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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. ChippindaleDepartment of Biology, University of Texas at Arlington, Arlington, TX 76019, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2010Revised 12 December 2010Accepted 23 December 2010Available online 19 January 2011

Keywords:ToadsHybridizationGene flowSpeciationAFLPsNuclear–mitochondrial discordance

1055-7903/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ympev.2010.12.018

⇑ Corresponding author. Fax: +1 817 272 2855.E-mail address: [email protected] (B.E.

1 Present address: Department of Biostatistics, UniversBirmingham, AL 35294, United States.

Natural hybridization among recently diverged species has traditionally been viewed as a homogenizingforce, but recent research has revealed a possible role for interspecific gene flow in facilitating speciesradiations. Natural hybridization can actually contribute to radiations by introducing novel genes orreshuffling existing genetic variation among diverging species. Species that have been affected by naturalhybridization often demonstrate patterns of discordance between phylogenies generated using nuclearand mitochondrial markers. We used Amplified Fragment Length Polymorphism (AFLP) data in conjunc-tion with mitochondrial DNA in order to examine patterns of gene flow and nuclear–mitochondrial dis-cordance in the Anaxyrus americanus group, a recent radiation of North American toads. We found highlevels of gene flow between putative species, particularly in species pairs sharing similar male advertise-ment calls that occur in close geographic proximity, suggesting that prezygotic reproductive isolatingmechanisms and isolation by distance are the primary determinants of gene flow and genetic differenti-ation among these species. Additionally, phylogenies generated using AFLP and mitochondrial data weremarkedly discordant, likely due to recent and/or ongoing natural hybridization events between sympatricpopulations. Our results indicate that the putative species in the A. americanus group have experiencedhigh levels of gene flow, and suggest that their North American radiation could have been facilitatedby the introduction of beneficial genetic variation from admixture between divergent populations com-ing into secondary contact after glacial retreats.

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1. Introduction

Assessing gene flow between closely related species is a crucialcomponent to our understanding of the speciation process. Underthe allopatric speciation model, gene flow between diverging spe-cies is often thought to lead to homogenization, reduced fitness,and erosion of species boundaries (Mayr, 1942; Coyne and Orr,2004). However, recent studies have shown that some organismscan experience relatively large amounts of interspecific gene ex-change and still maintain significant differentiation from other spe-cies (Hey, 2006; Fitzpatrick et al., 2008; Linnen and Farrell, 2007;Strasburg and Rieseberg, 2008). The fact that some species remaindistinct despite hybridization has led some researchers to hypothe-size that the introduction of novel genetic variation from heterospe-cific mating could facilitate speciation by promoting the shuffling ofexisting variation and creation of novel allelic combinations, en-abling hybrids to take advantage of changing environmental condi-tions (Arnold, 1997; Coyne and Orr, 2004; Seehausen, 2004). Thepossible role of interspecific hybridization in speciation has been atopic of discussion among evolutionary biologists for many years,

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Fontenot).ity of Alabama at Birmingham,

but recent advances in molecular techniques have allowedresearchers to examine this phenomenon in a much more detailedfashion leading to a renewed emphasis on hybridization studies.

One of the predicted signatures of radiations facilitated byhybridization is discordance between genealogies created usingnuclear and cytoplasmic genetic markers (Petit and Excoffier,2009; Seehausen, 2004). Cytoplasmic markers like mitochondrialDNA (mtDNA) or chloroplast DNA are more likely than nuclearDNA (nDNA) markers to introgress between species due to differ-ences in their parental inheritance mechanism, effective popula-tion size, recombination (or lack thereof), and mutation rate(Avise, 1994; Currat et al., 2008; Leaché, 2009; Petit and Excoffier,2009). Chan and Levin (2005) modeled a wide range of introgres-sion scenarios and found that introgression rates of maternallyinherited cytoplasmic markers consistently exceed those of pater-nal cytoplasmic markers and nuclear genes, particularly in second-ary contact scenarios where one species is less abundant than theother. They attribute this difference to frequency-dependent pre-zygotic barriers in female choice-based mating systems wherethe maternal cytoplasmic marker has more opportunity for intro-gression from the rare species to the more abundant species. In thisscenario, rare females have more opportunity to mate with heter-ospecific males than with conspecific males, leading to increasedrates of mitochondrial introgression. Differential patterns ofintrogression can provide an explanation for the topological

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discordance often seen in phylogenetic trees generated frommtDNA and nDNA gene fragments of closely related species (Taylorand McPhail, 2000; Dopman et al., 2005; Mendelson and Simons,2006; Petit and Excoffier, 2009; Seehausen, 2004; Wiens et al.,2010); however, incomplete lineage sorting and the retention ofancestral polymorphisms could also result in similar patterns ofdiscordance.

