Mitochondrial Discordance and Gene Flow in a Recent Radiation of Toads

Molecular Phylogenetics and Evolution 59 (2011) 66–80

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Molecular Phylogenetics and Evolution

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Nuclear–mitochondrial discordance and gene ﬂow in a recent radiation of toads ⇑ Brian E. Fontenot , Robert Makowsky 1, Paul T. Chippindale

Department of Biology, University of Texas at Arlington, Arlington, TX 76019, United States article info abstract

Article history: Natural hybridization among recently diverged species has traditionally been viewed as a homogenizing Received 28 April 2010 force, but recent research has revealed a possible role for interspecific gene flow in facilitating species Revised 12 December 2010 radiations. Natural hybridization can actually contribute to radiations by introducing novel genes or Accepted 23 December 2010 reshuffling existing genetic variation among diverging species. Species that have been affected by natural Available online 19 January 2011 hybridization often demonstrate patterns of discordance between phylogenies generated using nuclear and mitochondrial markers. We used Amplified Fragment Length Polymorphism (AFLP) data in conjunc- Keywords: tion with mitochondrial DNA in order to examine patterns of gene flow and nuclear–mitochondrial dis- Toads cordance in the Anaxyrus americanus group, a recent radiation of North American toads. We found high Hybridization Gene flow levels of gene flow between putative species, particularly in species pairs sharing similar male advertise- Speciation ment calls that occur in close geographic proximity, suggesting that prezygotic reproductive isolating AFLPs mechanisms and isolation by distance are the primary determinants of gene flow and genetic differenti- Nuclear–mitochondrial discordance ation among these species. Additionally, phylogenies generated using AFLP and mitochondrial data were markedly discordant, likely due to recent and/or ongoing natural hybridization events between sympatric populations. Our results indicate that the putative species in the A. americanus group have experienced high levels of gene flow, and suggest that their North American radiation could have been facilitated by the introduction of beneficial genetic variation from admixture between divergent populations coming into secondary contact after glacial retreats. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction but recent advances in molecular techniques have allowed researchers to examine this phenomenon in a much more detailed Assessing gene flow between closely related species is a crucial fashion leading to a renewed emphasis on hybridization studies. component to our understanding of the speciation process. Under One of the predicted signatures of radiations facilitated by the allopatric speciation model, gene flow between diverging spe- hybridization is discordance between genealogies created using cies is often thought to lead to homogenization, reduced fitness, nuclear and cytoplasmic genetic markers (Petit and Excoffier, and erosion of species boundaries (Mayr, 1942; Coyne and Orr, 2009; Seehausen, 2004). Cytoplasmic markers like mitochondrial 2004). However, recent studies have shown that some organisms DNA (mtDNA) or chloroplast DNA are more likely than nuclear can experience relatively large amounts of interspecific gene ex- DNA (nDNA) markers to introgress between species due to differ- change and still maintain significant differentiation from other spe- ences in their parental inheritance mechanism, effective popula- cies (Hey, 2006; Fitzpatrick et al., 2008; Linnen and Farrell, 2007; tion size, recombination (or lack thereof), and mutation rate Strasburg and Rieseberg, 2008). The fact that some species remain (Avise, 1994; Currat et al., 2008; Leaché, 2009; Petit and Excoffier, distinct despite hybridization has led some researchers to hypothe- 2009). Chan and Levin (2005) modeled a wide range of introgres- size that the introduction of novel genetic variation from heterospe- sion scenarios and found that introgression rates of maternally cific mating could facilitate speciation by promoting the shuffling of inherited cytoplasmic markers consistently exceed those of pater- existing variation and creation of novel allelic combinations, en- nal cytoplasmic markers and nuclear genes, particularly in second- abling hybrids to take advantage of changing environmental condi- ary contact scenarios where one species is less abundant than the tions (Arnold, 1997; Coyne and Orr, 2004; Seehausen, 2004). The other. They attribute this difference to frequency-dependent pre- possible role of interspecific hybridization in speciation has been a zygotic barriers in female choice-based mating systems where topic of discussion among evolutionary biologists for many years, the maternal cytoplasmic marker has more opportunity for introgression from the rare species to the more abundant species. In this scenario, rare females have more opportunity to mate with heter- ⇑ Corresponding author. Fax: +1 817 272 2855. ospecific males than with conspecific males, leading to increased E-mail address: [email protected] (B.E. Fontenot). 1 Present address: Department of Biostatistics, University of Alabama at Birmingham, rates of mitochondrial introgression. Differential patterns of Birmingham, AL 35294, United States. introgression can provide an explanation for the topological

