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BMC Evolutionary Biology 2008, 8:56 http://www.biomedcentral.com/1471-2148/8/56

of green toads. Gordon [48] experimentally demonstrated
survival of B. viridis in seawater for several hours or days.
Breeding and swimming in brackish (beach or desert)
pools and waters [49] as well as estuaries has been
observed [38]. Therefore, green toads may have the poten-
tial for transmarine dispersal. The distance from Africa to
Sicily (~140 km), however, can be considered a very
strong barrier, even during low sea levels (>45 km). In
addition, external fertilization (and absence of brood
care) requires that several adults or larvae disperse in
order to found new populations. This and the early diver-
gence time (0.63 My < 1.83 My < 3.5 My) of African and
Sicilian lineages make it more plausible that toads with a
terrestrial life style dispersed via land connections than by
rafting. This hypothesis was supported in the recent study
of terrestrial Chalcides lizards (see above), which esti-
mated a similar divergence between Tunisian and Sicilian
clades (1.8 My [20]) to the one we propose for green
toads. However, several papers have suggested the ability
of anurans to overcome large sea barriers [50,51], espe-
cially in the tropics, where swimming islands may facili-
Result of a discriminant analysis with 20 morphometric traits
Figure 4 tate rafting. Therefore, comparative phylogeographic data
Result of a discriminant analysis with 20 morphomet- from other terrestrial species in Sicily and North Africa
ric traits. DF1: Discriminant function 1, DF2: Discriminant would be necessary to test the feasibility of competing
function 2 on four groups of green toads specified in the leg- hypotheses.
end.
Divergence time estimates based on molecular data nor-
mally rely on external calibration points from the fossil
appeared genetically almost identical to Sardinian Chal- record or from well-known paleogeographic events.
cides, interpreted as a human introduction from Tunisia Because neither fossil nor paleogeographic calibration
into Sardinia. Very recently, however, Giovannotti et al. points were available, we calculated divergence times
[20] found two Sicilian Chalcides mtDNA haplotypes to using an uncorrelated relaxed molecular clock and a range
have a sister relationship with Tunisian and Sardinian of substitution rates for the two mitochondrial fragments.
haplotypes (the latter data not shown in the Chalcides We acknowledge a potential limitation, that is, the use of
phylogeny) and interpreted this as a possible Lower Pleis- only mtDNA to determine divergence times, which may
tocene (1.8 My) colonization event of Sicily (and Italy) lead to overestimation of the splitting dates. This overesti-
from North Africa. (ii) Using allozymes and cytochrome mation may occur because the most recent common
b, Zangari et al. [25] assessed lineage relationships among ancestor (MRCA) of the haplotypes (their coalescent)
discoglossid frogs from multiple locations in the western does not necessarily correspond to the real temporal split
Mediterranean (including Sicily, Sardinia, Tunisia). These of the populations but may precede the actual divergence
authors showed a deep divergence of some mtDNA hap- of the populations [52]. We consider substitution rates
lotypes across the Strait of Sicily, but also some nearly between 1% and 3% per lineage per My to be reasonable
identical haplotypes across Sicily, Tunisia and Algeria. values for amphibian mtDNA (for references see Materials
While the authors commented imprecisely (i.e., they did and Methods) although they have not been empirically
not specify if the following referred to Gibraltar or Sicily assessed in green toads. When regions with highly repeti-
or both) that the "spread of Discoglossus between Europe tive motifs are absent, as in green toads, the control region
and Africa should have occurred at the end of the Messin- tends to show higher rates of substitution than the rest of
ian salinity crisis", they interpreted closely-related haplo- the mitochondrial molecule [53,54]. Thus, the substitu-
types across the Strait of Sicily as "little genetic tion rates selected here are conservative, higher than the
differentiation detected among Algerian and Tunisian D. rate for the rest of the mitochondrial molecule, but lower
auritus with respect to Maltese and Sicilian D. pictus sug- than the fastest observed rates for the control region in
gest [ing] a very recent isolation", without further inter- other taxa. Higher rates would imply younger dates for the
preting or dating their results. The significance of land splitting of the African and Sicilian lineages. This point is
connections for faunal exchanges between Africa and Sic- illustrated by our temporal estimates from the mitochon-
ily is mildly challenged by the well-known salt tolerance drial 16S gene which we obtained applying a slower sub-

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