Analyses of the genetic structure of mitochondrial and nuclear markers in the Rhinella crucifer species group revealed five distinct genetic units, three corresponding to the core geographic area of the group (N, C, and S), and two units with more isolated distributions (G and P). Overlap occurs between units N and C and between C and S, with evidence of some degree of admixture between those pairs. The concordance of genetic units with currently recognized species boundaries is very limited; the morphospecies with clear correspondence to the recovered genetic units are R. henseli, which is fully represented by unit S, and R. inopina, represented by unit P. Although we did find some correspondence between R. crucifer and unit N, and R. ornata and unit C, their distributions are not completely coincident. The distribution of R. pombali within the putative hybrid zone between N and C explains its intermediate morphology and corroborates that it does not have a distinct evolutionary history from neighboring units. Rhinella abei corresponds to a mtDNA sub-clade, but its correspondence to a genetic group in combined marker analyses is not well-supported and might depend on the inclusion of more markers. Finally, the morphological distinctiveness of populations in genetic units G remains to be tested.
Delimitation of genetic units
We found deep mitochondrial structure within the R. crucifer group; haplotypes cluster in subclades that belong within the larger well-supported major clades N, C, S, G and P. Geographic distributions of sub–clades overlap greatly while major clades have more exclusive areas of occurrence. The mtDNA tree presented here (Figure
2) differs from the previously published topology
. All main clades (N, C and S) identified earlier were recovered by our current analysis, but the addition of previously unsampled geographically isolated populations revealed two new clades with apparently narrow distributions (clades G and P). Additional sampling also extended the known geographic distribution of clade S considerably to the north, the southern limit of clade N to the south, and the distribution of the C clade towards the southwest (Figure
3). Our analyses also improved the support for relationships within clade C, especially for northern sub–clades that showed more restricted distributions than sub–clades in the state of São Paulo and western regions of the states of Paraná and Santa Catarina, which overlapped extensively. However, this pattern might reflect unbalanced sampling. In eastern regions of the states of Paraná and Santa Catarina individuals clustered within sub–clade c1, a pattern that was recovered before
. The overlapping geographic distributions of all N sub–clades confirmed that their allopatric distributions inferred in Thomé et al.
 was an artifact of less-extensive sampling.
The nuclear gene genealogies (Additional file
1) confirmed incomplete lineage sorting previously described for the Rhinella crucifer group
. The nuclear genetic structure was assessed from Bayesian individual assignment using the method of Pritchard et al.
 with three nuclear loci and a model without prior information on the origin of individuals. This model avoids circularity in cross–validating results with other evidence (i.e. geography and mtDNA structure) but the limited number of loci can potentially reduce the power of reliably inferring the optimal number of clusters. We therefore delimited an interval of possible values of K (K=2–6) by comparing scores produced by different criteria [ΔK, Ln Pr(X/K)]. Assignment results consistently recovered five genetic breaks and six demes could be delimited based on the nuclear data alone (Figure
5). Individuals with low average assignment coefficients were primarily restricted to the geographic boundaries of demes (Figures
5), which argues for admixture at contact zones between divergent lineages. An important exception are the two individuals from L104 that were assigned, albeit not strongly, to deme G. This is certainly not due to admixture resulting from secondary contact between diverged lineages because demes G and S do not share a contact zone. This result may be rather explained by shared ancestral polymorphism, especially at the rhodopsin locus (the case of widespread alleles 1 and 2; see Additional file
Clusters defined by assignment analyses of nuclear markers correspond well to the mtDNA topology; of the six inferred demes, G, N, P, and S correspond largely to main mitochondrial clades G, N, P, and S, and demes C1 and C2 together account for the mitochondrial clade C (Figure
3). Substructure in the mitochondrial clade C does not correspond to the genetic break between demes C1 and C2. However, at values of K ≥7 a cluster geographically coincident with the distribution of mtDNA sub–clade c1 was revealed (data not shown), which suggests that additional data (more nuclear markers) will likely uncover a nuclear genetic structure consistent with that of mtDNA for the eastern regions of the states of Paraná and Santa Catarina.
