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




            substantially different from other western Mediterranean  terior support: 97% and 100%) and demonstrated that
            green toad forms. These phylogeographic relationships  nuclear and mitochondrial markers show essentially the
            substantiate scarce knowledge on western Mediterranean  same signals.
            terrestrial biogeography and may  have  implications for
            comparative research on the phylogeography of other ter-  The control region tree also revealed three well-supported
            restrial animals in the region.                     subclades: one comprising  toads from Corsica  and Sar-
                                                                dinia which exhibit some geographic intermixing between
            Results                                             genetically differentiated lineages within the islands; a
            Phylogeographic structure reveals a relationship across the   second much-less structured clade including toads from
            Strait of Sicily                                    the entire Apennine Peninsula  from  Turin, Pavia  and
            We found genetic markers in green toads (Additional file  Marche (loc. 13, 14, 55, 56) in the north to Apulia (loc.
            1) within our geographic scope (Figure 1) to indicate five  17) and Calabria (loc. 19) in the far south; and a third
            major spatially structured lineages (Figure 2). (I) The first  subclade (nested between  the two others)  containing
            lineage was found  on  Corsica, Sardinia,  the Balearic  toads from the Balearic Islands. Subclade structure is evi-
            Islands, Apennine Peninsula and the northeastern    dent in the average F values between each of these subc-
                                                                                 ST
            extreme of Sicily. This clade (balearicus) was different from  lades (Table 1): Analyses  of population structure
            other Eurasian mainland green toads (lineages II and III:  confirmed high genetic differentiation  (pairwise  F ST   =
            viridis and variabilis), whose ranges border northeast to the  0.7585, p = 0.00000) between  Italian mainland and
            Po River drainage  and belong to  widespread  mono-  island  toads  (Corsica, Sardinia). Demographic analyses
            phyletic groups in Eurasia [28]. (IV) Although geographi-  performed in Fluctuate estimated a greater than ten-fold
            cally neighboring I, on most of Sicily and its surrounding  exponential growth rate for the clade on  the  Apennine
            islands another lineage (Bufo n. sp.) occurs that is substan-  Peninsula than for the mitochondrial group on Corsica
            tially different from the first. (V) The entire North African  and Sardinia (767.1 > 51.7), suggesting population
            range and the off-coast islands constitutes a fifth lineage  expansion on the mainland. However, log-likelihood tests
            (boulengeri). The details of the results defining each line-  did not reject a scenario of zero growth (Table 2). Growth
            age are described below.                            of the mainland  population was also not significantly
                                                                supported by the mismatch distribution analysis in which
            In particular, our analyses shows a deep sister relationship  the observed distribution matched an expected distribu-
            between African (V) and Sicilian (VI) groups and there-  tion for an expanding population; however, the shape and
            fore a rarely studied phylogeographic connection across  magnitude of the observed and expected distributions are
            the Strait of Sicily.                               very similar, suggesting some support for expansion on
                                                                the mainland (Figure 3c). By contrast, this analysis, both
            Characterization of the groups identified based on the   graphical and statistical, supports stable populations on
            Bayesian phylogram                                  Corsica and Sardinia (Figure 3a, b). Tajima's D estimates
            Here we consider and name lineages that maintain their  for all balearicus groupings were not significant, and thus
            evolutionary integrity with respect to other lineages  population expansion was not indicated by this test.
            through both time and space and name them as species
            under the phylogenetic species concept.             The Bayesian phylogenetic analysis shows B. balearicus to
                                                                be reciprocally monophyletic on the mitochondrial level,
            Bufo balearicus Boettger, 1880                      albeit using  a subsample of individuals within each
            For details on taxonomy and nomenclature of Bufo balear-  group, with respect to all other green toads; thus our data
            icus [45] see supporting data in Additional file 2 (d). Anal-  corroborate the proposal that B. balearicus be given the
            yses of mitochondrial 16S (not shown in tree) and control  status of a separate species (see Additional file 2 for details
            region sequences of 50 green toads from  the Balearic  on nomenclature).
            Islands (loc. 5–7), Corsica (loc. 8–9), Sardinia (loc. 10–
            12), the entire Apennine Peninsula (loc. 13–16), Apulia  Bufo boulengeri Lataste, 1879 [46]
            (loc. 17), Calabria (loc. 18, 19), Marche (loc. 55, 56) and  While exhibiting some internal structure, all North Afri-
            northeastern Sicily (loc. 20) form one very well-supported  can control  region (Figure  2) and 16S mitochondrial
            haplotype group (Bayesian posterior support: 100%; Fig-  sequences (Additional file 1) of toads between the Atlan-
            ure 1, 2). Cloned tropomyosine intron alleles (Figure 2)  tic coast of Morocco (loc. 1–3) and the  Nile  Valley  of
            from four representatives each from Sardinia (loc. 10),  Egypt (loc. 37–40), including  the  off-coast islands  of
            Calabria (loc. 18) and northeastern Sicily (loc. 20), as well  Kerkennah (loc. 30, 31), Djerba (loc. 32) and Lampedusa
            as fragments of RAG-1 in three individuals each from Cor-  (loc.  26), form a well-supported monophyletic cluster
            sica (loc. 9),  Sardinia (loc. 10), and Calabria (loc. 18)  (posterior probability = 99%) that is a sister clade to mito-
            formed similarly highly supported clusters (Bayesian pos-  chondrial sequences of toads from Sicily (loc. 21–25), but


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