To examine the effects of hybridization on the speciation pro-cess, it is necessary to examine species capable of producing viableand fertile offspring from natural hybridization. Some empiricalexamples of nuclear–mitochondrial discordance come from taxathat have experienced natural hybridization such as birds (Freelandand Boag, 1999; Grant and Grant, 1996), crickets (Shaw, 1996,2002), Hawaiian silversword plants (Barrier et al., 1999), butterflies(Beltran et al., 2002; Gilbert, 2003), turtles (Wiens et al., 2010), liz-ards (Leaché, 2009), salamanders (Highton, 1995; Wiens et al.,2006) several fish species (DeMarais et al., 1992; Seehausen et al.,1997; Taylor and McPhail, 2000) and several frog species (Green,1984; Green and Parent, 2003; Yanchukov et al., 2006; Mezhzherinet al., 2010; Stöck et al., 2010). True toads in the family Bufonidae, anearly cosmopolitan group with a likely origin in the Upper Creta-ceous, are also known to engage in natural hybridization (Blair,1972; Green, 1984; Green and Parent, 2003; Jones, 1973; Maloneand Fontenot, 2008; Masta et al., 2002; Pramuk et al., 2008). Consid-erable research has focused on hybridization among members ofthe widely distributed Anaxyrus (formerly Bufo) americanus group(Blair, 1941, 1942; Blair, 1955, 1959, 1961, 1972; Green, 1984;Green and Parent, 2003; Vogel and Johnson, 2008; Volpe, 1952,1959). The Anaxyrus americanus group, thought to have arisen as re-cently as 2 million years ago, includes A. americanus, Anaxyrus char-lesmithi, Anaxyrus houstonensis, Anaxyrus terrestris, Anaxyrushemiophrys, Anaxyrus microscaphus, Anaxyrus woodhousii, Anaxyrusfowleri, and Anaxyrus velatus (Blair, 1972; Conant and Collins,1998; Dixon, 2000; Pauly et al., 2004). These putative species tradi-tionally have been identified using qualitative morphological traitsand male advertisement calls. Previous studies of mtDNA, allozymevariation, morphology, ecology, and karyotypes have not provided astrongly supported hypothesis of interspecific relationships in theA. americanus group, and the taxonomic status of many species inthe group remains problematic (Blair, 1972; Bragg and Sanders,1951; Masta et al., 2002). The recent origin of this group and multi-ple areas of natural contact between putative species make the A.americanus group an excellent system for studying hybridizationand speciation. Additionally, toads are valuable subjects for specia-tion studies because they exhibit homomorphic sex chromosomes(however, see Abramyan et al., 2009), female heterogamety (con-firmed in Rhinella marina; Abramyan et al., 2009; and suspectedin Bufo bufo; Ponse, 1941; however, see Rostrand, 1952, 1953),and use male advertisement calls for mate selection. Males of somefrog species are subjected to strong selective pressures as femalechoice can drive male advertisement call evolution and may evencause speciation events through assortative mating (Boul et al.,2007; Gerhardt, 2005; Höbel and Gerhardt, 2003; Murphy andGerhardt, 2002; Ryan et al., 1990).

Our study is the first to examine genetic variation in the A.americanus group across the majority of their range in the UnitedStates using Amplified Fragment Length Polymorphism (AFLP) datain conjunction with mtDNA. By documenting levels of geneticdiversity, estimating gene flow between and among putative spe-cies, and comparing results generated using mtDNA and AFLPs,we sought to answer the following questions: (1) Could the radia-tion of the A. americanus group have been affected by naturalhybridization? (2) Are nuclear and mitochondrial genealogies ofthe A. americanus group concordant? and (3) How are geographyand male advertisement call type correlated with patterns of geneflow and genetic differentiation between species?

2. Methods

2.1. Taxon sampling

We collected specimens for molecular analyses based on pub-lished geographic ranges for the currently recognized species inthe United States including Texas, Louisiana, Alabama, Arkansas,Connecticut, Minnesota, Delaware, Maine, New York, New Hamp-shire, Mississippi, Michigan, Florida, South Carolina, Kansas, andOklahoma (Conant and Collins, 1998; Dixon, 2000). Four membersof the A. americanus group (A. charlesmithi, A. fowleri, A. velatus, andA. woodhousii) occur either sympatrically or parapatrically in thedeciduous hardwood forests of eastern Texas, southeastern Okla-homa, western Louisiana, and southwestern Arkansas (Fig. 1; Con-ant and Collins, 1998; Dixon, 2000), and some authors havehypothesized that this region contains populations of hybrid indi-viduals (Blair 1941, 1942; Blair, 1972; Bragg and Sanders, 1951). Toevaluate whether or not hybridization can be detected in this re-gion, we concentrated sampling of A. woodhousii, A. velatus, A, char-lesmithi, and A. fowleri along a transect spanning a region fromwestern Texas to eastern Louisiana (see Fig. 2A for collection local-ities). Individuals collected from the field (n = 231) were combinedwith samples (n = 48) obtained from the Peabody Museum of Nat-ural History (HERA) at Yale University, the University of AlabamaHerpetological Collection (UAHC), and the Bell Museum of NaturalHistory (JFBM) at the University of Minnesota (see Appendix A forspecimen ID numbers, full locality data, and Genbank accessionnumbers). Field-collected voucher specimens were sexed using acombination of presence of male advertisement call if capturedduring a chorus, presence of secondary sexual characters such asnuptial pads in males, and finally, dissection to visualize gonadcondition.

A priori species designations of field-caught individuals weremade using a combination of the morphological characters ofConant and Collins (1998), the species designations from Mastaet al. (2002) according to their identified geographic species bound-aries, and male advertisement call type. Species designationsfollowed the framework of the General Lineage species concept(de Queiroz, 1998), and for the purposes of this study, we view spe-cies as ‘‘separately evolving metapopulation lineages’’ (de Queiroz,2005). However, our intent is not to revise the taxonomy of thegroup, but rather to evaluate how the currently accepted taxonomy,based on morphology and mitochondrial DNA, would be affected byadditional evidence from anonymous AFLP nuclear markers.

Tissue samples obtained from museum collections were classi-fied according to the species designation in each sample’s museumcatalog. All members of the A. americanus group are representedexcept the allopatric A. microscaphus and the endangered A. hou-stonensis. Species that occur in the putative hybrid zone spanningparts of Texas and Louisiana (A. fowleri, A. charlesmithi, A. velatus,and A. woodhousii) were sampled most extensively. Anaxyrus cogn-atus, the sister species to the A. americanus group, was chosen asthe outgroup taxon for molecular analyses (Masta et al., 2002;Pauly et al., 2004). Whole genomic DNA was extracted from liveror muscle tissue using DNeasy tissue kits (QUIAGEN). All field-collected specimens were deposited in the amphibian collectionat the University of Texas at Arlington Amphibian and ReptileDiversity Research Center (Appendix A).

