Journal of Biogeography (J. Biogeogr.) (2006) 33, 1889–1904

ORIGINAL Historical biogeography of lowland ARTICLE species of toads (Bufo) across the Trans- Mexican Neovolcanic Belt and the Isthmus of Daniel G. Mulcahy*, Benson H. Morrill and Joseph R. Mendelson III

Department of Biology, Utah State University, ABSTRACT Logan, UT, USA Aim In this study, we investigate phylogeographic structure in two different species groups of lowland toads. First, we further investigate strict parapatry of the Pliocene-vicariant Bufo valliceps/B. nebulifer species pair. Secondly, we test for similar phylogeographic structure in the distantly related toad B. marinus,a species we hypothesize will show a Pleistocene dispersal across the same area. Location The eastern extension of the Trans-Mexican Neovolcanic Belt (TMNB) contacts the Atlantic Coast in central , . Although it is not a massive structure at this eastern terminus, the TMNB has nonetheless effected vicariance and subsequent speciation in several groups of animals. The Isthmus of Tehuantepec unites the North American with Nuclear and is also known to be a biogeographic barrier for many taxa. Methods We use sequence data from two mitochondrial DNA genes (c. 550 base-pairs (bp) of 16S and c. 420 bp of cyt b) from 58 individuals of the B. valliceps/nebulifer complex, collected from 24 localities. We also present homologous sequence data from 23 individuals of B. marinus, collected from 12 localities. We conduct maximum-parsimony, maximum-likelihood and Bayesian analyses to investigate phylogeographic structure. We then use parsimony- and likelihood-based topology tests to assess alternative phylogenetic hypotheses and use a previously calibrated molecular rate of evolution to estimate dates of divergence. Results Our results further define the parapatric contact zone across the TMNB between the Pliocene-vicariant sister species B. valliceps and B. nebulifer.In contrast, phylogenetic structure among populations of B. marinus across the TMNB is much shallower, suggesting a more recent Pleistocene dispersal in this species. In addition, we found phylogeographic structure associated with the Isthmus of Tehuantepec in both species groups. Main conclusions The existence of a Pliocene–Pleistocene seaway across the Isthmus of Tehuantepec has been controversial. Our data depict clades on either side of the isthmus within two distinct species (B. valliceps and B. marinus), although none of the clades associated with the isthmus, for either species, are *Correspondence: Daniel G. Mulcahy, reciprocally monophyletic. In the B. valliceps/B. nebulifer complex, the TMNB Department of Herpetology, California separation appears to predate the isthmian break, whereas in B. marinus dispersal Academy of Sciences, 875 Howard Street, San across the TMNB has occurred subsequent to the presence of a barrier at the Francisco, CA 94103-3009, USA. E-mail: [email protected] Isthmus of Tehuantepec.

Present address: Department of Herpetology, Keywords Zoo Atlanta, 800 Cherokee Ave SE, Atlanta, GA Bufo nebulifer, Bufo marinus, Bufo valliceps, Bufonids, Central America, Mex- 30315-1440, USA. ico, mitochondrial DNA, phylogeography.

ª 2006 The Authors www.blackwellpublishing.com/jbi 1889 Journal compilation ª 2006 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2006.01546.x D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

state of Veracruz, Mexico (Fig. 2). The imposing backbone of INTRODUCTION the TMNB began activity during the mid-Miocene to Pliocene The Trans-Mexico Neovolcanic Belt (TMNB) is one of the (de Cserna, 1989), with its greatest development during the predominant geographical features of Mexico, and its geolo- Pliocene in the eastern portion (Ferrusquia-Villafranca, 1993), gical development has been posited as a primary contributor to giving rise to the highest peaks of Mexico. However, the the biogeographic histories of many upland taxa in central easternmost fingers of the TMNB, where they contact the Gulf Mexico (e.g. Campbell & Frost, 1993; Darda, 1994; Sullivan of Mexico, are barely noticeable to the casual observer. During et al., 2000; Castoe et al., 2003). However, a recent series of much of the Pliocene, sea-level maxima caused by a warmer papers have independently demonstrated the considerable effectively covered the entire coastal plain of eastern influence of this transverse massif on the biogeography and Mexico (Bryant et al., 1991). Later during the Pleistocene, sea evolution of the lowland fauna on both the Pacific (Mateos, levels fluctuated with corresponding glacial and inter-glacial 2005) and the Atlantic coasts of Mexico (Mulcahy & cycles (Beard et al., 1982). Glacial-maxima lowered sea levels Mendelson, 2000; Hulsey et al., 2004; Zaldı´var-Rivero´n et al., exposing large sections of the continental shelf, which greatly 2004). These papers generally support earlier hypotheses based increased the aerial extent of the coastal plain (Fig. 1), whereas on observations of consistent disjunctions in the distribution glacial minima raised sea levels, inundating much of the coastal of fishes (Rosen, 1978), and of reptiles and mammals (Pe´rez- plain by about 300 m (Ewing & Lopez, 1991). The area of the Higareda & Navarro, 1980), specifically along the Atlantic Sierra de Los Tuxtlas (Fig. 1) is of volcanic origin from the late Versant of Mexico. Therefore, it is suggested that the TMNB Cenozoic (Ferrusquia-Villafranca, 1993), and may have been may form a common geographical barrier to lowland species in isolated from the mainland during the Pliocene and during this . Pleistocene glacial minima. Farther south, debate continues as The concept of a massive volcanic chain such as the TMNB to whether a seaway existed at the Isthmus of Tehuantepec, acting as a vicariant feature to lowland populations is easily separating from Nuclear Central America tractable, but the reality is that the TMNB withers to a tiny (Campbell, 1999). string of lava-rock strewn hills at its eastern terminus (Fig. 1). By comparing phylogenetic structure from mtDNA It makes final contact with the current coastline, as a series of sequences of lowland toads in this region, Mulcahy & small fingers of raised lava-rock (the northern-most being just Mendelson (2000) demonstrated that the prevailing concept south of the small town of Palma Sola, the southern-most just of a single wide-ranging species (Bufo valliceps Wiegmann) north of the city Cardel, with a small pocket of suitable habitat should be replaced by recognition of a species pair showing an occurring in the middle, near the town of El Viejo´n) all in the apparent parapatric distribution in the region: B. valliceps

Figure 1 Map of the study area, showing major geographic features discussed in the text. Grey shading from light to dark indicates elevations from 300–900 m, 900–2100 m and > 2100 (including black) m a.s.l., respectively. Dotted line shows extent of continental shelf, much of which was exposed during lower sea levels of the Pleistocene (from Bryant & Bryant, 1991).

