Zootaxa 3138: 1–34 (2011) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2011 · Magnolia Press ISSN1175-5334(online edition) A phylogeny and evolutionary natural history of mesoamerican toads (Anura: Bufonidae: Incilius) based on morphology, life history, and molecular data JOSEPH R. MENDELSON III1,2,5, DANIEL G. MULCAHY3,4, TYLER S. WILLIAMS3 & JACK W. SITES JR.3 1Department of Herpetology, Zoo Atlanta, 800 Cherokee Ave SE, Atlanta, GA, 30315, USA 2School of Biology, Georgia Institute of Technology, 301 Ferst Dr, Atlanta, GA, 30332, USA 3Department of Biology, Brigham Young University, Provo, UT, 84602, USA 4Current address: Smithsonian Institution, PO Box 37012, MRC 162, Washington, DC, 20013, USA 5Corresponding author. E-mail: [email protected] Abstract We combine mitochondrial and nuclear DNA sequence data with non-molecular (morphological and natural history) data to conduct phylogenetic analyses and generate an evolutionary hypothesis for the relationships among nearly every spe- cies of Mesoamerican bufonid in the genus Incilius. We collected a total of 5,898 aligned base-pairs (bp) of sequence data from mitochondrial (mtDNA: 12S–16S, cyt b, ND2–CO1, including tRNAsTRP–TYR and the origin of light strand replica- tion; total 4,317 bp) and nuclear (CXCR4 and RAG1; total 1,581 bp) loci from 52 individual toads representing 37 species. For the non-molecular data, we collected 44 characters from 29 species. We also include Crepidophryne, a genus that has not previously been included in molecular analyses. We present results of parsimony and Bayesian analyses for these data separately and combined. Relationships based on the non-molecular data were poorly supported and did not resolve a monophyletic Incilius (Rhinella marina was nested within). Our molecular data provide significant support to most of the relationships. Our combined analyses demonstrate that inclusion of a considerably smaller dataset (44 vs. 5,898 charac- ters) of non-molecular characters can provide significant support where the molecular relationships were lacking support. Our combined results indicate that Crepidophryne is nested within Incilius; therefore, we place the former in the synony- my of the latter taxon. Our study provides the most comprehensive evolutionary framework for Mesoamerican bufonids (Incilius), which we use as a starting point to invoke discussion on the evolution of their unique natural history traits. Key words: Amphibia, Crepidophryne, natural history, phylogeny, taxonomy Resumen Combinamos datos moleculares de DNA mitocondrial y nuclear con datos morfológicos y de historia natural para realizar análisis filogenéticos y proponer hipótesis para esclarecer las relaciones filogenéticas entre especies de bufonidae dentro del género Incilius provenientes de Mesoamérica. Colectamos un total de 5,898 pares de bases (bp) de secuencias alin- eadas de loci mitocondriales (mtDNA: 12S–16S, cyt b, ND2–CO1, incluyendo tRNAsTRP–TYR y el origen de la replicación de la cadena liviana; totales 4,317 bp) y nucleares (CXCR4 y RAG1; totales 1,581 bp), obtuvimos loci de 52 ejemplares de sapos que representan 37 especies. Para los datos no moleculares, registramos 44 caracteres de 29 especies. Incluimos al género Crepidophryne, que nunca ha sido usado en análisis moleculares previos. Presentamos los resultados de los análisis Bayesianos y de parsimonia que realizamos combinando los datos y luego separándolos. Las relaciones resul- tantes basadas sólo en datos no-moleculares no son claras y no presentan al género Incilius como un grupo monofilético (Rhinella marina resulta dentro del grupo). Nuestros datos moleculares muestran un soporte significativo para varias de las relaciones filogenéticas. Y la combinación de ambos datos demuestra que al incluir la pequeña base de datos (44 vs. 5,898 caracteres) de caracteres no-moleculares ayuda significativamente a hacer más fuertes las relaciones que parecían débiles en el análisis con solo datos moleculares. Los resultados del análisis combinado indican que Crepidophryne se incluye dentro de Incilius; además, proponemos esta forma como un sinónimo del taxón mas reciente. Nuestro estudio proporciona el más comprensivo marco evolucionario para los bunonidos de Mesoamérica (Incilius), en el cual se empieza a discutir la importancia de la evolución de caracteres únicos de historia natural. Accepted by M. Vences: 10 Nov. 2011; published: 21 Dec. 2011 1 Introduction Mesoamerica, here considered to be the continental area south of the United States and north of Colombia, is a complex region characterized by geographic and climatic extremes and a correspondingly diverse