es),” which comprises the Brazilian section of the Amazon River between the triple border of Brazil-Colombia-Peru and the city of Manaus and has an extension of approximately 1700 km (Van- zolini, 1981). Although we consider that phyloge- netic evidence, UPDs (see table 11), and absence of distinctive adult and larval differential charac- ters (see comparisons provided by Caldwell, 1991, and Menin et al., 2006) support their conspecific- NO. 447 ity, we continue recognizing both taxa pending a thorough revision, including additional samples and detailed comparison with the type material. The sister taxon of the clade including the problematic Rhinella castaneotica and R. probos- cidea, is well supported (JGC = 97%, JAF = 98%) and includes two successively diverging species (R. lescurei and R. hoogmoedi), and two sub- clades. One of these is well supported (JGC and JAF = 99%) and composed of R. paraguayensis, R. scitula, R. stanlaii, and two undescribed spe- cies: Rhinella sp. 14 from SE Peru (“Bufo sp. 6” sensu Vélez-Rodriguez, 2004b, and “Bufo cf. margaritifer 5” sensu Pramuk, 2006), and Rhi- nella sp. 15 from La Paz, Bolivia (which corre- sponds to Bufo sp. 1 of Lotters and Kohler, 2000). The included specimen of R. paraguayensis was recovered in a polytomy with the specimens of R. scitula; the UPDs among these specimens are low (0.13%-0.3% in the complete the 16S rRNA gene). These parapatric species were differenti- ated mainly by adult size, crest development, and skin texture. All these characters have been dem- onstrated to be subject to variation due to spe- cific environmental conditions throughout the distribution of some bufonids (see Kutrup et al., 2006; Bandeira et al., 2016). Thus, we consider Rhinella paraguayensis Avila et al., 2010, to be a junior synonym of R. scitula (Caramaschi and Niemeyer, 2003). The other subclade is also well supported (JGC and JAF = 98%) and includes Rhinella mar- garitifera s.s. and a poorly supported clade (JGC <25%, JAF <50%) composed of R. dapsilis, R. cf. dapsilis, R. gildae, and several divergent lineages of R. margaritifera s.l. (e.g., the lineages called Rhinella sp. A and Rhinella sp. B by Fouquet et al., 2007c). The clade includes specimens that vary in the degree of development of bony pro- trusions and cranial crests, dorsal coloration, occurrence of a developed proboscis, and dorsal skin texture (Myers and Carvalho, 1945; Dixon, 1976; Rodriguez and Duellman, 1994; Vaz-Silva et al., 2015; M.O.P., personal obs.). The UPDs within this clade are 0%-2.79% (mean UPD = 1.29%, see table 11). Thus, the absence of 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 69 unequivocal morphological differences and very low genetic distances indicate that Rhinella gildae Vaz-Silva et al., 2015, is a junior synonym of R. dapsilis (Myers and Carvalho, 1945). We could not obtain tissue samples of Rhi- nella cristinae to test its relationships. However, this species can be assigned to this group on the basis of the occurrence of the only putative phe- notypic synapomorphy of the group (acuminate anterior margins of nasals; char. 6.1) and a com- bination of characters typical of this group: (1) preorbital crest weak (char. 0.1), (2) medial ramus of the pterygoid fused and extending medially along approximately half the length of the parasphenoid ala (char. 21.2), (3) posterior lobe of the anterolateral process of hyoid absent (char. 25.0), (4) inguinal fat bodies absent (char. 51.0), and (5) tarsal fold absent (char. 65.0). HYBRIDIZATION, DEEP MITOCHONDRIAL DIVERGENCE, AND “GHOST INTROGRESSION” IN RHINELLA Reports on natural and artificial hybridization are well known in many bufonids including mul- tiple species of Rhinella (e.g., Blair, 1972; Green, 1996; Gergus et al., 1999; Malmos et al., 2001; Masta et al., 2002; Baldo and Basso, 2004; Yamazaki et al., 2008; Goebel et al., 2009; Fon- tenot etwals 2001; (Correa etaal., 2010%, 2013; Pereyra et al., 2016a; Betto-Colliard et al., 2018). Explosive breeding events with intense male competition for mates and passive female choice (i.e., scramble competition; see Wells, 2007; Pereyra et al., 2016b) is common in many species of several genera of Bufonidae, and premating isolating mechanisms seem to be insufficient to avoid interspecific amplexus in these species (see Blair, 1958; Guerra et al., 2011). Malone and Fontenot (2008) also demonstrated that bufonids require a substantial genetic divergence to achieve postzygotic reproductive isolation. Under this scenario, the common occurrence of hybridization in this family is not surprising. A particular situation of natural hybridiza- tion could happen in “Rhinella pombali” (Thomé et al., 2010, 2012), where all individuals of this taxon are considered hybrids between R. crucifer and R. ornata (Thomé et al., 2010, 2012) and our results are in agreement with this idea. The two included specimens of “R. pombali” in preliminary analyses were not recovered as monophyletic in the nuclear analysis and each of them has a unique mitochondrion (one from R. crucifer and the other from R. ornata). More- over, available evidence is insufficient to test whether “R. pombali” could represent a species of hybrid origin (see Avise, 2008; Darras and Aron, 2015, Lavanchy and Schwander, 2019) and more detailed studies are necessary to explore this possibility. Although we deliberately excluded the hybrid specimens from our analyses, the impact of natu- ral hybridization in Bufonidae could be currently underestimated due to the difficulties in recog- nizing hybrids and/or past hybridization events. Introgressive hybridization (both nuclear and mitochondrial) could have an impact on bufonid evolution allowing a faster accumulation of genetic novelties than through mutation alone. The incorporation of additional genetic diversity could impact the acquisition of adaptive pheno- typic traits and have a significant role in specia- tion as is common in diverse taxonomic groups (for reviews see Baack and Rieseberg, 2007; Schwenk et al., 2008; Toews and Brelsford, 2012; Abbott et al., 2016; Gopalakrishnan et al., 2018; Hill, 2019; Servedio and Hermisson, 2019). Mitochondrial introgressions are more com- monly reported than are nuclear introgressions and can be evidenced by genetic populational studies or by the discordance between phyloge- netic trees inferred from separate analyses of both genomes (Toews and Brelsford, 2012; Bonnet et al., 2017). Within Rhinella, putative events of mitochondrial introgression were documented for R. marina (Sequeira et al., 2011; but also see Val- linoto et al., 2017 and Bessa-Silva et al., 2020), R. bernardoi (Pereyra et al., 2016a), and R. horribilis s.l. (present study), and they occur without notice- able evidence of nuclear introgression, as was also reported in other vertebrates (Alves et al., 2006; Chen et al., 2009; Schwarzer et al., 2012). 70 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY TABLE 11 NO. 447 Percentage of uncorrected p-distances between 16S sequences among species of the Rhinella margaritifera Group Values reported are mean (range). 