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Thom et al. BMC Evolutionary Biology 2012, 12:242http://www.biomedcentral.com/1471-2148/12/242
RESEARCH ARTICLE Open Access
Delimiting genetic units in Neotropical toadsunder incomplete lineage sorting andhybridizationMaria Tereza C Thom1*, Kelly R Zamudio2, Clio FB Haddad1 and Joo Alexandrino1,3
Background: Delimiting genetic units is useful to enhance taxonomic discovery and is often the first step towardunderstanding evolutionary mechanisms generating diversification. The six species within the Rhinella crucifer groupof toads were defined under morphological criteria alone. Previous data suggest limited correspondence of thesespecies to mitochondrial lineages, and morphological intergradation at transitions between forms suggestshybridization. Here we extensively sampled populations throughout the geographic distribution of the groupand analyzed mitochondrial and nuclear sequence data to delimit genetic units using treebased and allelefrequencybased approaches.
Results: These approaches yielded complementary results, with allele frequency-based methods performingunexpectedly well given the limited number of loci examined. Both mitochondrial and nuclear markers supported agenetic structure of five units within the group, with three of the inferred units distributed within its main range,while two other units occur in separate isolates. The inferred units are mostly discordant with currently describedforms: unequivocal association exists for only two of the six species in the group. Genetic evidence for hybridizationexists for two pairs of units, with clear cytonuclear allele mixing observed in one case.
Conclusions: Our results confirmed that current taxonomy does not represent evolutionary units in the Rhinellacrucifer group. Correspondence between genetically distinguishable units and the currently recognized species isonly possible for Rhinella henseli and R. inopina. The recognition of other species relies on the reassessment of thegeographic range of R. crucifer, the examination of the type series of R. ornata for hybrids, and on the use ofadditional markers to verify the genetic distinctiveness of R. abei. We state that R. pombali should not remain a validspecies since its description appears to be based on hybrids, and that the name R. pombali should be considered asynonym of both R. crucifer and R. ornata. The fifth inferred but undescribed genetic unit may represent a newspecies. Our results underscore the potential of the R. crucifer species group to contribute to a better understandingof diversification processes and hybridization patterns in the Neotropics, and provide the basis for futureevolutionary and taxonomic studies.
Keywords: Rhinella crucifer group, Species complex, Bufonidae, Taxonomy
* Correspondence: firstname.lastname@example.orgDepartamento de Zoologia, Instituto de Biocincias, UNESP - Univ EstadualPaulista, Campus Rio Claro, Caixa Postal 19913506-900, Rio Claro, SP, BrazilFull list of author information is available at the end of the article
2012 Thom et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.
Thom et al. BMC Evolutionary Biology 2012, 12:242 Page 2 of 13http://www.biomedcentral.com/1471-2148/12/242
BackgroundMolecular data in taxonomic studies has profoundlyimpacted the field by bringing fresh perspectives on dis-cussions about species concepts and delimitation [1,2]harnessing the power of barcoding for biodiversity dis-covery [3,4], and leading to a more representative ta-xonomy [5-7]. Early efforts of genetic delimitation ofEvolutionary Significant Units  for conservation pur-poses  met with controversy, primarily because of thecriteria applied . At the core of these discussions isthe recognition of species as segments of populationlineages against a view of species as operational units ofa taxonomic rank . Regardless of the adopted view,most biodiversity researchers agree that delimitation ofgenetic units is i) a useful proxy for enhancing the rateof taxonomic discovery and ii) the first step towardsunderstanding evolutionary mechanisms contributing todiversification of closely related organisms, two mea-sures that are specially relevant for conservation plan-ning of taxa in threatened environments [12,13].Using genetic criteria for unit delimitation has pro-