biota (reviewed by Campbell, 1999, and Savage, 2002). There is general agreement that the region has clear biogeographical influ- ences from both North and South America, but these influences are relatively minimal in light of the predominant endemic radiations (Savage, 1982). Not surprisingly, Mesoamerica has attracted the attention of many biogeo- graphic studies (e.g., Stuart, 1954; Savage, 1982) and was the conceptual birthplace for the field of modern vicari- ance biogeography (Rosen, 1978). Exemplar studies have examined relationships among species in endemic highland squamate radiations (e.g., Crother et al., 1992; Campbell and Frost, 1993), phylogeographic patterns among allopatric highland populations of rodent species (e.g., Sullivan et al., 2007; Harris et al., 2000), lowland fishes (e.g., Hulsey et al., 2004), beetles (e.g., Marshall and Liebherr, 2000), and frogs (e.g., Zaldívar-Riverón, 2004). In this paper we present the most extensive phylogenetic study for the majority of bufonid toads from this region, and we take the opportunity to review the varied natural histories of these anurans in an explicit evolution- ary framework. A review of the amphibians of Mesoamerica (Campbell, 1999) listed 389 species of anurans; that list excluded part of the Central Plateau of Mexico, which certainly has its faunal associations with North America. Since that review, many additional species have been described and, relevant to this work, there currently are 62 species of bufonids recognized from Mesoamerica (Frost, 2010). These are allocated to the primarily North American clade Anaxyrus (which has many species occuring in Mexico), the primarily South American clades Atelopus, Rhinella and Rhaebo (which have relatively few species in lower Central America), and the autochthonous clade Incilius, which currently has 35 species known from across the entire Mesoamerican region. Our efforts here are focused on the relationships among species referred to Incilius. Graybeal (1997: fig. 13) and all subsequent studies have found a monophyletic Bufonidae comprising two major groups. One is a large monophyletic, but nameless, group containing the species formerly referred to Bufo, plus various genera that rendered that problematic taxon paraphyletic; the taxonomy of this group has continued to undergo revision since the initial efforts by Frost et al. (2006). The other group is a non-monophyletic assemblage usually referred to informally as the “atelopodids” (including the familiar Harlequin frogs—Atelopus, and a variety of other generally smaller montane toads such as Osornophryne). Frost et al. (2006a) provided an important review of Graybeal’s (1997) work. Additional efforts (Pauly et al., 2004; Frost et al., 2006a; Pramuk et al., 2006; Pramuk et al., 2008; Van Bocxlaer et al., 2009; Van Bocxlear et al., 2010) have found differing relationships among three major clades of New World bufonids. Pauly et al. (2004), Frost et al. (2006a), and Pramuk et al. (2006, 2008) and Pyron & Wiens (2011) found Rhinella, Anaxyrus, and Incilius to form a monophyletic group (albeit in differing positions with respect to one another). Van Bocxlaer et al. (2009, 2010) incorporated broader taxonomic and geo- graphic sampling of bufonids and discovered Anaxyrus and Incilius as sister taxa that were sister to a clade contain- ing a monophyletic Rhinella plus a large variety of Old World taxa. All studies agree with the placement of the South American clade Rhaebo lying outside any arrangement of Rhinella, Anaxyrus, and Incilius. The conflicting results of these broad-based studies represent an important problem in bufonid systematics and biogeography (e.g., Pramuk et al., 2008). Our study was not designed to address these issues, nor to test the existing hypotheses of rela- tionships among these genera, but rather to elucidate relationships among species of Incilius. The majority of species currently referred to Incilius were historically allocated to a Bufo valliceps group that has been presented in myriad of different forms; some treatments also included a Bufo coccifer group. However, there has never been agreement on the content of these various groups, and none were based on results of phyloge- netic analyses. Given that these groups have been mentioned so frequently in the literature, a brief review is appro- priate. Firschein (1950) proposed the Bufo valliceps group (content: Inciliuscristatus and I. valliceps) and the Bufo cristatus group (content: Incilius cavifrons and I. cristatus). Firschein (1950) did not consider the relationships of several other crested toads (e.g., I. mazatlanensis) in Middle America