1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.12.018 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 67 discordance often seen in phylogenetic trees generated from 2. Methods mtDNA and nDNA gene fragments of closely related species (Taylor and McPhail, 2000; Dopman et al., 2005; Mendelson and Simons, 2.1. Taxon sampling 2006; Petit and Excoffier, 2009; Seehausen, 2004; Wiens et al., 2010); however, incomplete lineage sorting and the retention of We collected specimens for molecular analyses based on pub- ancestral polymorphisms could also result in similar patterns of lished geographic ranges for the currently recognized species in discordance. the United States including Texas, Louisiana, Alabama, Arkansas, To examine the effects of hybridization on the speciation pro- Connecticut, Minnesota, Delaware, Maine, New York, New Hamp- cess, it is necessary to examine species capable of producing viable shire, Mississippi, Michigan, Florida, South Carolina, Kansas, and and fertile offspring from natural hybridization. Some empirical Oklahoma (Conant and Collins, 1998; Dixon, 2000). Four members examples of nuclear–mitochondrial discordance come from taxa of the A. americanus group (A. charlesmithi, A. fowleri, A. velatus, and that have experienced natural hybridization such as birds (Freeland A. woodhousii) occur either sympatrically or parapatrically in the and Boag, 1999; Grant and Grant, 1996), crickets (Shaw, 1996, deciduous hardwood forests of eastern Texas, southeastern Okla- 2002), Hawaiian silversword plants (Barrier et al., 1999), butterflies homa, western Louisiana, and southwestern Arkansas (Fig. 1; Con- (Beltran et al., 2002; Gilbert, 2003), turtles (Wiens et al., 2010), liz- ant and Collins, 1998; Dixon, 2000), and some authors have ards (Leaché, 2009), salamanders (Highton, 1995; Wiens et al., hypothesized that this region contains populations of hybrid indi- 2006) several fish species (DeMarais et al., 1992; Seehausen et al., viduals (Blair 1941, 1942; Blair, 1972; Bragg and Sanders, 1951). To 1997; Taylor and McPhail, 2000) and several frog species (Green, evaluate whether or not hybridization can be detected in this re- 1984; Green and Parent, 2003; Yanchukov et al., 2006; Mezhzherin gion, we concentrated sampling of A. woodhousii, A. velatus, A, char- et al., 2010; Stöck et al., 2010). True toads in the family Bufonidae, a lesmithi, and A. fowleri along a transect spanning a region from nearly cosmopolitan group with a likely origin in the Upper Creta- western Texas to eastern Louisiana (see Fig. 2A for collection local- ceous, are also known to engage in natural hybridization (Blair, ities). Individuals collected from the field (n = 231) were combined 1972; Green, 1984; Green and Parent, 2003; Jones, 1973; Malone with samples (n = 48) obtained from the Peabody Museum of Nat- and Fontenot, 2008; Masta et al., 2002; Pramuk et al., 2008). Consid- ural History (HERA) at Yale University, the University of Alabama erable research has focused on hybridization among members of Herpetological Collection (UAHC), and the Bell Museum of Natural the widely distributed Anaxyrus (formerly Bufo) americanus group History (JFBM) at the University of Minnesota (see Appendix A for (Blair, 1941, 1942; Blair, 1955, 1959, 1961, 1972; Green, 1984; specimen ID numbers, full locality data, and Genbank accession Green and Parent, 2003; Vogel and Johnson, 2008; Volpe, 1952, numbers). Field-collected voucher specimens were sexed using a 1959). The Anaxyrus americanus group, thought to have arisen as recombination of presence of male advertisement call if captured cently as 2 million years ago, includes A. americanus, Anaxyrus char- during a chorus, presence of secondary sexual characters such as lesmithi, Anaxyrus houstonensis, Anaxyrus terrestris, Anaxyrus nuptial pads in males, and finally, dissection to visualize gonad hemiophrys, Anaxyrus microscaphus, Anaxyrus woodhousii, Anaxyrus condition. fowleri, and Anaxyrus velatus (Blair, 1972; Conant and Collins, A priori species designations of field-caught individuals were 1998; Dixon, 2000; Pauly et al., 2004). These putative species tradi- made using a combination of the morphological characters of tionally have been identified using qualitative morphological traits Conant and Collins (1998), the species designations from Masta and male advertisement calls. Previous studies of mtDNA, allozyme et al. (2002) according to their identified geographic species bound- variation, morphology, ecology, and karyotypes have not provided a aries, and male advertisement call type. Species designations strongly supported hypothesis of interspecific relationships in the followed the framework of the General Lineage species concept A. americanus group, and the taxonomic status of many species in (de Queiroz, 1998), and for the purposes of this study, we view spe- the group remains problematic (Blair, 1972; Bragg and Sanders, cies as ‘‘separately evolving metapopulation lineages’’ (de Queiroz, 1951; Masta et al., 2002). The recent origin of this group and multi- 2005). However, our intent is not to revise the taxonomy of the ple areas of natural contact between putative species make the A. group, but rather to evaluate how the currently accepted taxonomy, americanus group an excellent system for studying hybridization based on morphology and mitochondrial DNA, would be affected by and speciation. Additionally, toads are valuable subjects for specia- additional evidence from anonymous AFLP nuclear markers. tion studies because they exhibit homomorphic sex chromosomes Tissue samples obtained from museum collections were classi- (however, see Abramyan et al., 2009), female heterogamety (con- fied according to the species designation in each sample’s museum firmed in Rhinella marina; Abramyan et al., 2009; and suspected catalog. All members of the A. americanus group are represented in Bufo bufo; Ponse, 1941; however, see Rostrand, 1952, 1953), except the allopatric A. microscaphus and the endangered A. hou- and use male advertisement calls for mate selection. Males of some stonensis. Species that occur in the putative hybrid zone spanning frog species are subjected to strong selective pressures as female parts of Texas and Louisiana (A. fowleri, A. charlesmithi, A. velatus, choice can drive male advertisement call evolution and may even and A. woodhousii) were sampled most extensively. Anaxyrus cogn- cause speciation events through assortative mating (Boul et al., atus, the sister species to the A. americanus group, was chosen as 2007; Gerhardt, 2005; Höbel and Gerhardt, 2003; Murphy and the outgroup taxon for molecular analyses (Masta et al., 2002; Gerhardt, 2002; Ryan et al., 1990). Pauly et al., 2004). Whole genomic DNA was extracted from liver Our study is the first to examine genetic variation in the A. or muscle tissue using DNeasy tissue kits (QUIAGEN). All field- americanus group across the majority of their range in the United collected specimens were deposited in the amphibian collection States using Amplified Fragment Length Polymorphism (AFLP) data at the University of Texas at Arlington Amphibian and Reptile in conjunction with mtDNA. By documenting levels of genetic Diversity Research Center (Appendix A). diversity, estimating gene flow between and among putative species, and comparing results generated using mtDNA and AFLPs, we sought to answer the following questions: (1) Could the radia- 2.2. Male advertisement call and geography tion of the A. americanus group have been affected by natural hybridization? (2) Are nuclear and mitochondrial genealogies of Toads in the A. americanus group have two major types of male the A. americanus group concordant? and (3) How are geography advertisement calls that differ primarily in pulse rate (Sullivan and male advertisement call type correlated with patterns of gene et al., 1996). Male advertisement calls were broadly classified into flow and genetic differentiation between species? two qualitative categories: ‘‘Whining calls’’ (lower pulse rate), 68 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80

Fig. 1. Geographic distribution of putative Anaxyrus americanus group species in the United States based on Conant and Collins (1998). A. houstonensis and A. microscaphus are not shown since they were not included in this study. present in A. woodhousii, A. fowleri, and A. velatus, and ‘‘Whistling (Applied Biosystems, Inc.). Fragments were scored using Genemar- calls’’ (higher pulse rate), present in A. americanus, A. charlesmithi, kerÒ (Softgenetics, LLC.), and fragments less than 100 base pairs in and A. terrestris (Conant and Collins, 1998). While these species length were removed to avoid complications in homolog assess- do exhibit some geographic variation in advertisement calls, this ment (Vekemans et al., 2002). The remaining scorable fragments variation often manifests as fine-scale differences in pitch and/or were assembled into a presence/absence matrix for statistical anal- duration that do not change the qualitative ‘‘Whining’’ or ‘‘Whis- ysis. GenemarkerÒ was used to delineate fragments using a thresh- tling’’ nature of the advertisement call, but rather serve to further old peak value of 100 following Makowsky et al. (2009).In differentiate between syntopic species at a fine scale (Sullivan addition, all presence/absence assignments were verified by eye. et al., 1996; Waldman, 2001). We chose to classify species’ adver- Furthermore, select AFLP profiles were duplicated to verify tisement calls using the broad-scale qualitative ‘‘Whining’’ and repeatability. ‘‘Whistling’’ designations since this would be more representative of the variation seen at large geographic scales. All field-confirmed advertisement call designations were concordant with the adver- 2.4. AFLP analyses tisement call type predicted from a priori species designations made using the morphological characters of Conant and Collins Analyses were conducted on all species to assess genetic diver- (1998); therefore, individuals for which we could not confirm sity and intraspecific population structure. Analyses were also advertisement call type in the field were categorized according to conducted for all pairwise interspecific comparisons to evaluate a combination of their occurrence within the geographic species genetic diversity, interspecific population structure, and possible boundaries identified by Masta et al. (2002) and their morpholog- interspecific gene flow. The program GENALEX 6.2 (Peakall and ical species designation. We then used the program POPGENE (Yeh Smouse, 2006) was used to create a matrix of genetic distances using Nei’s D (Nei, 1972), and to conduct Mantel tests for Isola- and Boyle, 1997) to estimate genetic differentiation (GST) and gene tion-by-Distance. Measures of genetic diversity and population flow (NM) for all pairwise species comparisons as well as subsequent Neighbor-joining and Bayesian analyses in light of a priori structure were obtained using the program HICKORY 1.0 advertisement call designations. This allowed us to evaluate pat- (Holsinger et al., 2002) using the f-free, full model option. Because terns of genetic diversity among species in the context of both geo- AFLP markers are scored as dominant characters, direct observa- graphic location and male advertisement call. tion of heterozygosity is impossible. To address this potential problem, HICKORY 1.0 (Holsinger et al., 2002) implements Bayes- ian methods that assume Hardy–Weinberg equilibrium and a 2.3. AFLP markers non-uniform prior distribution of allelic frequencies (Zhivotovsky, 1999). This approach uses the observed fragment frequencies to AFLP data were obtained following the methods of Vos et al. produce robust estimates of allelic frequencies, heterozygosity

(1995) and Makowsky et al. (2009) using an EcoR I + G preselective (H), and genetic differentiation (FST). These estimates have been primer and a Fam-6 labeled Mse I + GC selective primer. A random shown to be accurate even when derived from dominant markers sample of 75 individuals was chosen for duplicate AFLP ampliﬁca- (Krauss, 2000). HICKORY 1.0 and POPGENE (Yeh and Boyle, 1997) tion to assess repeatability. Selective PCR products were puriﬁed were used to obtain measures of estimated heterozygosity (HT), B using standard ethanol precipitation cleanup and sequenced using the FST analog h as a measure of genetic differentiation, the Shan- an ABI 3130xl capillary sequencer with a Rox 400 HD size standard non Information Index (I) as a measure of genetic diversity, Nei’s B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 69

(A)

Whining Species in “Whining Call” Group Whining Species in “Whistling Call” Groups Whistling Species in “Whistling Call” Groups Whistling Species in “Whining Call” Group

(B)

A. fowleri Clade 1

A. terrestris Clade A. fowleri Clade 2

A. americanus/hemiophrys Clade

A. woodhousii Clade

A. americanus/velatus/charlesmithi Clade

Fig. 2. Collection localities for all 178 individuals included in this study and distribution of individuals recovered from phylogenetic analyses using (A) AFLP markers and (B) mtDNA markers.