1890 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo)

The three species of toads in our study are ecologically similar in their general reproductive biology and overall natural history. All three species are invasive, ‘weedy’ species that are typically more abundant in secondary, degraded habitats than in undisturbed primary forests (Zug & Zug, 1979; Mendelson, 1994; Lee, 1996; Campbell, 1998; McCranie & Wilson, 2002; Savage, 2002). These attributes would suggest that they are suitably comparable to one another, in order to test hypotheses of historical biogeography and that their invasive, dispersal-prone tendencies would make them a conservative test of the effect of the TMNB on lowland species. In this paper, we use c. 970 base-pairs (bp) of sequence data from two mtDNA genes (cyt b and 16S) from recently collected samples along a geographic transect across the TMNB to address the historical biogeography of the Atlantic Versant lowlands of Mexico. Specifically, we test three main hypotheses Figure 2 Map of the eastern terminus of the Trans-Mexico Neovolcanic Belt (TMNB) in central Veracruz. Squares indicate regarding the relationships and distributions concerning reference towns used in text, circles indicate collecting localities for bufonid toads in this region: (1) the hypothesis that B. valliceps Bufo nebulifer (Mx8) B. valliceps (Mx9–10) and B. marinus (Mx8– and B. nebulifer are parapatric at the eastern terminus of the 10) in relation to the eastern-most portion of the TMNB (see TMNB (i.e. as proposed by Mulcahy & Mendelson, 2000); (2) Figs 3 & 4 for complete sampling of each taxon). Shaded contours the hypothesis that the sympatric toad B. marinus shows a indicate elevation following Fig. 1. Los Tuxtlas is the Pliocene pattern of more recent dispersal across the TMNB, consistent volcanic uplift on the coastal plain south of the eastern terminus of with the Pleistocene dispersal, followed by the vicariance the TMNB (see Discussion). hypothesis of Mulcahy & Mendelson (2000); and (3) the hypothesis that there is evidence of a phylogeographic signal within B. valliceps and/or B. marinus, which is consistent with ranging from central Veracruz, Mexico, southward to Costa a historical seaway across the Isthmus of Tehuantepec. We use Rica; and B. nebulifer Girard, ranging from central Veracruz both parsimony and likelihood topology tests to compare our northward to the of America. Mulcahy phylogenetic results with alternative hypotheses. We also & Mendelson (2000) proposed and tested two historical discuss genetic variation, in terms of sequence divergence hypotheses related to the timing of this speciation event: (1) and the rate of molecular evolution previously calibrated for Miocene–Pliocene vicariance associated with the orogeny of the B. valliceps/nebulifer group (Mulcahy & Mendelson, 2000), the TMNB; and (2) Pleistocene dispersal and vicariance caused between clades associated with either sides of the TMNB and by low sea levels initially exposing the continental margin, the Isthmus of Tehuantepec. followed by raised sea levels, which obliterated the coastal plain in this region. Their results supported the Miocene–Pliocene MATERIALS AND METHODS vicariance hypothesis. However, their sampling was insuffi- cient to demonstrate strict parapatry of these two species on Taxon sampling the narrow coastal plains on either side of the TMNB. A third species, B. marinus Linnaeus, ranges from the We collected sequence data for 19 new B. valliceps and of to the southern tip of Texas, B. nebulifer specimens along the eastern terminus of the USA, in . This species is largely sympatric with TMNB (Fig. 3). For consistency, we continued our numbering B. valliceps, partially sympatric with B. nebulifer, and has an system from that of Mulcahy & Mendelson (2000). Ten of the apparently continuous distribution across the eastern terminus new specimens represent B. nebulifer (locality Mx8, Figs 2 & 3) of the TMNB. Bufo marinus is of a South American origin and were collected just north of the town of Palma Sola, seven (Pauly et al., 2004; Pramuk, 2006), and most likely entered represent B. valliceps (locality Mx10, Figs 2 & 3) collected Central America during the Pliocene (Slade & Moritz, 1998). A south of Cardel, and two were collected in the middle of the broad-scale phylogeographic study of B. marinus (Slade & eastern-most reaches of the TMNB, near the town of El Viejo´n, Moritz, 1998) indicated that this complex showed dramatic tentatively assigned to B. valliceps (locality Mx9, Figs 2 & 3). historical effects of the orogeny of the in South America, We compared our new sequences with those of Mulcahy & and lower levels of genetic divergence between samples from Mendelson (2000), and used one representative of each unique Costa Rica and Mexico. These results suggest that a finer-scale haplotype in the phylogenetic analyses. This resulted in a total study may show that the biogeographic history of northern of 28 specimens of B. nebulifer (11 haplotypes) and 30 B. marinus is more consistent with the Pleistocene dispersal specimens of B. valliceps (21 haplotypes; see Results). Multiple and vicariance hypothesis of Mulcahy & Mendelson (2000). specimens from the same locality were individually labelled

Journal of Biogeography 33, 1889–1904 1891 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

Figure 3 Map of the geographic distribution and sampling for the Bufo valliceps and B. nebulifer used in this study. Squares indicate new sample localities, circles represent those from Mulcahy & Mendelson (2000); grey circles indicate identical haplotypes omitted from phylogenetic analyses. Box indicates area of the TMNB transect highlighted in Fig. 2. alphabetically (e.g. Mx8a–Mx8j). We also collected sequence Laboratory protocols data from 23 specimens of B. marinus from Central America and northern Mexico (Fig. 4). Again, for sake of convenience, We collected sequence data from c. 550 bp region of the we kept the same numbering system as the B. valliceps/ ribosomal gene 16S and c. 420 bp region of the protein-coding B. nebulifer data because many of our sampling localities were gene cyt b. The gene- have been demonstrated to be the same (e.g. Mx8–Mx10). Outgroup taxa were chosen accurate markers for resolving recent divergences (Graybeal, following phylogenetic hypotheses of Mulcahy & Mendelson 1993, 1997; Mulcahy & Mendelson, 2000). For all specimens, (2000; D.G. Mulcahy & J.R. Mendelson, unpubl. data); Pauly tissue was taken from liver, muscle, skin or toe clips and stored et al. (2004) and Pramuk (2006). Outgroup taxa for analyses of either at )80 C or in 95% ethanol. Extraction of DNA B. nebulifer and B. valliceps included B. mazatlanensis, followed standard phenol/chloroform extraction methods B. campbelli (AY008253–4 of Mulcahy & Mendelson, 2000), (Maniatis et al., 1982). Primers used for PCR amplification and B. coccifer (AY927856 and AY927863 of Mendelson et al., and sequence reactions (for cyt b, MVZ43: GAGTCTGCCT[A/ 2005). Outgroup taxa for analyses of B. marinus included T]AT[T/C]GC[C/T]CA[A/G]AT, 3¢ and MVZ28: B. crucifer, B. schneideri and a specimen of B. marinus from CGAGGC[G/C]CC[T/C]GCAAT[A/G]ATAA, 3¢; for 16S, South America (Pramuk, 2006). Slade & Moritz (1998) showed 16Sar: CGCCTGTTTATCAAAAACAT, 3¢ and 16Sbr: that B. marinus may be paraphyletic with respect to South CCGGTCTGAACTCAGATCACGT 3¢). Amplifications for American samples and B. schneideri, therefore we considered PCR followed that of Mulcahy & Mendelson (2000). Sequence the South American individual of B. marnius as an outgroup. reactions were done in both directions using the same primers Voucher specimens for this study are deposited at the used for PCR, with BigDyeTM Terminator Cycle Sequencing following institutions: University of Texas at Arlington Kits (Version 2, ABI Part No. 4303152; Foster City, CA, USA) (UTA); Museo de Zoologia, Facultad de Ciencias, Universidad in 10–12 mL reactions following recommended protocols. Autonoma de Mexico (MZFC), and University of Kansas Sequence-reaction products were cleaned with SephadexTM (KU). Voucher numbers, specific locality information and (Piscataway, NJ, USA) and run on an ABI 377 automated GenBank accession numbers for each specimen are listed in sequencer. Individual sequences were aligned with compli- Table 1. mentary strands in SequencherTM 4.1.2 (Gene Codes Corp.,

1892 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo)

Figure 4 Map of the northern distribution and sampling for Bufo marinus (squares) in this study. Sample localities follow the same numbering system from the B. valliceps and B. nebulifer, with the addition of several new localities for B. marinus only. Grey squares indicate identical haplotypes omitted from phylogenetic analyses. Box indicates area of the TMNB transect highlighted in Fig. 2.