and his taxonomic arrangement is problem- atic because he placed I. cristatus simultaneously in two different groups. Subsequently Blair (1959, 1961) alluded to a Bufo valliceps group, but did not define it. Based on osteology, Tihen (1962) provided an explicit proposal of the content of the Bufo valliceps group and divided it into “South American” and “Mexican” sections. Blair (1966) disagreed with Tihen, claiming that he (Blair, 1959, 1963) already had proposed the content of a Bufo valliceps group—a claim that is not justified in Blair's earlier papers. Blair (1966) provided a summary of the group whose 2 · Zootaxa 3138 © 2011 Magnolia Press MENDELSON ET AL. content somewhat matches that of Tihen's (1962) “Mexican Section.” Porter (1962, 1964) provided a thorough review of the species then recognized in Mexico. Martin (1972) provided definitions, based on osteology, for the following: B. valliceps group, B. alvarius group, B. coccifer group, B. canaliferus group, B. occidentalis group, B. marmoreus group, B. bocourti group, B. periglenes group, and B. holdridgei group; considered altogether, these groups all include species currently referred to Incilius (discounting recently described species).In major works on the Bufo valliceps group since 1950, 20 species have been assigned to the group by one or more authors; the most recent formal application of the concept of a B. valliceps group was that of Duellman & Schulte (1992), who pro- vided a definition but did not list the content of the group. The current phylogenetic concept of the clade Incilius is based on the works of Pauly et al. (2004), Frost et al. (2006a), and Van Bocxlaer et al. (2010). Frost et al. (2006a) resurrected Cranopsis Cope, 1875, for the Mesoamerican toads, in error, corrected to Ollotis Cope, 1875 by Frost et al. (2006b), and later to Incilius Cope, 1863, by Frost et al. (2009a). Frost et al. (2006a) provided both a diagno- sis and content for this clade. Most previous studies (cited above) have found Incilius to be monophyletic. Van Bocxlaer et al. (2010) found Incilius to be non-monophyletic because of the placement of the taxon bocourti as sis- ter to Anaxyrus. Pauly et al.’s (2004) parsimony analysis found bocourti sister to Rhinella + Anaxyrus with weak support; their likelihood analyses resolved a monophyletic Incilius (including I. bocourti) with strong support. Pyron & Wiens (2011) found I. bocourti within Incilius, with low support (58% bootstrap). Since the efforts by Firschein (1950) and Porter (1962, 1964), many new species in this complex have been described or resurrected from synonymy: Mendelson (e.g., 1994, 1997a, b), Mulcahy & Mendelson (2000), McCranie & Wilson (2000), and O’Neill & Mendelson (2004), Mendelson & Mulcahy (2010), and Mendelson et al. (in press), and Santos-Barrera & Flores Villela (2011). Although the aforementioned recent phylogenetic studies including bufonids (e.g., Pramuk et al., 2008) included species of crested toads that have been historically referred to some concept of an Incilius (= Bufo) valliceps group, the only studies specific to the group were Mulcahy & Mendelson (2000) and Mulcahy et al. (2006). In these latter papers, a clade containing a group of species ecologi- cally associated with mostly upland moist forests was discovered and informally referred to as the “Forest toads,” along with the well supported lowland species pair I. valliceps +I. nebulifer. Mendelson et al. (2005) reviewed and revised the Incilius (=Bufo) coccifer group, including descriptions of several new species, and presented a prelim- inary phylogeny for some of the species in the group. Considered together, species of Incilius show a remarkable ecological and biogeographical diversity that argu- ably exceeds that of any comparable clade of Neotropical amphibians. These toads include micro-endemic species fully restricted to undisturbed cloud forest habitat (e.g., I. spiculatus; Mendelson, 1997b; Mendelson et al., 1999) and widespread lowland species that prefer disturbed habitats (e.g., I. valliceps; Mendelson, 1998; Mendelson et al., 1999; McCranie & Wilson, 2002). The genus includes species largely restricted to subhumid habitats (e.g., I. luetkenii; Savage, 2002), rainforests (e.g., I. campbelli; Mendelson, 1994), or upland pine-oak forests (e.g., I. cycladen; Mendelson et al., 2005). Collectively, the group has representatives in every major biogeographic region of Mesoamerica (Campbell, 1999; Duellman & Sweet, 1999), although the Central Plateau of Mexico is only peripherally occupied by I. occidentalis and I. mccoyi. The anuran faunas of North America and Mesoamerica share remarkably