GST as a measure of genetic differentiation among populations, Joining (NJ) tree with nodal support from 10,000 bootstrap and NM as the estimated number of migrants among populations pseudoreplicates was created using PAUP 4.0 beta (Swofford, to characterize gene ﬂow (Holsinger et al., 2002). A Neighbor- 1999). 70 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80

Table 1 from the ML analysis as the starting tree in a subsequent Modeltest Comparison of partitioning strategies used in Bayesian analysis of mtDNA. 3.7 search to optimize the model of evolution. This process was re- Analysis Description of Number of ln likelihood of peated until the best tree likelihoods in sequential iterations were partitions partitions harmonic mean comparable. Nodal support was assessed from 100 bootstrap pseu- 1 16S + leu + ND1 1 4277.41 doreplicates using PhyML 3.0 (http://www.atgc-montpellier.fr/ 2 16S + leu, ND1 2 4276.56 phyml/). 3 16S, leu, ND1 3 4257.85 The best ML tree was used to determine the appropriate model 4 16S + leu, ND1 (by 4 3895.64 of evolution for Bayesian analyses in MrModelTest 2 (Nylander, codon) 5 16S, leu, ND1 (by 5 3895.23 2004). These model parameters were used for analyses in MrBayes codon) 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) for at least 10 million generations with four chains, two repetitions, two swaps per generation, and sampling every 1000 generations for both standard and partitioned (by codon position) 2.5. Mitochondrial markers analyses. Analyses were run until the standard deviation of split frequencies was <0.01 and convergence was further checked in Mitochondrial markers were amplified using the Polymerase AWTY (Nylander et al., 2008). The first 2.5–5.0 million generations 0 Chain Reaction (PCR). Primers (Bw16S-L – 5 -ATTTTTTCTAGT were discarded as burnin. We compared five partitioning strategies 0 0 ACGAAAGGAC-3 and B-Ile-H – 5 -GCACGTTTCCATGAAATTGG for the Bayesian analysis (Table 1) using Bayes factors (Nylander 0 TGG-3 ) obtained from Masta et al. (2002) were used to amplify et al., 2008), which were calculated as twice the difference of –ln 0 1029 base pairs including the 3 end of the 16S ribosomal subunit likelihood harmonic means between competing hypotheses using LEU gene, all of tRNA , and part of the NADH dehydrogenase subunit the harmonic mean output in MrBayes 3.1.2. We interpreted Bayes 1 (ND1) gene. PCR was carried out in 10 ll reactions containing factor values <0 as no evidence (0–2), positive support (3–6), 1 ll DNA, 1 ll 10X Buffer, 1 ll dNTPs, 0.6 ll of each primer, strong support (7–10), or very strong support (> 10) in favor of 0.1 ll MgCl2, and 0.05 ll Taq polymerase under the following ther- the more parameterized model. mal cycling conditions: 94° for 3 min followed by 35 cycles of denaturation at 94° for 30 s, annealing at 54° for 45 s, and extension at 72° for 75 s, followed by a final extension at 72° for 3. Results 7 min. PCR samples were cleaned by ExoSapIt (United States Bio- chemical, Cleveland, OH) and sequenced on an ABI 3130xl sequen- 3.1. AFLP analyses cer (Applied Biosystems Inc.) using standard protocols. Both strands were sequenced for all samples. Forward and reverse se- AFLP profiles were generated for 231 individuals, with 178 quences were compared using Sequencher 4.8 (Gene Codes Corpo- showing sufficient clarity to allow accurate profiling (Appendix ration) and consensus sequences were aligned using ClustalW A). One specimen of A. hemiophrys was also profiled, but we ex- (Chenna et al., 2003; Larkin et al., 2007) in Macvector 9.0 (MacVec- cluded it from all analyses except the NJ and Bayesian analyses, tor Inc.). No gaps were found and alignment was unambiguous. as it was the only individual of that species for which data were obtained. Inspection of AFLP profiles of the 75 individuals randomly chosen for duplication revealed that 71/75 (95%) gave profiles 2.6. Mitochondrial analyses identical to the original, indicating sufficient repeatability of our AFLP amplification protocol. The four profiles that did differ upon Aligned sequences totaled 1029 bases and redundant haplo- replication had profiles differing in fragment sizes or numbers of types were removed for all analyses involving mitochondrial mark- fragments ranging from 2 to 12 fragments. After removal of frag- ers. PAUP 4.0 beta (Swofford, 1999) was used for maximum ments under 100 bp and editing of the AFLP profiles to remove likelihood (ML) analyses with 10 random addition sequences and ambiguous fragments, 100 scorable loci were obtained for 178 TBR swapping with 10 repetitions. To obtain an appropriate model individuals and 100% of these loci were polymorphic (Table 2). of evolution for each gene, we used Modeltest 3.7 (Posada and Visual examination of the AFLP profiles revealed no fixed diagnos- Crandall, 1998) with the model chosen based on the Akaike Infor- tic loci for any samples assigned to our a priori designated species. mation Criterion (AIC). Following the initial model selection (ob- With respect to overall AFLP diversity, the Shannon Information tained with a NJ starting tree), we used the best tree obtained Index (I) ranged from 0.31 to 0.55, average heterozygosity (HT)

Table 2 Descriptive statistics of 100 AFLP loci from the Anaxyrus americanus group.

B Species n Number of Loci Na (s.d.) Ne (s.d.) I (s.d.) HT (s.d.) h (s.d.) Percent polymorphic loci americanus 18 100 1.87 (0.34) 1.56 (0.34) 0.48 (0.23) 0.36 (0.01) 0.23 (0.05) 87 charlesmithi 19 100 1.86 (0.35) 1.68 (0.33) 0.54 (0.23) 0.41 (0.01) 0.13 (0.03) 86 fowleri 44 100 1.89 (0.31) 1.64 (0.32) 0.53 (0.21) 0.33 (0.04) 0.16 (0.04) 89 terrestris 17 100 1.88 (0.33) 1.71 (0.33) 0.55 (0.22) 0.41 (0.02) 0.14 (0.04) 88 velatus 50 100 1.93 (0.26) 1.65 (0.30) 0.54 (0.18) 0.30 (0.04) 0.19 (0.04) 93 woodhousii 23 100 1.82 (0.39) 1.63 (0.34) 0.50 (0.25) 0.36 (0.03) 0.14 (0.04) 82 cognatus 7 100 1.59 (0.49) 1.34 (0.36) 0.31 (0.28) 0.41 (0.02) 0.09 (0.04) 59 All Populations 178 100 2.00 (0.00) 1.76 (0.25) 0.61 (0.12) 0.41 (0.04) 0.27 (0.02) 100 n: number of individuals. Na: number of observed alleles. Ne: number of expected alleles. I: Shannon Index (a measure of gene diversity).