2.2. Male advertisement call and geography

Toads in the A. americanus group have two major types of maleadvertisement calls that differ primarily in pulse rate (Sullivanet al., 1996). Male advertisement calls were broadly classified intotwo qualitative categories: ‘‘Whining calls’’ (lower pulse rate),

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 arenot shown since they were not included in this study.

68 B.E. Fontenot et al. / Molecular Phylogenetics and Evolution 59 (2011) 66–80

present in A. woodhousii, A. fowleri, and A. velatus, and ‘‘Whistlingcalls’’ (higher pulse rate), present in A. americanus, A. charlesmithi,and A. terrestris (Conant and Collins, 1998). While these speciesdo exhibit some geographic variation in advertisement calls, thisvariation often manifests as fine-scale differences in pitch and/orduration that do not change the qualitative ‘‘Whining’’ or ‘‘Whis-tling’’ nature of the advertisement call, but rather serve to furtherdifferentiate between syntopic species at a fine scale (Sullivanet al., 1996; Waldman, 2001). We chose to classify species’ adver-tisement calls using the broad-scale qualitative ‘‘Whining’’ and‘‘Whistling’’ designations since this would be more representativeof the variation seen at large geographic scales. All field-confirmedadvertisement call designations were concordant with the adver-tisement call type predicted from a priori species designationsmade using the morphological characters of Conant and Collins(1998); therefore, individuals for which we could not confirmadvertisement call type in the field were categorized according toa combination of their occurrence within the geographic speciesboundaries identified by Masta et al. (2002) and their morpholog-ical species designation. We then used the program POPGENE (Yehand Boyle, 1997) to estimate genetic differentiation (GST) and geneflow (NM) for all pairwise species comparisons as well as subse-quent Neighbor-joining and Bayesian analyses in light of a prioriadvertisement call designations. This allowed us to evaluate pat-terns of genetic diversity among species in the context of both geo-graphic location and male advertisement call.

2.3. AFLP markers

AFLP data were obtained following the methods of Vos et al.(1995) and Makowsky et al. (2009) using an EcoR I + G preselectiveprimer and a Fam-6 labeled Mse I + GC selective primer. A randomsample of 75 individuals was chosen for duplicate AFLP amplifica-tion to assess repeatability. Selective PCR products were purifiedusing standard ethanol precipitation cleanup and sequenced usingan ABI 3130xl capillary sequencer with a Rox 400 HD size standard

(Applied Biosystems, Inc.). Fragments were scored using Genemar-ker� (Softgenetics, LLC.), and fragments less than 100 base pairs inlength were removed to avoid complications in homolog assess-ment (Vekemans et al., 2002). The remaining scorable fragmentswere assembled into a presence/absence matrix for statistical anal-ysis. Genemarker� was used to delineate fragments using a thresh-old peak value of 100 following Makowsky et al. (2009). Inaddition, all presence/absence assignments were verified by eye.Furthermore, select AFLP profiles were duplicated to verifyrepeatability.

2.4. AFLP analyses

Analyses were conducted on all species to assess genetic diver-sity and intraspecific population structure. Analyses were alsoconducted for all pairwise interspecific comparisons to evaluategenetic diversity, interspecific population structure, and possibleinterspecific gene flow. The program GENALEX 6.2 (Peakall andSmouse, 2006) was used to create a matrix of genetic distancesusing Nei’s D (Nei, 1972), and to conduct Mantel tests for Isola-tion-by-Distance. Measures of genetic diversity and populationstructure were obtained using the program HICKORY 1.0(Holsinger et al., 2002) using the f-free, full model option. BecauseAFLP markers are scored as dominant characters, direct observa-tion of heterozygosity is impossible. To address this potentialproblem, HICKORY 1.0 (Holsinger et al., 2002) implements Bayes-ian methods that assume Hardy–Weinberg equilibrium and anon-uniform prior distribution of allelic frequencies (Zhivotovsky,1999). This approach uses the observed fragment frequencies toproduce robust estimates of allelic frequencies, heterozygosity(H), and genetic differentiation (FST). These estimates have beenshown to be accurate even when derived from dominant markers(Krauss, 2000). HICKORY 1.0 and POPGENE (Yeh and Boyle, 1997)were used to obtain measures of estimated heterozygosity (HT),the FST analog hB as a measure of genetic differentiation, the Shan-non Information Index (I) as a measure of genetic diversity, Nei’s

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

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

A. americanus/hemiophrys Clade

A. fowleri Clade 1

A. woodhousii Clade

A. fowleri Clade 2

A. terrestris Clade

A. americanus/velatus/charlesmithi Clade

(A)

(B)

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,and NM as the estimated number of migrants among populationsto characterize gene flow (Holsinger et al., 2002). A Neighbor-

Joining (NJ) tree with nodal support from 10,000 bootstrappseudoreplicates was created using PAUP� 4.0 beta (Swofford,1999).

Table 1Comparison of partitioning strategies used in Bayesian analysis of mtDNA.

Analysis Description ofpartitions

Number ofpartitions

�ln likelihood ofharmonic mean

1 16S + leu + ND1 1 �4277.412 16S + leu, ND1 2 �4276.563 16S, leu, ND1 3 �4257.854 16S + leu, ND1 (by

codon)4 �3895.64

5 16S, leu, ND1 (bycodon)

5 �3895.23


2.5. Mitochondrial markers

Mitochondrial markers were amplified using the PolymeraseChain Reaction (PCR). Primers (Bw16S-L – 50-ATTTTTTCTAGTACGAAAGGAC-30 and B-Ile-H – 50-GCACGTTTCCATGAAATTGGTGG-30) obtained from Masta et al. (2002) were used to amplify1029 base pairs including the 30 end of the 16S ribosomal subunitgene, all of tRNALEU, and part of the NADH dehydrogenase subunit1 (ND1) gene. PCR was carried out in 10 ll reactions containing1 ll DNA, 1 ll 10X Buffer, 1 ll dNTPs, 0.6 ll of each primer,0.1 ll MgCl2, and 0.05 ll Taq polymerase under the following ther-mal cycling conditions: 94� for 3 min followed by 35 cycles ofdenaturation at 94� for 30 s, annealing at 54� for 45 s, and exten-sion at 72� for 75 s, followed by a final extension at 72� for7 min. PCR samples were cleaned by ExoSapIt (United States Bio-chemical, Cleveland, OH) and sequenced on an ABI 3130xl sequen-cer (Applied Biosystems Inc.) using standard protocols. Bothstrands were sequenced for all samples. Forward and reverse se-quences were compared using Sequencher 4.8 (Gene Codes Corpo-ration) and consensus sequences were aligned using ClustalW(Chenna et al., 2003; Larkin et al., 2007) in Macvector 9.0 (MacVec-tor Inc.). No gaps were found and alignment was unambiguous.

2.6. Mitochondrial analyses

Aligned sequences totaled 1029 bases and redundant haplo-types were removed for all analyses involving mitochondrial mark-ers. PAUP� 4.0 beta (Swofford, 1999) was used for maximumlikelihood (ML) analyses with 10 random addition sequences andTBR swapping with 10 repetitions. To obtain an appropriate modelof evolution for each gene, we used Modeltest 3.7 (Posada andCrandall, 1998) with the model chosen based on the Akaike Infor-mation Criterion (AIC). Following the initial model selection (ob-tained with a NJ starting tree), we used the best tree obtained

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

Species n Number of Loci Na (s.d.) Ne (s.d.)

americanus 18 100 1.87 (0.34) 1.56 (0.34)charlesmithi 19 100 1.86 (0.35) 1.68 (0.33)fowleri 44 100 1.89 (0.31) 1.64 (0.32)terrestris 17 100 1.88 (0.33) 1.71 (0.33)velatus 50 100 1.93 (0.26) 1.65 (0.30)woodhousii 23 100 1.82 (0.39) 1.63 (0.34)cognatus 7 100 1.59 (0.49) 1.34 (0.36)

All Populations 178 100 2.00 (0.00) 1.76 (0.25)

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 prophB: Estimated genetic differentiation (comparable to Wright’s Fst).

from the ML analysis as the starting tree in a subsequent Modeltest3.7 search to optimize the model of evolution. This process was re-peated until the best tree likelihoods in sequential iterations werecomparable. Nodal support was assessed from 100 bootstrap pseu-doreplicates using PhyML 3.0 (http://www.atgc-montpellier.fr/phyml/).

The best ML tree was used to determine the appropriate modelof evolution for Bayesian analyses in MrModelTest 2 (Nylander,2004). These model parameters were used for analyses in MrBayes3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck,2003) for at least 10 million generations with four chains, two rep-etitions, two swaps per generation, and sampling every 1000 gen-erations for both standard and partitioned (by codon position)analyses. Analyses were run until the standard deviation of splitfrequencies was <0.01 and convergence was further checked inAWTY (Nylander et al., 2008). The first 2.5–5.0 million generationswere discarded as burnin. We compared five partitioning strategiesfor the Bayesian analysis (Table 1) using Bayes factors (Nylanderet al., 2008), which were calculated as twice the difference of –lnlikelihood harmonic means between competing hypotheses usingthe harmonic mean output in MrBayes 3.1.2. We interpreted Bayesfactor values <0 as no evidence (0–2), positive support (3–6),strong support (7–10), or very strong support (> 10) in favor ofthe more parameterized model.

3. Results

3.1. AFLP analyses

AFLP profiles were generated for 231 individuals, with 178showing sufficient clarity to allow accurate profiling (AppendixA). One specimen of A. hemiophrys was also profiled, but we ex-cluded it from all analyses except the NJ and Bayesian analyses,as it was the only individual of that species for which data were ob-tained. Inspection of AFLP profiles of the 75 individuals randomlychosen for duplication revealed that 71/75 (95%) gave profilesidentical to the original, indicating sufficient repeatability of ourAFLP amplification protocol. The four profiles that did differ uponreplication had profiles differing in fragment sizes or numbers offragments ranging from 2 to 12 fragments. After removal of frag-ments under 100 bp and editing of the AFLP profiles to removeambiguous fragments, 100 scorable loci were obtained for 178individuals and 100% of these loci were polymorphic (Table 2).Visual examination of the AFLP profiles revealed no fixed diagnos-tic loci for any samples assigned to our a priori designated species.

With respect to overall AFLP diversity, the Shannon InformationIndex (I) ranged from 0.31 to 0.55, average heterozygosity (HT)

I (s.d.) HT (s.d.) hB (s.d.) Percent polymorphic loci

0.48 (0.23) 0.36 (0.01) 0.23 (0.05) 870.54 (0.23) 0.41 (0.01) 0.13 (0.03) 860.53 (0.21) 0.33 (0.04) 0.16 (0.04) 890.55 (0.22) 0.41 (0.02) 0.14 (0.04) 880.54 (0.18) 0.30 (0.04) 0.19 (0.04) 930.50 (0.25) 0.36 (0.03) 0.14 (0.04) 820.31 (0.28) 0.41 (0.02) 0.09 (0.04) 59

0.61 (0.12) 0.41 (0.04) 0.27 (0.02) 100

ortions.

http://www.atgc-montpellier.fr/phyml/

http://www.atgc-montpellier.fr/phyml/

Table 3Estimated pairwise hB (genetic differentiation) with 95% credibility intervals below diagonal and pairwise genetic distances (Nei’s D) above diagonal. Values in bold indicatesignificant 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 0.45690.01–0.26 �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 4Nei’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 2.9charlesmithi 0.08 1.2 5.2 5.8 5.9 6.2cognatus 0.29 0.29 1.0 1.4 1.0 1.0fowleri 0.15 0.09 0.32 6.5 21.1 12.1terrestris 0.09 0.08 0.26 0.07 5.4 5.6velatus 0.15 0.08 0.33 0.02 0.08 12.6woodhousii 0.15 0.08 0.33 0.04 0.08 0.04


ranged from 0.30 to 0.41, genetic differentiation (hB) ranged from0.09 to 0.23, and percentage of polymorphic loci ranged from59% to 93% (Table 2). Pairwise genetic distances (Nei’s D) betweenspecies ranged from 0.02 to 0.46 with the only significant differ-ence occurring between A. americanus and A. cognatus (Table 3).Pairwise GST ranged from 0.02 to 0.33 and pairwise NM ranged from1.0 to 21.1 (Table 4). Mantel tests for Isolation-by-distance (IBD)were significant for all pairwise species comparisons (Supplemen-tal Table S1).

An NJ tree depicting relationships among all individuals is givenin supplemental Fig. S1. Fig. 3 represents the NJ tree with somegroups collapsed for simplified viewing. NJ analysis of the AFLPdata broadly separates the species into three groups; one com-posed primarily of species from our a priori defined ‘‘whining call’’category and two smaller groups composed primarily of speciesfrom our a priori defined ‘‘whistling call’’ category. Only 14/125(11.2%) individuals from the ‘‘whistling call’’ category were placedin the ‘‘whining call’’ group (Fig. 3). In the two ‘‘whistling call’’groups, only 5/42 (11.9%) individuals from the ‘‘whining call’’ cat-egory were placed in either of the ‘‘whistling call’’ groups (Fig. 3).Overall bootstrap support was low, but six nodes were supported(supplemental Fig. S1). Geographic localities for individuals inthe NJ analysis are shown in Fig. 2A.

3.2. Mitochondrial analyses

The final mtDNA alignment revealed 88 unique haplotypes and261 variable sites, of which 165 were parsimony informative forthe ingroup. The model of evolution (for likelihood searches) forall genes combined was TrN + G: Base frequencies of A = 0.3276,C = 0.1171, G = 0.2552, T = 0.3002; substitution rate parametersA–C = 1.000, A–G = 11.568, A–T = 1.000, C–G = 1.000, C–T = 24.720,

G–T = 1.0000; gamma distribution shape parameter = 0.1250. ForBayesian analyses, a partitioning scheme with four partitions(16S + tRNALEU and ND1 partitioned by codon) was chosen aftercomparison of Bayes factors (Table 1). The model chosen for eachgene in the Bayesian analysis was GTR + G.

The phylogeny recovered from the Bayesian analysis containssix major clades with strong support (Fig. 4). Two clades containingindividuals designated as A. fowleri were found to be paraphyleticwith respect to a clade containing individuals designated as A. ter-restris. The paraphyly of A. fowleri and A. terrestris is congruent withrelationships obtained in other studies using mtDNA markers(Masta et al., 2002; Pauly et al., 2004). Along with the A. fowleriand A. terrestris grouping are three other major clades, one contain-ing northern individuals designated as A. americanus and A. hemi-ophrys, another containing A. woodhousii, and a large cladecontaining individuals designated as A. americanus, A. charlesmithi,and A. velatus. The sister relationship of A. americanus and A. wood-housii is congruent with previous studies (Masta et al., 2002 andPauly et al., 2004). However, the larger clade containing individualsdesignated as A. americanus, A. charlesmithi, and A. velatus is some-what unexpected, and differs from the results of previous studiesprimarily due to the inclusion of individuals from northeasternlocalities designated as A. americanus (Pauly et al., 2004). The geo-graphic localities for individuals in the clades recovered from theBayesian analysis are shown in Fig. 2B.


Analysis of the species that occur sympatrically and/or parapat-rically in the putative hybrid zone (A. woodhousii, A. fowleri,A. velatus, and A. charlesmithi) resulted in a GST value of 0.08 andan NM value of 5.4 indicating little genetic differentiation and high

Fig. 3. Neighbor-joining tree generated using AFLP data. Yellow clade contains putative species with whining male advertisement calls and red clades contain putativespecies with whistling male advertisement calls. Scale bar represents Nei’s (1978) genetic distance measure. �Indicates individual designated as having whistling maleadvertisement call type. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


0.70.7

BF180-cognatus (TX)

JWS001-americanus (VA)

BF167-charlesmithi (OK)BF166-charlesmithi (OK)

BF011-fowleri (LA)

UAHC14993-americanus (WV)

BF183-cognatus (TX)

JFBM14328-hemiophrys (ND)

BF141COG-cognatus (TX)

BF107-woodhousii (TX)

BF154-fowleri (MS)

BF050-velatus (TX)

BF013-fowleri (LA)

UAHC14625-fowleri (AL)

CMW1028-terrestris (GA)