few species, a pattern illustrated roughly by the few species that occur in both USA and Mexico (Campbell, 1999; Duellman & Sweet, 1999; Mulcahy & Mendelson, 2000). It is noteworthy then that only three species of Incilius straddle the Neotropical/Nearctic boundary—viz., I. alvarius in the Sonoran Desert, I. mazatlan- ensis along the Pacific Coast of Mexico, and I. nebulifer along on the Gulf Coast of Mexico and the USA. Simi- larly, to the South, only the species I. coniferus penetrates the South American continent, occurring in the Choco region of Colombia and Ecuador. As future endeavors to reconstruct the bewildering complexity of Mesoamerican biological evolution proceed, we offer Incilius as a group comparable to both highland clades with restricted spe- cies’ distributions, such as squamates in the genera Bothriechis, Atropoides, and Abronia (Crother et al., 1992; Chippindale et al., 1998; Castoe et al., 2009), and clades of more widespread lowland species, such as cichlid fishes (Hulsey et al., 2004).Using data from morphology, natural history, and both nuclear and mtDNA sequence data, we here contribute a phylogenetic hypothesis of the relationships among species of Incilius, including nearly all extant species. We then use this phylogenetic hypothesis as a basis for a brief discussion of the evolutionary nat- ural history and biogeography of these toads. EVOLUTION OF MESOAMERICAN TOADS Zootaxa 3138 © 2011 Magnolia Press · 3 Material and methods In this paper we follow the taxonomic recommendations of Frost et al. (2006a), Pramuk et al. (2008), Frost et al. (2008), and Frost et al. (2009a). Marginal disagreement on bufonid taxonomy was reviewed by Frost et al. (2009b). Taxon sampling. In order to examine the phylogenetic relationships among Mesoamerican bufonids, we examined two datasets: I) 44 non-molecular characters from morphology and life history; and II) 5,898 base-pairs (bp) of mitochondrial and nuclear sequence data. For the molecular data, we sampled a total of 52 individuals rep- resenting 37 species (with outgroups), including most species of Mesoamerican bufonids (excluding Atelopus). However, no tissues samples were available for five taxa (Incilius gemmifer, I. mccoyi, I. holdridgei, I. periglenes, and I. peripatetes). Where possible, we sampled multiple individuals for species with broad geographic ranges (e.g., I. valliceps). For outgroup taxa, we used Anaxyrus boreas from the “North American” clade (Pauly et al., 2004) and five species [Rhinella marina, R. festae R. schneideri, R. margaritifera (= typhonius),and Rhaebo hae- matiticus] from the “South American” clade discussed by Pramuk (2006). Note: we mention the invalid name R. typhonius here because our tissue sample is listed by some authors in GenBank under that name. We rooted all of our trees post-analyses between Incilius and the outgroup taxa Rhinella marina, R. margaritifera, Rhaebo haemati- ticus, and Anaxyrus boreas. We did not designate Rhinella (= Rhamphophryne) festae nor Crepidophryne as out- groups, so as to test their phylogenetic position with respect to the other included taxa (see below). The non- molecular dataset contained 29 species; osteological specimens for some species were not available (e.g., some recently described species such as I. signifer). In the non-molecular dataset, we included four South American taxa (Rhinella marina, R. festae, R. margaritifera, and Rhaebo haematiticus) and Anaxyrus boreas as outgroups. We first present the molecular and non-molecular data separately, followed by combined analyses. The combined anal- yses of both datasets include one representative from each of the 37 species. We were unable to obtain all data for some specimens and some sequences were taken from GenBank for taxa that were already available from the same specimens used in our study (Pauly et al., 2004; Pramuk et al., 2008). Table 1 shows all samples used in the molec- ular analyses, including voucher information (see Appendix I for GenBank accession numbers, and Appendix II for non-molecular vouchers). Because of specimen and tissue availability, we had to use a specimen of Crepidophyrne chompipe for molecular data and C. epiotica for non-molecular data; our original sampling efforts occurred prior to the taxonomic revision by Vaughan & Mendelson (2007), when all populations were referred to C. epiotica. Thus, in our phylogenetic analyses we use the terminal “Crepidophryne” to represent what we consider a monophyletic group (see Discussion). TABLE 1. Voucher specimens used for molecular analyses. Note that our sample of Rhinella margaritifera is listed by some in GenBank as “Bufo