Morphological and genetic differentiation of Bufo toads: two cryptic species in Western Europe (Anura, Bufonidae)

Abstract

The Common toad Bufo bufo sensu lato is a widespread, morphologically conserved taxon. Recent studies have uncovered deep genetic differentiation between population groups, highlighting the need to revise the current taxonomy of the group and recognize additional species. Here we investigate patterns of variation in molecular (a mitochondrial DNA restriction enzyme assay and sequence data for two nuclear DNA fragments totalling 979 bp) and 17 morphological variables in Northern France where two of these groups meet (B. bufo sensu stricto and B. spinosus), in order to delineate their contact zone and uncover characters that would allow discrimination of the two taxa. Mitochondrial DNA data show an abrupt transition from areas where B. bufo is present to those inhabited by B. spinosus, with a narrow area of overlap east of the city of Caen. Morphometric characters, particularly those related to the positioning of the parotoid glands and metatarsal tubercle shape and size, proved useful in discriminating between species (AUC ≥ 0.97, kappa ≥ 0.79). We then used the differentiating character states to allocate over 300 museum specimens from Western Europe to either species with consistent results, including comparable values of AUC and kappa of the identification models, indicating that models could successfully be applied across datasets. We summarize available evidence relevant to the delineation of the distribution of B. bufo and B. spinosus in France and discuss the characters differentiating both species in an evolutionary context. In view of the observed morphological and genetic differentiation and the absence of unequivocal evidence for widespread hybridization we support the view that B. bufo and B. spinosus are best considered different species. Finally, we propose that ‘parotoids in parallel position’ and a thin and smooth skin are derived character states for B. bufo over the northern part of its range.

Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France

“…[28]). Initial assessments of patterns of allele sharing across species in this system were inconclusive as to whether gene flow or incomplete lineage sorting best explained observed discordance between mtDNA and nDNA data [14, 16, 17, 19]. This, and the characterization of a hybrid population based on morphological and molecular genetic data [18], raised questions about the extent of reproductive isolation in this species pair.…”

“…1) adjacent to a locality where we documented a B. spinosus – B. bufo hybrid population [18]. Morphological characters studied were those that help to distinguish B. spinosus from B. bufo [16], namely Snout-Urostyle length (SUl), anterior and posterior Parotoid distance (Pda, Pdp), length and width of the inner Metatarsal Tubercle (MTl, Mtw) and Paratoid angle (Pa) and the derived measures Paratoid divergence (Pd = Pda/Pdp), Metatarsal Tubercle size (MTsize = MTl/SUl) and shape (MTshape = MTw/MTl). For illustrations of these characters see [16].…”

“…Adult males ( N  = 190) and adult females ( N  = 76) were analyzed separately. For each individual we calculated the probability to belong to B. spinosus (P s ), with the logistic regression formulae derived for this part of France (see [16]).…”

“…Recent molecular studies on the systematics of the Bufo bufo species group have resulted in the recognition of four species, two of which were formerly regarded as subspecies of B. bufo [14–16]. One of these, B. spinosus, present in Iberia and North Africa, contacts B. bufo in France, forming an extensive contact zone along a NW-SE line, approximately from Caen to Lyon [17].…”

“…These species split in the Late Miocene at around 9.2 MYA [14], but are morphologically similar. They can, however, be differentiated in their contact zone and recent studies have shown overall agreement between morphological characters, mitochondrial and nuclear DNA markers in delineating species boundaries in France [16]. Occasional discordance is mostly restricted to slowly evolving nuclear markers and interpreted as resulting from incomplete lineage sorting, although hybridization-mediated gene flow cannot be completely ruled out.…”