Ann Arbor, MI, USA). The protein-coding region of cyt b was genes model parameters were used in paup* for the ML translated into amino acid sequence and the G-C content was analyses of both genes combined for 100 step-wise random inspected to verify that authentic mtDNA sequences were addition replicates. obtained, using MacClade 4.0 (Maddison & Maddison, 2000). Three Bayesian Inference (BI) analyses were conducted on combined (gene) data sets for each group using the program Mr. Bayes version 10.3 (Huelsenbeck & Ronquist, 2001). Each Phylogenetic analysis gene-region was run under its own model using data To determine if the two gene regions, 16S and cyt b, partitions. Each analysis was run for 5 · 106 generations, each contained similar phylogenetic signals, we conducted parti- using four-heated Markov chains (using program defaults) and tion-homogeneity tests. We ran 100 replicates with 10 each was sampled every 100 generations. Posterior-probabil- random additions at each replicate between 16S and cyt b. ities were estimated by conducting a 50% majority-rules This test determines whether the data sets are giving consensus tree after discarding the burn-in trees. Runs were significantly different signals. Maximum parsimony (MP) determined to reach stationarity visually by plotting likelihood and Maximum-likelihood (ML) analyses were conducted in scores against generations. Nodes with 95% or greater paup* beta version 4.0b10 (Swofford, 1999). All MP analyses posterior-probabilities were considered significant. were conducted with Tree-bisection reconnection branch- We use the rate of molecular evolution of 0.33% per lineage, swapping, ACCTRAN optimization, with 100 random step- per million years, (Mulcahy & Mendelson, 2000) to estimate wise additions. Non-parametric bootstrap analyses under the dates of divergence in the B. valliceps/nebulifer complex. MP criterion were conducted in paup* with 1000 replicates, However, we could not confidently calibrate a rate in the using random step-wise additions with 25 replicates for each B. marinus group because the B. valliceps/nebulifer and B. mar- bootstrap replicate. Nodes with indices of 75% or greater inus data are not clock-like when compared with other Middle were considered well supported. The program Modeltest 3.06 American bufonids (D.G. Mulcahy & J.R. Mendelson, unpubl. (Posada & Crandall, 1998) was used to select appropriate data). Therefore, we report average pair-wise sequence models of evolution, and was conducted on each gene divergence between major clades in both groups to estimate separately, as well as both genes combined for each data set the relative age of divergences, but acknowledge those in the (B. valliceps/B. nebulifer and B. marinus). The combined B. marinus group may be less accurate.

Journal of Biogeography 33, 1889–1904 1893 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

Table 1 Specimen information for sequence data collected for this study (other specimens used in this study are from Mulcahy & Mendelson, 2000). The first column list the species and locality number from maps (Figs 2–4), and names in phylogenetic trees (Figs 5 & 6). The second column gives general locality followed by museum specimen number and GenBank accession numbers for 16S and cyt b

Taxon ID Locality Voucher No. 16S Cyt b

Bufo nebulifer Mx8a Mexico: Veracruz: north of Palma Sola UTA A-54860 DQ415599 DQ415619 Mx8b Mexico: Veracruz: north of Palma Sola UTA A-54861 DQ415600 DQ415620 Mx8c Mexico: Veracruz: north of Palma Sola UTA A-54862 DQ415601 DQ415621 Mx8d Mexico: Veracruz: north of Palma Sola UTA A-54863 DQ415602 DQ415622 Mx8e Mexico: Veracruz: north of Palma Sola UNAM-JRM 4851 DQ415603 DQ415623 Mx8f Mexico: Veracruz: north of Palma Sola UNAM-JRM 4852 DQ415604 DQ415624 Mx8g Mexico: Veracruz: north of Palma Sola UTA A-54864 DQ415605 DQ415625 Mx8h Mexico: Veracruz: north of Palma Sola UTA A-54865 DQ415606 DQ415626 Mx8i Mexico: Veracruz: north of Palma Sola UNAM-JRM 4855 DQ415607 DQ415627 Mx8j Mexico: Veracruz: north of Palma Sola UTA A-54859 DQ415608 DQ415628 B. valliceps Mx9a Mexico: Veracruz: near El Viejo´n UNAM-JRM 4824 DQ415609 DQ415629 Mx9b Mexico: Veracruz: near El Viejo´n UNAM-JRM 4825 DQ415610 DQ415630 Mx10a Mexico: Veracruz: south of Cardel UNAM-JRM 4799 DQ415611 DQ415631 Mx10b Mexico: Veracruz: south of Cardel UNAM-JRM 4801 DQ415612 DQ415632 Mx10c Mexico: Veracruz: south of Cardel UTA A-54855 DQ415613 DQ415633 Mx10d Mexico: Veracruz: south of Cardel UTA A-54856 DQ415614 DQ415634 Mx10e Mexico: Veracruz: south of Cardel UTA A-54858 DQ415615 DQ415635 Mx10f Mexico: Veracruz: south of Cardel UTA A-54854 DQ415616 DQ415636 Mx10g Mexico: Veracruz: south of Cardel UTA A-54857 DQ415617 DQ415637 B. mazatlanensis Mexico: Sinaloa: near Cosala UNAM-JRM 4491 DQ415618 DQ415638 B. marinus Mx8a Mexico: Veracruz: north of Palma Sola UTA A-54875 DQ415547 DQ415573 Mx8b Mexico: Veracruz: north of Palma Sola UNAM-JRM 4846 DQ415548 DQ415574 Mx8c Mexico: Veracruz: north of Palma Sola UNAM-JRM 4848 DQ415549 DQ415575 Mx8d Mexico: Veracruz: north of Palma Sola UNAM-JRM 4844 DQ415550 DQ415576 Mx9a Mexico: Veracruz: near El Viejo´n UTA A-54882 DQ415551 DQ415577 Mx9b Mexico: Veracruz: near El Viejo´n UTA A-54873 DQ415552 DQ415578 Mx9c Mexico: Veracruz: near El Viejo´n UNAM-JRM 4834 DQ415553 DQ415579 Mx9d Mexico: Veracruz: near El Viejo´n UNAM-JRM 4835 DQ415554 DQ415580 Mx9e Mexico: Veracruz: near El Viejo´n UTA A-54881 DQ415556 DQ415582 Mx9f Mexico: Veracruz: near El Viejo´n UTA A-54879 DQ415555 DQ415581 Mx9g Mexico: Veracruz: near El Viejo´n UTA A-54877 DQ415557 DQ415583 Mx10a Mexico: Veracruz: south of Cardel UTA A-54878 DQ415558 DQ415584 Mx10b Mexico: Veracruz: south of Cardel UTA A-54871 DQ415559 DQ415585 Mx11a Mexico: Guerrero: near Atoyac UTA A-54869 DQ415560 DQ415586 Mx11b Mexico: Guerrero: near Atoyac UTA A-54870 DQ415561 DQ415587 Mx12 Mexico: Sinaloa: near Cosala UTA A-54868 DQ415562 DQ415588 C1 Costa Rica: Heredia: at Chilamate toe-clip only DQ415563 DQ415589 E1 El Salvador: Ahuachapan: El Imposible KU 289750 DQ415564 DQ415590 E2 El Salvador: Ahuachapan: El Imposible KU 289772 DQ415565 DQ415591 G1 : Huehuetenango: near Nenton UTA A-50876 DQ415566 DQ415592 G5 Guatemala: Izabal: Montanas del Mico UTA A-50870 DQ415567 DQ415593 H3 Honduras: El Paraiso: Las Manos UTA A-50638 DQ415568 DQ415594 H4 Honduras: Colon: Quebrada Machin USNM 534124 DQ415569 DQ415595 B. crucifer Brazil: Sao Paulo USNM 303015 DQ415570 DQ415596 B. marinus Ecuador: Loja NA Vilcabamba KU 217482 DQ415571 DQ415597 B. schneideri Paraguay: San Luis de la Sierra KU 289057 DQ415572 DQ415598

We tested alternative phylogenetic hypotheses to support or test putative biogeographic barriers inferred from the uncon- refute particular biogeographic scenarios under both parsi- strained phylogenetic hypotheses. Alternative topologies were mony and likelihood conditions. Constraints were designed to constructed using MacClade and implemented as constraints