HT: expected heterozygosity estimate calculated under Hardy–Weinberg genotypic proportions. hB: Estimated genetic differentiation (comparable to Wright’s Fst). B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 71

Table 3 Estimated pairwise hB (genetic differentiation) with 95% credibility intervals below diagonal and pairwise genetic distances (Nei’s D) above diagonal. Values in bold indicate signiﬁcant differences in hB between species.

americanus charlesmithi cognatus fowleri terrestris velatus woodhousii americanus 0.0836 0.3314 0.2001 0.1047 0.2007 0.1810 charlesmithi 0.10 0.3808 0.1075 0.0938 0.0947 0.0820 0.01–0.21 cognatus 0.14 0.04 0.4620 0.3150 0.4773 0.4569 0.01–0.26 0.07–0.15 fowleri 0.06 0.04 0.07 0.0834 0.0215 0.0373 0.05–0.17 0.14–0.06 0.19–0.04 terrestris 0.09 0.02 0.05 0.02 0.1053 0.0939 0.03–0.19 0.11–0.08 0.16–0.06 0.07–0.13 velatus 0.04 0.07 0.10 0.03 0.05 0.0365 0.09–0.15 0.18–0.04 0.22–0.01 0.14–0.08 0.16–0.06 woodhousii 0.09 0.01 0.05 0.03 0.01 0.05 0.03–0.20 0.10–0.09 0.15–0.06 0.07–0.13 0.10–0.10 0.05–0.17

Table 4

Nei’s GST (genetic differentiation) below the diagonal and NM (gene ﬂow) above the diagonal for all pairwise species comparisons.

americanus charlesmithi cognatus fowleri terrestris velatus woodhousii americanus 5.6 1.2 2.7 4.7 2.8 2.9 charlesmithi 0.08 1.2 5.2 5.8 5.9 6.2 cognatus 0.29 0.29 1.0 1.4 1.0 1.0 fowleri 0.15 0.09 0.32 6.5 21.1 12.1 terrestris 0.09 0.08 0.26 0.07 5.4 5.6 velatus 0.15 0.08 0.33 0.02 0.08 12.6 woodhousii 0.15 0.08 0.33 0.04 0.08 0.04

ranged from 0.30 to 0.41, genetic differentiation (hB) ranged from G–T = 1.0000; gamma distribution shape parameter = 0.1250. For 0.09 to 0.23, and percentage of polymorphic loci ranged from Bayesian analyses, a partitioning scheme with four partitions 59% to 93% (Table 2). Pairwise genetic distances (Nei’s D) between (16S + tRNALEU and ND1 partitioned by codon) was chosen after species ranged from 0.02 to 0.46 with the only signiﬁcant differ- comparison of Bayes factors (Table 1). The model chosen for each ence occurring between A. americanus and A. cognatus (Table 3). gene in the Bayesian analysis was GTR + G.

Pairwise GST ranged from 0.02 to 0.33 and pairwise NM ranged from The phylogeny recovered from the Bayesian analysis contains 1.0 to 21.1 (Table 4). Mantel tests for Isolation-by-distance (IBD) six major clades with strong support (Fig. 4). Two clades containing were significant for all pairwise species comparisons (Supplemen- individuals designated as A. fowleri were found to be paraphyletic tal Table S1). with respect to a clade containing individuals designated as A. ter- An NJ tree depicting relationships among all individuals is given restris. The paraphyly of A. fowleri and A. terrestris is congruent with in supplemental Fig. S1. Fig. 3 represents the NJ tree with some relationships obtained in other studies using mtDNA markers groups collapsed for simplified viewing. NJ analysis of the AFLP (Masta et al., 2002; Pauly et al., 2004). Along with the A. fowleri data broadly separates the species into three groups; one com- and A. terrestris grouping are three other major clades, one contain- posed primarily of species from our a priori defined ‘‘whining call’’ ing northern individuals designated as A. americanus and A. hemi- category and two smaller groups composed primarily of species ophrys, another containing A. woodhousii, and a large clade from our a priori defined ‘‘whistling call’’ category. Only 14/125 containing individuals designated as A. americanus, A. charlesmithi, (11.2%) individuals from the ‘‘whistling call’’ category were placed and A. velatus. The sister relationship of A. americanus and A. wood- in the ‘‘whining call’’ group (Fig. 3). In the two ‘‘whistling call’’ housii is congruent with previous studies (Masta et al., 2002 and groups, only 5/42 (11.9%) individuals from the ‘‘whining call’’ cat- Pauly et al., 2004). However, the larger clade containing individuals egory were placed in either of the ‘‘whistling call’’ groups (Fig. 3). designated as A. americanus, A. charlesmithi, and A. velatus is some- Overall bootstrap support was low, but six nodes were supported what unexpected, and differs from the results of previous studies (supplemental Fig. S1). Geographic localities for individuals in primarily due to the inclusion of individuals from northeastern the NJ analysis are shown in Fig. 2A. localities designated as A. americanus (Pauly et al., 2004). The geographic localities for individuals in the clades recovered from the 3.2. Mitochondrial analyses Bayesian analysis are shown in Fig. 2B.

The ﬁnal mtDNA alignment revealed 88 unique haplotypes and 3.3. Male advertisement call and geography 261 variable sites, of which 165 were parsimony informative for the ingroup. The model of evolution (for likelihood searches) for Analysis of the species that occur sympatrically and/or parapat- all genes combined was TrN + G: Base frequencies of A = 0.3276, rically in the putative hybrid zone (A. woodhousii, A. fowleri,

C = 0.1171, G = 0.2552, T = 0.3002; substitution rate parameters A. velatus, and A. charlesmithi) resulted in a GST value of 0.08 and A–C = 1.000, A–G = 11.568, A–T = 1.000, C–G = 1.000, C–T = 24.720, an NM value of 5.4 indicating little genetic differentiation and high 72 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80

Fig. 3. Neighbor-joining tree generated using AFLP data. Yellow clade contains putative species with whining male advertisement calls and red clades contain putative species with whistling male advertisement calls. Scale bar represents Nei’s (1978) genetic distance measure. Indicates individual designated as having whistling male advertisement call type. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 73

BF013-fowleri (LA) JWS008-terrestris (VA) TG00019-americanus (MS)* TG00020-fowleri (MS) * BF019-fowleri (LA) JWS001-americanus (VA) 1 BF163-terrestris (SC) * A. fowleri CMW1003-fowleri* (AL) 1.0 UAHC14625- (AL) 1.0 fowleri UAHC15113-fowleri (AL) UAHC15115-fowleri (AL) HERA10493-fowleri (DE) HERA10313-fowleri (CT) BF134-terrestris (FL) 0.95 BF135-terrestris (FL)* BF172-terrestris (FL) * BF169- (AL) 0.99 terrestris * BF141TERR-terrestris* (FL) BF171-terrestris (FL) * A. terrestris BF173-terrestris (FL)* 0.98 BF155-terrestris (FL)* BF156-terrestris (FL)* BF174-terrestris (FL) 0.96 * 1.0 CMW1028-terrestris (GA)* UAHC14621B-terrestris (AL) * BF145-fowleri (SC)* 1.0 BF146B-fowleri (SC) 2 BF154-fowleri (MS) A. fowleri 1.0 CMW1027-fowleri (SC) BF188-charlesmithi (OK) BF022-velatus* (TX) JFBM14332-americanus (MN) JFBM14318- (MN) 1.0 americanus * A. americanus JFBM14319-americanus (MN) * JFBM14320-americanus (MN)* JFBM14326-americanus (MN)* A. hemiophrys JFBM14328-hemiophrys (ND) * JFBM14331-americanus (MN)* BF043-woodhousii (TX)* BF044-woodhousii (TX) BF102-woodhousii (TX) BF064-charlesmithi (OK) BF107-woodhousii (TX)* A. woodhousii BF133- (KA) 1.0 woodhousii BF179-woodhousii (TX) BF186-charlesmithi (OK) BF066-charlesmithi (OK)* 0.95 BF003-velatus (TX) BF114-fowleri (LA) * BF122-velatus (TX) BF017-fowleri (LA) BF116-fowleri (LA) BF166-charlesmithi (OK) 1.0 BF167-charlesmithi (OK)* BF131-fowleri (LA) BF008-charlesmithi (OK) * BF009-charlesmithi (OK)* BF010-charlesmithi (OK) * BF011-fowleri (LA) * BF018-fowleri (LA) BF032-velatus (TX) BF045-velatus (TX) 1.0 BF123-velatus (TX) 0.95 MG0002-velatus (TX) BF089-americanus (MI) A. americanus JFBM14366-americanus* (MN) BF090-americanus (MI) * HERA10372-americanus* (NY) A. velatus HERA10491-fowleri (DE) 1.0 * HERA9853-americanus (CT) A. charlesmithi UAHC14993-americanus (WV)* HERA10214-americanus (CT) * JFBM14347-americanus (MN) * BF038-charlesmithi (TX) * BF049-velatus (TX)* TG00025-fowleri (MS) BF061-velatus (TX) 1.0 BF105-velatus (TX) BF130-fowleri (LA) RLG2637-velatus (TX) BF109-fowleri (LA) BF002-velatus (TX) BF015-fowleri (LA) BF050-velatus (TX) BF052-velatus (TX) BF067-charlesmithi (OK) MG0001-velatus (TX) * 0.96 BF141COG-cognatus (TX) BF180-cognatus (TX) 1.0 BF181-cognatus (TX) BF182-cognatus (TX) BF185-cognatus (TX) BF183-cognatus (TX)