cf. typhonius” and Rhinella festae is listed as “Rhamphophryne” in GenBank. out Locality Museum No. Outgroup: A. boreas USA: California: Los Angeles Co., San Dimas Canyon MVZ 223292 Rhaebo haematiticus Costa Rica: Cartago: I.C.E. Plant in Rio Macho MVZ 164805 Rhinella marina Ecuador: Loja, Vilcabamba KU 217482 R. schneideri Paraguay: Parque Nacional San Luis de la Sierra KU 289057 R. margaritifera Peru: Madre de Dios: Cuzco Amazonico, 15 km E Puerto Maldonado KU 215146 (= typhonius) R. festae Ecuador: Pastaza: Petrolera Garza 1, NE Montalvo KU 217501 Mesoamerican bufonids: Incilius alvarius USA: Arizona: Cochise County UTA A-53924 I. aucoinae_1 Costa Rica: Golfito, Quebrada Canaza UCR 14323 I. aucoinae_2 " UCR 14324 I. bocourti Guatemala: Escuintla UTA A-50920 I. campbelli_1 Belize: Toledo: El Tigre/Columbia River FR USNM 326155 continued next page 4 · Zootaxa 3138 © 2011 Magnolia Press MENDELSON ET AL. TABLE 1. (continued) out Locality Museum No. I. campbelli_2 " USNM 326161 I. campbelli_3 Guatemala: Izabal; Sierra de Caral, San Miguelito KU 221203 I. campbelli_4 Guatemala: Izabal: Montanas del Mico, Las Escobas UTA A-50902 I. canaliferus Guatemala: Escuintla: Palín, Finca Medio Monte UTA A-47640 I. cavifrons Mexico: Veracruz: Sierra de los Tuxtlas, Volcan San Martin UTA A-ENS 10384 I. coccifer_1 El Salvador: Morazan KU 290030 I. coccifer_2 Costa Rica: San Jose: Montanas Jamaic TCWC 83998 I. coniferus Costa Rica: Prov. Cartago: Cabina Tapanti MVZ 203775 Crepidophryne Costa Rica: Cerro Dantas, Cordillera Volcanic Central UCR 16075 chompipe I. cristatus Mexico: Puebla: Municipio de Zacapoaxtla, Apulco EBUAP 544 I. cycladen Mexico: Guerrero: near Agua de Obispo UTA A-54847 I. fastidiousus Costa Rica: Puntarenas: Rio Coton below La Casita MVZ 217438 I. ibarrai_1 Honduras: Ocotepeque UTA A-53662 I. ibarrai_2 Guatemala: Quiche UTA A-52528 I. karenlipsae Panama: Cocle: Parque Nacional G. D. Omar Torrijos UTA-A-59522 I. leucomyos_1 Honduras: Atlantida: La Ceiba, Cordillera Nombre de Dios UTA A-50642 I. leucomyos_2 Honduras: Olancho: Quebrada, El Pinol, Parque Nacional La Muralla USNM 559731 I. leucomyos_3 Honduras: Francisco Morazan UTA A-MEA 892 I. luetkenii_1 Guatemala: Zacapa: S Teculutan UTA A-50877 I. macrocristatus_1 Mexico: Oaxaca: Santa Maria Chimalapa MZFC-EPR 37 I. macrocristatus_2 Mexico: Oaxaca: Santa Maria Chimalapa MZFC-259 I. macrocristatus_3 Mexico: Chiapas: 10.0 mi NW Pueblo Nuevo Solistahuacan UTA-JAC 7993 I. marmoreus Mexico: Oaxaca: 0.7 mi NE Tapanatepec UTA A-13032 I. mazatlanensis Mexico: Sonora: Alamos MVZ 132967 I. melanochlorus Costa Rica: Prov. Heredia: La Selva Biological Station MVZ 229635 I. nebulifer_1 USA: Louisiana: Tangipahoa Parish; Hammond UTA A-52489 I. nebulifer_2 Mexico: Veracruz: road to Hueytepec UTA A-54860 I. porteri Honduras: Francisco Morazan: Reserva Biologica Cerro Uyuca, Cabot UF-JHT 2249 Biological Station I. occidentalis Mexico: Oaxaca: El Tejacate UTA A-13543 I. perplexus Mexico: Guerrero: Rio Zopilote, N of Zumpango de Rio UTA A-54851 I. pisinna Mexico: Michoacan: Hwy 37 S of Lombardia UTA A-JAC 26118 I. signifer Panama: Cocle: El Cope UTA A-JRM 4968 I. spiculatus Mexico: Oaxaca: S of Vista Hermosa UTA A-54853 I. tacanensis Mexico: Chiapas: Colonia Talquian, Union Juarez, Volcan Tacana MVZ 170329 I. tutelarius_1 Mexico: Oaxaca: Cerro Baul MZFC 5262 I. tutelarius_2 Mexico: Oaxaca: Cerro Baul MZFC 5277 I. valliceps_1 Mexico: Veracruz: Catemaco MZFC JRM-3868 I. valliceps_2 Honduras: Cortes: Tegucigalpita USNM 530601 I. sp. nov._1 Guatemala: Huehuetenango: Nenton, Aldea Yalambojoch UTA A-52597 I. sp. nov._2 Guatemala: Huehuetenango: Nenton, Aldea Yalambojoch UTA A-52591 I. sp. nov._3 Guatemala: Huehuetenango, on Ridge ca. 2km NW Barillas MVZ 143380 EVOLUTION OF MESOAMERICAN TOADS Zootaxa 3138 © 2011 Magnolia Press · 5 Frost et al. (2006a) noted several potential morphological synapomorphies between Crepidophryne and South American toads of the genus Rhamphophryne—a taxon later placed in the synonymy of Rhinella by Chapparo et al. (2007; see also Pramuk, 2006, and Pramuk et al. 2008; and Van Bocxlaer et al., 2010). Indeed, all data, including both molecules and morphology, that have been brought to bear on Rhamphophryne and Rhinella suggests a close relationship (see Discussion); however, our study is the first to include Crepidophryne. Therefore, we included Rhinella [= Rhamphophryne] festae to test the relationship between Crepidophryne and Rhamphophryne. For the molecular data, we used sequences of Rhinella (= Rhamphophryne)festae (KU 217501) from GenBank [CXCR4: DQ306521 (Pramuk et al., 2008); RAG-1: DQ158349 and 12S–16S: DQ158423 (Pramuk, 