1894 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo) in paup* and run for 100 random-addition replicates, in the between the two trees was the placement of a sub-clade same manner as the unconstrained analyses (see Appendix 1 containing samples of B. nebulifer from Texas. This clade was for constraint topologies). Under parsimony conditions, placed basal to the remaining samples of B. nebulifer in one alternative hypotheses were tested by finding the most tree, and nested among them in another. A strict consensus of parsimonious trees compatible with the imposed constraints the two trees was nearly identical to the ML and BI analyses and comparing them with the unconstrained MP trees using (see below), and supports two monophyletic clades referable to the Wilcoxon signed-rank tests (Templeton, 1983; Felsenstein, B. valliceps and B. nebulifer. These two clades were supported 1985). One- and two-tailed Wilcoxon signed-rank tests were by bootstrap values of 97 and 100, respectively (Fig. 5). There conducted because one-tailed probabilities are close to the was relatively little phylogenetic structure among samples of exact but are not always conservative, whereas two-tailed test B. nebulifer; however, two sub-clades within B. valliceps show are always conservative (Felsenstein, 1985). We used the S–H some suggestion of a phylogenetic break that is geographically (Shimodaira & Hasegawa, 1999) topology test to compare the consistent with the Isthmus of Tehuantepec. Two of the three constrained and unconstrained ML trees, with one-tailed samples from Los Tuxtlas, Veracruz (north of the isthmus, (RELL) distributions and 1000 bootstrap replicates. This test is samples Mx3a and b), were placed in a clade with all other sam- preferred over the K–H (Kishino & Hasegawa, 1989) test ples found south of the Isthmus of Tehuantepec, while one (Goldman et al., 2000). However, it may be too conservative in (Mx3c) grouped with those north of the isthmus (Fig. 5). some cases (Buckley et al., 2001), and borderline results should Modeltest selected TrN + G (gamma shape parameter) be used with caution (Townsend et al., 2004). model under the hierarchical likelihood ratio test (hLRT) with base frequencies of A ¼ 0.3016; C ¼ 0.2366; G ¼ 0.1789; T ¼ 0.2828, six substitution types with a rate-matrix of RESULTS A–G ¼ 7.3187; C–T ¼ 11.4953 (all others equal to 1.0), and a gamma shape parameter: G ¼ 0.0713. The ML analysis run Phylogenetic analyses of B. valliceps and B. nebulifer with these parameters produced one tree with a score of Of the 19 recently collected individuals included from the )ln L ¼ 2723.60976 (Fig. 5). The results of this analysis were B. valliceps/nebulifer data set, 14 represented unique haplo- similar to those of the MP analysis, with both B. nebulifer and types when sampled across both genes (i.e. some individuals B. valliceps being monophyletic, and with some support for an were identical for the 16S sequence, but had different cyt b isthmian break in B. valliceps (again slightly obscured by sequences, and vice versa). When compared with the data of samples from Los Tuxtlas, Veracruz). In the Bayesian analyses, Mulcahy & Mendelson (2000) this resulted in a total of 32 the three searches each appeared to reach stationarity near unique haplotypes for the B. valliceps/nebulifer data set. Two 15,000 generations, therefore the first 2000 trees representing individuals from locality Mx8 were identical (Mx8d and h), conservatively the first 20,000 generations were discarded as two individuals (Mx8i and j) were identical to samples Mx1 the burn-in process. Average tree scores ()ln L ¼ 2826.1891) to Mx2, and four unique haplotypes were recovered from were taken from each of the 48,000 trees. The 50% majority- locality Mx10 (Mx10d, f, g were identical; Mx10e was rules consensus trees were largely consistent with the MP, and identical to Mx3c from Los Tuxtlas). All phylogenetic identical to the ML analyses; results for the first BI run are analyses were conducted on a data set consisting of the 32 shown in Fig. 5. The outgroup taxon B. coccifer was removed unique haplotypes and three outgroup taxa (B. coccifer, in Fig. 5 to focus on branch-lengths within the ingroup. B. mazatlanensis and B. campbelli). Analysis of the 16S data Uncorrected sequence divergence between B. valliceps and set (562 characters, 73 variable, 26 parsimony-informative) B. nebulifer ranged from 2.8% to 4.0%; similarly, the corrected produced 3767 trees (103 steps each); tree not shown. A strict differences ranged from 2.8% to 4.1% (Table 2). Corrected consensus of these trees contained one clade, consisting of all sequence divergence (HKY85) for the two clades of B. valliceps the samples of B. nebulifer, with the remaining samples in an on either side of the isthmus ranged from 1.0% to 2.0% unresolved polytomy (tree not shown). The cyt b data set (Table 2). It is worth noting that the model of evolution used (421 characters, 105 variable, 57 parsimony-informative) in the ML phylogenetic analyses of Mulcahy & Mendelson produced six trees (155 steps); tree not shown. A strict (2000) was the Hasegawa–Kishino–Yano (HKY85; Hasegawa consensus of these trees is generally consistent with the et al., 1985). That model was chosen to account for among-site overall combined MP tree (see below). The partition-homo- rate variation in the data (Mulcahy & Mendelson, 2000), prior geneity tests revealed the two data sets were significantly to the wide use of Modeltest. In the present study, we used the different (P ¼ 0.04); however, this test may be too conser- program Modeltest and a different model of evolution was vative at the a ¼ 0.05 level and combining weakly-incongru- selected for the data (TrN + G; see above). ent data sets generally provides a more accurate estimation The results from the phylogenetic analyses based on the two (Cunningham, 1997). Therefore, we combined the two gene- different models are very similar; however, the corrected regions into a single analysis. sequence divergences between taxa are very different. For The MP analysis (100 random additions) of 35 operational example, the corrected sequence divergence average between taxonomic units (OTUs) resulted in two most-parsimonious B. valliceps and B. nebulifer is 3.3% using the HKY85 model trees (262 steps, RI ¼ 0.88, CI ¼ 0.79). The only difference (Table 2), whereas the average difference using the TrN + G

Journal of Biogeography 33, 1889–1904 1895 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

Figure 5 Bayesian analysis phylogram for the Bufo valliceps/nebulifer data set. Topology was nearly identical to the MP and ML ana- lyses. The outgroup B. coccifer was second- arily removed to focus on branch lengths within the ingroup. Parsimony bootstrap values are shown above and Bayesian pos- terior probabilities (· 100) are shown below significant clades. Bars on right indicate geographic distribution of haplotypes relative to the TMNB and the Isthmus of Tehua- ntepec.