0.7 substitutions

Fig. 4. Bayesian phylogenetic tree generated using mtDNA with posterior probabilities for nodal support. Yellow clades contain putative species with whining male advertisement calls and red clades contain putative species with whistling male advertisement calls. Indicates individual designated as having whistling male advertisement call type. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

levels of gene flow among populations. Analysis of the species that value of 4.0 indicating moderate genetic differentiation and high possess similar male advertisement calls and occur sympatrically levels of gene flow among populations. and/or parapatrically (A. woodhousii, A. fowleri, A. velatus) resulted in a GST value of 0.04 and an NM value of 10.8 indicating very little genetic differentiation and very high levels of gene flow among 4. Discussion populations. Analysis of the species that possess similar male advertisement calls and occur allopatrically (A. americanus, A. char- The use of AFLP and mtDNA markers has revealed previously lesmithi, and A. terrestris) resulted in a GST value of 0.11 and an NM undetected patterns of nuclear–mitochondrial discordance and a 74 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 link between levels of gene flow and male advertisement call in the (e.g. fixed for presence in one species and absent in the other). A. americanus group. Our analyses provide evidence that (1) natural While this approach has proven useful in identifying F1 hybrids hybridization has affected the radiation of the A. americanus group, in other taxa (Bonin et al., 2007; Meudt and Clarke, 2006), the large (2) nuclear and mitochondrial genealogies are discordant for these numbers of polymorphic loci in the A. americanus group provide lit- putative species, and (3) levels of interspecific gene flow between tle information regarding identification of hybrid individuals, as putative species increase with geographic proximity and/or simi- there are no fixed diagnostic loci between any of the putative spe- larity of male advertisement calls. Many studies have shown sub- cies. However, some of our results can still provide information stantial genetic differentiation among amphibian populations regarding the role of hybridization in the radiation of the A. amer- across a wide range of taxonomic and geographic scales (Barber, icanus group. We interpret the high levels of gene flow and genetic 1999; Chippindale et al., 2000; Lampert et al., 2003; Highton, similarity between species that occur in close geographic proxim- 1995; Masta et al., 2003; Phillips, 1994; Rowe et al., 1998; Rowe ity as evidence in support of genetic exchange between putative et al., 2000; and Schaffer et al., 2000). While species pairs in the species due to past, and possibly present, hybridization events. A. americanus group that do not share similar male advertisement Moreover, we find this a plausible scenario because members of calls appear to be valid species, species pairs that do share similar the A. americanus group are known to produce viable and fertile male advertisement calls appear to persist as weakly differentiated hybrid offspring in interspecific crosses (Blair, 1972; Malone and metapopulations connected by at least some gene flow. This pat- Fontenot, 2008). Seehausen (2004) suggested that hybridization tern is consistent with recent range expansions coupled with sec- could facilitate and enhance rapid species radiations by reshuffling ondary contact preceding the acquisition of complete reproductive alleles and introducing novel genetic variation at the beginning of a isolation (Coyne and Orr, 2004; Seehausen, 2004; Petit and radiation. However, Wiens et al. (2006) found evidence that in- Excoffier, 2009; Wiens et al., 2006). Prezygotic isolation has been creased hybridization may simply be a consequence of multiple, shown to evolve more rapidly than postzygotic isolation in areas recent speciation events in which species pairs do not have suffi- of sympatry for both Drosophila fruit flies and Angiosperm plants cient time to achieve proper reproductive isolation rather than a (Coyne and Orr, 1989, 1997, 2004; Moyle et al., 2004), and diverg- cause of rapid radiations. Our data do not allow for a distinction ing populations coming into secondary contact could rapidly between the above scenarios, but do suggest that natural hybrid- reduce heterospecific gene flow by assortative mating. While ization likely featured prominently in the radiation of the A. amer- insects and plants are quite different biologically from toads, the icanus group. fact that prezygotic isolation acts in much the same fashion between these disparate taxa argues that it may act in a similar 4.2. Nuclear–mitochondrial discordance fashion in toads. It is possible that this recent radiation of toads is in the process of acquiring prezygotic isolating barriers in the Similar to other taxa that have been affected by natural hybrid- form of male advertisement calls, and these data appear to support ization, the topological differences between the mtDNA and AFLP the notion that mitochondrial introgression occurs more fre- trees we generated for the A. americanus group are striking. No quently than nuclear introgression in species that have unequal clades are identical between the two trees and nodal support is abundance and prezygotic isolating barriers that create female high in the mtDNA tree and very low in the AFLP tree. The mtDNA choice-based mating systems (Chan and Levin, 2005). Studies have phylogeny does not follow the same pattern with regard to male shown that frog species pairs with low levels of genetic divergence advertisement call type as the AFLP phylogeny. In fact, the mtDNA often have not attained postzygotic isolation, therefore prezygotic phylogeny suggests that species in the A. americanus group have isolating mechanisms likely play a role in reproductive isolation experienced multiple episodes of convergence in advertisement among the recently radiated A. americanus group, the members call type. Additionally, our estimates of genetic differentiation of which have not attained postzygotic reproductive isolation (GST) and gene flow (NM) are lower and higher respectively be- (Malone and Fontenot, 2008; Sasa et al., 1998). tween species that share similar male advertisement calls, a pattern that is not concordant with the relationships obtained from 4.1. Hybridization in the A. americanus group the mtDNA phylogeny. The discordance between AFLP and mtDNA data may indicate Natural hybridization is thought to occur among members of either incomplete lineage sorting or a recent division of one or the A. americanus group based on: (1) observations of putatively more species into several subpopulations that persist despite re- intermediate morphology in some populations (Blair, 1941, 1942; cent and possibly ongoing gene flow. Seehausen (2004) and Petit Blair, 1956, 1959, 1961, 1963a, 1963b, 1964, 1966, 1972; Green and Excoffier (2009) hypothesized that hybridization between clo- and Parent, 2003; Jones, 1973; Volpe, 1952, 1959), (2) allozyme sely related species at the beginning of a radiation would result in a variation in A. americanus, A. fowleri, and A. hemiophrys (Green pattern of discordance between cytoplasmic and nuclear markers 1983 and 1984) and (3) evidence of mtDNA introgression among due to differential fixation of mitochondrial haplotypes after sec- lineages (Masta et al., 2002; Vogel and Johnson, 2008). Although ondary contact (also predicted from Chan and Levin, 2005). Incom- these lines of evidence suggest that natural hybridization may oc- plete lineage sorting among recently diverged species could also cur, they can be uninformative or even misleading. Overlap in mor- result in the retention of a large number of ancestral polymor- phological variation among and between members of this species phisms among species, resulting in similar patterns of nuclear– group is considerable, making identification of intermediate (i.e. mitochondrial discordance and complicating species delimitation ‘‘hybrid’’) morphology difficult. Allozyme variation has only been (Petit and Excoffier, 2009; Stelkens and Seehausen, 2009). If nucle- examined at relatively small geographic scales and has not been ar–mitochondrial discordance is due to incomplete lineage sorting, examined in all members of the group (Green 1983, 1984). Finally, then the mitochondrial data would be expected to show more using only maternally inherited mtDNA markers can fail to identify reciprocally monophyletic lineages than the nuclear data owing certain instances of hybridization. In crosses where a hybrid indi- to its’ smaller effective population size and mode of inheritance vidual is morphologically similar to the maternal species, the (Linnen and Farrell, 2007, 2008). Conversely, if introgression has mtDNA will match the morphological species diagnosis and recently occurred, the mitochondrial data would be expected to hybridization will remain undetected. show high levels of nonmonophyly (Linnen and Farrell, 2007, One method of identifying hybridization using AFLP markers in- 2008). Our data show very little reciprocal monophyly in the mito- volves examining loci that are differentially fixed between species chondrial data set, as several species appear to be paraphyletic B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 75 and/or polyphyletic (Fig. 4); providing support for the notion that 5. Conclusions hybridization has impacted the A. americanus group. Furthermore, we interpret the fact that species with similar male advertisement The members of the A. americanus group are characterized by calls in close geographic proximity share higher amounts of gene very little genetic divergence and no intrinsic postzygotic repro- flow as additional evidence supporting the occurrence of hybrid- ductive isolation (Malone and Fontenot, 2008; Sasa et al., 1998). ization as male advertisement call is a prezygotic isolating barrier Hybrid offspring of both sexes are viable and fertile through multi- in anurans (Waldman, 2001). Linnen and Farrell (2008) found a ple generations, suggesting that admixture between species could similar pattern in Neodiprion sawflies that used similar host plant be frequent (W.F. Blair, 1972; Green and Parent, 2003; Jones, 1973; species, a prezygotic isolating barrier in this group. However, di- Malone and Fontenot, 2008). Our analyses support the idea that rectly testing whether discordance is due to incomplete lineage natural hybridization has led to confusing patterns of genetic var- sorting or hybridization requires comparing gene flow rates in both iation in the A. americanus group. Our results provide further proof mitochondrial and nuclear DNA. Software exists that allows for this that taxonomic revision of the A. americanus group may be needed type of test, but is designed to work with species pairs that are as- as they demonstrate that classifications of species in this group sumed not to have shared genetic material with a third taxon (Hey based solely on morphological or mitochondrial data have failed and Nielsen, 2004). We cannot confidently make this assumption to fully reflect their evolutionary history. Given the weight of the with any species pair in the A. americanus group as several of these evidence, at least some of the putative species in this recent radi- species occur with one or more congener in sympatry or close ation may be more appropriately characterized as metapopulations parapatry. Additionally, these analyses require sequence data and exchanging varying levels of gene flow depending on geographic our AFLP data are not appropriate for this type of analysis. Further proximity and the strength of prezygotic isolating barriers. We data collection should focus on obtaining nuclear sequence data suggest that the discordance between the evolutionary relation- from allopatric species pairs in order to rule out incomplete lineage ships supported by AFLP and mtDNA data could be the signature sorting as the cause of nuclear–mitochondrial discordance. of secondary contact leading to multiple hybridization events Discordance between AFLP and mtDNA genealogies could also and differential fixation of regionally dominant mitochondrial hap- be attributed to dispersal effects as sex-biased dispersal is ex- lotypes as populations colonized newly available postglacial habi- pected to result in nuclear–mitochondrial discordance (Petit and tats. To better characterize the evolutionary history of the A. Excoffier, 2009). Due to their male polygynous reproductive sys- americanus group, we suggest conducting fine scale phylogeo- tem, where female choice determines mating success rather than graphic analyses of each putative species using both nuclear and male-male resource competition, toads are expected to have mitochondrial markers and quantitative analysis of male adver- male-biased dispersal; however, Smith and Green (2006) found tisement calls to further assess the relationship between gene flow no sex-biased dispersal among A. fowleri from Canada. Additional and male advertisement call. If future research corroborates the re- study is required to determine whether or not sex-biased dispersal sults of our study, a thorough taxonomic revision of the A. americ- exists in other members of the A. americanus group before any con- anus group will be necessary. clusions can be made regarding the role of sex-biased dispersal in nuclear–mitochondrial discordance. Acknowledgments