The limited number of loci used in the STRUCTURE analysis limits the power of our results; clearly more conclusive inferences will require additional markers. Nonetheless, we were surprised at the performance of this method with only three loci, suggesting that they contain the signature of geographic history of the major lineages. Other studies with similar number of markers coded from sequence data have found similar results: assignment analyses based on five nuclear loci successfully revealed cryptic species in the West African forest gecko Hemidactylus fasciatus. Success in detecting relevant genetic structure with a reduced number of loci in assignment analysis (up to two orders of magnitude less that in regular analysis) may depend on marker choice, sufficient sampling of individuals, as well as the specific history of the focal species; optimal clustering may be achievable as long as the choice of markers is efficient in targeting the question to be addressed
. In the present case, the use of more conserved markers may have prevented revealing further genetic structure (e.g., at the population level) that would be predictable for an amphibian
. Finer scale genetic structure, albeit interesting in itself, could have resulted in oversplitting the species group into more genetic units with no association with lineage/species diversification. In this context, a comparative study of the sensitivity of genetic assignment analyses to marker choice should be of great utility in studies concerning the boundaries of closely related species.
The combination of mitochondrial and nuclear data in FCA corroborated the results obtained for these two marker classes when analyzed individually (Figure
6). Therefore, the existence of five genetically-defined units within the Rhinella crucifer group is strongly supported by the overall genetic data. The units C, N and S are parapatrically distributed spanning the main range of the group. Units G and P have very restricted distributions at the northern and western limits of the range, and appear to be geographically isolated. These allopatric distributions likely reflect the patchy nature of the habitat in the transition zones between the Atlantic Forest and neighboring biomes (Caatinga to the north and Cerrado to the west), but broader-scale sampling might also explain this isolation pattern to some degree.
Two potential hybrid zones
Geographic overlap of units occurs along the borders of units N and C and more extensively between units C and S (Figure
3). Strong evidence of admixture was found for the N/C contact zone, with four individuals showing hybrid cyto–nuclear allele combinations. These individuals were distributed in localities along an east–west axis near the limits of the states of Espírito Santo, Rio de Janeiro, and Minas Gerais. Although these four samples are spatially restricted relative to the ranges of units C and N, the hybrid zone between those units may be wider across the transition zone. This is supported at the level of both mtDNA and nuclear data given the distribution of admixed individuals in areas of clade overlap (Figure
3) and by the lack of clear delimitation in the FCA groups in combined analysis (Figures
6). We therefore predict that the hybrid zone between units N and C extends across most of the states of Espírito Santo and southern Rio de Janeiro, in addition to the central region of the state of Minas Gerais.
Evidence for a second hybrid zone between units C and S is less conclusive. The co–occurrence of individuals belonging to mitochondrial clades C and S was detected at two localities (L100 and 101, Figure
1), with individuals of both clades reproducing synchronously at the same pond at locality L100 (L. M. Giassom, personal communication). We sampled seven individuals at this pond but obtained a complete nuclear dataset for only one individual, showing no signal of hybridization both with STRUCTURE and FCA. The remaining six individuals with incomplete nuclear datasets (two of the three nuclear fragments sequenced) were not included in STRUCTURE and FCA analyses but their allelic compositions provided some evidence of mixing at this locality (Additional file
2). Sharing of nuclear alleles occurred exclusively within members of the same mitochondrial clade with the exception of one individual from clade C that contained alleles otherwise exclusively found in members of clade S (Additional file
2). This signal of admixture was evident at the two nuclear markers available for that individual (rhodopsin and alpha polypeptide), suggesting that incomplete sorting of lineages is, in this case, a much less parsimonious explanation than recent hybridization. Contrastingly, the two individuals from locality L104 (Figure
1) with low assignment to deme S (moderately assigned to deme G) cannot be taken as evidence of admixture, as discussed above.
Admixture between the closely related genetic units in the Rhinella crucifer group is not surprising; natural hybridization in bufonids is very frequent and cases of natural hybrids have been reported in genetic studies for almost every continent in which this cosmopolitan family occurs
[38–41]. In our case, we found stronger evidence for hybridization between units N and C than between units C and S, a result that might be explained by the divergences among these clades. Malone & Fontenot
 revisited a large dataset of experiments crossing species from all major clades of bufonids
 and re–interpreted results in the light of newly available phylogenies. The authors found intrinsic postzygotic reproductive isolation to be a gradual and likely outcome related to the degree of divergence, with high levels of divergence (~9% 12S and 16S) being required for hybrid offspring mortality during early developmental stages. Because the levels of divergence found in the R. crucifer complex are lower than those found by Malone & Fontenot
, and considering that the 12S and 16S have lower mutation rates than the mitochondrial markers used in this study (0.0025 substitutions per site per million years for 12S and 16S, compared to 0.0096 for the ND2 fragment
[44, 45], the relevant question is if the levels of mtDNA divergence observed between the S unit and all other units (6.3% ND2, Figure
2) are sufficient to produce genetic isolation. If so, the expectation that fitness should be lower for crosses between C and S than N and C could be tested in a future study comparing levels of hybridization at the respective hybrid zones. Colliard et al.
 found slight fitness reduction in F1 hybrids, strong hybrid breakdown in backcrossed offspring, and complete mortality in F2 hybrids for Plio–Pleistocene diverged populations of toads from the Pseudepidalea viridis group. However, hybridization was a weak indicator of phylogenetic relationship for the African 20-chromosome toads, and seems to be widespread among all species in that group independent of phylogenetic distance
A problematic taxonomy
Six species of the Rhinella crucifer group are currently recognized on the basis of morphological and morphometric data
[30, 31, 34], although diagnosis of these morphospecies is not always straightforward. The results of this study underscore the fact that current classification does not reflect evolutionary relationships within the group
, and further identifies several groups or populations that are especially problematic. Unequivocal correspondence between genetically cohesive units and recognized species exists only between unit P and R. inopina, and between S and R. henseli. Rhinella henseli corresponds to a deeply divergent mitochondrial lineage
 and is the most clearly diagnosable species in this complex
. Strong but problematic associations are evident between unit N and R. crucifer, and between unit C and R. ornata. These species are easily diagnosable morphologically if one compares individuals from across the core ranges of units C and N (i.e., not including individuals from the putative hybrid zone). The difficulty is that other named species appear to be nested within these 2 units. This is the case for R. abei, a species that occurs within the distribution of unit C, and which shows very subtle diagnostic characters
. The morphospecies Rhinella abei does not correspond to a genetic unit but its distribution roughly coincides to subclade c1. Interestingly, Baldissera et al.
 found a similar pattern in morphometric analysis in that the distribution of R. abei occurs within the polygon of the wider distribution of R. ornata. Despite the morphological data, we cannot decidedly comment on the status of R. abei, because the limited number of nuclear markers in our analysis may contribute to our inability to detect this species (see above). A second difficulty arises in the correspondence between unit C and R. ornata. The type locality for this species ("probably Rio de Janeiro" according to Bokermann
) and type localities for all its synonyms
 lies within the putative hybrid zone between unit N and C; thus, the type series may actually include hybrids. A careful examination of the type series is necessary to solve this issue.
Hybridization is probably the cause of the most problematic case in the taxonomy of the R. crucifer species group. Rhinella pombali was originally described based on individuals from the central region of the state of Minas Gerais
. This species’ distribution has since been expanded to the state of Rio de Janeiro near the border with Espírito Santo
, resulting in an overall area of occurrence that is largely concordant with the putative hybrid zone between units N and C. The morphology of R. pombali shows characters that are intermediate between R. crucifer and R. ornata (tarsal tubercles forming a row in R. ornata and in small individuals of R. pombali; tarsal tubercles forming a fringe in R. crucifer and in large individuals of R. pombali)
. This species has an intermediate size as well, which can mislead the interpretations of traditional morphometric analysis (Figure 27 in Baldissera et al.
). Projected principal component scores of R. pombali occupied a distinct area of the morphometric space but most of the variation contained in the canonical axis is explained by body size and not by significant changes in shape. Not surprisingly, the advertisement call of allopatric populations of R. pombali and R. ornata overlap in call duration and dominant frequency
, but there is no available data on the advertisement call of R. crucifer. Our data argue in favor of the hypothesis that R. pombali was described based on hybrids between R. crucifer and R. ornata. Similar difficulties in inferring species within closely related Bufonidae have been previously attributed to hybridization by taxonomists and phylogeneticists
[38, 49] with toads being referred as a test–case in anuran systematics
. The International Code for Zoological Nomenclature
 states that names proposed for hybrid specimens are excluded from the provisions of the Code (article 1.3.3) and must not be used as the valid name for either of the parental species, regardless of precedence, achieving a condition of homonymy (article 23.8). Therefore, the name R. pombali (and combinations) should be considered a synonym of both R. crucifer and R. ornata. Similarly, the name proposed for hybrids resulting from crosses between Pelophylax lessonae and P. ridibundus ("Rana esculenta"), remains under synonymy of the names of both parental species
[31, 51], although in this case the molecular evidence confirming the hybrid condition came latter
Further investigation of the status and distribution of genetic unit G will be another requirement to reach a taxonomical consensus for this group. If careful inspection of specimens from this region reveals considerable morphological divergence, then this population may justify the description of a new species under morphological and phylogenetic criteria.
Implication for evolutionary studies and conservation
Genetic diversification in the Rhinella crucifer group dates back to the Plio-Pleistocene and earlier studies based on the geographic distribution of mtDNA lineages suggested a major role of geographic barriers as promoters of divergence
. However, that first broad survey of genetic diversity included limited and uneven sampling, and thus necessarily precluded inferences on the demographic history of the group. Recent studies of Atlantic Forest taxa support a scenario of regional differences in evolutionary forces promoting diversification in this biome
[53, 54] resulting in patterns that are hard to interpret or generalize for the whole biome (but see Carnaval et al.
). Both fine scale sampling and the delimitation of units in this study will enable inferences of demographic patterns at regional scales, contributing to a better understanding and hypothesis testing of the microevolutionary processes affecting the R. crucifer group.
The genetic delimitation of units within the R. crucifer also revealed two hybrid zones that may have originated through differentiation under gene flow or from secondary contact after a long period of isolation. While recent studies suggest a history of past habitat fluctuations for the Brazilian Atlantic Forest biome throughout the Pleistocene to the Holocene
[35, 55], it is reasonable to consider a role of more recent human-induced habitat alterations in bringing toads into sympatry
, even though the Rhinella crucifer group occurs exclusively in forested habitat. In a landscape ecology study, Dixo et al.
 noted that R. ornata occurs at higher densities in medium sized fragments than in continuous forest or small fragments, raising the possibility that populations increase and expand distributions under moderate disturbance. Hybridization in Bufonids has been useful for studying mechanisms of reproductive isolation and speciation
[42, 43, 46]. In Neotropical toads. it was shown by Haddad et al.
 and suggested by karyotype data
, but as of yet, we still do not have a good description of hybrid zone dynamics in the Neotropics. The two putative hybrid zones we identified enable experiments and hypothesis testing on hybrid fitness relative to genetic divergences, population history, and on the role of anthropogenic disturbances. Thus, the Rhinella crucifer species complex represents an excellent candidate system for studies of how hybridization contributes to diversity and to biological complexity in the Atlantic Forest biome.
The six species currently recognized in the Rhinella crucifer group are endemic to one of the most biodiverse and endangered global hotspots
. None of the species are considered threatened or endangered according to the IUCN red list
, but our results indicate that there are still cryptic lineages and evolutionary processes to be described and preserved in this group. The most recent survey of remaining areas of the Brazilian Atlantic Forest points to a loss of at least 85% relative to the original distribution, and raises concerns about the inefficiency of the current system of conservation units in keeping fragments of considerable size and connectivity
. The maintenance of genetic diversity, and especially of continued evolutionary potential, has been typically neglected in conservation policies in this biome
. Our study contributes the identification of a previously unidentified new lineage with a restricted distribution.