Table 2 Pair-wise percentage sequence divergence within and between Bufo nebulifer and B. valliceps samples (excluding identical hapl- otypes); averages followed by ranges in parentheses. Comparisons with HKY85-corrected distances (see text) are shown in italics, others are uncorrected. Comparisons within B. valliceps are shown within and between those found north and south of the Isthmus of Tehuantepec (with Mx3a–b in the south)

B. valliceps

B. nebulifer B. valliceps North of Isthmus South of Isthmus

B. nebulifer 0.4% (0.1–0.8%) 3.2% (2.8–4.0%) – – B. valliceps 3.3% (2.8–4.1%) 1.0% (0.1–2.1%) –– North of Isthmus – – 0.3% (0.1–0.6%) 1.5% (1.0–2.0%) South of Isthmus – – 1.5% (1.0–2.1%) 0.7% (0.1–1.6%)

model is 5.3% (range ¼ 4.1–7.4%), which changes interpre- and the HKY85-corrected data, we calculate – from Table 2, tations using the molecular clock. We used HKY85-corrected 3.3% divided by 2 (per lineage), divided by 0.33 (the rate of distances to compare sequence data between lineages in this evolution) – an average c. 5.0 ma (range ¼ 4.2–6.2 ma using study (Table 2) to be consistent with our previous study; min.–max., Table 2) separation of B. valliceps and B. nebulifer however, we present the TrN + G model calculations for a and c. 8.0 ma (range ¼ 6.2–11.2 ma) using the TrN + G comparison. Using the rate of molecular evolution (0.33% per corrected data. Using the HKY85 calculations, the divergence lineage, per million year) from Mulcahy & Mendelson (2000) at the Isthmus of Tehuantepec in B. valliceps is estimated to be

1896 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo) c. 2.3 ma (range ¼ 1.5–3.2 ma), and c. 2.7 ma (range ¼ 1.7– (Mx9; Fig. 6). The remaining samples along the transect 4.1 ma) using the TrN + G corrected data (1.8%, (Mx10) were placed in a polytomy with those from Guerrero range ¼ 1.1–2.7% sequence divergence). (Mx11) and Sinaloa (Mx12), Mexico. In the MP analysis, we found support for a break associated with the Isthmus of Tehuantepec (Fig. 6), with the exception of one sample (G1) Phylogenetic analyses of B. marinus taken from the head of the Grijalva Valley, in Guatemala. This Of the 23 individuals from the B. marinus data set, 19 had sample was placed among the westerly samples from Mexico, unique haplotypes when sampled across both genes. Analysis and the support for this clade was weak (< 50%; Fig. 6); of the 16S data set (548 characters, 50 variable, 30 parsimony- however, the clade found southeast of the Isthmus was well- informative) produced 4 trees (56 steps); tree not shown. supported. Analysis of the cyt b data set (425 characters, 93 variable, For the two genes combined, Modeltest selected TrN + G 42 parsimony-informative) produced 70 trees (131 steps), the (gamma shape parameter) as the best-fit model under the strict consensus of which was generally consistent with the hierarchical likelihood ratio test (hLRT) criteria, with base combined MP tree (see below). The partition-homogeneity frequencies of A ¼ 0.2855; C ¼ 0.2417; G ¼ 0.1824; tests revealed that the two data sets were not significantly T ¼ 0.2904, six substitution types with a rate-matrix of different (P ¼ 0.90) and the two were combined into a single A–G ¼ 7.8102; C–T ¼ 14.1003 (all others equal to 1.0), and analysis. The combined MP analysis (100 random additions) of a gamma shape parameter: G ¼ 0.1042. The ML analysis run 22 OTUs resulted in 50 most-parsimonious trees (189 steps, with these parameters produced a tree a score of RI ¼ 0.81, CI ¼ 0.80). Bootstrap values for the major clades )ln L ¼ 2355.4517. Our ingroup samples all had relatively are shown in Fig. 6. We found phylogenetic signal associated short branch lengths, and the results of this analysis were with the break across the TMNB, however the clades were only generally similar to that of the MP analysis. We found similar weakly supported, and showed very little genetic divergence support for a phylogenetic break across the TMNB and the across this break (see below). One clade, containing the Isthmus of Tehuantepec, with sample G1 placed in the clade of samples north of the TMNB (Mx8) had low bootstrap support samples from Mexico (west of the isthmus). In this analysis, (63), and was sister to the clade of samples from the middle B. schneideri rendered B. marinus paraphyletic with respect to

Figure 6 Bayesian analysis phylogram for Bufo marinus. Topology was nearly identical to the MP and ML analyses. Parsimony bootstrap values are shown above and Baye- sian posterior probabilities (· 100) are shown below for significant clades. Bars on right indicate geographic distribution of haplotypes relative to the TMNB and the Isthmus of Tehuantepec.

Journal of Biogeography 33, 1889–1904 1897 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III our sample from South America and the remaining Meso- B. valliceps/nebulifer group showed divergence across the american samples (not shown). TMNB prior to separation across the Isthmus of Tehuantepec, For each gene-region tested separately, Modeltest selected while the B. marinus group showed the opposite (Fig. 7). the HKY + G (gamma shape parameter) model under the However, in both groups, clades associated with either side of hierarchical likelihood ratio test (hLRT) criteria. Therefore, the the isthmus were not reciprocally monophyletic. three Bayesian analyses were run, each with a 2-substitution- We tested alternative phylogenies in order to validate the type (transition/transversion) plus gamma model, and searches strength of the effect of these barriers on our data. First, in appeared to reach stationarity near 10,000–12,000 generations. order to test the effect of the TMNB on the B. valliceps/ The first 2000 trees representing conservatively the first 20,000 nebulifer group, we constrained two B. valliceps haplotypes generations were discarded as the burn-in process. Average tree (Mx9a–b) to be in the B. nebulifer clade and conducted the scores ()ln L ¼ 2400.1138) were taken from the 48,000 trees same analyses that were performed on the unconstrained data. of each run. The 50% majority-rules consensus tree and This resulted in six equally-parsimonious trees, each of which posterior-probabilities for the first run are shown in Fig. 6. were significantly different from either of the unconstrained The results were largely consistent with those of the MP and trees and the ML constrained topology was also significantly ML analyses, with strong support for a monophyletic Meso- different (‘TMNB’, Table 4). Therefore we could reject para- american clade of B. marinus low support for a break across phyly of B. valliceps with respect to the samples at locality the TMNB, and strong support for an isthmian break, again Mx9. Secondly, to test the affect of an isthmian break in with the exception of the position of G1. B. valliceps, we constrained haplotypes Mx3a–c to be in the Sequence divergence for B. marinus across the TMNB ranged clade north-west of the isthmus, but south of the TMNB (with from 0.4% to 0.7% (Table 3). Using the difference between Mx9–10). This resulted in 12 trees that were each significantly north and south of the TMNB, we estimated a date of divergence different from the unconstrained trees. However, the ML of c. 0.9 ma (range ¼ 0.6–1.1). Sequence divergence between constrained tree had an )ln L score difference of 21.740, samples of B. marinus on either side of the Isthmus of which was not significantly different (‘Isthmus’, Table 4). Tehuantepec were slightly larger, ranging from 0.8% to 2.2% Here, we reject reciprocally monophyly of B. valliceps haplo- (Table 3), estimated as c. 2.7 ma (range ¼ 1.2–3.3). Sequence types on either side of the Isthmus of Tehuantepec, but with variation was much greater among samples southeast of the caution. Thirdly, to test for the alternative scenario recovered Isthmus of Tehuantepec (0.1–2.2%) as compared to variation in the B. marinus group, we constrained the B. valliceps/ found north-west of the isthmus (0.1–1.3%), where the greatest nebulifer group to have the divergence associated with the variation was found between sample Mx12 and Mx8–10 (0.8– isthmus to precede that of the TMNB; i.e., enforcing the 1.3%); Mx12 differed from Mx11 by 0.8%. The sample from topology of Fig. 7b on the B. valliceps/nebulifer group, conse- South America was very different from the Mesoamerican quently rendering B. valliceps paraphyletic: (B. valliceps S. of samples (6.3–7.4%; Table 3), with an estimated date of diver- Isthmus (B. valliceps N of Isthmus + B. nebulifer)). This gence ranging from c. 10.9 ma (range ¼ 9.5–11.2). analysis resulted in four MP trees that were significantly different from either of the unconstrained trees based on the parsimony analysis (‘MNB vs. Isthmus’, Table 4). However, Topology tests for both species groups the constrained ML topology had a likelihood score that Although clades associated with two major geographic features differed by 27.37, which was not significantly different based (putative barriers) were recovered in both species groups, the on the S–H topology test (Table 4). Based on these results, we overall structure among those clades was strikingly different. tentatively reject the hypothesis that the B. valliceps/nebulifer The timing of divergence associated with each of the respective group shows structure similar to that of B. marinus associated geographic barriers was different for each species group. The with the TMNB and the Isthmus of Tehuantepec, because the