4.3. Male advertisement call and geography We acknowledge the following institutions and individuals for sharing tissue samples, laboratory and museum resources, and In the A. americanus group, putative species sharing similar specimens used in this study: J.A. Campbell, E.N. Smith, and C.J. male advertisement calls have elevated levels of gene flow and lit- Franklin at the University of Texas at Arlington Amphibian and tle genetic differentiation, suggesting that similar male advertise- Reptile Diversity Research Center, G. Watkins-Colwell at the Pea- ment calls may be less effective in preventing interspecific gene body Museum of Natural History at Yale University, K. Kozak, B. flow. At small geographic scales, female A. americanus and A. wood- Lowe, and T. Gamble at the Bell Museum of Natural History at housii appear to select mates on the basis of dominant frequency or the University of Minnesota, L. Rissler and T. York at the University high effort calls (i.e. longer calls or faster pulse rates), potential of Alabama Herpetological Collection, and S. Peterson, C. Watson, J. indicators of fitness (Sullivan, 1983a,b, 1992; Gatz, 1981; Licht, Meik, C. Roelke, J. Streicher, C. Cox, J. Malone, R. Calisi, A. Lawing, J. 1976). Additionally, Waldman (2001) found that females of A. Morse, S. Braman, M. Gifford, and R.L. Gutberlet. We also acknowl- americanus can discriminate between males that are closely or dis- edge A. Carrillo for laboratory assistance. J. Demuth, J. Meik, J. Strei- tantly related to themselves on the basis of their advertisement cher, and S. Schaack provided valuable help commenting on early calls, thus avoiding inbreeding and indicating the fine scale varia- versions of this manuscript. Funding for this project was provided tion that female frogs are capable of detecting. Hybrids between A. by the University of Texas at Arlington Phi Sigma Biological Honor americanus and A. fowleri possess a male advertisement call inter- Society Research Grant to BEF. mediate between that of the parental species and this could poten- tially reduce their attractiveness to females of either species, resulting in selective pressure to evolve prezygotic isolating barriers to avoid hybridization (Green and Parent, 2003). Boul et al. Appendix A (2007) found that sexual selection greatly influenced speciation in the frog Physalaeumus petersi with females choosing to mate Voucher specimen ID numbers (HERA = Peabody Museum of with males that possess advertisement calls similar to those Natural History at Yale University; JFBM = Bell Museum of Natural from the female’s local population. Advertisement call variation History at the University of Minnesota; UAHC = University of Ala- among populations resulted in differential female preferences, bama Herpetological Collection; BF = B. Fontenot field series; furthering divergence in male advertisement call and increasing CMW = C. Watson field series; TG = T. Gamble field series; JWS = J. behavioral isolation as a response to female choice. Boul et al. Streicher field series; CJF = C. Franklin field series; RMC = R. Calisi (2007) also found that gene flow was higher between species field series; SCB = S. Braman field series; MG = M. Gifford field ser- that shared similar male advertisement calls as seen in the ies; and RLG = R. Gutberlet field series). Sex, Genbank accession A. americanus group. numbers, and locality data for individuals in this study. 76 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80

