BackgroundLittle attention has been paid to the consequences of the last landbridge between Africa and Sicily on Mediterranean biogeography. Previous paleontological and scarce molecular data suggest possible faunal exchange later than the well-documented landbridge in the Messinian (5.3 My); however, a possible African origin of recent terrestrial Sicilian fauna has not been thoroughly tested with molecular methods. To gain insight into the phylogeography of the region, we examine two mitochondrial and two nuclear markers (one is a newly adapted intron marker) in green toads (Bufo viridis subgroup) across that sea barrier, the Strait of Sicily.ResultsExtensive sampling throughout the western Mediterranean and North Africa revealed a deep sister relationship between Sicilian (Bufo siculus n.sp.) and African green toads (B. boulengeri) on the mitochondrial and nuclear level. Divergence times estimated under a Bayesian-coalescence framework (mtDNA control region and 16S rRNA) range from the Middle Pliocene (3.6 My) to Pleistocene (0.16 My) with an average (1.83 to 2.0 My) around the Pliocene/Pleistocene boundary, suggesting possible land connections younger than the Messinian (5.3 My). We describe green toads from Sicily and some surrounding islands as a new endemic species (Bufo siculus). Bufo balearicus occurs on some western Mediterranean islands (Corsica, Sardinia, Mallorca, and Menorca) and the Apennine Peninsula, and is well differentiated on the mitochondrial and nuclear level from B. siculus as well as from B. viridis (Laurenti), whose haplotype group reaches northeastern Italy, north of the Po River. Detection of Calabrian B. balearicus haplotypes in northeastern Sicily suggests recent invasion. Our data agree with paleogeographic and fossil data, which suggest long Plio-Pleistocene isolation of Sicily and episodic Pleistocene faunal exchange across the Strait of Messina. It remains unknown whether both species (B. balearicus, B. siculus) occur in sympatry in northern Sicily.ConclusionOur findings on green toads give the first combined mitochondrial and nuclear sequence evidence for a phylogeographic connection across the Strait of Sicily in terrestrial vertebrates. These relationships may have implications for comparative phylogeographic research on other terrestrial animals co-occurring in North Africa and Sicily.

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Species complexes undergoing rapid radiation present a challenge in molecular systematics because of the possibility that ancestral polymorphism is retained in component gene trees. Coalescent theory has demonstrated that gene trees often fail to match lineage trees when taxon divergence times are less than the ancestral effective population sizes. Suggestions to increase the number of loci and the number of individuals per taxon have been proposed; however, phylogenetic methods to adequately analyze these data in a coalescent framework are scarce. We compare two approaches to estimating lineage (species) trees using multiple individuals and multiple loci: the commonly used partitioned Bayesian analysis of concatenated sequences and a modification of a newly developed hierarchical Bayesian method (BEST) that simultaneously estimates gene trees and species trees from multilocus data. We test these approaches on a phylogeny of rapidly radiating species wherein divergence times are likely to be smaller than effective population sizes, and incomplete lineage sorting is known, in the rodent genus, Thomomys. We use seven independent noncoding nuclear sequence loci (total approximately 4300 bp) and between 1 and 12 individuals per taxon to construct a phylogenetic hypothesis for eight Thomomys species. The majority-rule consensus tree from the partitioned concatenated analysis included 14 strongly supported bipartitions, corroborating monophyletic species status of five of the eight named species. The BEST tree strongly supported only the split between the two subgenera and showed very low support for any other clade. Comparison of both lineage trees to individual gene trees revealed that the concatenation method appears to ignore conflicting signals among gene trees, whereas the BEST tree considers conflicting signals and downweights support for those nodes. Bayes factor analysis of posterior tree distributions from both analyses strongly favor the model underlying the BEST analysis. This comparison underscores the risks of overreliance on results from concatenation, and ignoring the properties of coalescence, especially in cases of recent, rapid radiations.