2006). Additionally, Van Bocxlaer et al. (2010) included three samples of the widespread species I. valliceps, and one sample from its putative sister-taxon I. nebulifer (all from GenBank). Their analyses found I. valliceps to be non- monophyletic, with one sample from Honduras (USNM 534129) placed as sister to the Mexican–Guatemalan spe- cies I. macrocristatus, and the other two samples as sister to I. nebulifer. Likewise, Pyron & Wiens (2011) included the 12S–16S, CXCR4, and RAG1 data from this individual as well, and recovered a chimeric “I. valliceps” sister to a I. macrocristatus + I. campbelli clade. Two previous analyses (Mulcahy & Mendelson, 2000; Mulcahy et al., 2006), with greater geographic sampling, have recovered a monophyletic I. valliceps as sister to I. nebulifer. Thus, we compared the 12S–16S (DQ158493), CXCR4 (DQ306545.1), and RAG1 (DQ158409.1) sequences of USNM 534129 with our data and examined the specimen (USNM 534129) to verify its identity. TABLE 2. Primers used in this study. Locus Name Sequence '5 to '3 cyt b MVZ43 GAGTCTGCCTWATYGCYCARAT cyt b CB3H GGCAAATAGGAARTATCATTC 16S 16Sar CGCCTGTTTATCAAAAACAT 16S 16Sbr CCGGTCTGAACTCAGATCACGT 12S 12StPhe AAAGCACRGCACTGAAGATGC 12S 12Se GTRCGCTTACCWTGTTACGACT ND2 metF6 AAGCTTTCGGGCCCATACC ND2 CO1r1 AGRGTGCCAATGTCTTTGTGRTT ND2 IncAsnF1 AAACGCTCAATCCAGCGAGCT ND2 IncAsnR1 AGCTCGCTGGATTGAGCGTTT ND2 IncND2f1 TGCYCAAGAAATARTTAAACA ND2 IncND2r1 TGTTTAAYTATTTCTTGRGCA CO1 dgLCO-1490 GGTCAACAAATCATAAAGAYATYGG CO1 dgHCO-2198 TAAACTTCAGGGTGACCAAARAAYCA CXCR4 CXCR4C GTCATGGGCTAYCARAAGAA CXCR4 CXCR4F TGAATTTGGCCCRAGGAARGC RAG1 RAG1F AGYCARTAYCATAARATGTA RAG1 RAG1R GCRTTNCCDATRTCRCARTG Molecular data sampling. We collected sequence data from six mitochondrial gene regions: 12S, 16S, cyt b, ND2, tRNATRP, tRNAALA, tRNAASN, OL (origin of light strand replication), tRNACYS, and tRNATYR (all treated as one partition), and CO1. In addition, we collected sequence data from two nuclear loci (CXCR4 and RAG1) for a total of eight markers. The protein-encoding nuclear loci were previously used in a world-wide bufonid study and we followed their protocols for PCR and sequence reactions (Pramuk et al., 2008). The mitochondrial genes 12S–16S are frequently used in anuran studies (Pauly et al., 2004; Pramuk et al., 2008; Frost et al., 2006) and cyt b is used in studies focused on Incilius (Mulcahy & Mendelson, 2000; Mendelson et al., 2005; Mulcahy et al., 2006). These loci were collected using primers and protocols similar to our previous studies referenced above. The ND2 and tRNA regions are frequently used in general amphibian and reptile studies (e.g., Macey et al., 1997; 1998) and 6 · Zootaxa 3138 © 2011 Magnolia Press MENDELSON ET AL. were collected using their primers as well as internal primers designed specifically for Incilius, and CO1 is used in the DNA Barcode of Life Project (e.g., Crawford et al., 2010), and was obtained using primers from Meyer (2003). A complete list of primers used for this study is shown in Table 2. Profiles for PCR reactions were run similar to Mulcahy & Mendelson (2000) with annealing temperature varying from 45–51.0° C for the mitochondrial DNA, while the nuclear loci were obtained with the following profile: step 1: 94.0° C for 2:45 min; step 2: 94.0° C for 0:15 s, step 3: 51.0* C for 0:20 s (where * reduces the temp by 0.3° C each cycle); Step 4: 72.0° C for 1:00 min, back to step 2, for 35 cycles, and a final elongation of 72.0° C for 7:00 min. Products of PCR were purified using Millipore microplates, sequence reactions were conducted in both directions with PCR primers, using BigDye® Terminator v3.1 Cycle Sequencing Kit using manufacturers protocols. Sequenced products were purified using Sephadex columns and run out on an ABI 3730xl sequencer at the BYU DNA Sequencing Center. Complimentary chromatograms were assembled and annotated in Sequencher™ v4.7, alignments were done by eye and adjusted in MacClade v4.08 by translated amino acid sequence for protein encoding loci. The ribosomal (12S and 16S) regions were aligned using the ClustalW (v1.4) default option in MacVector. This method uses an open gap penalty of 10, extended gap penalty 5, delay divergent 40%, and weighted transitions in order to minimize gaps. The tRNAs were aligned by secondary structure (Macey et al., 1997). There were no insertions or deletions in the protein-encoding loci, and very few (<40) in the ribosomal and tRNA gene regions, gaps were treated as missing data. Phylogenetic analyses. Phylogenetic analyses of non-molecular data. We examined phylogenetic relation- ships among Mesoamerican bufonids using parsimony (separately and combined) and Bayesian