Species Sex ID# Genbank Latitude Longitude State Anaxyrus americanus Female BF089 HM135206 43.3250 85.3103 MI Anaxyrus americanus Female BF090 HM135207 43.3250 85.3103 MI Anaxyrus americanus Unknown HERA010214 HM135208 41.3320 73.2064 CT Anaxyrus americanus Unknown HERA010347 HM135209 45.5913 69.9999 ME Anaxyrus americanus Unknown HERA010372 HM135210 42.1795 79.4495 NY Anaxyrus americanus Unknown HERA010512 HM135211 44.1105 71.2177 NH Anaxyrus americanus Unknown HERA9853 HM135212 41.3267 72.8043 CT Anaxyrus americanus Unknown JFBM14318 HM135213 45.1084 95.9051 MN Anaxyrus americanus Unknown JFBM14319 HM135214 45.1085 95.9052 MN Anaxyrus americanus Unknown JFBM14320 HM135215 45.1086 95.9053 MN Anaxyrus americanus Unknown JFBM14326 HM135216 45.3082 93.8993 MN Anaxyrus americanus Unknown JFBM14331 HM135217 46.3595 95.6243 MN Anaxyrus americanus Unknown JFBM14332 HM135218 46.3595 95.6243 MN Anaxyrus americanus Unknown JFBM14347 HM135219 45.2212 92.7724 MN Anaxyrus americanus Unknown JFBM14366 HM135220 44.0876 92.0189 MN Anaxyrus americanus Unknown JWS001 HM135221 38.8462 77.2469 VA Anaxyrus americanus Unknown TG0019 HM135222 34.5761 89.4778 MS Anaxyrus americanus Unknown UAHC14993 HM135223 39.0208 80.5435 WV Anaxyrus charlesmithi Female BF008 HM135224 34.6833 94.6352 OK Anaxyrus charlesmithi Female BF009 HM135225 34.6833 94.6352 OK Anaxyrus charlesmithi Male BF010 HM135226 34.6833 94.6352 OK Anaxyrus charlesmithi Female BF038 HM135227 33.6835 95.5981 TX Anaxyrus charlesmithi Male BF039 HM135228 33.6835 95.5981 TX Anaxyrus charlesmithi Female BF064 HM135229 33.9008 95.2888 OK Anaxyrus charlesmithi Female BF065 HM135230 33.9008 95.2888 OK Anaxyrus charlesmithi Female BF066 HM135231 33.9008 95.2888 OK Anaxyrus charlesmithi Male BF067 HM135232 33.9008 95.2888 OK Anaxyrus charlesmithi Female BF069 HM135233 34.5452 96.5918 OK Anaxyrus charlesmithi Female BF166 HM135234 35.9962 96.3334 OK Anaxyrus charlesmithi Male BF167 HM135235 35.9962 96.3334 OK Anaxyrus charlesmithi Female BF170 HM135236 36.5979 95.5325 OK Anaxyrus charlesmithi Male BF186 HM135237 34.7423 94.7238 OK Anaxyrus charlesmithi Female BF187 HM135238 34.7122 94.6648 OK Anaxyrus charlesmithi Female BF188 HM135239 34.7122 94.6648 OK Anaxyrus charlesmithi Juvenile BF189 HM135240 34.7147 94.6768 OK Anaxyrus charlesmithi Juvenile BF190 HM135241 34.7147 94.6768 OK Anaxyrus charlesmithi Juvenile BF191 HM135242 34.7147 94.6768 OK Anaxyrus cognatus Juvenile BF141(cog) HM135377 34.9270 102.1061 TX Anaxyrus cognatus Juvenile BF180 HM135378 34.9270 102.1061 TX Anaxyrus cognatus Juvenile BF181 HM135379 34.9270 102.1061 TX Anaxyrus cognatus Juvenile BF182 HM135380 34.9270 102.1061 TX Anaxyrus cognatus Juvenile BF183 HM135381 34.9270 102.1061 TX Anaxyrus cognatus Female BF184 HM135382 34.9270 102.1061 TX Anaxyrus cognatus Female BF185 HM135383 34.9270 102.1061 TX Anaxyrus fowleri Male BF011 HM135243 30.4095 91.1974 LA Anaxyrus fowleri Male BF013 HM135244 30.3886 91.2116 LA Anaxyrus fowleri Juvenile BF014 HM135245 30.3886 91.2116 LA Anaxyrus fowleri Juvenile BF015 HM135246 30.3886 91.2116 LA Anaxyrus fowleri Female BF017 HM135247 30.3848 91.2149 LA Anaxyrus fowleri Male BF018 HM135248 30.3848 91.2149 LA Anaxyrus fowleri Male BF019 HM135249 30.3848 91.2149 LA Anaxyrus fowleri Male BF020 HM135250 30.3859 91.2149 LA Anaxyrus fowleri Male BF021 HM135251 30.3859 91.2149 LA Anaxyrus fowleri Female BF109 HM135252 32.5313 92.4734 LA Anaxyrus fowleri Female BF110 HM135253 32.5313 92.4734 LA Anaxyrus fowleri Juvenile BF111 HM135254 32.5363 92.3592 LA Anaxyrus fowleri Female BF112 HM135255 32.5363 92.3592 LA Anaxyrus fowleri Male BF113 HM135256 32.5036 92.2524 LA Anaxyrus fowleri Male BF114 HM135257 32.5036 92.2524 LA Anaxyrus fowleri Male BF115 HM135258 32.5036 92.2524 LA Anaxyrus fowleri Male BF116 HM135259 32.6129 93.0558 LA B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 77