Table 3 Pair-wise percentage sequence North of TMNB Middle South of TMNB divergence within and between Bufo marinus samples (excluding identical haplotypes); North of TMNB 0.1% (0.1–0.2%) 0.4% (0.3–0.5%) 0.6% (0.4–0.7%) averages followed by ranges in parentheses. Middle 0.4% (0.3–0.5%) 0.2% (0.1–0.3%) 0.7% (0.5–0.8%) Comparisons with HKY85-corrected distan- South of TMNB 0.6% (0.4–0.7%) 0.7% (0.5–0.8%) 0.62% ces are shown in italics, others are uncor- North of Isthmus South of Isthmus rected. Samples from north and south of the TMNB represent localities Mx8 and Mx10, North of Isthmus 0.6% (0.1–1.3%) 1.7% (0.8–2.2%) respectively, whereas those in the middle are South of Isthmus 1.8% (0.8–2.2%) 1.1% (0.1–2.2%) from locality Mx9. There is only one within- comparison of those south of the TMNB. Central America South America Comparisons made with those north and south of the Isthmus of Tehuantepec include Central America 1.2% (0.1–2.2%) 6.8% (6.3–7.4%) Mx11–12 as north of the Isthmus South America 7.2% (6.7–7.9%) –

1898 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo)

monophyletic and obtained 25 trees, none of which were significantly different from the 50 unconstrained MP trees. The ML constrained topology was also not significantly different (‘TMNB’, Table 4). Therefore we could not reject a separation of haplotypes across the TMNB; however, we note that the level of divergence across the TMNB in B. marinus haplotypes is much less than that observed in the B. valliceps/nebulifer group (0.6% vs. 3.3%, respectively; Tables 2 & 3). Secondly, we constrained clades on either side of the Isthmus of Tehuante- pec to be monophyletic by forcing G1 with the samples originally recovered in the clade south of the isthmus (G3, E1– Figure 7 Area cladograms recovered for the two species groups 2, H3–4, C1). This resulted in 35 MP trees and an ML tree with in this study. (a) Area-cladogram for the Bufo valliceps/nebulifer a likelihood score that had a difference of 7.08; none of these species group, showing that the TMNB divergence occurred prior trees was significantly different from the unconstrained trees to the separation of B. valliceps haplotypes at the Isthmus of (‘Isthmus’, Table 4). Therefore, we could not reject an effect of Tehuantepec. (b) Area-cladogram for B. marinus, showing the the Isthmus of Tehuantepec on B. marinus haplotypes. Lastly, opposite pattern of the B. valliceps/nebulifer species group. we constrained the B. marinus group to show the same overall topological structure as the B. valliceps/nebulifer group (i.e. ML test was marginally non-significant (Buckley et al., 2001; enforcing the topology of Fig. 7a on B. marinus: (Mx8 (Mx9– Townsend et al., 2004). 12, G1) + (G3, E1–2, H3–4, C1)). The results of both the MP Likewise, we conducted similar topology tests for the and ML tests were significantly different from the uncon- B. marinus group. Here, we constrained samples north of the strained trees (‘TMNB vs. Isthmus’, Table 4). Therefore we TMNB (Mx8–9) to be monophyletic, and those south of could reject the hypothesis that B. marinus shows an overall the TMNB and north of the isthmus (Mx10–12, and G1) to be pattern similar to that of the B. valliceps/nebulifer group.

Table 4 Results of the topology tests. Tree scores and number of equally parsimonious trees recovered in the unconstrained phylogenies are shown for both groups in the left column. Columns to the right show results for the constrained phylogenies, with the range of summary statistics (see text for explanation of constraint topologies and Appendix 1 for actual constraints enforced).

Unconstrained phylogenies Constrained phylogenies

TMNB Isthmus TMNB vs. Isthmus

Bufo valliceps/nebulifer (6 trees, 274 steps) (12 trees, 271 steps) (4 trees, 280 steps)

Parsimony n Zà P§ nZ P nZP

(2 trees, 262 steps) 11 3.21 0.0007** 11 2.18 0.0147** 17 4.02 0.0001** 12 3.46 0.0003** 19 3.38 0.0007**

Likelihood Constrain Diff. P Constrain Diff. P Constrain Diff. P

)ln L ¼ 2723.6098 2744.3887 20.78 0.035* 2745.3506 21.740 0.085 2750.9784 27.37 0.057

Bufo marinus (25 trees, 190 steps) (35 trees, 192 steps) (20 trees, 194 steps)

Parsimony nZP nZ P nZP

(50 trees, 189 steps) 1 1.00 0.1587 5 1.34 0.0899 5 2.24 0.0127** 3 0.58 0.2819 7 1.13 0.1284 7 1.89 0.0294* 5 0.45 0.3274

Likelihood Constrain Diff. P Constrain Diff. P Constrain Diff. P

)ln L ¼ 2355.4517 2359.5382 4.08 0.303 2362.5321 7.08 0.143 2374.5949 19.14 0.045*

Number of characters differing in minimum number of changes on paired topologies. àNormal approximation for Wilcoxon signed-ranks tests. §Asterisks indicate a significant difference between the overall shortest tree and the constrained topologies. One asterisk denotes significance using the one-tailed probability and two asterisks denote significance using the two-tailed probability. One-tailed probabilities are shown, two-tailed are twice the values.

Journal of Biogeography 33, 1889–1904 1899 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