analyses of the combined datasets (molecular and non-molecular). We chose these methods because parsimony offers simple, un- weighted analyses of the data and Bayesian analyses offer more complex model for the molecular data, while main- taining a simple model for the non-molecular data (Felsenstein, 2004). Based partly on the analysis presented by Mendelson (1997c) and information in Mendelson et al. (1999), we identified and scored 44 characters drawn from osteology, soft tissue, larval morphology, and natural history (Appendix III). The non-molecular data matrix was evaluated using a maximum parsimony analysis using PAUP* v4.0b10 (Swofford, 2000). Heuristic searches (1000 random addition replicates) were performed using tree-bisection-reconnection branch swapping, saving all mini- mal length trees at each replicate; the starting seed used was 1. The tree was rooted between the outgroup taxa and Incilius, because of the ambiguity involved in the sister group to Incilius (Pauly et al., 2004; Frost et al., 2006a; Pramuk et al., 2008; Van Bocxlaer et al., 2009; Van Boxclaer et al., 2010).Agreement among the shortest trees was assessed by strict consensus. Bremer/decay indices (Bremer, 1994) were measure by keeping step-by-step longer trees, and taking a strict consensus of each run, until all nodes were collapsed, and recording the number of steps required to collapse nodes. Bootstrap analyses were conducted to test nodal support, based on 100 replicates, each with 100 random step-wise additions per replicate. Characters 2, 7, 8, and 37 were treated as ordered, following the reasoning proposed by Wilkinson (1992) and Campbell & Frost (1993); exceptions to this treatment are discussed with the character descriptions (Appendix III). All characters were equally weighted. When possible, multiple specimens were examined in order to assess individual variation. In the few cases in which multiple character states were observed among individuals, the character was coded as polymorphic to account for all observed conditions. Variation in character states among specimens examined were coded as polymorphic in the data matrix (e.g., 0/1; see Wiens, 2000). In almost every case, we had only one or two skeletal specimens available, so we were unable to employ any frequency-based parsimony methods (e.g., Smith & Gutberlet, 2001). The complete non-molecular data matrix appears in Table 3; descriptions of each character are presented in Appendix III. Missing data were coded as “?” in all analyses. Phylogenetic analyses of molecular data. Analyses of the molecular data were based on parsimony and Bayesian inference because of the simplistic model in parsimony, particularly for the less-informative nuclear loci, and Bayesian for the convenience of combining the non-molecular data. Parsimony analyses were conducted in PAUP* using the heuristic search options with 100 random, step-wise additions, tree bisection-reconnection branch swapping algorithm, saving multiple best-trees. Bootstrap analyses were conducted to test nodal support, based on 100 replicates, each with 10 random step-wise additions per replicate for the mtDNA and nuclear data separately. The nuclear data were set to have maximum number of trees saved at 10,000 for both heuristic and bootstrap searches. For the combined molecular data, parsimony bootstrap analyses were based on 1000 replicates, each with 100 random step-wise additions per replicate. Bayesian analyses were conducted in MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). Each partition was analyzed in MrModeltest v2.2 (Nylander, 2004) to determine the best model of nucleotide substitution under the Akaike Information Criterion, because this method penalizes increased parameters, thus favors a more simplistic model than the hierarchical likelihood ratio test (Felsenstein, 2004). The EVOLUTION OF MESOAMERICAN TOADS Zootaxa 3138 © 2011 Magnolia Press · 7 8 · Zootaxa 3138 © 2011 Magnolia Press MENDELSON ET AL. EVOLUTION OF MESOAMERICAN TOADS Zootaxa 3138 © 2011 Magnolia Press · 9 molecular data were separated into 18 partitions, one for each codon position of each protein-encoding loci (CXCR4, RAG1, cyt b, ND2, and CO1 because a different model was selected for each codon, demonstrating they are evolving under different models) and one each for the RNA 12S, 16S, and tRNA/OL region. Two analyses were run for 50 million generations each, saving trees every 1000, with four heated chains (user defaults). Stationarity was assessed by the average standard deviation of split frequencies (ASDSF < 0.01) and visual plots of log-likeli- hood by generation in Tracer v1.2 (Rambaut and