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Rhinella sp. 5 (N = 2) R. ocellata (N = 4) R. iserni (N = 4) R. magnussoni (N=1) Rhinella sp. 6 (N = 1) Rhinella sp. 7 (N = 1) Rhinella sp. 8 (N = 1) Rhinella sp. 9 (N = 3) R. sclerocephala (N= 1) “R. alata” (N = 5) Rhinella sp. 10 (N= 1) Rhinella sp. 11 (N= 1) Rhinella sp. 12 (N = 3) R. acutirostris (N = 5) Rhinella sp. 13 (N = 6) “R. castaneotica” (N = 4) R. proboscidea (N = 3) R. lescurei (N = 3) R. hoogmoedi (N = 4) Rhinella sp. 14 (N = 7) Rhinella sp. 15 (N = 1) R. scitula (N = 2) R. stanlaii (N = 4) R. margaritifera (N = 3) R. dapsilis (N = 15) 1 0.00 7.58 (6.71-8.99) 5.34 (5.29-5.39) 6.12 (6.08-6.16) 5.34 (5.29-5.39) 6.95 (6.91-6.98) 13.45 (13.42- 13.49) 5.87 (5.69-6.01) 7.81 (7.80-7.82) 5.75 (6.68- 6.94) 7.11 (7.07-7.14) 7Al (7.28-7.53) 7.90 (7.07- 9.31) 7.31 (6.90- 7.73) 6.81 (6.69- 7.09) 6.46 (5.69- 7.87) 6.94 (6.08- 8.61) 7.57 (6.47- 8.41) 7.03 (6.48- 8.33) 6.32 (6.09- 6.57) 5,93 (5.89-5.97) 6.12 (6.09- 6.16) 6.01 (5.71- 6.16) 5.99 (4.91- 6.52) 6.64 (5.69- 8.59) 0.49 (0.00-0.96) 4.63 (4.23-5.27) 6.11 (5.75-6.83) 4.95 (4.62-5.58) 7.97 (7.24-9.86) 15.55 (15.04- 16.39) 6.88 (6.40-8.04) 8.64 (7.99-9.84) 6.99 (8.42-) 5.76 (5.24-6.78) 6.12 (5.57-7.03) 6.14 (5.19- 7.08) 7.09 (5.97- 8.46) 6.55 (5.59- 7.92) 5.81 (4.79- ea) 6.53 (5.18- 8.21) 6.05 (4.80- 6.91) 6.33 (5.38- 7.40) 5.14 (4.22- 6.52) 5,86 (5.18-6.78) 6.48 (5.76- 7.55) 5.98 (5.19- 6.99) 6.22 (4.99- 7.13) 6.04 (4.80- 7.42) 3.97 11.65 3.33 9.66 2.77 (3.85-4.39) (3.25-3.82) (3.26-3.44) (2.61-2.90) 3 4 0.00 (0.00) 3.24 (3.24) i 3.44 (5.44) 2.10 4.58 (4.58) 250) 12.25 (12.25) The? 4.17 3.58 5.34 a 5.13 3.98 4.48 (3.68- (4.20- 4.21) 4.78) 4.45 (4.45) 5.21 4.78 rey 5.93 4.95 6.56 (4.40- (5.92- 5.83) 7.63) 4.99 6.20 (4.62- (5.76- 5.37) 6.51) 5.45 6.39 (5.19- (6.12- 5.83) 6.76) 3.47 4.59 (2.87- (4.01- 4.86) 5.92) 4.11 4.98 (3.25- (4.21- 5.33) 6.38) 5.79 6.03 (4.77- (5.15- 6.46) 6.50) 4.78 4.93 (4.40- (4.59- 5.57) 5.61) 4.06 4,46 (3.83- (4.21- 4.40) 4.83) 3.82 ee 4.39 4.01 4.58 (4.01) (4.58) 3.88 4.50 (3.64- (4.40- 4.01) 4.58) 4.63 4.01 (3.82- (3.44- 5.04) 4.30) 4.10 4.74 (3.63- (4.01- 5.06) 5.88) 6.46 4.90 (4.63- 5.15) 4.45 4.97 5.41 (4.79- 6.43) 5.83 (5.56- 6.14) 5.51 (5.18- 5.82) 4.33 (3.83- 5.45) 4.79 (3.83- 6.20) 5.44 (4.59- 6.02) 4.81 (4.40- 5.67) All (4.01- 4.27) 4.02 4.21 (4.21) 4.17 (4.08- 4.21) 4.47 (3.63- 4.89) 4.91 (4.09- 6.20) 6.94 6.11 (5.60- 6.42) 6.90 7.24 8.40 (7.26- 10.42) 8.00 (7.58- 8.42) 8.07 (7.78- 8.57) 5.66 (4.64- 7.68) 6.36 (4.81- 7.93) 8.67 (6.77- 9.93) 7.10 (6.40- 8.60) 5.90 (5.72- 6.25) 5.95 6.16 (6.16) 5.98 (5.17- 6.14) 5.74 (4.63- 6.30) 5.99 (5.10- 7.68) 7 8 9.93 (9.63- 0.66 10.29) —_ (0.58-0.80) 7.20 ee (6.85-7.51) 14.01 5.91 (13.86- (5.31- 14.19) 6.30) 6.09 pak (6.01-6.17) 15.17 oe (6.30-6.49) 15.87 7.00 (15.17- (6.40- 17.06) 8.09) 15.43 7.04 (15.02- (6.62- 15.58) 7.35) 15.58 7.29 (15.38- (6.98- 15.82) 7.72) 13.42 4.80 (12.65- (3.86- 14.94) 6.12) 13.91 5.64 (13.25- (4.47- 14.90) 7.12) 14.50 6.51 (13.58- (5.61- 15.17) 7.41) 13.99 6.25 (13.65- (5.50- 14.63) 7.39) 13.35 5.71 (13.03- (5.10- 13.42) 6.18) 5.35 a (5.09-5.53) 13.23 Pe (13.23) Oe 5.72) 12.81 5.41 (12:715 (4.88- 12.84) 5.72) 12.77 5.14 (12.25- (4.29- 13.03) 5.83) 13.40 5.77 (12.18- (4.90- 14.35) 7.12) 1.41 (1.28- 1.50) 3.82 4.46 4.64 (4.25- 5.19) 4.28 (4.11- 4.52) 4.77 (4.55- 5.20) 4.61 (4.08- 5.60) 4.69 (4.06- 5.53) 6.53 (5.74— 6.95) 5.34 (4.95- 6.09) 4.97 (4.70- 5.19) 4.71 4.71 (4.71) 4.64 (4.50- 4.69) 5.70 (4.95- 6.07) 5.10 (4.30- 6.40) 10 0.96 (0.57- 1.35) 3.21 (2.90-3.67) 3.63 (3.28-4.00) 3.74 (2.90- 5.14) 3.72 (3.26-) 4.13 (3.63- 4.85) 3.47 (2.70- 4.97) 3.79 (2.70- 4.91) 5.26 (4.39- 5.76) 4.24 (3.82- 5.17) 3.74 (3.43- 4.25) 3.48 (3.44-3.63) 3.56 (3.44— 3.63) 3.52 (3.29- 3.63) 4.45 (3.63- 4.91) 3.93 (3.05- 5.19) 11 0.78 1.36 (1.16- 1.55) 2.12 (1.93- 2.31) 2.83 (2.51- 3.09) 3.69 (3.28- 4.35) 4.05 (3.09- 5.11) 5.72 (4.82- 6.19) 4.98 (4.45- 5.87) 3.83 (3.47- 4.06) 3.47 3.86 (3.86) 3.43 (3.33- 3.47) 5.85 (4.82— 6.37) 4.92 (4.07- 6.40) 12 1.68 (1.53- 1.79) 2.88 (2.68- 3.07) 3.58 (3.26- 3.88) 4.02 (3.65- 4.60) 4.46 (3.45- 5.37) 5.99 (5.16- 6.44) 5.26 (4.79- 6.12) 4.18 (3.81- 4.45) 3.82 4.20 (4.20) 3.78 (3.68- 3.82) 6.13 (5.16- 6.62) 5.21 (4.27- 6.65) 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) TABLE 11 continued 13 14 15 16 17 18 19 20 21 22 23 24 25 Rhinella sp. 12 (N = 3) R. acutirostris (N = 5) Rhinella sp. 13 (N = 6) “R. castaneotica” (N = 4) R. proboscidea (N = 3) R. lescurei (N = 3) R. hoogmoedi (N = 4) Rhinella sp. 14 (N = 7) Rhinella sp. 15 (N = 1) R. scitula (N = 2) R. stanlaii (N = 4) R. margaritifera (N = 3) R. dapsilis (N = 15) 13 0.06 (0.00- 0.19) 2.86 (2.29- 3.66) 3.63 (2.86- 4.70) 4.40 (3.64- 5.34) 4.91 (3.44- 5.90) 5.99 (4.77- 6.46) 5.80 (4.79- 6.68) 4.90 (4.20- 6.15) 4.76 (4.20- 5.65) 4.75 (4.20- 5.65) 471 (4.07- 5.62) 6.54 (5.15- 6.94) 5.80 (4.58- 7.10) 14 0.69 (0.19- 1.15) 1.67 (1.34- 2.13) 4.85 (4.23- 5.79) 4.99 (4.02- 6.26) 6.50 (5.35- 7.09) 5.56 (4.97- 6.77) 4.46 (4.02- 5.04) 4.55 (4.40- 4.78) 4.55 (4.40- 4.78) 4.53 (4.28- 4.79) 6.63 (5.36- 7.27) 5.67 (4.78- 7.29) 15 0.49 (0.00- 0.77) 5.09 (4.23- 6.58) 5.20 (4.03- 6.80) 6.09 (4.96- 7.10) 5.31 (4.60- 6.78) 4.12 (3.63- 4,46) 4.66 (4.40- 5.03) 4.66 (4.40- 5.02) 4.62 (4.28- 5.04) 6.37 (4.97- 7.27) 5.65 (4.78- 7.54) 16 1.19 (0.19- 2.28) 1.82 (0.76- 2.83) 4.16 (3.24- 4.90) 3.30 (2.57- 4.43) 3.51 (2.29- 4.91) 2.91 (2.29- 4.39) 3.01 (2.48- 4.40) 3.25 @7i- 4.37) 4.00 (2.86- 5.20) 3.60 (2.29- 6.04) 17 1.31 (0.98- 1.78) 4.80 (3.82- 5.20) 3.92 (3.25- 4.38) 3.77 (2.87- 4.86) 3.28 (2.86- 3.82) 3.22 (2.87- 3.84) 3.48 (2,79- 4.33) 4.63 (3.44- 5.15) 431 (3.25- 5.47) 18 0.85 (0.00- 1.28) 2.90 (2.49- 3.17) 3.48 (2.48- 4.14) 3.83 (3.25- 4.14) 3.61 (3.06- 3.90) 3.82 (3.05- 4.36) 4.07 (3.25- 4.27) 3.45 (2.48- 4.50) 19 0.46 (0.00- 0.79) 2.39 (1.53- 2.82) 2.48 (2.29- 2.78) 2.28 (2.10- 2.54) 2.65 (2.33- 3.04) 3.04 (2.48- 3,33) 2.49 (1.72- 3.42) 20 0.57 (0.00- 0.97) 1.50 (1.15- 1.74) 2.24 (7 2= 2.51) 1.79 (1.55- 2.33) 2.83 (2.10- 3.32) 2.72 (1.53- 3.88) 21 1.15 (1.15) 1.01 (0.78- 1.34) 3.49 (2.86- 3.80) 2.90 (2.29- 3.56) 22 0.00 (0.00) 0.96 (0.78- 1.15) 3.72 (3.05- 4.05) 2.98 (2.29- 3.84) 23 0.42 (0.00- 0.77) 3.54 (2.68- 4.02) 3.19 (2.30- 4,33) 24 24 0.00 (0.00) 2.31 1.22 (1.33- (0.00- 3.14) 2.60) 72 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY Several populations of Rhinella marina from south of the Amazon River seem to have similar mitochondrial lineages as R. diptycha, in contrast to populations northward. As nuclear loci of spec- imens of both populations of R. marina were similar, and divergent from R. diptycha, the occur- rence of an extensive mtDNA unidirectional introgression from R. diptycha into R. marina was hypothesized (Sequeira et al., 2011). However, this hypothesis was not conclusively corroborated in a subsequent study because an additional mito- chondrial clade, found for some populations of R. marina, obscured the direction of the introgres- sion between these species (Vallinoto et al., 2017). A similar situation of possible unidirectional mitochondrial introgression from R. dorbignyi to R. bernardoi was reported by Pereyra et al. (2016a). Evidence that supports this hypothesis comes from the well-supported incongruence between the independent analyses of the mitochondrial and nuclear genes: R. bernardoi is deeply nested within R. dorbignyi in the mitochondrial analysis, but not in the nuclear analysis. Our results from independent mitochondrial and nuclear analyses (rMD and rND, respec- tively) also show incongruence in the position of the specimens of Rhinella horribilis s.1. We recover this species deeply nested within (mor- phologically similar) species of the R. marina Group in the rND analysis, whereas in the rMD analysis it is recovered as sister of all the species of the R. crucifer + R. marina Groups. Another striking characteristic of this case of hybridiza- tion is the origin of these mitochondria, which is not traceable to any known extant species. These particular forms of deep mitochondrial divergence were denominated “ghost introgres- sions’ (see Zhang et al., 2019). This kind of event involving deep mitochondrial divergence that implies past mitochondrial introgression from an unknown and not closely related spe- cies is uncommon in anurans. Historical inter- specific introgressions events were reported in several groups of Anura: Ameerega (Dendro- batidae; Brown and Twomey, 2009); Anaxyrus, Bufo, and Bufotes (Bufonidae; Malmos et al., NO. 447 2001; Yamazaki et al., 2008; Colliard et al., 2010; Dufresnes et al., 2019); Bombina (Bombinato- ridae; Hofman and Szymura, 2007; De Cahsan et al., 2019); Dyscophus (Microhylidae; Orozco- terWengel et al., 2013); Hyla (Hylidae; Lamb and Avise, 1986; Bryson et al., 2010, 2014; Klymus et al., 2010); Mantella (Mantellidae; Crottini et al., 2019); Pelophylax and Rana (Ranidae; Liu et al., 2010; Zhou et al., 2012; Eto et al., 2013); Quasipaa (Dicroglossidae; Zhang et al., 2018); and Scutiger (Megophryidae; Chen et al., 2009). However, most of these events (except in Bombina, Bufotes, Quasipaa, and Scutiger) occurred among closely related species. Another striking characteristic of this phenomenon in Rhinella horribilis s.l. is that after the ancient introgression, the GIM (i.e., the mitochondrial DNA) diversified into two divergent clades (UPDs >3.33%). We consider most plausible the hypothesis that these mito- chondrial clades represent two different species (R. horribilis s.s. and Rhinella sp. 1) that are not fully detectable (e.g., recovered as monophyletic) with our limited nuclear dataset. More intense genomic and phylogeographic sampling will be necessary to eventually solve the taxonomic sta- tus and puzzling history of R. horribilis and its lineages. The reports of hybridization and mitochon- drial introgression in Rhinella suggest the need for an extensive and careful exploration of these phenomena in other lineages of Bufonidae. The particular reproductive biology (i.e., scramble competition), the occurrence of broad sympatric areas between related species, and genetic fea- tures (i.e., complete reproductive isolation with relatively high levels of genetic divergence; Malone and Fontenot, 2008) of many bufonids may facilitate the occurrence of these phenom- ena. The identification of foreign mitochondrial genomes is particularly relevant to avoid errors both in phylogeographic and taxonomic studies (especially DNA barcoding studies) and phyloge- netic inferences (Ballard and Whitlock, 2004; Alves et al., 2006; Obertegger et al., 2018; Barley et al., 2019). Moreover, the identification of mitochondrial introgressions will serve, among 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 73 other things, as a base for future studies on adap- tive coevolution between these organelles and the nuclear components of the oxidative metabo- lism of the cell (Hill, 2019). COMMENTS ON THE PHENOTYPIC EVIDENCE CONSIDERED FOR RHINELLA Our phenotypic sampling results in some syn- apomorphic and/or diagnostic characters for several internal clades of Rhinella, including most of the species groups. However, an evalua- tion of the available information for the charac- ter systems used clearly shows large gaps in the knowledge within each species group/clades (see fig. 15). In general, there is relatively more informa- tion available for species of the Rhinella marina Clade. Within the R. margaritifera Clade, char- acters on adult osteology and musculature, natu- ral history, and larval morphology are poorly known, and characters of larval chondrocra- nium, cytogenetics, and embryonic morphology are virtually unknown. This is a source of ambi- guity in the reconstruction of ancestral character states for many characters that optimize in more inclusive nodes (e.g., oviposition mode within the R. festae and R. veraguensis Groups). With the exceptions of foot and hand muscu- lature, external larval, and embryonic morpholo- gies described for several species of Rhinella (e.g., Mercés et al., 2009; Tolledo and Toledo, 2010; Blotto et al., 2014; Vera Candioti et al., 2016, 2020; Grosso et al., 2020; B.L.B., personal obs.), detailed descriptions considering ontoge- netic variation, sexual dimorphism, and inter- population variations are still largely necessary. It must be noticed that these and many other character systems are promising as additional sources of evidence to be included in future phy- logenetic analyses. Some examples of variation within species of Rhinella were reported on bio- acoustics (W.F. Martin, 1972; De la Riva et al., 1996; Guerra et al., 2011; Andrade et al., 2015; Valencia-Zuleta et al. 2020); integument and parotoid macroglands structure (O’Donohoe et al., 2019); anatomy of urogenital and digestive systems (Stohler, 1932; Lynch and Renjifo, 1990; but see Grant, 2000); clutch and egg size (Liedtke et al., 2014; Pereyra et al., 2015); mandibular, pel- vic, and thigh musculature (Noble, 1922; Lime- ses, 1964, 1965; Trueb, 1971; Winokur and Hillyard, 1992; Grant and Bolivar-G., 2014); and secretions (Cei et al., 1968; Maciel et al., 2006; Rodriguez et al., 2017). An inclusive sampling considering all these characters will contribute to the study of patterns of evolution of different character systems and their functional and adap- tive significance. THE FossIL RECORD OF RHINELLA AND CALI- BRATION POINTS As is common for most neobatrachian anuran families, the pre-Pleistocene fossil record of Bufoni- dae is deficient, and most of these specimens lack an apomorphy-based diagnosis to unambiguously assign them to particular nodes or species (see Par- ham et al., 2012). The currently known pre-Pleisto- cene fossils of Rhinella are phylogenetically concentrated within the R. marina Group: (1) R. arenarum (as R. pisanoi) from Pliocene outcrops (3.9-3.2 Ma) of coastal Buenos Aires Province, Argentina (Casamiquela, 1967; Pérez-Ben et al., 2014); (2) R. loba, an extinct species putatively related to R. arenarum, from the late Pliocene (4.5- 3.2 Ma) from the Chapadmalal Formation of Argentina (Pérez-Ben et al., 2019); (3) R. marina from the mid Miocene (13.8-11.8 Ma) from La Venta fauna of Colombia (Estes and Wassersug, 1963); and (4) R. aff. arenarum and Rhinella sp. marina Group from the upper Oligocene (29-26 Ma) of Salla, Bolivia (Baez and Nicoli, 2004). Another fossil from the upper Paleocene (59.2-56 Ma) from Itaborai, Brazil (Estes, 1970) was also considered as related to some of the South Ameri- can species groups of the former Bufo, but all pro- posed associations are vague and tentative (see Estes, 1970; Estes and Reig, 1973; Baez and Gaspa- rini, 1977); even an association with Rhinella is controversial. Only the Miocene specimen of R. marina has been used as a calibration point (along with few other non-Rhinella bufonid fossils) in divergence dating analyses of Bufonidae or its inter- 74 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 fo) ae Sk WSR qr? 0 atacamensis arunco rubropunctata vellardi cf. amabilis limensis gallardoi achalensis papillosa altiperuviana R. ARUNCO GROUP R. SPINULOSA GROUP spinulosa trifolium bernadoi dorbignyi pygmaea major AADVANVANADVANVAADVANA R. GRANULOSA R. mirandaribeiroi GROUP R. merianae “R. humbolati” centralis beebei henseli casconi crucifer inopina ornata poeppigii veredas marina diptycha horribilis AqV1O VNIAVA 'Y R. CRUCIFER GROUP R. MARINA arenarum GROUP achavali “R. icterica” R. cerradensis R. rubescens sternosignata inca leptoscelis fissipes rumbolli justinianoi veraguensis quechua manu nesiotes tacana lilyrodriguezae yanachaga chavin cf. multiverrucosa arborescandens festae paraguas cf. nicefori ruizi lindae acrolopha tenrec macrorhina ocellata R. iserni R. magnussoni “R. alata” R. sclerocephala R. acutirostris R. proboscidea “R. castaneotica” R. lescurei R. hoogmoedi R. margaritifera R. dapsilis R. stanlaii R. scitula DADADNVADAADNAAA R. VERAGUENSIS GRoupP R. FESTAE GROUP DNDANDADANADNANDADNADNNANDADNANAA AQV1O VYssILINVOYVN “Y R. MARGARITIFERA GROUP NNSENN INNNNNNSNSN NNN NNN NNN NNN _ NS NeENee +, ie | See ee eee ee ee ee eee eee ee ee | hee | ls SSSR SERS SS Resse E Es See SEs G SSS NGS SES See eo NERS sees Bee ee _ eee _| eee See | ee | |) lhe 2h he ee ee eee eee eee See ee le ee ees eee |) he ee 6 lL EN_NNNS SER NeNNNEE NNN NN Ne |__| eee Ne FIG. 15. Main character systems scored for each included species of Rhinella. References: Filled squares, most characters of the considered character system scored (> 50%); half-filled squares, most characters not scored (<40%); empty squares, no character scored. Abbreviations: Ad. ext. morphol., Adult external morphology; Ad. musc., Adult musculature; Ad. osteol., Adult osteology; Emb. morphol., Embryonic morphology; Larv. chondrocr., Larval chondrocranium; Larv. morphol., Larval external morphology; Nat. hist., Natural history. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 75 nal clades (e.g., Pramuk et al., 2008; Maciel et al., 2010; van Bocxlaer et al., 2010; Liedtke et al., 2016, 2017). These studies differ in the sampled taxa and genes, and their results are not fully congruent, but the divergence-time estimates, considering relaxed molecular clocks and similar calibration points, indicate a split time between Rhinella and Anaxyrus + Incilius around 41 Ma (34—47 Ma; Pramuk et al., 2008) and 38.7 Ma (28.5-51.8; van Bocxlaer et al., 2010). However, the absence of an understanding of the interspecific osteological variation in species of Rhinella and the absence of an apomorphy-based determination of the fossils could result in the asso- ciation of fossils to a lower-level taxon than the data can demonstrate (see Bever, 2005; Parham et al., 2012). Consequently, a critical reexamination of the available pre-Pleistocene fossils of Rhinella, along with an extensive study of living species of all the species groups is necessary before their defensible use as calibration points in divergence dating analy- ses. If material from Itaborai can be unambiguously associated with Rhinella, its inclusion will provide a crucial point of calibration that could modify extensively our current understanding of the pat- terns of diversification of Rhinella and also of Bufonidae. CONCLUSIONS Our results provide a general framework for bet- ter understanding the evolution and taxonomy of Rhinella and its internal clades. The main results of our work include: (1) the generation of a well-sup- ported phylogenetic hypothesis of the genus result- ing from a total evidence analysis of most of its specific diversity, (2) the redefinition and morpho- logical diagnosis of its species groups, (3) the dem- onstration of hybridization and mitochondrial introgression between some species, and (4) evalu- ation of the taxonomic status of several species. Nevertheless, many challenges are still pending. For example: (1) the taxonomic revision of many clades, including the designation of neotypes for several taxa; (2) the evaluation of the ontogenetic and intersexual variation in several problematic taxa; (3) the use of denser gene sampling (with high throughput sequencing) to better understand the evolutionary relationships in poorly supported clades and evaluate the role of the introgressive hybridization in the evolution of some lineages of Rhinella; and (4) the incorporation of more pheno- typic characters to better understand their evolu- tion in this group and define many morphologically and ecologically diverse clades of the genus. Future studies addressing these problems would result in crucial contributions in the knowledge of the diver- sity of Rhinella. ACKNOWLEDGMENTS We sincerely thank José M. Padial (Bronx Com- munity College, New York); Thelia M. Céspedes Alejabo (CINBIOTYC, Peru); Guarino Colli and Helga Wiederhecker (Colecao Herpetoldgica da Universidade de Brasilia, Brazil); Jimena Grosso (Fundacion Miguel Lillo, Argentina); Juan M. Daza (Grupo Herpetoldgico de Antioquia, Colombia); Diego A. Barrasso and Leonardo Cotichelli (Insti- tuto de Diversidad y Evolucién Austral, Centro Nacional Patagénico, Argentina); Albertina P. Lima (Instituto Nacional de Pesquisas da Amazonia, Bra- zil); Antoine Fouquet (Laboratoire Evolution & Diversité Biologique, Université Toulouse, France); Juan M. Boeris, Andrés E. Brunetti, Dario E. Car- dozo, and Juan M. Ferro (Laboratorio de Genética Evolutiva, Instituto de Biologia Subtropical, Argen- tina); Sebastian Barrionuevo, Nadia G. Cervino, Laura Nicoli, and Santiago J. Nenda (Museo Argen- tino de Ciencias Naturales, Argentina); César Agui- lar (Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Pert); Bea- triz Alvarez Dorda and Isabel Rey Fraile (Museo Nacional de Ciencias Naturales, Spain); personnel of the herpetological collection of Museo de Biodi- versidad (MUBI, Pert); Andrés Pansonato and Tiago Pezzuti (Universidade Federal de Minas Gerais, Brazil); Francisco Brusquetti, Ariadne Fares Sabbag, and Mariana Lyra (Universidad Estadual Paulista, Brazil); and Pedro H. Dias, Juliana Jordao, Julia S. Parreiras, Paulo D.P. Pinheiro, and Miguel Rodrigues (Universidade de Sao Paulo, Brazil) for sharing with us specimens, tissue samples and/or 76 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY DNA sequences. For access to collections and spec- imen loans we thank Ariane Silva and Fernanda Werneck (Instituto Nacional de Pesquisas da Ama- zonia, Brazil); Rafe Brown, Richard Glor, Linda Trueb, and Luke Welton (Kansas University, Law- rence); Marta Calvo (Museo Nacional de Ciencias Naturales, Spain); Aline Staskowian Benetti (Museu de Zoologia da Universidade de Sao Paulo, Brazil); Carol Spencer and Natasha Stepanova (Museum of Vertebrate Zoology, Berkeley, California); and Eric Smith (University of Texas at Arlington). Pedro H. Dias; Ana Duport Bru, M. Laura Ponssa, and M. Florencia Vera Candioti (Fundacién Miguel Lillo, Argentina); and Marinus S. Hoogmoed (Museu Paraense Emilio Goeldi, Brazil), contributed with valuable information on taxonomy or morphology of several specimens. Michael Jowers (Porto Uni- versity) helped us to clarify the identity of some sequences of R. beebei and R. humboldti. M. Jimena Gomez Fernandez, M. Daniela Pereyra, and Araceli Seiffe (Museo Argentino de Ciencias Naturales, Argentina) provided technical laboratory assis- tance. Several colleagues kindly shared with us photographs and/or data of species of Rhinella: César L. Barrio-Amoros (R. sternosignata), Michelle Castellanos (R. sclerocephala), Andrés Cecconi (R. diptycha), Pedro H. Dias (R. acutirostris), José Gerardo Espinoza (Rhinella sp. 14), Peter Janzen (R. crucifer), Konstantin D. Milto (type material of R. margaritifera), Arturo Mufioz (R. quechua), Roberto L.M. Novaes (R. icterica), Mirco Solé (R. hoogmoedi), Mauro Texeira Jr. (R. ornata), Rodrigo Tinoco (R. ocellata), Vicente Valdés Guzman (R. atacamensis), and Mario Yafiez-Munoz (R. cf. roqueana). David Blackburn, contributors, and the staff of Morphosource (Duke Library Digital Repository) very kindly made available CT images of relevant species. TNT was provided free by the Willi Hennig Society. We thank Consejo Nacional de Investigaciones Cientificas y Técnicas (CONI- CET), ANPCyT, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo for financial support: PIP 11220110100889; PICT 2013-0404, 2015-0813, 2015-0820, 2015-2381, 2017-2437, and 2018-3349; and grants #2012/10000-5, #2013/20423-3, #2013/50741-7, #2014/03585-2, #2015/11237-7, NO. 447 #2016/25070-0, and #2018/15425-0 from Sao Paulo Research Foundation (FAPESP). M.R. was sup- ported by a PNPD post-doctoral fellowship from the Brazilian Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES grant 2016.1.263.41.6). J.M. thanks Ministry of Culture of the Czech Republic (grant DKRVO 2021/6.VIL.c National Museum Prague, 00023272). S.C.E was supported by CNPq (grant 312744/ 2017-0) and the PrInt program of Coordenacao de Aperfeicoa- mento de Pessoal de Nivel Superior, CAPES, Brazil (grant 88887.508359/2020-00). T.G. was supported by research fellowships from the Brazilian Con- selho Nacional de Desenvolvimento Cientifico e Tecnoldgico (CNPq grant 306823/2017-9). C.F.B.H. thanks CNPq for a research fellowship (grant 306623/2018-8). The Centro de Estudos de Insetos Sociais, I.B., UNESP, Rio Claro allowed access to its molecular laboratory facilities for the production of some sequences used in this study. Field and labo- ratory work in Ecuador were funded by Secretaria Nacional de Educacién Superior, Ciencia, Tec- nologia e Innovacion del Ecuador SENESCYT (Arca de Noé initiative; $.R.R. and Omar Torres principal investigators) and grants from Pontificia Universidad Catolica del Ecuador, Direccioén Gen- eral Académica. B.L.B. acknowledges Esteban O. Lavilla for his support during initial studies on the R. spinulosa Group. We greatly appreciate the criti- cal reviews of the manuscript by Aaron Bauer and Joseph Mendelson. REFERENCES Abbott, R.J., N.H. Barton, and J.M. Good. 2016. 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Speciation in the Rana chensi- nensis species complex and its relationship to the uplift of the Qinghai-Tibetan Plateau. Molecular Ecology 21: 960-973. 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 97 APPENDIX 1 LOCALITY DATA OF VOUCHER SPECIMENS AND SOURCES OF THE SEQUENCES Collection abbreviations are as follow: AG, Anna Goebel field series; AACRG, African Amphib- ian Conservation Research Group, North-West University, Potchefstroom, South Africa; AF, Antoine Fouquet field series; AJC, Andrew J. Crawford field series; AML, Alejandro Montoya L. field series; AMNH, American Museum of Natural History, New York; AMNH-FS, American Museum of Natural History field series, New York; ANDES, Museo de Historia Natural Andes, Universidad de los Andes, Bogota, Colombia; APL, Albertina P. Lima field series; AR, Alexander Robertson field series; BB, Boris Blotto field series; to be accessioned in MACN; BM, Michel Blanc field series; BMNH, Natural History Museum, London, UK; CAS, California Academy of Sciences, San Francisco, California; CBA, César Barrio-Amoros field series; CFBH, Collection Célio EB. Haddad, Universidade Estadual Pau- lista, Rio Claro, Sao Paulo, Brazil; CFBH-T, Célio EB. Haddad tissue collection, Universidade Estadual Paulista, Rio Claro, Sao Paulo, Brazil; CH, Circulo Herpetologico de Panama, Panama, Panama; CHUNB, Colecao Herpetolégica da Universidade de Brasilia, Brasilia, Brazil; CORBIDI, Centro de Ornitologia y Biodiversidad, Lima, Peru; CTGA-UFAM, tissues collection of Universidade Federal do Amazonas, Manaus, Amazonas, Brazil; CZUT, Coleccién Zooldégica, Facultad de Ciencias, Universi- dad del Tolima, Tolima, Colombia; DCC, David C. Cannatella field series; DPL, Dwight P. Lawson field series; ESTR, locality code; (Miguel Trefaut Rodrigues field series) FML, Fundacion Miguel Lillo, San Miguel de Tucuman, Argentina; FMNH, Field Museum, Chicago, IL; ICN, Universidad Nacional de Colombia, Instituto de Ciencias Naturales, Museo de Historia Natural, Bogota, Colombia; IDLR, Ignacio de la Riva field series; IIBP, Instituto de Investigacién Bioldgica del Paraguay, Asuncion; I[WU, Illinois Wesleyan University, Bloomington, IN; IZUA, Instituto de Zoologia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile; JMP, José M. Padial field series; KMH, Kim M. Howell field series; KRL, Karen R. Lips field series; KU, University of Kansas Natural History Museum, Law- rence, Kansas, KS; LAJ, locality code; LGE, Laboratorio de Genética Evolutiva, Universidad Nacional de Misiones, Argentina; LSUMZ, Louisiana State University, Museum of Natural Science, Baton Rouge, Louisiana, LA; MACN, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia’- CONICET, Buenos Aires, Argentina, MAR, Marco A. Rada field series; MC, Christian Marty field series; MCZ, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, MA; MHNLS, Museo de Historia Natural La Salle, Fundacion La Salle de Ciencias Naturales, Caracas, Venezuela; MHUA, Museo de Herpetologia de la Universidad de Antioquia, Medellin, Colombia; MJH, Martin J. Henzl field series; MNCN, Museo Nacional de Ciencias Naturales, Madrid, Spain; MNCN-ADN, Museo Nacional de Ciencias Naturales tissue collection, Madrid, Spain; MNHN-Fr, Muséum national d'Histoire naturelle, Paris, France; MNHN-Uy, Museo Nacional de Historia Natural, Montevideo, Uruguay; MNRJ, Museu Nacional do Rio de Janeiro, Rio de Janeiro, Brazil; MTD, Senck- enberg Naturhistorische Sammlungen Dresden, Dresden, Germany; MTR, Miguel Trefaut Rodrigues field series; MTSN, Museo Tridentino di Scienze Naturali, Trento, Italy; MUBI, Museo de Biodivers- idad del Peru, Cusco, Peru; MUSM, Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru; MVUP, Museo de Vertebrados, Universidad de Panama, Panama, Panama; MVZ, University of California, Museum of Vertebrate Zoology, Berkeley, CA; MW, Mark Wilkinson field series; MZUSP, Museu de Zoologia, Universidade de Sao Paulo, Sao Paulo, Brazil; NB, Néstor Basso field series; MNK, Museo de Historia Natural “Noel Kempff Mercado’, Santa Cruz de la Sierra, Bolivia; NMP, Narodni muzeum National Museum, Prague, Czech Republic; NP, Nikolai Poyarkov field series; 98 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 447 PD, Pedro Dias field series; PG, Philippe Gaucher field series; QCAZ, Museo de Zoologia, Pontificia Universidad Catélica del Ecuador, Quito, Pichincha, Ecuador; RGP, Roberto Gutierrez Poblete field series; ROM, Royal Ontario Museum, Department of Natural History, Toronto, Canada; SBH, S. Blair Hedges field series; SMF, Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt am Main, Germany; TG, Taran Grant field series; TWR, Tod W. Reeder field series; UFMT, Universidade Fed- eral de Mato Grosso, Cuiaba, Mato Grosso, Brazil; UFRGS, Universidade Federal do Rio Grande do Sul, Departamento de Zoologia, Rio Grande do Sul, Brazil; UNSJ, Universidad Nacional de San Juan, San Juan, Argentina; URCA, Universidade Regional do Cariri, Crato, Ceara, Brazil; USNM, National Museum of Natural History, Smithsonian Institution, Washington DC; UTA, University of Texas at Arlington Department of Biology, Arlington, TX; UWIZM, University of the West Indies, Zoology Museum, Department of Life Sciences, St. Augustine, Saint George, Trinidad and Tobago; VG, Vaclav Gvozdik field series; VUB, Vrije Universiteit Brussel, Belgium; ZUEC, Museu de Historia Natural, Universidade Estadual de Campinas, Campinas, Brazil; ZUFG, Universidade Federal de Goias, Goia- nia, state of Goias, Brazil; and ZVC, Coleccién de Zoologia Vertebrados de la Facultad de Ciencias, Montevideo, Uruguay. Abbreviations: nd, no data; nv, no voucher specimen; —, no change in the taxonomy of the species. RHINELLA Current taxonomy Updated taxonomy Voucher Locality Sources CEBH 18141 Brazil: Parana: Quatro ‘This study; Thome et Barras al., 2010 Brazil: ina: R. abei R. ornata MACN 46672 soe arc a This study Garopaba MZUSP 128425 Brazil: Fane Wenc- This study; Thomé et eslau Bras al., 2010 A ina: Luis: R. achalensis — MACN 52406 ees eer This study La Carolina Uruguay: Treinta y MNHN-Uy 9301 Tres: Quebrada de los ‘This study Cuervos R. achavali — Uruguay: Treinta y ZVC 3801 Tres: Estancia Dona Vallinoto et al., 2010 Alba 1 ia: Choco: MAR 1425 oY amie oe This study Unguia R. acrolopha — Behe Canes MAR 1426 ieee ei This study Unguia CORBIDI 4635 Peru: Loreto: Andoas _This study MTR 36593 ae Amazonas: Rio hig Study: Brazil: Amazonas: Rio ; R. acutirostris “R. acutirostris” MTR 36684 Icd This study Ecuador: Orellana: Parque Nacional Yas- Pramuk, 2006; Pramuk uni, Estacién Cienti- et al., 2008 fica Yasuni PUCE QCAZ 10601 2021 APPENDIX 1 continued RHINELLA Current taxonomy R. acutirostris R. alata R. amabilis R. amboroensis R. arborescandens R. arenarum R. arenarum arenarum R. arenarum men- docina R. arequipensis R. arunco Updated taxonomy “R. acutirostris” “R. alata” R. cf. amabilis R. quechua R. arenarum R. arenarum R. spinulosa Voucher QCAZ 28379 CH 9192 MAR 2574 MHUA 8415 QCAZ 11597 QCAZ 13896 QCAZ 68471 MNK 5302 CORBIDI 2020 MUBI 14076 MUBI 14082 AR 305 MNHN-Uy 9935 MACN 38639 MNCN-ADN 5972 MACN 49141 KU 214792 LGE 2516 KU 217369 Locality Ecuador: Sucumbios: Reserva de Produccion Faunistica Cuyabeno, Playas de Cuyabeno Panama: Coldn: Parque Nacional Soberania Colombia: Tolima: Rioblanco Colombia: Antioquia: Sonsén Ecuador: Esmeraldas: Bosque Protector La Chiquita Ecuador: Canar: Manta Real Peru: Cajamarca Bolivia: Santa Cruz: Parque Nacional Amboré Peru: Amazonas: Bagua, Cataratas de Camfopite Peru: Amazonas: Bon- gara Peru: Amazonas: Bon- gara Argentina Uruguay: Cerro Largo: Laguna Merin Argentina: San Luis: Lomas Blancas Bolivia: Tarija: Reserva Tariquia Argentina: Mendoza: Tunuyan Peru: Arequipa: Zama- cola, Cerro Colorado Peru: Arequipa: Canon del Colca Chile: Santiago: Run- gue PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 99 Sources This study Santos et al., 2015 This study This study Pramuk, 2006; Pramuk et al., 2008 Pramuk, 2006; Pramuk et al., 2008 This study Frost et al., 2006 This study This study This study Pramuk, 2006; Pramuk et al., 2008 This study This study; Faivovich et al., 2005; Frost et al., 2006 This study This study Pramuk, 2006; Pramuk et al., 2008 This study Pramuk, 2006; Pramuk et al., 2008 100 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. atacamensis R. azarai R. beebei R. bergi R. bernardoi R. casconi R. castaneotica R. cf. castaneotica Updated taxonomy rs) . castaneotica” Voucher AMNH 168401 RU 217352 LGE 8710 LGE 8711 CBA 5732 ICN 55776 ICN 55784 nv UWIZM 2012.27.72.3 LGE 8723 MACN 46555 FML 23921 UNSJ 5046 CFBH 22863 CFBH 22865 LSUMZ 17429 NMP6V 74261 BM 131 ZUFG 8171 Locality n/d Chile: Coquimbo: Cuesta Pajonales Argentina: Misiones: Candelaria Argentina: Misiones: Candelaria Venezuela: Bolivar: Chivaton, Gran Sabana Colombia: Casanare: Paz de Ariporo, Vereda La Colombina, Finca El Porvenir Colombia: Casanare: Trinidad, Vereda La Canada, Finca La Pal- mita Venezuela: Amazonas: Puerto Ayacucho Trinidad and Tobago: Trinidad: Trincity Central Road Argentina: Formosa: Pilcomayo, Palma Sola Argentina: Chaco: San Fernando Argentina: San Juan: Parque Provincial Ischigualasto Argentina: San Juan: Caucete Brazil: Ceara: Guaramiranga Brazil: Ceara: Guaramiranga Brazil: Para: 100 km S Santarém Bolivia: Pando, Fed- erico Roman: Santa Crucito French Guiana: Mataroni Brazil: Acre: Boca do Acre NO. 447 Sources Frost et al., 2006 Pramuk, 2006; Pramuk et al., 2008 Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a Murphy et al., 2017 Murphy et al., 2017 Pereyra et al., 2016a Murphy et al., 2017 Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a This study This study Pramuk, 2006; Pramuk et al., 2008 Moravec et al., 2014 Fouquet et al., 2007c This study 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 101 APPENDIX 1 continued RHINELLA Current taxonomy R. cf. castaneotica R. centralis R. ceratophrys R. cerradensis R. aff. cerradensis R. chavin R. crucifer R. dapsilis R. cf. dapsilis Updated taxonomy R. proboscidea Rhaebo ceratophrys R. dapsilis Voucher MTR 10003 CH 9383 MVUP 2305 JMP 2284 QCAZ 40240 CFBH 20517 CHUNB 38671 CHUNB 39953 LGE 19096 LGE 19103 MNHN-Uy 9514 MTD 43789 CFBH 2867 CFBH 24630 QCAZ 17719 QCAZ 38892 QCAZ 43967 CORBIDI 1969 MTR 6313 MZUSP 139598 QCAZ 38621 Locality Brazil: Amazonas: Lago Cipotuba Panama: Coclé: Valle de Anton Panama: Coclé: Valle de Antén Colombia: Amazonas: Leticia Ecuador: Sucumbios: Sansa Huari, Comuna Singue 1 Brazil: Bahia: Jabo- randi Brazil: Distrito Fed- eral: Brasilia Brazil: Distrito Fed- eral: Brasilia Argentina: Misiones: Posadas Argentina: Misiones: Posadas Uruguay: Rivera: Pueblo Madera Peru: Huanuco: Pachitea, Palma Pampa Brazil: Espirito Santo: Aracruz Brazil: Bahia: Cama- can Ecuador: Napo: Cando Ecuador: Pastaza: Vil- lano Ecuador: Orellana: Parque Nacional Yas- uni, Comunidad Anangu, Rio Napo Peru: Amazonas: Bagua, Chonza Alta Brazil: Para: Serra do Kukoinhokren Brazil: Para Ecuador: Pastaza, Vil- lano Sources Fouquet et al., 2012a Pereyra et al., 2016a Pereyra et al., 2016a This study This study This study This study This study This study This study This study Pramuk, 2006; Pramuk et al., 2008 This study; Thome et al., 2010 This study; Thomé et al., 2010 Pramuk, 2006; Pramuk et al., 2008 Santos et al., 2015 This study This study Fouquet et al., 2012a This study Santos et al., 2015 102 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. cf. dapsilis R. diptycha R. cf. diptycha R. dorbignyi R. cf. dorbignyi R. fernandezae R. festae R. fissippes R. gallardoi Updated taxonomy R. dapsilis R. diptycha R. dorbignyi R. dorbignyi Voucher QCAZ 39474 KU 289057 MACN 51118 MNCN-ADN 6044 LGE 9867 MACN 43695 MNHN-Uy 9492 CFBH 14062 LGE 8717 LGE 8718 CORBIDI 7505 KU 217501 QCAZ 18203 QCAZ 41490 QCAZ 46457 MNCN-ADN 6310 LGE 4546 LGE 4735 Locality Ecuador: Orellana: Alta Florencia, 6.5 km NO de Nuevo Roca- fuerte, Rio Napo Paraguay: Concepcion: Parque Nacional Ser- rania San Luis Argentina: Santiago del Estero: Guasayan Bolivia: La Paz: San José de Uchupiamonas Argentina: Misiones: Capital, Fachinal Argentina: Buenos Aires: Dolores Uruguay: Treinta y Tres: Bafiado de los Oliveras Brazil: Rio Grande do Sul: Rio Grande Argentina: Corrientes: General Paz, Ita Ibaté Argentina: Santa Fe: 9 de Julio, Tostado Peru: Loreto: Datem del Marafion, Morona Ecuador: Pastaza: Montalvo Ecuador: Napo: Estacion Bioldgica Jatun Sacha Ecuador: Zamora: Miazi Alto Ecuador: Morona: Santiago, Nuevo Israel Bolivia: Beni-Cocha- bamba: Santo Domingo, Parque Nacional Isiboro- Sécure Argentina: Jujuy: Man- uel Belgrano Argentina: Jujuy: Abra Colorada NO. 447 Sources This study Mulcahy et al., 2006 This study; Frost et al., 2006 This study; Vallinoto et al., 2010 This study Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a Pereyra et al., 2016a This study Pramuk, 2006; Pramuk et al., 2008; Mendelson et al., 2011 Santos et al., 2015 This study This study This study This study This study 2021 APPENDIX 1 continued RHINELLA Current taxonomy R. gildae R. granulosa R. henseli R. hoogmoedi R. horribilis R. humboldti Updated taxonomy R. dapsilis Rhinella sp. 1 “R. humboldti” Voucher CFBH 11400 ESTR-173 URCA 12651 CFBH 7341 CFBH 18706 CFBH 20117 MNRJ 33006 UFRGS 3569 CFBH 13286 CFBH 15962 MTR 16199 ZUECDCC3393 KRL 744 KU 289750 MAR 2057 UTA 54882 KU 202274 KU 217482 QCAZ 47444 QCAZ 50698 AJC 3533 Locality Brazil: Tocantins: Babaculandia Brazil: Maranhao: Carolina Brazil: Maranhao: Sao Pedro da Agua Branca Brazil: Alagoas: Passo de Camaragibe Brazil: Espirito Santo: Linhares Brazil: Rio Grande do Sul: Catipora Brazil: Rio Grande do Sul: Mato Castelhano Brazil: Rio Grande do Sul: Nova Roma do Sul Brazil: Bahia: Una Brazil: Sao Paulo: San- tos Brazil: Bahia: Serra Bonita, Camacan Brazil: Rio de Janeiro: Mage, Santo Aleixo Panama: Coclé: El Cope El Salvador: Ahuacha- pan: Parque Nacional El Imposible Colombia: Valle del Cauca: Dagua Mexico: Veracruz Ecuador: Pichincha: Tinalandia Ecuador: Loja: Vilca- bamba Ecuador: Loja: San Bernabé Ecuador: Manabi: Puerto Cayo Colombia: Santander: San Vicente de Chu- curi, Reserva El Arbo- retum PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 103 Sources This study Fouquet et al., 2012b Avila et al., 2018 Pereyra et al., 2016a Pereyra et al., 2016a This study Thomé et al., 2010; Pereyra et al., 2016a This study This study This study Fouquet et al., 2012b Pauly et al., 2004 Crawford et al., 2010 Mulcahy et al., 2006; Pramuk, 2006; Pramuk et al., 2008 This study Mulcahy et al., 2006 Pauly et al., 2004 Mulcahy et al., 2006; Pramuk, 2006; Pramuk et al., 2008 This study This study Guarnizo et al., 2015 104 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. humboldti R. icterica R. cf. icterica R. inca R. inopina R. jimi R. justinianoi R. leptoscelis Updated taxonomy “R. humboldti” “R. icterica” “R. icterica” R. diptycha Rhinella sp. 2 Voucher CZUT 1717 CFBH 11027 CFBH 13965 CFBH 27410 CFBH 38392 MACN 43789 CORBIDI 6920 LGE 2554 MNCN 44405 MNCN 44406 CHUNB 51110 MZUSP 142356 MZUSP 142094 CFBH 19335 CFBH 19523 MNCN-ADN 6065 MUBI 5976 MUBI 5989 CORBIDI 7266 Locality Colombia: Tolima: Prado, Vereda El Cai- man, Represa hidro- eléctrica Hidroprado Brazil: Santa Catarina: Bom Jardim da Serra, Serra do Rio do Rastro Brazil: Rio de Janeiro: Petropolis Brazil: Rio de Janeiro: Parque Estadual dos Trés Picos Brazil: Minas Gerais: Rio Preto Argentina: Misiones: San Vicente Peru: Ayacucho: San Antonio, La Mar Peru: Cusco: Urubamba Peru: Cusco: La Con- vencion, Rio Kimbiri, Comunidad Machiguenga Pomoreni Peru: Cusco: La Con- vencion: Rio Kimbiri, Comunidad Machiguenga Pomoreni Brazil: Bahia: Sao Desidério Brazil: Minas Gerais: Januaria Brazil: Minas Gerais: Januaria Brazil: Bahia: Maracas Brazil: Bahia: Maracas Bolivia: Santa Cruz: Florida, La Yunga de Mairana Peru: Puno: Carabaya Peru: Puno: Carabaya Peru: Oxapampa: Huan- cabamba, Huampal NO. 447 Sources Murphy et al., 2017 This study; Thomé et al., 2010 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 2021 APPENDIX 1 continued RHINELLA Current taxonomy R. leptoscelis R. lescurei R. lilyrodriguezae R. limensis R. lindae R. macrorhina Updated taxonomy Rhinella sp. 2 Voucher MUBI 14523 MUSM 31150 NMP6V 74749 AF 1613 MC5 MNHN-Fr 2006.2611 CORBIDI 6778 CORBIDI 6780 CORBIDI 8839 MUSM 32205 nv KU 215587 RGP 4719 MAR 3330 MAR 3431 MAR 3432 MAR 2867 MAR 2903 Locality Peru: Pasco: Oxa- pampa Peru: Pasco: Oxa- pampa, Oxapampa Peru: Pasco: Oxa- pampa, Quebrada San Alberto French Guiana: St Laurent Du Maroni Saul French Guiana: Cis- ame French Guiana: Haute Wanapi Peru: San Martin: Mariscal Caceres Peru: San Martin: Mariscal Caceres Peru: San Martin: Alto Biavo, Parque Nacional Cordillera Azul Peru: San Martin: Alto Biavo, Parque Nacional Cordillera Azul Peru: Lima: Lima Peru: Ancash: Casma, Rio Casma, Casma Peru: Arequipa: Islay Colombia Antioquia: Parque Nacional Natu- ral Las Orquideas Colombia: Antioquia: Parque Nacional Natu- ral Las Orquideas Colombia: Antioquia: Parque Nacional Natu- ral Las Orquideas Colombia: Caldas: Parque Nacional Selva de Florencia Colombia: Caldas: Parque Nacional Selva de Florencia PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 105 Sources This study Moravec et al., 2014 Moravec et al., 2014 This study Fouquet et al., 2007c Fouquet et al., 2012a This study This study This study Cusi et al., 2017 This study Pramuk, 2006; Pramuk et al., 2008 This study This study This study This study This study This study 106 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. macrorhina R. magnussoni R. major R. manu R. cf. margaritifera Updated taxonomy Rhinella sp. 3 R. dapsilis Rhinella sp. 6 Rhinella sp. 7 Rhinella sp. 10 Rhinella sp. 11 Rhinella sp. 12 Rhinella sp. 13 Rhinella sp. 14 Voucher MHUA 8319 MHUA 10262 APL 20530 LGE 8720 MNCN-ADN 6232 MUBI 11372 MNCN-ADN 20672 CORBIDI 5152 MUBI 10487 IWU 334 MUSM 32715 ANDES 1723 PD 16 QCAZ 42269 CHUNB 32342 NMP6V 74260 ROM 40103 USNM 268828 CORBIDI 5840 CORBIDI 5848 MUBI 6374 MUBI 14775 Locality Colombia: Antioquia: Vereda Santa Rita, Guatapé Colombia: Antioquia: Vereda La Esperanza, El Carmen de Viboral Brazil: Para: Treviso Argentina: Salta: Rivadavia, El Ocultar Bolivia: Cochabamba: Chapare Peru: Cusco: Trocha Union Peru: Cusco: Parque Nacional Manu Peru: Madre de Dios: Tambopata, Baltimore Peru: Cusco: La Con- vencion Peru: Junin: Chan- chamayo, Ayte, Bosque de Proteccién Pui Pui Peru: Cusco: Oxa- pampa Colombia: Amazonas: Leticia Brazil: Amazonas: Rio Ica Ecuador: Napo: Reserva Ecoldgica Yachana Brazil: Amazonas: Humaita Bolivia: Pando: Manuripi, San Antonio Peru: Madre de Dios: Tambopata Peru: Madre de Dios: Reserva Tambopata Peru: Loreto: Curupa Peru: Loreto: Curupa Peru: Loreto: Mayna Peru: Ucayali NO. 447 Sources This study This study This study Pereyra et al., 2016a Pereyra et al., 2016a This study This study This study This study This study; Cusi et al., 2017 This study; Cusi et al., 2017 This study This study This study This study Moravec et al., 2014 Fouquet et al., 2012b Pramuk, 2006; Pramuk et al., 2008 Santos et al., 2015 This study This study This study 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) APPENDIX 1 continued RHINELLA Current taxonomy R. cf. margaritifera R. marina R. martyi R. merianae R. mirandaribeiroi R. multiverrucosa R. nesiotes R. nicefori Updated taxonomy Rhinella sp. 14 R. margaritifera R. cf. multiverrucosa R. cf. nicefori Voucher MUBI 14776 CORBIDI 5468 KU 215145 KU 215146 MNCN-ADN 20639 NMP6V 74915 MAR 1982 SBH 190696 VUB 1965 MC 156 MNHN-Fr 2006.2602 MW 1006 CFBH 16641 MTR 20517 CFBH 10254 CFBH 13849 MUBI 11455 CORBIDI 8122 CORBIDI 13953 MHUA 4793 Locality Peru: Ucayali Peru: Cusco: La Con- vencion Peru: Madre de Dios: Cusco Amazonico, Puerto Maldonado Peru: Madre de Dios: Cusco Amazonico, Puerto Maldonado Peru: Puno: Carabaya, between Puerto Leguia and San Gaban Peru: Ucayali: Pucallpa, Masisea Colombia: Caqueta: Florencia Jamaica: St. Mary: Galina Suriname French Guiana: Tri- jonction Suriname: Brownsberg Nature Park Guyana Brazil: Amazonas: Manaus, Reserva Ducke Brazil: Roraima: Esta- cao Ecoldégica de Maraca Brazil: Tocantins: Ara- guacema Brazil: Maranhao: Parque Nacional dos Lencdis Maranhenses Peru: Huantinco Peru: Cusco: La Con- vencion, Echarati Peru: Huanuco: Puerto Inca, Yuyapichis Colombia: Antioquia: Belmira 107 Sources This study This study Pramuk, 2006; Pramuk et al., 2008 Mendelson et al., 2011 This study Moravec et al., 2014 This study Pramuk et al., 2001 van Bocxlaer et al., 2009; Liedtke et al., 2016 Fouquet et al., 2007c Fouquet et al., 2012a van Bocxlaer et al., 2009; van Bocxlaer et al., 2010 Pereyra et al., 2016a This study Pereyra et al., 2016a Pereyra et al., 2016a This study This study This study This study 108 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. ocellata R. ornata R. paraguas R. paraguayensis R. cf. paraguayensis R. poeppigii Updated taxonomy R. scitula R. stanlaii Voucher CFBH 26592 LAJ 210 MZUSP 103261 ZUFG 8519 CFBH 18815 CFBH 38375 LGE 6503 LGE 8729 LGE 19020 LGE 19027 USNM 303015 ZVECDCG@ 3392 TG 1415 TG 1480 UFMT 1876 SME 88237 MUBI 6863 MUBI 6864 USNM 268824 Locality Brazil: Maranhao: Barreirinhas Brazil: Tocantins: Parque Estadual do Lajeado Brazil: Tocantins: Peixe Brazil: Goias: Morrin- hos Brazil: Rio de Janeiro: Parque Nacional da Serra dos Orgaos Brazil: Rio de Janeiro: Visconde de Maua Argentina: Misiones: Cunha Piri Argentina: Misiones: Profundidad Argentina: Misiones: El Soberbio Argentina: Misiones: Capital Brazil: Sao Paulo: Salesdpolis, Serra do Mar Brazil: Rio de Janeiro: Magé, Campo de Escoteiros, Santo Aleixo Colombia: Valle del Cauca: El Cairo Colombia: Valle del Cauca: El] Cairo Brazil: Mato Grosso: Poconé Bolivia: Santa Cruz: Nuflo de Chavez, San Sebastian Peru: Cusco: Quispi- canchis Peru: Cusco: Quispi- canchis Peru: Madre de Dios: Puerto Maldonado NO. 447 Sources This study Fouquet et al., 2012b Pramuk, 2006; Pramuk et al., 2008 This study This study; Thomé et al., 2010 This study This study This study This study This study Mulcahy et al., 2006; Pramuk, 2006 Pauly et al., 2004; Brandvain et al., 2014 This study This study This study Jansen et al., 2011 This study This study Pramuk, 2006; Pramuk et al., 2008, Brandvain et al., 2014 2021 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 109 APPENDIX 1 continued RHINELLA Current taxonomy R. proboscidea R. pygmaea R. quechua R. rubescens R. rubropunctata R. ruizi R. rumbolli R. scitula R. cf. scitula R. sclerocephala Updated taxonomy Rhinella sp. 8 Rhinella sp. 9 R. scitula Voucher AMNH-FS 20085 CTGA-UFAM 5602 QCAZ 28573 CORBIDI 102 CORBIDI 5835 MNCN-ADN 26559 CFBH 2894 CFBH-T 15163 MNCN-ADN 3927 CFBH 5836 CFBH 7696 MACN 52275 AML 39 AML 40 MACN 53782 MACN 43719 IIBP 849 CFBH 42359 MHNLS 7495 Locality Brazil: Roraima: Flo- resta Brazil: Amazonas: Universidade Federal do Amazonas, near Manaus Ecuador: Sucumbios: Sucumbios: Campo Vinita, via Palma Roja- Pto El Carmen de Putumayo Peru: Loreto: Maynas Peru: Loreto: Maynas Peru: Loreto: Maronal, Rio Ampiyacu Brazil: Rio de Janeiro: Sao Joao da Barra Brazil: Espirito Santo: Mimoso do Sul Bolivia: Cochabamba: Parque Nacional Carrasco, Sehuencas Brazil: Minas Gerais: Pocos de Caldas Brazil: Goias: Cocalz- inho de Goias Argentina: Chubut: Cushamen Colombia: Antioquia: Medellin Colombia: Antioquia: Medellin Argentina: Salta: Santa Victoria, Parque Nacional Baritt Argentina: Salta: Santa Victoria, Parque Nacional Baritu Paraguay: Concepcion Brazil: Mato Grosso do Sul: Corumba Venezuela: Cojedes: Cerro Azul, fila La Blanquera Sources This study Motta et al., 2018 This study This study This study This study Pereyra et al., 2016a This study, Pereyra et al., 2016a This study This study This study; Thomé et al., 2010 This study This study This study This study This study This study This study This study 110 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. spinulosa papillosa R. spinulosa spinulosa R. spinulosa trifolium R. stanlaii R. aff. stanlaii R. sternosignata R. cf. sternosignata Updated taxonomy R. papillosa R. spinulosa R. altiperuviana R. trifolium Rhinella sp. 15 Rhinella sp. 13 Voucher BB 983 BB 1032 MACN 49782 NB 96-23 IDLR 3837 MUBI 10737 MUBI 10770 nv MACN 49701 MNCN 41989 CORBIDI 5530 nv ZUFG 6456 MNCN-ADN 4160 MNCN-ADN 6274 MNCN-ADN 4159 nv MAR 1314 MAR 1955 Locality Argentina: Neuquén: Minas Argentina: Rio Negro: Bariloche, Pampa Linda Argentina: Chubut: Lago Puelo Argentina: Neuquén: Laguna Blanca Bolivia: La Paz: stream between Charazani and Curva Peru: Cusco: Colcha Peru: Cusco: Chumbivilcas Peru: Puno: Acocollo Argentina: Jujuy: Tum- baya, Quebrada de Sepultura Bolivia: La Paz: Inqui- sivi, Quebrada entre Quime e Inquisivi Peru: Lima: Huancaya, Vilca Peru: Junin: Huancayo Brazil: Mato Grosso: Tangara da Serra Bolivia: Santa Cruz: Amboré, Ichilo Bolivia: Cochabamba: Carrasco, Chaquisacha Bolivia: La Paz: Parque Nacional Madidi, Ser- rania Sadiri Venezuela: Barinas: Cano Los Monos, Acequias Colombia: Boyaca: Pajarito Colombia: Caqueta: Florencia NO. 447 Sources This study Frost et al., 2006 This study Pauly et al., 2004, Brandvain et al., 2014 Pramuk, 2006; Pramuk et al., 2008 This study This study This study This study This study This study This study This study This study This study This study Pereyra et al., 2016a This study This study 2021 APPENDIX 1 continued RHINELLA Current taxonomy R. tacana R. tenrec R. vellardi R. veraguensis R. veredas R. yanachaga Updated taxonomy Voucher MUBI 6950 MUBI 7409 MUBI 7007 MNK 7187 UTA 53310 MAR 3584 MAR 3585 CORBIDI 7626 KU 211765 MUBI 14281 MUBI 14291 IDLR 3820 MNCN-ADN 5808 MUBI 5946 USNM 346048 CFBH 20516 CHUNB 44609 CORBIDI 7269 MUBI 7119 MUBI 7121 Locality Peru: Cusco: Quispi- canchis Peru: Cusco: Quispi- canchis Peru: Cusco: Quispi- canchis, Camanti Bolivia: La Paz: Parque Nacional Madidi Bolivia: La Paz Colombia: Antioquia: Parque Nacional Las Orquideas Colombia: Antioquia: Parque Nacional Las Orquideas Peru: La Libertad: Pataz, Vijus Peru: Cajamarca: Caja- bamba Peru: La Libertad: Sanchez Carrion Peru: La Libertad: Sanchez Carrion Bolivia: La Paz Bolivia: Cochabamba: Parque Nacional Carrasco, Kharahuasi Peru: Puno: Santo Domingo Peru: Cusco: Paucart- ambo Brazil: Bahia: Sao Desidério Brazil: Minas Gerais: Buritizeiro Peru: Pasco: Oxa- pampa Peru: Pasco: Oxa- pampa Peru: Pasco: Oxa- pampa PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 111 Sources This study This study This study This study Pramuk, 2006; Pramuk et al., 2008 This study This study This study Pramuk, 2006; Pramuk et al., 2008 This study This study Pramuk, 2006; Pramuk et al., 2008 This study This study Pramuk, 2006; Pramuk et al., 2008 This study This study; Maciel et al., 2010 This study This study This study 112 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued RHINELLA Current taxonomy R. yunga R. sp. “gr. acrolopha” R. sp. gr. margaritifera R. sp. gr. marina OUTGROUPS Amazophrynella aff. minuta Anaxyrus americanus Anaxyrus boreas Anaxyrus quercicus Anaxyrus woodhousii Ansonia longidigita Updated taxonomy R. iserni Rhinella sp. 4 R. dapsilis Rhinella sp. 5 Rhinella sp. 13 “R. icterica” Voucher MJH 7095 CAS 207258 CAS 201586 MVZ 223370 KU 224658 VUB 666 Voucher NMP6V 75552 MUSM 31096 MUSM 31097 MUSM 31950 ih Coed bbs MC 204 PG 144 QCAZ 53072 QCAZ 53142 MHNLS 21837 LGE' 19195 Locality Peru: Huanuco: Rio Llullapi- chis, Panguana i prings Rd c= Malaysia: Borneo US.: Mississippi: Boswell Lake .S.: California: Grover Hot .S.: Florida: Charlotte .S.: Kansas: Barber, Sharon Locality Peru: Junin: Chan- chamayo, Bosque de Proteccion Pui Pui Peru: Cusco: Oxa- pampa, Quebrada San Alberto, Parque Nacio- nal Yanachaga-Chemi- llén Peru: Cusco: Oxa- pampa, Quebrada San Alberto, Parque Nacio- nal Yanachaga-Chemi- llén Peru: Junin: Chan- chamayo, Bosque de Proteccion Pui Pui Colombia: Tolima: Murillo, km 22 carret- era Murillo-Manizales French Guiana: Saul French Guiana: Patawa Ecuador: Pastaza: Comunidad Puka Yaku Ecuador: Pastaza: Comunidad Campus Venezuela: Miranda: Rio Araira Argentina: Misiones: Posadas Sources NO. 447 Sources This study; Cusi et al., 2017 This study This study This study Machado et al., 2016 Fouquet et al., 2007c Fouquet et al., 2007c This study This study This study This study Faivovich et al., 2005; Grant et al., 2006 van Bocxlaer et al., 2009; Liedtke et al., 2016 2016 van Bocxlaer et al., 2009; Liedtke et al., Pauly et al., 2004; Brandvain et al., 2014 Pramuk, 2006; Pramuk et al., 2008 van Bocxlaer et al 2009; Liedtke et al 2016 2021 APPENDIX 1 continued OUTGROUPS Bufo bufo Bufo gargarizans Bufotes luristanicus Duttaphrynus melanostictus Incilius alvarius Incilius coniferus Incilius nebulifer Incilius valliceps Ingerophrynus galeatus Leptophryne borbonica Melanophryniscus stelzneri Mertensophryne loveridgei Nannophryne variegata Nectophrynoides tornieri Pelophryne misera Peltophryne empusa Peltophryne lemur Phrynoidis juxtaspera Voucher VUB 982 MVZ 230209 CAS 228184 USNM 292081 NP 13-1 VUB 52 USNM 320001 TWR 628 KU 217480 UTA 52489 DEC. 3170 UTA 13097 FMNH 256443 VUB 673 VUB 985 KMH 26653 MCZ 32084 IZUA 3198 MW 1822 BMNH 2005.1375 VUB 641 nv SBH 193517 AG nd SBH 190657 VUB 649 Locality Belgium Turkey: Bursa: Bursa China: Yunnan: Fugong- Gongshan rd, N of Fugong China: Sichuan: Shimian nd India US.: Arizona: Continental US.: Arizona: Pima, Why Ecuador: Pichincha: Vicente Maldonado US.: Louisiana: Tangipahoa US.: Texas: Gulf Coast Mexico: Chiapas Lao PDR: Khammouan: Nakai Malaysia nd Tanzania Tanzania Chile: XII Region: Puerto Edén, Isla Wellington Tanzania Tanzania Malaysia: Borneo Cuba: Isla de la Juventud: Los Indios Cuba: Granma: Bartolome Maso Puerto Rico Puerto Rico Malaysia: Borneo PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 113 Sources van Bocxlaer et al., 2009 Pramuk, 2006; Pramuk et al., 2008 van Bocxlaer et al., 2009; Liedtke et al., 2016 Pramuk, 2006; Pramuk et al., 2008 van Bocxlaer et al., 2010 Bossuyt and Milinkovitch, 2000; Biju and Bossuyt, 2003; Roelants et al., 2007; van Bocxlaer et al., 2009 Pramuk, 2006; Pramuk et al., 2008 Wiens et al., 2005 Pramuk, 2006; Pramuk et al., 2008 Mulcahy and Mendelson, 2000; Mendel- son et al., 2011 Santos and Cannatella, 2011 Mulcahy and Mendelson, 2000, Pauly et al., 2004; Brandvain et al., 2014 Pramuk, 2006; Pramuk et al., 2008 Roelants et al., 2007; van Bocxlaer et al., 2009; Liedtke et al., 2016 Roelants et al., 2007; van Bocxlaer et al., 2009; Liedtke et al., 2016 van Bocxlaer et al., 2009 Liedtke et al., 2016 Pramuk, 2006; Pramuk et al., 2008 van Bocxlaer et al., 2009 Roelants et al., 2007 van Bocxlaer et al., 2009; Liedtke et al., 2016 Alonso et al., 2012 Pramuk et al., 2001; Landestoy et al., 2018 Pramuk, 2006; Pramuk et al., 2008 Pramuk et al., 2001 van Bocxlaer et al., 2009, Liedtke et al., 2016 114 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY APPENDIX 1 continued OUTGROUPS Rentapia hosii Rhaebo ecuadorensis Rhaebo guttatus Rhaebo nasicus Schismaderma carens Sclerophrys brauni Sclerophrys garmani Sclerophrys mauritanica Sclerophrys regularis Sclerophrys steindachneri Vandijkophrynus robinsoni Wolterstorffina parvipalmata Voucher VUB 661 QCAZ 13234 LSUMZ 17418 ROM 20650 MW 4279 MW 3840 BMNH 2002.350 CAS 214829 NP 22-1 VG 7-025 KU 290435 CAS 214839 CAS 193549 AACRG 68 DPL 5101 MTSN 5895 Locality Malaysia Ecuador: Napo: Talag Alto Brazil: Rond6énia: Parque Estadual Guajara-Mirim Guyana: Mazaruni-Potaro: Tukeit Tanzania Tanzania Tanzania Kenya: Coast Province: Watamu Morocco Morocco Ghana: Eastern Region: Win- neba Kenya: Coast: Kilifi, Karara- cha pond South Africa: Northern Cape: Richtersveld National Park nd Cameroon Cameroon NO. 447 Sources Roelants et al., 2007; van Bocxlaer et al., 2009; Liedtke et al., 2016 Pramuk, 2006; Pramuk et al., 2008 Pramuk, 2006; Pramuk et al., 2008 Pramuk, 2006; Pramuk et al., 2008 van Bocxlaer et al., 2009 Van Bocxlaer et al., 2009 Roelants et al., 2007; Liedtke et al., 2016 Pramuk, 2006; Pramuk et al., 2008; van Bocxlaer et al., 2009; Brandvain et al., 2014 van Bocxlaer et al., 2009 Liedtke et al., 2016 Pramuk, 2006; Pramuk et al., 2008 Pramuk, 2006; Pramuk et al., 2008; van Bocxlaer et al., 2009; Liedtke et al., 2016 van Bocxlaer et al., 2010 Liedtke et al., 2016 Frost et al., 2006 van Bocxlaer et al., 2009 115 PEREYRA ET AL.: EVOLUTION IN RHINELLA (ANURA: BUFONIDAE) 2021 9T9SOOMIN ST9COOMWN VI9ECOOMWN SvCLO6ND cT9COOMN 0¢€cZL06ND [pu cvVPcOOMWN ChVcOOMWN IvPvVeOOMN OvPEOOMWN 86Z8ZTND L8Z8ZTND 6cVCOOMWN ScVEOOMWN LEve0OMWN 9CPEOOMWN ScveooOMWN SOISGT 6088ZTND eZecOOMWN cLECOOMWN IZEcOOMWN qua souas [elLIpuoyso} yy 8ZTEOOMWN LLTEOOMWN 9LTEOOMWN SZTCOOMIN bLTCOOMWN [DgIs are 6S9€ WLW arS€9V IdIguoo ar9CP1 WVW aLagscvi UVW ar l08€ DAZ ALL ploe6 AN -NHNW aru90VTS NOVI atuSCV8T1 dSNZW atcL99V NOVI aL l Pit Hado CvV8COOMWN 66TSOOMIN i 861TC00MN a aa — IV8cOOMN L6IZOOMW 8OTLOOMN SEOSOOMN TZ6COOMN OF8TOOMINN SOVZ06ND + LOTSOOMWN PEOCOOMN OL6TOOMN 6E8700MN 96TSOOMWN SOTEOOMWN ecOcCOOMN = = I8€Z06ND SOTEOOMN CEOLOOMN 696C00MN 8E8TOOMN oys q- [804 R-[ ODL gumod PsIXI ayono souas IeaponN aL doses SLISOMNNID U,, « DIVULO “Y Aurouoxe} parepdq SLAJSOANJNIV “Y vydojo19w “Y YVABYIV “Y SISUgJDYID “Y foqv °U AUIOUOXe} yuarmMy) VTITANIHY ‘(op'ds'ps/T Ess 01/810 1op//:sdyyy ye syusuTUsTTe 998) ssouanbas aja[dur09 §9I-SZI YIM 194}080} pousiye usyM sde are] aye19U98 pue ‘sited suid Burddey -IQAOUOU Y}IM poyesiouasd a1aMm Ady} se ‘(yURequUay UI suOT}dtIDsap sduaNbas 9as) YISUIT d1TJUS ITaY} BUOTE sNONsT}UOD JOU oe sI9qUUNU UOTssad0e 9say} JopuN saouanbas # *((‘UNWIUIOD [eUOsIad ‘sIaMmo{) YURGUey 0} payyTuqns o19M Ady} UdYM pextur AT[eyUapTOse 919M (ZIZT LAZO) Upjoquny -y pue 9ZS¢ NDI 12qG22q 'Yy) usIs snjd e YIM paysreul suswtdeds Jo saouanbas + ‘suUOTSIZOIIUT [eIIPUOYIO}U JO 9dUdIINIIO ay} 0} anp ‘sasATeuR IUAPIAd [e}0} dy} UT APJUspuedaput pozdjeue a19yM sotiouas [eLIpUOYIO UI pue Ieap—nu jo saouanbas a19YM [CUTULIA] soyeoIpuT , ‘sasdyeue (POOYTayT] WinuiTxeut pue Auourisied winuirxeur) JOUIPIAD [e}0} UT Pakoydurs o19M JeY} S[PUTUTIA} sayeoIpuT ,, ‘sasATeue (NJ) Jeapnu pure (VJ) [eIpuOYPoyW pozdtsei UT podoTdura o19M Jey} STeUTUI} SdJVOIPUI y “TW Joquiour g ApTurey JoT1Ied aynJos :;7vgsjs ‘ouss UIsdopoyL :oys <(q a}eTOST)