precisely parapatric distribution across this ancient geographic DISCUSSION barrier. Our data support a role for two major geographic features in Our increased sampling south of the TMNB greatly improved shaping the historical biogeography in two independent the phylogenetic support for the monophyly of B. valliceps, lineages of lowland toads. However, the sequence of events which was previously lacking (Mulcahy & Mendelson, 2000). appears different for each group (Fig. 7). The TMNB has been We attribute this difference to the increased sampling between shown to represent a barrier to gene flow in other lowland taxa the TMNB and the Isthmus of Tehuantepec, and the (Hulsey et al., 2004; Zaldı´var-Rivero´n et al., 2004), while the phylogenetic signal associated with the isthmus. The sampling Isthmus of Tehuantepec has been more typically invoked as a of Mulcahy & Mendelson (2000) contained only three samples barrier for upland taxa (Marshall & Liebherr, 2000; Sullivan of B. valliceps north of the isthmus (Mx3a–c), all from Los et al., 2000). In our study, we confirmed that the B. valliceps/ Tuxtlas. These three haplotypes were scattered across the nebulifer species boundary lies along the eastern terminus of distribution of other haplotypes of B. valliceps, with Mx3c the TMNB, consistent with the Pliocene-vicariance hypothesis recovered in the most basal position (Mulcahy & Mendelson, of Mulcahy & Mendelson (2000) and identified subsequent 2000). With increased sampling (this study), Mx3c was placed in phylogeographic structure in B. valliceps associated with the a clade of haplotypes found north of the Isthmus of Tehuantepec Isthmus of Tehuantepec. In contrast, B. marinus showed (Fig. 5), a clade estimated to be of late Pliocene–Pleistocene phylogeographic structure along either side of the Isthmus of origin. However, samples Mx3a–b remain nested among Tehuantepec, prior to that of the TMNB. At the TMNB, the haplotypes in the clade south of the isthmus. We were able to signal in the B. marinus data is more consistent with the reject the alternative hypothesis that samples north of the ‘Pleistocene-dispersal, followed by vicariance hypothesis’ of isthmus formed a monophyletic clade (Table 4). At this time, Mulcahy & Mendelson (2000). We evaluated the veracity of we can not discriminate between incomplete lineage sorting vs. a these contrasting patterns between the B. valliceps/nebulifer recent dispersal for the occurrence of haplotypes Mxa–b at Los and B. marinus groups by using alternative topology tests. We Tuxtlas. However, because the Los Tuxtlas landscape is of late imposed the B. marinus phylogenetic structure (Fig. 7b) on Pliocene volcanic origin (Ferrusquia-Villafranca, 1993) – the the B. valliceps/nebulifer group, and vice versa, and the approximate divergence time for the B. valliceps isthmian clades resulting topologies were significantly different, based on the – we favour a more recent dispersal explanation for the MP analyses, from the shortest trees recovered in each group occurrence of haplotypes from both clades at Los Tuxtlas. (Table 4). The ML topology test recovered marginally non- Our analyses of the B. marinus data do not support a significant results for the B. valliceps/nebulifer group with hypothesis that the Pliocene uprising of the TMNB caused a respect to the isthmus (Table 4). However, this result is in major vicariant event among populations of this species, as it conflict with a well-supported node based on the MP and did in the B. valliceps/nebulifer group. The data from B. mar- Bayesian analyses (97 and 96 respectively; Fig. 5), which inus do contain phylogeographic structure associated with the suggests that the ML-based (S–H) topology test may be more TMNB; however, the branch lengths are very shallow (Fig. 6) conservative than the MP topology test (Wilcoxon signed- and the sequence divergences are low (Table 3). Based on these rank). Therefore, we reject the hypothesis that these sympatric results, we suggest that B. marinus dispersed across this barrier species groups, with similar natural histories, show the same during lower sea levels associated with glacial maxima during phylogeographic structure. the Pleistocene. Our rate of evolution estimated from the Previous molecular studies invoking vicariance for related B. valliceps/nebulifer group corroborates this hypothesis. If we taxa distributed on either side of the TMNB consisted of data calibrate a rate for B. marinus based on a Pliocene (5.4 ma) sampling far to the north and south, and inferred clade divergence across the TMNB, consistent with that of the boundaries to be at the eastern terminus of the TMNB B. valliceps/nebulifer group (Mulcahy & Mendelson, 2000; this (Mulcahy & Mendelson, 2000; Hulsey et al., 2004; Zaldı´var- study), we obtain a rate of c. 1.6%, per lineage, per million Rivero´n et al., 2004). Our additional samples of B. valliceps years, which is twice the expected rate for ectotherms (Tan & and B. nebulifer across the TMNB (Figs 2 & 3) demonstrate Wake, 1995), and far beyond that estimated for other bufonid the strict parapatry on either side of this now seemingly minor mtDNA (Graybeal, 1993; Macey et al., 1998). Bufo marinus is geographic barrier. Our sample size in the middle of the known to be of South American origin (i.e. all of its closest TMNB is relatively small (n ¼ 2), such that future work may relatives are included in clades restricted to South America; reveal these two species to be sympatric in the small area of Slade & Moritz, 1998; Pauly et al., 2004; Pramuk, 2006). Our suitable habitat near the town of El Viejo´n (Mx9, Fig. 2). Based results are consistent with a model in which B. marinus on the fact that species of Bufo are frequently interfertile (Blair, dispersed into Central America during the early Pliocene, and 1963; Masta et al., 2002), it would not be surprising to us if later into southern Mexico, subsequent to the uplift of the these species were found to hybridize in this area. If this were TMNB. A fossil record referred to B. marinus from the the case, we may not be able to detect the presence of both Miocene of Kansas, USA, (Wilson, 1968) refutes this scenario. species with mtDNA as our only genetic marker. Nonetheless, However, recent concern has questioned the reliability of our data strongly indicate that these northern and southern precise taxonomic assignment based on fragmentary fossil data clades represent distinct evolutionary species, showing a in anurans (Bever, 2005; see also Pauly et al., 2004), and the