mpted a recent change of paradigm in systematics .Newly available computational tools based on multilocusmethods are now applied to 'species' delimitation ,inference of the relationships among previously definedspecies [16-18], or both [19,20]. However, the delimita-tion of genetic units with treebased methods is notstraightforward because it often requires previous as-signment of individuals to hypothetical species. Inaddition, the absence of horizontal gene flow is an as-sumption for most of these methods . Alternatively,analyses of allele frequencies at multilocus markerscan jointly explore genetic structure and infer levelsof migration without a priori information , andhave been used to delimit closely related species thatpotentially hybridize [23-27]. These methods have dis-advantages over species-tree methods, such as limitedphylogenetic signal, potential sensitivity to very recentgenetic isolation , and a tradeoff between the costsof populationlevel sampling and the number of markersrequired to detect genetic structure, especially when theefficiency of the markers  is not known a priori.The Rhinella crucifer species group is a widespread
group of toads that occurs throughout the Brazilian At-lantic Forest, a highly endangered biome  distributedalong 30 lat degrees along the eastern coastline of SouthAmerica. This widespread distribution includes highmorphological variation, both within and among popu-lations , which has led to a confused taxonomichistory for the group . In a taxonomic review,Baldissera et al.  recognized five species based onmorphological and morphometric variation: Rhinellacrucifer occurs from the State of Cear to northernState of Esprito Santo; R. pombali is restricted to the
State of Minas Gerais with isolated records in the stateof Rio de Janeiro; R. ornata occurs from southern Stateof Esprito Santo to northern state of Paran; R. abei isdistributed from the State of Paran to the State ofSanta Catarina; and R. henseli occurs from southernState of Santa Catarina to the State of Rio Grande doSul, with isolated records in the state of Paran [30-33].More recently, VazSilva et al.  described the sixthspecies in the group, Rhinella inopina, with an allopatricdistribution in forest enclaves within the Cerrado biomein the limits of the States of Gois, Tocantins and Bahia (Figure 1). A broad scale phylogeographical surveyrevealed that mitochondrial clades do not fully corres-pond to the current taxonomy . Rhinella henseli cor-responds to a highly divergent mitochondrial clade atthe southern limit of the species range; a northern cladeincludes R. crucifer and part of R. pombali, and a centralclade includes remaining populations of R. pombali,along with R. ornata and R. abei. Rhinella pombali isfound along an eastwest axis where the northern andcentral clades meet, ranging from the coast throughoutthe inland plateau. Its morphology is intermediate be-tween R. crucifer and R. ornata , which taken to-gether with the mitochondrial DNA is suggestive of ahybrid origin. Hybridization between the morphospeciesR. abei and R. henseli is also possible as individualsoccur in syntopy, and are thus likely to interbreed.Nuclear sequence data from this study showed onlyevidence of incomplete lineage sorting .The lack of fine-scale sampling in Thom et al. 
prevented both detailed mapping of mtDNA lineagesand description of geographic patterns of nuclear allelefrequencies, resulting in an incomplete description ofthe spatial genetic structure in the Rhinella crucifergroup. Here we greatly increase both the geographic andpopulation sampling and use sequence data from a setof three mitochondrial and three nuclear genes to de-limit genetic units in the Rhinella crucifer group. Giventhe widespread distribution of the group, and evidenceof recent divergences and hybridization, we employ acombination of tree and allele frequencybased me-thods. We discuss the results in the context of availableinformation on the taxonomy and history of the group,and provide a perspective on the potential for futureevolutionary studies.
ResultsMitochondrial DNA genetic lineagesWe obtained 386, 401, and 398 sequences for controlregion, ND1, and ND2 fragments, respectively. Afterconcatenation we found 305 unique haplotypes amongthe 404 sampled individuals. The topology of the mito-chondrial tree (Figure 2) recovers the monophyly ofthe Rhinella crucifer group with high support. Two
Figure 1 Species distribution and sampling. Distribution of morphological species in the Rhinella crucifer group, and localities sampled for thisstudy: Rhinella crucifer in yellow, R. in