BF134-terrestris (FL)

BF002-velatus (TX)

JFBM14332-americanus (MN)

BF066-charlesmithi (OK)

BF045-velatus (TX)

BF105-velatus (TX)

BF090-americanus (MI)


UAHC14621B-terrestris (AL)

BF169-terrestris (AL)


CMW1027-fowleri (SC)


MG0002-velatus (TX)


HERA9853-americanus (CT)

BF017-fowleri (LA)


BF146B-fowleri (SC)

JWS008-terrestris (VA)

BF019-fowleri (LA)




HERA10372-americanus (NY)


HERA10491-fowleri (DE)

BF061-velatus (TX)

MG0001-velatus (TX)


BF032-velatus (TX)


BF022-velatus (TX)

BF123-velatus (TX)

BF181-cognatus (TX)


CMW1003-fowleri (AL)

BF185-cognatus (TX)

TG00019-americanus (MS)

BF109-fowleri (LA)RLG2637-velatus (TX)

BF116-fowleri (LA)


BF163-terrestris (SC)

BF049-velatus (TX)TG00025-fowleri (MS)

BF003-velatus (TX)

BF052-velatus (TX)



BF130-fowleri (LA)

TG00020-fowleri (MS)


BF122-velatus (TX)


BF141TERR-terrestris (FL)

BF145-fowleri (SC)

BF133-woodhousii (KA)

HERA10493-fowleri (DE)





BF015-fowleri (LA)




BF114-fowleri (LA)

BF018-fowleri (LA)

BF089-americanus (MI)

BF038-charlesmithi (TX)


HERA10214-americanus (CT)JFBM14347-americanus (MN)

BF182-cognatus (TX)

HERA10313-fowleri (CT)


BF131-fowleri (LA)

0.950.95

0.990.99

0.950.95

0.960.96

0.960.96

0.980.98

1.01.0

0.950.95

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

1.01.0

A. terrestrisA. terrestris

A. fowleri A. fowleri 1

A. fowleri A. fowleri 2

A. americanusA. americanusA. hemiophrysA. hemiophrys

A. woodhousiiA. woodhousii

* ***

**** ********* *******

***

*****

* ** ******

*

A. americanusA. americanusA. velatusA. velatusA. charlesmithiA. charlesmithi

substitutions

Fig. 4. Bayesian phylogenetic tree generated using mtDNA with posterior probabilities for nodal support. Yellow clades contain putative species with whining maleadvertisement calls and red clades contain putative species with whistling male advertisement calls. �Indicates individual designated as having whistling male advertisementcall 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 thatpossess similar male advertisement calls and occur sympatricallyand/or parapatrically (A. woodhousii, A. fowleri, A. velatus) resultedin a GST value of 0.04 and an NM value of 10.8 indicating very littlegenetic differentiation and very high levels of gene flow amongpopulations. Analysis of the species that possess similar maleadvertisement calls and occur allopatrically (A. americanus, A. char-lesmithi, and A. terrestris) resulted in a GST value of 0.11 and an NM

value of 4.0 indicating moderate genetic differentiation and highlevels of gene flow among populations.

4. Discussion

The use of AFLP and mtDNA markers has revealed previouslyundetected patterns of nuclear–mitochondrial discordance and a


link between levels of gene flow and male advertisement call in theA. americanus group. Our analyses provide evidence that (1) naturalhybridization has affected the radiation of the A. americanus group,(2) nuclear and mitochondrial genealogies are discordant for theseputative species, and (3) levels of interspecific gene flow betweenputative species increase with geographic proximity and/or simi-larity of male advertisement calls. Many studies have shown sub-stantial genetic differentiation among amphibian populationsacross a wide range of taxonomic and geographic scales (Barber,1999; Chippindale et al., 2000; Lampert et al., 2003; Highton,1995; Masta et al., 2003; Phillips, 1994; Rowe et al., 1998; Roweet al., 2000; and Schaffer et al., 2000). While species pairs in theA. americanus group that do not share similar male advertisementcalls appear to be valid species, species pairs that do share similarmale advertisement calls appear to persist as weakly differentiatedmetapopulations connected by at least some gene flow. This pat-tern is consistent with recent range expansions coupled with sec-ondary contact preceding the acquisition of complete reproductiveisolation (Coyne and Orr, 2004; Seehausen, 2004; Petit andExcoffier, 2009; Wiens et al., 2006). Prezygotic isolation has beenshown to evolve more rapidly than postzygotic isolation in areasof sympatry for both Drosophila fruit flies and Angiosperm plants(Coyne and Orr, 1989, 1997, 2004; Moyle et al., 2004), and diverg-ing populations coming into secondary contact could rapidlyreduce heterospecific gene flow by assortative mating. Whileinsects and plants are quite different biologically from toads, thefact that prezygotic isolation acts in much the same fashionbetween these disparate taxa argues that it may act in a similarfashion in toads. It is possible that this recent radiation of toadsis in the process of acquiring prezygotic isolating barriers in theform of male advertisement calls, and these data appear to supportthe notion that mitochondrial introgression occurs more fre-quently than nuclear introgression in species that have unequalabundance and prezygotic isolating barriers that create femalechoice-based mating systems (Chan and Levin, 2005). Studies haveshown that frog species pairs with low levels of genetic divergenceoften have not attained postzygotic isolation, therefore prezygoticisolating mechanisms likely play a role in reproductive isolationamong the recently radiated A. americanus group, the membersof which have not attained postzygotic reproductive isolation(Malone and Fontenot, 2008; Sasa et al., 1998).