1900 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd Biogeography of lowland toads (Bufo) specific designation of this record remains questionable & Navarro (1980) point out some 16 species of reptiles that (Holman, 2003). straddle this barrier and many others that show subspecific Data from both B. valliceps and B. marinus show phylo- delineations on either side, yet these remain to be tested with genetic structure associated with the Isthmus of Tehuantepec. molecular data. Both B. valliceps and B. marinus show low While our sampling is not fully adequate to test for an effect of levels of intraspecific variation, possibly associated with a mid- a Pliocene seaway across the Isthmus of Tehuantepec, our data Pliocene barrier across the Isthmus of Tehuantepec. The do provide two independent lines of evidence to suggest that it question remains as to why neither B. nebulifer nor B. valliceps may have been present. Our estimated dates of divergence have secondarily dispersed over the TMNB, concurrent with from clades on either side of the isthmus were approximately B. marinus. Perhaps our limited sampling of the area in the 2–3 ma, in both species groups. This suggests a common middle (or our choice of molecular marker) was not sufficient barrier of some type existed for lowland taxa at the Isthmus of to detect sympatry in these species. Nevertheless, we were able Tehuantepec during the mid-Pliocene. Beard et al. (1982) to pin-point a putative area of contact between these two suggest sea-level fluctuations associated with continental species for future investigation. In a logical sequence then, our glaciation began approximately 3 ma, indicating high sea results demonstrate an ancient vicariant event, a more recent levels prior to the first glacial cycle beginning c. 2.8 ma. This dispersal event, and the area of possible ongoing biological corresponds to our timing of divergence in both B. valliceps influences maintaining species boundaries (viz., between and B. marinus at the isthmus. Sullivan et al. (2000) also found B. nebulifer and B. valliceps). sequence divergence, in harvest mice (Reithrodontomys), consistent with a late Pliocene trans-isthmus marine barrier. ACKNOWLEDGEMENTS We were able to reject monophyletic lineages on either side of the isthmus for B. valliceps; however, we were not able to reject We would like to thank the following people, institutions and this alternative in B. marinus. Bufo marinus is abundant in the curators for providing tissue samples and locality data for this lowlands of the isthmus and indeed in all adjacent lowland study: Jonathan A. Campbell, Kevin de Queiroz, Oscar Flores- areas (Duellman, 1960; J.R. Mendelson, pers. obs.). Bufo Villela, Carl J. Franklin, Eli B. Greenbaum, John H. Malone, valliceps however, is common along the Atlantic lowlands Jenny B. Pramuk, Mahmood Sasa M., John E. Simmons, Eric northeast of the isthmus, absent along the Pacific Coast of N. Smith, Larry D. Wilson, UTACV, KUNHM, USNM and and Guerrero to the southwest of the isthmus, and is UNAM. We thank Paul Wolf, Mike Pfrender, Eric O’Neill, and inexplicably uncommon along the Pacific Coastal Plain of Chris Feldman for assistance in the lab, and Chris Feldman and Guatemala to the southeast of the isthmus and Bob Macey for assistance in data analysis, Karen R. Lips (Mendelson, 2001). This biogeographical contrast between the and Ron M. Bonett for field assistance, and Ted Papenfuss for two species suggests that B. marinus may disperse more readily use of his Mac (G5) for phylogenetic analyses. USU Depart- into new regions than does B. valliceps. The exception to the ment of Biology and an Undergraduate Research and Creative isthmian clades in our B. marinus data (sample G1) may be an Opportunities Grant (URCO), awarded to B. H. Morrill, artifact of poor sampling in this area, or may represent a recent provided funding the data collection. Fieldwork along the dispersal from the northwest into the Grijalva Valley, similar to transect of the TMNB was funded by a Theodore Roosevelt that observed in B. valliceps (cf. Fig. 3). Further sampling of Memorial Grant from the American Museum of Natural multiple taxa, including B. valliceps and B. marinus should be History awarded to D. G. Mulcahy and a National Geographic conducted across this region, in order specifically to test grant to J. R. Mendelson and K. R. Lips. hypotheses associated with a Pliocene seaway across the Isthmus of Tehuantepec, and subsequent dispersal routes REFERENCES across this barrier. 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Villela), pp. 10–19. The University of Texas at El Paso, El Contributions from the Museum of Paleontology, University of Paso, TX. Michigan, 2, 75–126. Mendelson, J.R., III, Williams, B.L., Sheil, C.A. & Mulcahy, Zaldı´var-Rivero´n, A., Leon-Regagnon, V. & Nieto-Montes D.G. (2005) Systematics of the Bufo coccifer complex (An- de Oca, A. (2004) Phylogeny of the Mexican coastal ura: Bufonidae) of . Scientific Papers, University leopard frogs of the Rana berlandieri group based on of Kansas Natural History Museum, 38, 1–27. mtDNA sequences. Molecular Phylogenetics and Evolution, Mulcahy, D.G. & Mendelson, J.R., III (2000) Phylogeo- 30, 38–49. graphy and speciation of the morphologically variable, Zug, G.R. & Zug, P.B. (1979) The marine toad, Bufo marinus:a widespread species Bufo valliceps, based on molecular natural history resume of native populations. Smithsonian evidence from mtDNA. Molecular Phylogenetics and Evo- Contributions to Zoology, 284, 1–58. lution, 17, 173–189. Pauly, G.B., Hillis, D.M. & Cannatella, D.C. (2004) The history of a nearctic colonization: molecular phylogenetics and biogeography of the nearctic toads (Bufo). Evolution, 58, BIOSKETCHES 2517–2535. Pe´rez-Higareda, G. & Navarro, L.D. (1980) The faunistic dis- Daniel G. Mulcahy is a PhD candidate in the Department of tricts of the low plains of Veracruz, Mexico, based on rep- Biology, at Utah State University (USU). His graduate research tilian and mammalian data. Bulletin of Maryland involves biogeography and systematics of Neotropical and Herpetological Society, 16, 54–69. western North American amphibians and reptiles. Posada, D. & Crandall, K.A. (1998) Modeltest: testing the Joseph R. Mendelson III received his PhD in 1997 from The model of DNA substitution. Bioinformatics, 14, 817–818. University of Kansas, and has been conducting research and Pramuk, J.B. (2006) Phylogeny of South American Bufo (An- conservation studies on Neotropical amphibians and reptiles ura: Bufonidae) inferred from combined evidence. Zoologi- for 16 years. He is now Curator of Herpetology at Zoo Atlanta, cal Journal of the Linnaean Society, 146, 407–452. and Adjunct Associate Professor of Biology at Utah State Rosen, D.E. (1978) Vicariant patterns and historical explan- University. ation in biogeography. Systematic Zoology, 27, 159–188. Savage, J.M. (2002) The amphibians and reptiles of Costa Rica. Benson H. Morrill is a PhD student in L. Rickords’ molecular The University Chicago Press, Chicago, IL. biology laboratory at Utah State University. He was an Shimodaira, H. & Hasegawa, M. (1999) Multiple comparisons undergraduate at USU and completed his honours thesis as of log-likelihoods with applications to phylogenetic in- part of this study. His academic interests are herpetology and ference. Molecular Biology and Evolution, 16, 1114–1116. genetics. Slade, R.W. & Moritz, C. (1998) Phylogeography of Bufo marinus from its natural and introduced ranges. Proceedings Editor: Brett Riddle of the Royal Society of London Series B, Biological Sciences, 265, 769–777. Sullivan, J., Arellano, E. & Rogers, D.S. (2000) Comparative APPENDIX 1 TOPOLOGIES USED IN phylogeography of Mesoamerican Highland Rodents: con- CONSTRAINT TEST. certed versus independent response to past climatic fluc- tuations. The American Naturalist, 155, 755–768. Constrained topologies are labelled following Table 4 for the Swofford, D.L. (1999) PAUP*: Phylogenetic Analysis Using Par- three tests for each group: TMNB, Isthmus, and TMNB vs. simony (*and other methods), Beta Version 4.0b10. Sinauer, Isthmus. For the Bufo valliceps/nebulifer data, the following Sunderland, MA. translation is used: 1 B. coccifer,2B. mazatlanensis,3B. camp- Tan, A.-M. & Wake, D.B. (1995) MtDNA phylogeography belli, 4 Bneb Lou1, 5 Bneb Tx2, 6 Bneb Tx4, 7 Bneb Mx1, 8 of the California newt Taricha torosa (Caudata, Sala- Bneb Mx8b, 9 Bneb Mx8c, 10 Bneb Mx8d, 11 Bneb Mx8e, 12 mandridae). Molecular Phylogenetics and Evolution, 4, 383– Bneb Mx8a, 13 Bneb Mx8f, 14 Bneb Mx8g, 15 Bval Mx3a, 16 394. Bval Mx3b, 17 Bval Mx3c, 18 Bval Mx5, 19 Bval Mx6, 20 Bval Templeton, A.R. (1983) Phylogenetic inference from restric- Mx7, 21 Bval B1, 22 Bval B2, 23 Bval H1, 24 Bval H2, 25 tion endonuclease cleavage site maps with particular refer- Bval G1, 26 Bval G2, 27 Bval G3, 28 Bval G4a, 29 Bval G4b, 30 ence to the evolution of humans and the apes. Evolution, 37, Bval Mx10a, 31 Bval Mx10b, 32 Bval Mx10c, 33 Bval Mx10d, 221–244. 34 Bval Mx9a, 35 Bval Mx9b, with the following topologies Townsend, T.M., Larson, A., Louis, E. & Macey, R. (2004) were used for the constrained phylogenetic analyses: TMNB ¼ Molecular phylogenetics of Squamata: the position of (1, ((2, ((4–14, 34–35), (15– 33))), 3)); Isthmus ¼ (1, ((2, ((4– snakes, amphisbaenians, and dibamids, and the root of the 14), ((18–29,), (15–17, 30–35)))), 3)); TMNB vs. Isth- squamate tree. Systematic Biology, 53, 735–757. mus ¼ (1, ((2, ((18–29), ((15–35), (4–14)))), 3)); For the Wilson, R.L. (1968) Systematic and faunal analysis of a Lower B. marinus data, the following translation is used: 1 B. crucifer, Pliocene vertebrate assemblage from Trego County, Kansas. 2 B. schneideri,3B. marinus S. A., 4 Bmar Mx11b, 5 Bmar

Journal of Biogeography 33, 1889–1904 1903 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd D. G. Mulcahy, B. H. Morrill and J. R. Mendelson III

Mx9f, 6 Bmar Mx9a, 7 Bmar Mx9d, 8 Bmar G3, 9 Bmar Mx9b, constrained phylogenetic analyses: TMNB ¼ (1, (2, (3, (((5–7, 10 Bmar Mx11a, 11 Bmar C1, 12 Bmar Mx12, 13 Bmar Mx8a, 9, 13, 16, 19, 20), (4, 10, 12, 18, 21, 22)), (8, 11, 14, 15, 17))))); 14 Bmar H3, 15 Bmar E1, 16 Bmar Mx9c, 17 Bmar H4, 18 Isthmus ¼ (1, (2, (3, ((4–7, 9, 10, 13, 16, 19, 20, 22, 12, 21), (8, Bmar G1, 19 Bmar Mx8b, 20 Bmar Mx8c, 21 Bmar Mx10b, 22 11, 14, 15, 17, 18))))); TMNB vs. Isthmus ¼ (1, (2, (3, ((13, Bmar Mx10a, with the following topologies were used for the 19, 20), ((4–7, 9, 10, 12, 16, 22, 18, 21), (8, 11, 14, 15, 17)))))).

1904 Journal of Biogeography 33, 1889–1904 ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd