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Testing assertions of widespread introgressive hybridization in a clade of neotropical toads with low mate selectivity (Rhinella granulosa species group)

Heredity volume 130, pages 14–21 (2023)Cite this article

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Abstract

Discordance between different genomic regions, often identified through multilocus sequencing of selected markers, presents particular difficulties in identifying historical processes which drive species diversity and boundaries. Mechanisms causing discordance, such as incomplete lineage sorting or introgression due to interspecific hybridization, are better identified based on population-level genomic datasets. In the toads of the Rhinella granulosa species group, patterns of mito-nuclear discordance and potential hybridization have been reported by several studies. However, these patterns were proposed based on few loci, such that alternative mechanisms behind gene-tree heterogeneity cannot be ruled out. Using genome-wide ddRADseq loci from a subset of species within this clade, we found only partial concordance between currently recognized species-level taxon boundaries and patterns of genetic structure. While most taxa within the R. granulosa group correspond to clades, genetic clustering analyses sometimes grouped distinct taxonomic units into a single cluster. Moreover, levels of admixture between inferred clusters were limited and restricted to a single taxon pair which is best explained by incomplete lineage sorting as opposed to introgressive hybridization, according to D-statistics results. These findings contradict previous assertions of widespread cryptic diversity and gene flow within the R. granulosa clade. Lastly, our analyses suggest that diversification events within the Rhinella granulosa group mostly dated back to the early Pliocene, being generally younger than species divergences in other closely related clades that present high levels of cross-species gene flow. This finding uniquely contradicts common assertions that this young clade of toads exhibits interspecific hybridization.

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Fig. 1: A comparison of the Rhinella granulosa clade 16S and ddRAD phylogenies (IQTree/MrBayes).
Fig. 2: Population structure of the Rhinella granulosa clade.
Fig. 3: Locality map of focal species depicting average ADMIXTURE cluster assignments per locality (K = 4).
Fig. 4: The fb statistic (summary of f4 admixture ratios).

Data availability

Sequence data are available on GenBank (ON692445- ON692509) and demultiplexed sequence files are available on the Sequence Read Archive (PRJNA858546). Associated scripts and input files have been made available on dryad: https://doi.org/10.5061/dryad.p2ngf1vtc.

References

  • Abreu LM, Santos JEM, Corrêa AT, Ribeiro MV (2021) Interspecific amplexus between two sympatric species, Rhinella granulosa (Spix, 1824) and Rhinella crucifer (Wied-Neuwied, 1821) (Anura: Bufonidae) in Ilhéus, Bahia, Brazil. HNO 14:869–871

    Google Scholar 

  • Alexander DH, Novembre J, Lange K (2009) Fast model-based estimation of ancestry in unrelated individuals. Genome Res 19:1655–1664

    Article  CAS  Google Scholar 

  • Alonso R, Crawford AJ, Bermingham E (2012) Molecular phylogeny of an endemic radiation of Cuban toads (Bufonidae: Peltophryne) based on mitochondrial and nuclear genes: origin and diversification of Cuban toads. J Biogeogr 39:434–451

    Article  Google Scholar 

  • Arntzen JW, de Vries W, Canestrelli D, Martínez-Solano I (2017) Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads. Mol Ecol 26:5663–5675

    Article  CAS  Google Scholar 

  • Azevedo MFC, Foresti F, Ramos PRR, Jim J (2003) Comparative cytogenetic studies of Bufo ictericus, B. paracnemis (Amphibia, Anura) and an intermediate form in sympatry. Genet Mol Biol 26:289–294

    Article  Google Scholar 

  • Blanco-Pastor JL, Vargas P, Pfeil BE (2012) Coalescent simulations reveal hybridization and incomplete lineage sorting in Mediterranean Linaria. PLoS ONE 7:e39089

    Article  CAS  Google Scholar 

  • Bonnet T, Leblois R, Rousset F, Crochet P-A (2017) A reassessment of explanations for discordant introgressions of mitochondrial and nuclear genomes. Evolution 71:2140–2158

    Article  Google Scholar 

  • Caye K, Jay F, Michel O, Francois O (2018) Fast inference of individual admixture coefficients using geographic data. Ann Appl Stat 12:586–608

    Article  Google Scholar 

  • Caye K, Deist T, Martins H, Michel O, Francois O (2016) TESS3: fast inference of spatial population structure and genome scans for selection. Mol Ecol Resour 16:540–548

    Article  CAS  Google Scholar 

  • Chifman J, Kubatko L (2014) Quartet inference from SNP data under the coalescent model. Bioinformatics 30:3317–3324

    Article  CAS  Google Scholar 

  • Chiocchio A, Arntzen JW, Martínez-Solano I et al. (2021) Reconstructing hotspots of genetic diversity from glacial refugia and subsequent dispersal in Italian common toads (Bufo bufo). Sci Rep. 11:260

    Article  CAS  Google Scholar 

  • Cvijović I, Good BH, Desai MM (2018) The effect of strong purifying selection on genetic diversity. Genetics 209:1235–1278

    Article  Google Scholar 

  • Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA et al. (2011) The variant call format and VCFtools. Bioinformatics 27:2156–2158

    Article  CAS  Google Scholar 

  • Degnan JH, Rosenberg NA (2009) Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends Ecol Evol 24:332–340

    Article  Google Scholar 

  • Doronina L, Churakov G, Shi J, Brosius J, Baertsch R, Clawson H et al. (2015) Exploring massive incomplete lineage sorting in Arctoids (Laurasiatheria, Carnivora). Mol Biol Evol 32:3194–3204

    CAS  Google Scholar 

  • Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7:214

    Article  Google Scholar 

  • Dufresnes C, Nicieza AG, Litvinchuk SN, Rodrigues N, Jeffries DL, Vences M et al. (2020) Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs. Mol Ecol 29:986–1000

    Article  Google Scholar 

  • Durand EY, Patterson N, Reich D, Slatkin M (2011) Testing for ancient admixture between closely related populations. Mol Biol Evol 28:2239–2252

    Article  CAS  Google Scholar 

  • Eaton DAR, Overcast I (2020) ipyrad: Interactive assembly and analysis of RADseq datasets. Bioinformatics 36:2592–2594

    Article  CAS  Google Scholar 

  • Edwards RJ, Tuipulotu DE, Amos TG, O’Meally D, Richardson MF, Russell TL et al. (2018) Draft genome assembly of the invasive cane toad, Rhinella marina. GigaScience 7:1–13

    Article  Google Scholar 

  • Firneno Jr TJ, Townsend JH (2019) Evaluation of species boundaries in sympatric and parapatric populations of Mesoamerican toads. Zool Scr 48:454–465

    Article  Google Scholar 

  • Firneno Jr TJ, O’Neill JR, Portik DM, Emery AH, Townsend JH, Fujita MK (2020) Finding complexity in complexes: assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads. Mol Ecol 29:3543–3559

    Article  CAS  Google Scholar 

  • Fujita MK, Leaché AD, Burbrink FT, McGuire JA, Moritz C (2012) Coalescentbased species delimitation in an integrative taxonomy. Trends Ecol Evol 27:480–488

    Article  Google Scholar 

  • Gama V, dos Santos Protázio A, dos Santos, Protázio A (2020) Interspecific amplexus between male Rhinella granulosa (Spix, 1824) and a metamorph of Ceratophrys joazeirensis Mercadal, 1986 (Amphibia: Anura) in a temporary pond in the Caatinga, Bahia State, Northeastern Brazil. Herpetol Notes 13:749–751

    Google Scholar 

  • Gronau I, Hubisz MJ, Gulko B, Danko CG, Siepel A (2011) Bayesian inference of ancient human demography from individual genome sequences. Nat Genet 43:1031–1034

    Article  CAS  Google Scholar 

  • Guerra C, Baldo D, Rosset S, Borteiro C, Kolenc F (2011) Advertisement and release calls in Neotropical toads of the Rhinella granulosa group and evidence of natural hybridization between R. bergi and R. major (Anura: Bufonidae). Zootaxa 3092:26–42

    Article  Google Scholar 

  • Hill GE, Havird JC, Sloan DB, Burton RS, Greening C, Dowling DK (2019) Assessing the fitness consequences of mitonuclear interactions in natural populations. Biol Rev Camb Philos Soc 94:1089–1104

    Article  Google Scholar 

  • Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35:518–522

    Article  CAS  Google Scholar 

  • Ivanov V, Lee KM, Mutanen M (2018) Mitonuclear discordance in wolf spiders: genomic evidence for species integrity and introgression. Mol Ecol 27:1681–1695

    Article  CAS  Google Scholar 

  • Jakob SS, Blattner FR (2006) A chloroplast genealogy of Hordeum (Poaceae): long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference. Mol Biol Evol 23:1602–1612

    Article  CAS  Google Scholar 

  • Jombart T, Ahmed I (2011) adegenet 1.3-1: new tools for the analysis of genomewide SNP data. Bioinformatics 27:3070–3071

    Article  CAS  Google Scholar 

  • Jombart T, Devillard S, Balloux F (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet 11:94

    Article  Google Scholar 

  • Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587–589

    Article  CAS  Google Scholar 

  • Leaché AD, Portik DM, Rivera D, Rödel MO, Gvoždík V, Penner J et al. (2019) Exploring rainforest diversification using demographic model testing in the African foam-nest treefrog (Chiromantis rufescens). J Biogeogr 46:2706–2721

    Article  Google Scholar 

  • Letunic I, Bork P (2019) Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res 47:W256–W259

    Article  CAS  Google Scholar 

  • Lever C (2001) The cane toad: the history and ecology of a successful colonist. Westbury Academic and Scientific Publishing, Otley, Yorkshire, UK

  • Liu L, Yu L, Kubatko L, Pearl DK, Edwards SV (2009) Coalescent methods for estimating phylogenetic trees. Mol Phylogenet Evol 53:320–328

    Article  CAS  Google Scholar 

  • Malinsky M, Matschiner M, Svardal H (2020) Dsuite—Fast D‐statistics and related admixture evidence from VCF files. Mol Ecol Resour 21:584–595

    Article  Google Scholar 

  • Malinsky M, Svardal H, Tyers AM, Miska EA, Genner MJ, Turner GF et al. (2018) Whole-genome sequences of Malawi cichlids reveal multiple radiations interconnected by gene flow. Nat Ecol Evol 2:1940–1955

    Article  Google Scholar 

  • Martin SH, Dasmahapatra KK, Nadeau NJ, Salazar C, Walters JR, Simpson F et al. (2013) Genome-wide evidence for speciation with gene flow in Heliconius butterflies. Genome Res 23:1817–1828

    Article  CAS  Google Scholar 

  • McGuire JA, Linkem CW, Koo MS, Hutchison DW, Lappin AK, Orange DI et al. (2007) Mitochondrial introgression and incomplete lineage sorting through space and time: phylogenetics of crotaphytid lizards. Evolution 61:2879–2897

    Article  CAS  Google Scholar 

  • McKay BD, Zink RM (2010) The causes of mitochondrial DNA gene tree paraphyly in birds. Mol Phylogenet Evol 54:647–650

    Article  Google Scholar 

  • de Medeiros BAS, Farrell BD (2018) Whole-genome amplification in doubledigest RADseq results in adequate libraries but fewer sequenced loci. PeerJ 6:e5089

    Article  Google Scholar 

  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE), p 1–8

  • Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A et al. (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37:1530–1534

    Article  CAS  Google Scholar 

  • Morales HE, Pavlova A, Joseph L, Sunnucks P (2015) Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance. Mol Ecol 24:2820–2837

    Article  CAS  Google Scholar 

  • Narvaes P, Rodrigues MT (2009) Taxonomic revision of Rhinella granulosa species group (Amphibia, Anura, Bufonidae), with a description of a new species. Arquivos De Zoologia 40:1–73

    Article  Google Scholar 

  • Patterson N, Moorjani P, Luo Y, Mallick S, Rohland N, Zhan Y et al. (2012) Ancient admixture in human history. Genetics 192:1065–1093

    Article  Google Scholar 

  • Pereyra MO, Baldo D, Blotto BL, Iglesias PP, Thomé MTC, Haddad CFB et al. (2016) Phylogenetic relationships of toads of the Rhinella granulosa group (Anura: Bufonidae): a molecular perspective with comments on hybridization and introgression. Cladistics 32:36–53

    Article  Google Scholar 

  • Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS ONE 7:e37135

    Article  CAS  Google Scholar 

  • Pollard DA, Iyer VN, Moses AM, Eisen MB (2006) Widespread discordance of gene trees with species tree in Drosophila: evidence for incomplete lineage sorting. PLoS Genet 2:e173

    Article  Google Scholar 

  • Portik DM, Leaché AD, Rivera D, Barej MF, Burger M, Hirschfeld M et al. (2017) Evaluating mechanisms of diversification in a Guineo-Congolian forest frog using demographic model selection. Mol Ecol 26:5245–5263

    Article  Google Scholar 

  • Pramuk JB, Robertson T, Sites JW, Noonan BP (2007) Around the world in 10 million years: biogeography of the nearly cosmopolitan true toads (Anura: Bufonidae). Glob Ecol Biogeogr 17:72–83

    Google Scholar 

  • Prates I, Rivera D, Rodrigues MT, Carnaval AC (2016) A mid-Pleistocene rainforest corridor enabled synchronous invasions of the Atlantic Forest by Amazonian anole lizards. Mol Ecol 25:5174–5186

    Article  Google Scholar 

  • Prates I, Penna A, Rodrigues MT, Carnaval AC (2018) Local adaptation in mainland anole lizards: Integrating population history and genome-environment associations. Ecol Evol 8:11932–11944

    Article  Google Scholar 

  • Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 67:901–904

    Article  CAS  Google Scholar 

  • Rivera D, Prates I, Rodrigues MT, Carnaval AC (2020) Effects of climate and geography on spatial patterns of genetic structure in tropical skinks. Mol Phylogenet Evol 143:106661

    Article  Google Scholar 

  • Rivera D, Prates I, Firneno Jr. TJ, Rodrigues MT, Caldwell JP, Fujita MK (2022) Phylogenomics, introgression, and demographic history of South American true toads (Rhinella). Mol Ecol 31:978–992

    Article  Google Scholar 

  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S et al. (2012) MRBAYES 3.2: Efficient Bayesian phylogenetic inference and model selection across a large model space. Syst Biol 61:539–542

    Article  Google Scholar 

  • Sambrook J, Russell DW (2006) Purification of nucleic acids by extraction with phenol:chloroform. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot4455.

  • Sequeira F, Sodré D, Ferrand N, Bernardi JAR, Sampaio I, Schneider H et al. (2011) Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers. BMC Evol Biol 11:264

    Article  CAS  Google Scholar 

  • Simon MN, Machado FA, Marroig G (2016) High evolutionary constraints limited adaptive responses to past climate changes in toad skulls. Proc Biol Sci 283:20161783

    Google Scholar 

  • Singhal S, Moritz C (2013) Reproductive isolation between phylogeographic lineages scales with divergence. Proc Biol Sci 280:20132246

    Google Scholar 

  • Singhal S, Bi K (2017) History cleans up messes: the impact of time in driving divergence and introgression in a tropical suture zone. Evolution 71:1888–1899

    Article  Google Scholar 

  • Swofford DL (2002) PAUP: phylogenetic analysis using parsimony (and other methods), Version 4.0 Beta 10. Sinauer Associates, Sunderland

  • Thielsch A, Knell A, Mohammadyari A, Petrusek A, Schwenk K (2017) Divergent clades or cryptic species? Mito-nuclear discordance in a Daphnia species complex. BMC Evol Biol 17:227

    Article  Google Scholar 

  • Toews DPL, Brelsford A (2012) The biogeography of mitochondrial and nuclear discordance in animals. Mol Ecol 21:3907–3930

    Article  CAS  Google Scholar 

  • Wang K, Lenstra JA, Liu L, Hu Q, Ma T, Qiu Q et al. (2018) Incomplete lineage sorting rather than hybridization explains the inconsistent phylogeny of the wisent. Commun Biol 1:169

    Article  Google Scholar 

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Acknowledgements

We thank all the Brazilian collectors who made this study possible and particularly students in MTR’s laboratory for field collection and support. Brazil’s Instituto Chico Mendes de Conservação da Biodiversidade issued collecting permits (SISBIO 36753-1, 36753-4, and 27290-3). This work was co-funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; BIOTA 2013/50297-0), the National Science Foundation (DEB 1343578), and the National Aeronautics and Space Administration through the Dimensions of Biodiversity Program. JPC was funded by the National Science Foundation (DEB 9200779 and DEB 9505518). MTR acknowledges additional funding from FAPESP grants 2003/10335-8, 2011/50146-6, 2011/50206‐9, 2012/15754-8, and 2017/08357-6. DR was funded by NSF GRFP. IP was funded by NSF grant DEB 1754398.

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Authors and Affiliations

  1. Department of Applied Ecology, North Carolina State University, Raleigh, NC, USA

    Danielle Rivera

  2. Amphibian and Reptile Diversity Research Center, University of Texas at Arlington, Arlington, TX, USA

    Danielle Rivera & Matthew K. Fujita

  3. Department of Ecology and Evolutionary Biology and Museum of Zoology, University of Michigan, Ann Arbor, MI, USA

    Ivan Prates

  4. Sam Noble Museum and Department of Biology, University of Oklahoma, Norman, OK, USA

    Janalee P. Caldwell

  5. Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil

    Miguel Trefaut Rodrigues

  6. Department of Biology and Amphibian and Reptile Diversity Research Center, University of Texas at Arlington, Arlington, TX, USA

    Matthew K. Fujita

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  1. Danielle Rivera
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  2. Ivan Prates
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  3. Janalee P. Caldwell
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  4. Miguel Trefaut Rodrigues
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Contributions

Contributions DR, IP, and MKF discussed and designed the study. IP provided expertise for many analyses. DR compiled the dataset, ran the analyses, and wrote the first draft and revised versions of the manuscript. IP provided significant input to the first draft and revised versions of the manuscript. JPC, MTR, and MKF contributed to the manuscript.

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Correspondence to Danielle Rivera.

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Rivera, D., Prates, I., Caldwell, J.P. et al. Testing assertions of widespread introgressive hybridization in a clade of neotropical toads with low mate selectivity (Rhinella granulosa species group). Heredity 130, 14–21 (2023). https://doi.org/10.1038/s41437-022-00571-9

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