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

time estimates were also derived using the 16S data (see from 0.164 to 3.603 My (mean 2.051) for the 16S rRNA,
Material & Methods for details). Results were similar to probably excludes human introduction and reveals an
those obtained using the mtDNA control region (diver- infrequently-studied phylogeographic relationship in ter-
gence between African and Sicilian haplotypes: 95% HPD restrial vertebrates. This evolutionary connection may be
from 0.164 to 3.603 My (mean: 2.051); split of balearicus/ significant for the phylogeography of the Mediterranean
viridis clades: 95% HPD from 0.18 and 3.795 (mean: and have implications for research on other terrestrial ani-
2.123). All the dates estimated in our analysis seem to rep- mals. While some biogeographic studies have suggested
resent post-Messinian divergence events. common ranges for terrestrial vertebrates (for example the
"Siculo-Maltese-Maghrebin" range type of Turrisi and Vac-
Multivariate morphometric comparisons carro [42]), and paleontologists discuss a possible early
A discriminant analysis using all 20 characters demon- Pleistocene faunal exchange with North Africa (see intro-
strated the distinctness of toads from Sicily by correct duction), so far, very few molecular studies have suggested
reclassification of 100% of individuals into the four any genetic relationships within terrestrial vertebrates
groups from: (i) Sicily, (ii) N-Africa, (iii) Corsica and Sar- across the Strait of Sicily. Two examples come from spe-
dinia, and (iv) the Apennine Peninsula. Although cies (e.g. Discoglossus [25]; Chalcides [47,20]), for which
encoded as belonging to different geographic groups, the human introduction elsewhere at circum-Mediterranean
genetically more closely-related groups (iii) and (iv) of sites has been demonstrated and for which African-Sicil-
the B. balearicus clade showed fewer differences (Figure 4), ian relationships have never been demonstrated using any
making us confident of the power of the analyses' and this nuclear sequence data. Despite some recent speculation
morphometric dataset. However, in order to test for (e.g. Crocidura sicula, potential Messinian origin from
potential over-parameterization of the analysis, we also North Africa [16]), a possible African origin of terrestrial
reduced the number of morphometric characters to five, Sicilian fauna has rarely been thoroughly tested with
which again reclassified 100% of the individuals into their molecular methods, and never using any nuclear
four respective groups. sequence, and thus supported by both mitochondrial and
nuclear sequence data. To our knowledge, a genetic Afri-
Discussion can-Sicilian link has only been inferred in two other ter-
A phylogeographic relationship between Africa and Sicily restrial species, but even these have ambiguous
We have demonstrated a sister relationship on the mito- information on timing and direction: (i) Phylogenetic
chondrial and nuclear levels between green toads from inference based on allozymes regarding relationships
Sicily (B. siculus, see below) and those from the entire among Chalcides lizards from Sicily, Africa, the Apennine
North African range (B. boulengeri). The phylogenetic Peninsula and Sardinia [47] showed Sicilian skinks most
depth of this divergence, which may range between 0.63 closely related to Italian mainland skinks (Calabria in the
My and 3.5 My (mean 1.83 My) for the control region and south to Liguria in the north), while lizards from Tunisia

Table 3: Within-clade (numbers in italics) and between-clade (numbers in regular font) divergence time estimates obtained using a
coalescent-Bayesian framework as implemented in BEAST v1.4.6 applied to the mitochondrial control region and 16S sequences (not
available for B. variabilis). The African-Sicilian divergence time estimates (boulengeri to siculus) are printed in bold. Estimates are
shown in My. Values in parentheses show the 95% highest posterior density intervals (95% HPD); they represent the shortest intervals
that contain 95% of the posterior sampled values.
MtDNA clade(s) Divergence time estimate MtDNA clade(s) and/or Geographic region

Control region
16S
boulengeri 1.2 (0.394–2.351) boulengeri N-Africa
1.57 (0.099–2.80)
siculus 0.677 (0.115–1.53) siculus Sicily
1.56 (0.101–2.69)
balearicus 0.914 (0.227–1.875) balearicus Apennine Peninsula, Corsica, Sardinia, Balearic Islands
1.84 (0.15–3.365)
variabilis 0.85 (0.172–1.838) variabilis Balkan, Anatolia
viridis 0.527 (0.096–1.152) viridis Central, E-Europe
1.44 (0.196–2.785)
balearicus-variabilis-viridis 1.956 (0.66–3.795) balearicus-variabilis-viridis
boulengeri 1.833 (0.635–3.509) siculus
2.05 (0.16–43.60)
boulengeri-siculus 2.749 (1.188–4.906) balearicus-viridis-variabilis

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