4.1. Hybridization in the A. americanus group

Natural hybridization is thought to occur among members ofthe A. americanus group based on: (1) observations of putativelyintermediate morphology in some populations (Blair, 1941, 1942;Blair, 1956, 1959, 1961, 1963a, 1963b, 1964, 1966, 1972; Greenand Parent, 2003; Jones, 1973; Volpe, 1952, 1959), (2) allozymevariation in A. americanus, A. fowleri, and A. hemiophrys (Green1983 and 1984) and (3) evidence of mtDNA introgression amonglineages (Masta et al., 2002; Vogel and Johnson, 2008). Althoughthese lines of evidence suggest that natural hybridization may oc-cur, they can be uninformative or even misleading. Overlap in mor-phological variation among and between members of this speciesgroup is considerable, making identification of intermediate (i.e.‘‘hybrid’’) morphology difficult. Allozyme variation has only beenexamined at relatively small geographic scales and has not beenexamined in all members of the group (Green 1983, 1984). Finally,using only maternally inherited mtDNA markers can fail to identifycertain instances of hybridization. In crosses where a hybrid indi-vidual is morphologically similar to the maternal species, themtDNA will match the morphological species diagnosis andhybridization will remain undetected.

One method of identifying hybridization using AFLP markers in-volves examining loci that are differentially fixed between species

(e.g. fixed for presence in one species and absent in the other).While this approach has proven useful in identifying F1 hybridsin other taxa (Bonin et al., 2007; Meudt and Clarke, 2006), the largenumbers of polymorphic loci in the A. americanus group provide lit-tle information regarding identification of hybrid individuals, asthere are no fixed diagnostic loci between any of the putative spe-cies. However, some of our results can still provide informationregarding the role of hybridization in the radiation of the A. amer-icanus group. We interpret the high levels of gene flow and geneticsimilarity between species that occur in close geographic proxim-ity as evidence in support of genetic exchange between putativespecies due to past, and possibly present, hybridization events.Moreover, we find this a plausible scenario because members ofthe A. americanus group are known to produce viable and fertilehybrid offspring in interspecific crosses (Blair, 1972; Malone andFontenot, 2008). Seehausen (2004) suggested that hybridizationcould facilitate and enhance rapid species radiations by reshufflingalleles and introducing novel genetic variation at the beginning of aradiation. However, Wiens et al. (2006) found evidence that in-creased hybridization may simply be a consequence of multiple,recent speciation events in which species pairs do not have suffi-cient time to achieve proper reproductive isolation rather than acause of rapid radiations. Our data do not allow for a distinctionbetween the above scenarios, but do suggest that natural hybrid-ization likely featured prominently in the radiation of the A. amer-icanus group.

4.2. Nuclear–mitochondrial discordance

Similar to other taxa that have been affected by natural hybrid-ization, the topological differences between the mtDNA and AFLPtrees we generated for the A. americanus group are striking. Noclades are identical between the two trees and nodal support ishigh in the mtDNA tree and very low in the AFLP tree. The mtDNAphylogeny does not follow the same pattern with regard to maleadvertisement call type as the AFLP phylogeny. In fact, the mtDNAphylogeny suggests that species in the A. americanus group haveexperienced multiple episodes of convergence in advertisementcall type. Additionally, our estimates of genetic differentiation(GST) and gene flow (NM) are lower and higher respectively be-tween species that share similar male advertisement calls, a pat-tern that is not concordant with the relationships obtained fromthe mtDNA phylogeny.

The discordance between AFLP and mtDNA data may indicateeither incomplete lineage sorting or a recent division of one ormore species into several subpopulations that persist despite re-cent and possibly ongoing gene flow. Seehausen (2004) and Petitand Excoffier (2009) hypothesized that hybridization between clo-sely related species at the beginning of a radiation would result in apattern of discordance between cytoplasmic and nuclear markersdue to differential fixation of mitochondrial haplotypes after sec-ondary contact (also predicted from Chan and Levin, 2005). Incom-plete lineage sorting among recently diverged species could alsoresult in the retention of a large number of ancestral polymor-phisms among species, resulting in similar patterns of nuclear–mitochondrial discordance and complicating species delimitation(Petit and Excoffier, 2009; Stelkens and Seehausen, 2009). If nucle-ar–mitochondrial discordance is due to incomplete lineage sorting,then the mitochondrial data would be expected to show morereciprocally monophyletic lineages than the nuclear data owingto its’ smaller effective population size and mode of inheritance(Linnen and Farrell, 2007, 2008). Conversely, if introgression hasrecently occurred, the mitochondrial data would be expected toshow high levels of nonmonophyly (Linnen and Farrell, 2007,2008). Our data show very little reciprocal monophyly in the mito-chondrial data set, as several species appear to be paraphyletic


and/or polyphyletic (Fig. 4); providing support for the notion thathybridization has impacted the A. americanus group. Furthermore,we interpret the fact that species with similar male advertisementcalls in close geographic proximity share higher amounts of geneflow as additional evidence supporting the occurrence of hybrid-ization as male advertisement call is a prezygotic isolating barrierin anurans (Waldman, 2001). Linnen and Farrell (2008) found asimilar pattern in Neodiprion sawflies that used similar host plantspecies, a prezygotic isolating barrier in this group. However, di-rectly testing whether discordance is due to incomplete lineagesorting or hybridization requires comparing gene flow rates in bothmitochondrial and nuclear DNA. Software exists that allows for thistype of test, but is designed to work with species pairs that are as-sumed not to have shared genetic material with a third taxon (Heyand Nielsen, 2004). We cannot confidently make this assumptionwith any species pair in the A. americanus group as several of thesespecies occur with one or more congener in sympatry or closeparapatry. Additionally, these analyses require sequence data andour AFLP data are not appropriate for this type of analysis. Furtherdata collection should focus on obtaining nuclear sequence datafrom allopatric species pairs in order to rule out incomplete lineagesorting as the cause of nuclear–mitochondrial discordance.