Appendix A (continued) Species Sex ID# Genbank Latitude Longitude State Anaxyrus fowleri Male BF117 HM135260 32.6129 93.0558 LA Anaxyrus fowleri Male BF118 HM135261 32.6129 93.0558 LA Anaxyrus fowleri Female BF119 HM135262 32.6129 93.0558 LA Anaxyrus fowleri Female BF125 HM135263 31.2458 93.3511 LA Anaxyrus fowleri Female BF126 HM135264 31.2458 93.3511 LA Anaxyrus fowleri Female BF127 HM135265 31.2458 93.3511 LA Anaxyrus fowleri Female BF128 HM135266 31.2458 93.3511 LA Anaxyrus fowleri Female BF129 HM135267 31.2458 93.3511 LA Anaxyrus fowleri Female BF130 HM135268 31.2598 92.7601 LA Anaxyrus fowleri Female BF131 HM135269 31.2598 92.7601 LA Anaxyrus fowleri Male BF145 HM135270 34.6868 82.8141 SC Anaxyrus fowleri Male BF146B HM135271 34.6868 82.8141 SC Anaxyrus fowleri Female BF154 HM135272 31.4394 89.4301 MS Anaxyrus fowleri Male CMW1003 HM135273 32.5930 86.9871 AL Anaxyrus fowleri Unknown CMW1027 HM135274 33.7398 80.8562 SC Anaxyrus fowleri Unknown HERA010313 HM135275 41.7665 72.6732 CT Anaxyrus fowleri Unknown HERA010491 HM135276 38.9687 75.6174 DE Anaxyrus fowleri Unknown HERA010493 HM135277 38.9688 75.6175 DE Anaxyrus fowleri Unknown TG0020 HM135278 34.5780 89.3316 MS Anaxyrus fowleri Unknown TG0024 HM135279 34.4800 89.2689 MS Anaxyrus fowleri Unknown TG0025 HM135280 34.4900 89.2690 MS Anaxyrus fowleri Unknown TG0146 HM135281 37.7150 90.5968 MO Anaxyrus fowleri Unknown UAHC14625 HM135282 34.0173 87.3593 AL Anaxyrus fowleri Unknown UAHC14629 HM135283 34.0172 87.3592 AL Anaxyrus fowleri Unknown UAHC15113 HM135284 33.1985 87.4045 AL Anaxyrus fowleri Unknown UAHC15115 HM135285 33.1986 87.4046 AL Anaxyrus fowleri Unknown UAHC15193 HM135286 31.4541 86.7871 AL Anaxyrus hemiophrys Unknown JFBM14328 HM135376 48.9803 97.5559 ND Anaxyrus terrestris Female BF134 HM135287 30.7180 85.9342 FL Anaxyrus terrestris Female BF135 HM135288 30.7180 85.9342 FL Anaxyrus terrestris Female BF141(terr) HM135289 30.7180 85.9342 FL Anaxyrus terrestris Juvenile BF155 HM135290 30.6426 84.2029 SC Anaxyrus terrestris Female BF156 HM135291 30.6426 84.2029 FL Anaxyrus terrestris Male BF163 HM135292 32.7863 79.7872 SC Anaxyrus terrestris Male BF169 HM135294 32.4775 85.3902 AL Anaxyrus terrestris Male BF171 HM135295 30.3073 86.0960 FL Anaxyrus terrestris Male BF172 HM135296 30.3073 86.0960 FL Anaxyrus terrestris Male BF173 HM135297 30.3073 86.0960 FL Anaxyrus terrestris Male BF174 HM135298 30.3073 86.0960 FL Anaxyrus terrestris Male CMW1028 HM135299 33.2974 81.6545 GA Anaxyrus terrestris Unknown JWS008 HM135300 36.7114 76.2396 VA Anaxyrus terrestris Unknown UAHC14621A HM135301 34.0172 87.3592 AL Anaxyrus terrestris Unknown UAHC14621B HM135302 34.0173 87.3593 AL Anaxyrus velatus Juvenile BF001 HM135303 32.2561 95.1847 TX Anaxyrus velatus Male BF002 HM135304 32.2561 95.1847 TX Anaxyrus velatus Juvenile BF003 HM135305 32.2561 95.1847 TX Anaxyrus velatus Juvenile BF004 HM135306 32.2561 95.1847 TX Anaxyrus velatus Juvenile BF005 HM135307 32.2561 95.1847 TX Anaxyrus velatus Juvenile BF006 HM135308 32.2561 95.1847 TX Anaxyrus velatus Male BF007 HM135309 32.2561 95.1847 TX Anaxyrus velatus Male BF022 HM135310 31.3231 95.1009 TX Anaxyrus velatus Male BF023 HM135311 31.3231 95.1009 TX Anaxyrus velatus Male BF026 HM135312 31.8770 95.6051 TX Anaxyrus velatus Male BF027 HM135313 31.8770 95.6051 TX Anaxyrus velatus Male BF028 HM135314 31.8770 95.6051 TX Anaxyrus velatus Female BF029 HM135315 31.9113 95.8972 TX Anaxyrus velatus Male BF030 HM135316 31.9113 95.8972 TX Anaxyrus velatus Male BF031 HM135317 31.9113 95.8972 TX Anaxyrus velatus Juvenile BF032 HM135318 32.1424 95.8569 TX Anaxyrus velatus Male BF034 HM135319 32.1424 95.8569 TX

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Appendix A (continued) Species Sex ID# Genbank Latitude Longitude State Anaxyrus velatus Female BF035 HM135320 32.2713 94.5882 TX Anaxyrus velatus Female BF036 HM135321 32.2713 94.5882 TX Anaxyrus velatus Male BF037 HM135322 32.2713 94.5882 TX Anaxyrus velatus Female BF040 HM135323 33.0618 95.0248 TX Anaxyrus velatus Female BF041 HM135324 33.0618 95.0248 TX Anaxyrus velatus Female BF042 HM135325 33.0618 95.0248 TX Anaxyrus velatus Juvenile BF045 HM135326 31.2236 93.6750 TX Anaxyrus velatus Juvenile BF047 HM135327 31.2236 93.6750 TX Anaxyrus velatus Juvenile BF048 HM135328 31.2236 93.6750 TX Anaxyrus velatus Female BF049 HM135329 32.7100 94.1203 TX Anaxyrus velatus Female BF050 HM135330 32.7038 94.1157 TX Anaxyrus velatus Male BF051 HM135331 32.7181 94.1357 TX Anaxyrus velatus Female BF052 HM135332 32.6378 94.2015 TX Anaxyrus velatus Female BF053 HM135333 32.6378 94.2015 TX Anaxyrus velatus Female BF054 HM135334 32.7032 94.0708 TX Anaxyrus velatus Female BF061 HM135335 33.4019 94.4254 TX Anaxyrus velatus Female BF062 HM135336 33.4019 94.4254 TX Anaxyrus velatus Male BF063 HM135337 33.4019 94.4254 TX Anaxyrus velatus Male BF068 HM135338 31.2236 93.6750 TX Anaxyrus velatus Female BF085 HM135339 31.0424 94.3269 TX Anaxyrus velatus Male BF088 HM135340 31.1836 93.8125 TX Anaxyrus velatus Female BF105 HM135341 31.5212 94.7672 TX Anaxyrus velatus Male BF121 HM135342 31.2212 93.6796 TX Anaxyrus velatus Male BF122 HM135343 31.2212 93.6796 TX Anaxyrus velatus Female BF123 HM135344 31.2212 93.6796 TX Anaxyrus velatus Female BF124 HM135345 31.2212 93.6796 TX Anaxyrus velatus Unknown MG0001 HM135346 32.2561 95.1847 TX Anaxyrus velatus Unknown MG0002 HM135347 32.2561 95.1847 TX Anaxyrus velatus Female RLG2634 HM135348 32.5883 95.8567 TX Anaxyrus velatus Male RLG2637 HM135349 32.5883 95.8567 TX Anaxyrus velatus Female RLG2638 HM135350 32.5883 95.8567 TX Anaxyrus velatus Female RLG2639 HM135351 32.5883 95.8567 TX Anaxyrus velatus Female SCB006 HM135352 32.3757 94.8491 TX Anaxyrus woodhousii Male BF024 HM135353 33.5220 101.8460 TX Anaxyrus woodhousii Female BF043 HM135354 32.8729 96.7561 TX Anaxyrus woodhousii Female BF044 HM135355 32.8729 96.7561 TX Anaxyrus woodhousii Male BF095 HM135356 32.6825 97.5084 TX Anaxyrus woodhousii Male BF096 HM135357 32.6825 97.5084 TX Anaxyrus woodhousii Male BF097 HM135358 32.6825 97.5084 TX Anaxyrus woodhousii Female BF098 HM135359 32.6825 97.5084 TX Anaxyrus woodhousii Female BF099 HM135360 32.7428 97.1998 TX Anaxyrus woodhousii Female BF101 HM135361 32.6182 97.1526 TX Anaxyrus woodhousii Female BF102 HM135362 32.6182 97.1526 TX Anaxyrus woodhousii Female BF107 HM135363 32.9010 98.5560 TX Anaxyrus woodhousii Female BF108 HM135364 32.8950 98.5310 TX Anaxyrus woodhousii Female BF132-B HM135365 33.2446 98.3290 TX Anaxyrus woodhousii Female BF133 HM135366 38.8265 98.4747 KA Anaxyrus woodhousii Male BF162 HM135367 33.6969 95.6854 TX Anaxyrus woodhousii Male BF165 HM135293 33.3642 97.6839 TX Anaxyrus woodhousii Male BF168 HM135368 32.6051 98.0339 TX Anaxyrus woodhousii Male BF175 HM135369 33.8330 97.4950 TX Anaxyrus woodhousii Female BF176 HM135370 33.4580 97.6120 TX Anaxyrus woodhousii Juvenile BF177 HM135371 34.9270 102.1061 TX Anaxyrus woodhousii Juvenile BF178 HM135372 34.9270 102.1061 TX Anaxyrus woodhousii Juvenile BF179 HM135373 34.9270 102.1061 TX Anaxyrus woodhousii Unknown CJF3182 HM135374 32.6051 98.0339 TX Anaxyrus woodhousii Juvenile RMC00001 HM135375 32.7834 97.1400 TX B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80 79

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