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




            Table 1: Pairwise F ST  between groups of West Mediterranean green toads based on sequences of the mitochondrial d-loop. All F ST  -
            values are significant (p < 0.0000).
                                           B. balearicus   B. balearicus   B. balearicus   B. boulengeri Offshore   B. boulengeri North
                                          Corsica+Sardinia  Balearic Islands  Apennine Peninsula  Islands, N. Africa  African Mainland
                 B. balearicus Balearic Islands  0.60662
                B. balearicus Apennine Peninsula  0.75859  0.49501
             B. boulengeri Offshore Islands, N. Africa  0.91530  0.91812  0.95058
              B. boulengeri North African Mainland  0.77888  0.74269  0.82886         0.16073
                        B. siculus           0.97543     0.98188      0.98815         0.96950         0.87081


            population  growth  for  the green toads on the  African  ported (posterior support = 100%) clade that differs from
            mainland, as well as for all boulengeri considered together;  all sampled African and Eurasian green toads (Figure 2).
            log likelihood tests rejected no growth scenarios for both  For all mitochondrial and nuclear markers,  Bufo  n.sp.
            groupings (Table 2). Tajima's D calculated for the main-  shows a much greater genetic distance from Italian main-
            land group indicated some support for population expan-  land B. balearicus than from B. boulengeri, which inhabits
            sion (-1.63869, 0.10 > p > 0.05); however, the mismatch  all of North Africa. Taken together, as for the two preced-
            distribution analysis did not support a growth model.  ing taxa, phylogenetic divergence of Bufo n. sp. is evident
                                                                and we acknowledge this by describing it as new species
            Tropomyosine intron sequences in African samples from  (Bufo siculus, see below).
            Libya (loc. 36), Tunisia (loc. 29) and Lampedusa (loc. 26)
            exhibit the same tree topology as the mitochondrial mark-  Bufo viridis (Laurenti, 1768) and old isolates
            ers and constitute a highly supported clade (posterior  The  haplotype  group representing this taxon, as previ-
            probability = 96%) that differs substantially from tropo-  ously shown by Stöck et al. [28], was found exclusively in
            myosine sequences from two Sicilian green toads (loc.  the very northeastern part of Italy, northeast of  the  Po
            21). The very conserved RAG-1 marker shows two African  River (loc. 41, 42). So far, two very well supported mono-
            samples in a polytomy with Eurasian green toads, from  phyletic and geographically widespread mtDNA groups of
            which both B. balearicus and two samples each from Sicily  green toads are known from the Balkan region. The hap-
            and Lampedusa differ.                               lotype  group  dominating Asia Minor (B. variabilis) and
                                                                that of  B. viridis  (Figure  1: blue  and light green; [28])
            Bufo n. sp                                          apparently co-occur  in Greece.  However, neither  of the
            This taxon was identified on most of Sicily (loc. 21-23a)  two was found off the Adriatic coast on  the Croatian
            and two close islands (Favignana, loc. 25; Ustica, loc. 24).  islands of Krk and Cres (loc. 43, 44). Instead, a very well
            Demographic analyses performed in Fluctuate estimated θ  supported separate clade (posterior support = 100%)
            to be an order of magnitude lower than that estimated for  formed by two sequences  of toads from those islands
            all other green toad groups examined except the mainland  revealed an apparent relict group, which is more closely
            balearicus  group (Table  2). Although the log likelihood  related to B. viridis than to B. variabilis (Figure 2), but fur-
            test did not permit rejection of a no growth scenario, the  ther data are required to confirm this relationship.
            very low initial θ estimated by the analysis indicates that
            few individuals may have founded the lineage. Similarly,  Age of African-Sicilian vicariant separation
            the observed mismatch distribution is not statistically  Divergence time estimates for the principal mitochondrial
            identical to the  expected distribution under a  sudden  (control  region and 16S rRNA)  clades recovered  are
            growth model (p = 0.077). However, the unimodal peak  shown in Table 3. We provide the 95% highest posterior
            is shifted to the left of the distribution and very tightly  density intervals (95% HPD, that is, the shortest intervals
            matches the expected distribution for a recently expanded  that include 95% of the posterior sampled values) as well
            population (Figure 3f). Finally, Tajima's D did not indi-  as the mean of  the sampled values. Time estimates are
            cate deviation from neutrality, and thus a scenario of pop-  based on the reconstruction of the most common recent
            ulation expansion cannot be invoked with this measure.  ancestor for the mtDNA control regions under the coales-
                                                                cent (see Material & Methods section for details). The 95%
            Bufo n.sp. exhibits reciprocal monophyly for both mito-  HPD,  although overlapping, show  consistent values
            chondrial markers  (and  using the tropomyosine intron  within and between clades. Divergence between African
            marker  on a subsample  of individuals) with all  other  and Sicilian haplotypes falls within the range of 0.6 and
            groups/taxa (Figure  2). The RAG-1 phylogram shows  3.5 My [mean 1.8 My, around the Pliocene/Pleistocene
            toads from Sicily and Lampedusa forming a well-sup-  boundary (~1.8 My)] for the control region. Divergence


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