Drummond, 2004); the first 10,000 trees (of 50,000) were dis- carded as the burn-in. A 50% majority-rule with compatible groups (“allcompat”) consensus was taken from the remaining trees and posterior probabilities of 0.95 or above were considered significant. Phylogenetic analyses of molecular and non-molecular data. The combined datasets were analyzed under parsimony and Bayesian conditions. Parsimony analyses were conducted in PAUP* under the same conditions as the molecular data alone (see above), with 1000 bootstrap replicates, each with 100 random additions per replicate, with the morphological characters 2, 7, 8, and 37 coded as ordered, all others unordered. Bayesian analyses were conducted in MrBayes with 19 partitions, 18 for the molecular data (as above) and the 19th for the morphological data with characters 2, 7, 8, and 37 coded as ordered, all others unordered, and using the Mk model (Lewis, 2001) with a parameter (Γ) for rate variation among characters. Two analyses were run for 50 million generations each, saving trees every 1000, with four heated chains (user defaults). Stationarity was assessed by the ASDSF (<0.01) and by plotting log-likelihood by generation Tracer; the first 10,000 trees were discarded. An “allcompat” consen- sus was taken from the remaining trees and posterior probabilities of 0.95 or above were considered significant. Alignments were deposited in the Dryad Repository (doi:10.5061/dryad.t1r37b7v). Results Phylogenetic analyses of non-molecular data. Parsimony analysis of the 44 non-molecular data discovered 149 equally most parsimonious trees (170 steps; CI = 0.353; RI = 0.637). A strict consensus of these trees failed to recover a monophyletic Incilius, with the taxon Rhinella marina being placed therein. Among the outgroups, the following taxa formed a clade: Rhaebo haematiticus, Rhinella margaritifera, plus the taxon pair Crepidophryne + Rhinella festae (Fig. 1). A clade containing all of the “Forest toad” species (sensu Mendelson et al., 1999, and Mulcahy & Mendelson, 2000) was recovered with68% bootstrapsupport. The majority of other species tradition- ally referred to the valliceps and coccifer groups, and Rhinella marina were in a polytomy, including the Forest toad clade. The remaining species of Incilius were placed in poorly resolved basal clades, with the most basal divergence being I. fastidiosus. Phylogenetic analyses of molecular data.We obtained 5,898 aligned base pairs (bp), 4,317 bp of mtDNA and 1,581 bp of nuclear DNA from 52 individual specimens, characteristics of each locus, including size and number of parsimony-informative sites can be found in Table 4. Parsimony analyses of the mtDNA recovered 27 equally par- simonious trees, each 6,827 steps. A strict consensus of these trees (Appendix IV, Fig. A) recovered a monophyl- etic Incilius, with I. bocourti as the most basal divergence, with a clade containing I. tacanensis, and I. alvarius + I. occidentalis as the next most basal divergence. The I. valliceps group was resolved as monophyletic and weakly supported as sister to a (marmoreus (canaliferus + perplexus)) clade. Crepidophryne was nested within Incilius. The coccifer group is strongly placed sister to a (fastidiosus (Crepidophryne (coniferus +karenlipsae))) clade—the I. coniferus group. Parsimony analyses of the nuclear loci (CXCR4 and RAG1) recovered 33,275 equally parsimo- nious trees of 529 steps, with generally poor resolution. A strict consensus tree (Appendix IV, Fig. B) showed most of the valliceps group as monophyletic with the exception of a clade containing luetkenii + mazatlanensis and mel- anochlorus + aucoinae, which was placed in a basal polytomy among all other clades. Crepidophryne was again nested within Incilius, and placed sister to I. coniferus. Parsimony analyses of the 5,898 aligned bp from combined nuclear and mtDNA data contained 1,413 parsimony-informative characters and resulted in two trees, 7,388 steps in length. A strict consensus tree (Fig. 2) has a topology with I. bocourti as the first divergence in Incilius, then a clade consisting of I.tacanensis,I. alvarius + I. occidentalis that is sister to all remaining species. Clades referable to I. coniferus, I. coccifer, and I. valliceps groups were recovered, with I. coniferus group and I. coccifer group sis- ter to one another. A (canaliferus (marmoreus + perplexus)) clade was also found, in a basal polytomy with the I. coniferus + I. coccifer groups, and the I. valliceps group. Within the I. valliceps group, Forest toads were rendered paraphyletic with respect to the Lowland toads (Fig. 2). 10 · Zootaxa 3138 © 2011 Magnolia Press MENDELSON ET AL.