Discordance between AFLP and mtDNA genealogies could alsobe attributed to dispersal effects as sex-biased dispersal is ex-pected to result in nuclear–mitochondrial discordance (Petit andExcoffier, 2009). Due to their male polygynous reproductive sys-tem, where female choice determines mating success rather thanmale-male resource competition, toads are expected to havemale-biased dispersal; however, Smith and Green (2006) foundno sex-biased dispersal among A. fowleri from Canada. Additionalstudy is required to determine whether or not sex-biased dispersalexists in other members of the A. americanus group before any con-clusions can be made regarding the role of sex-biased dispersal innuclear–mitochondrial discordance.


In the A. americanus group, putative species sharing similarmale advertisement calls have elevated levels of gene flow and lit-tle genetic differentiation, suggesting that similar male advertise-ment calls may be less effective in preventing interspecific geneflow. At small geographic scales, female A. americanus and A. wood-housii appear to select mates on the basis of dominant frequency orhigh effort calls (i.e. longer calls or faster pulse rates), potentialindicators of fitness (Sullivan, 1983a,b, 1992; Gatz, 1981; Licht,1976). Additionally, Waldman (2001) found that females of A.americanus can discriminate between males that are closely or dis-tantly related to themselves on the basis of their advertisementcalls, thus avoiding inbreeding and indicating the fine scale varia-tion that female frogs are capable of detecting. Hybrids between A.americanus and A. fowleri possess a male advertisement call inter-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 barri-ers to avoid hybridization (Green and Parent, 2003). Boul et al.(2007) found that sexual selection greatly influenced speciationin the frog Physalaeumus petersi with females choosing to matewith males that possess advertisement calls similar to thosefrom the female’s local population. Advertisement call variationamong populations resulted in differential female preferences,furthering divergence in male advertisement call and increasingbehavioral isolation as a response to female choice. Boul et al.(2007) also found that gene flow was higher between speciesthat shared similar male advertisement calls as seen in theA. americanus group.

5. Conclusions

The members of the A. americanus group are characterized byvery little genetic divergence and no intrinsic postzygotic repro-ductive isolation (Malone and Fontenot, 2008; Sasa et al., 1998).Hybrid offspring of both sexes are viable and fertile through multi-ple generations, suggesting that admixture between species couldbe frequent (W.F. Blair, 1972; Green and Parent, 2003; Jones, 1973;Malone and Fontenot, 2008). Our analyses support the idea thatnatural hybridization has led to confusing patterns of genetic var-iation in the A. americanus group. Our results provide further proofthat taxonomic revision of the A. americanus group may be neededas they demonstrate that classifications of species in this groupbased solely on morphological or mitochondrial data have failedto fully reflect their evolutionary history. Given the weight of theevidence, at least some of the putative species in this recent radi-ation may be more appropriately characterized as metapopulationsexchanging varying levels of gene flow depending on geographicproximity and the strength of prezygotic isolating barriers. Wesuggest that the discordance between the evolutionary relation-ships supported by AFLP and mtDNA data could be the signatureof secondary contact leading to multiple hybridization eventsand differential fixation of regionally dominant mitochondrial hap-lotypes as populations colonized newly available postglacial habi-tats. To better characterize the evolutionary history of the A.americanus group, we suggest conducting fine scale phylogeo-graphic analyses of each putative species using both nuclear andmitochondrial markers and quantitative analysis of male adver-tisement calls to further assess the relationship between gene flowand male advertisement call. If future research corroborates the re-sults of our study, a thorough taxonomic revision of the A. americ-anus group will be necessary.

Acknowledgments

We acknowledge the following institutions and individuals forsharing tissue samples, laboratory and museum resources, andspecimens used in this study: J.A. Campbell, E.N. Smith, and C.J.Franklin at the University of Texas at Arlington Amphibian andReptile Diversity Research Center, G. Watkins-Colwell at the Pea-body Museum of Natural History at Yale University, K. Kozak, B.Lowe, and T. Gamble at the Bell Museum of Natural History atthe University of Minnesota, L. Rissler and T. York at the Universityof Alabama Herpetological Collection, and S. Peterson, C. Watson, J.Meik, C. Roelke, J. Streicher, C. Cox, J. Malone, R. Calisi, A. Lawing, J.Morse, S. Braman, M. Gifford, and R.L. Gutberlet. We also acknowl-edge A. Carrillo for laboratory assistance. J. Demuth, J. Meik, J. Strei-cher, and S. Schaack provided valuable help commenting on earlyversions of this manuscript. Funding for this project was providedby the University of Texas at Arlington Phi Sigma Biological HonorSociety Research Grant to BEF.

Appendix A

Voucher specimen ID numbers (HERA = Peabody Museum ofNatural History at Yale University; JFBM = Bell Museum of NaturalHistory at the University of Minnesota; UAHC = University of Ala-bama Herpetological Collection; BF = B. Fontenot field series;CMW = C. Watson field series; TG = T. Gamble field series; JWS = J.Streicher field series; CJF = C. Franklin field series; RMC = R. Calisifield series; SCB = S. Braman field series; MG = M. Gifford field ser-ies; and RLG = R. Gutberlet field series). Sex, Genbank accessionnumbers, and locality data for individuals in this study.

Species Sex ID# Genbank Latitude Longitude State

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


Appendix A (continued)


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

(continued on next page)


Appendix A (continued)


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



Appendix B. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2010.12.018.

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