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Colliard et al. BMC Evolutionary Biology 2010, 10:232 Page 7 of 16
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Axis 2: 13.13% inertia, P=0.001 20 19 16 21 3 5 6 2 11 4 10 1 9 8 13 14

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Axis 1: 40.36% inertia, P= 0.001
Figure 4 Principal component analysis based on pairwise F ST over all populations. Both axes are significant (P < 0.001). Samples are
encoded as in Table 1. Colored ellipsoids correspond to clusters shown in Figure 1b and were only drawn for better visualization.

B. balearicus) showed much lower survival after meta- divergence was also sufficient to bring the speciation
morphosis (Table 3). process close to completion.
We raised about 160 F 1 -metamorphs from the 2007 On the one hand, the endemic B. siculus,ofNorth-
cross to a snout-vent-length of ca. 2 cm, and kept 50 African origin [home of its sister clade B. boulengeri;
of them until secondary sexual characters became visi- [23]], occupies the western and southern parts of Sicily,
ble (paler coloration and nuptial pads of males). plus two small islands off the north-western coasts
Though sex ratio was approximately even, we noticed (Ustica and Favignana) [23]. On the other hand,
about 30% of dwarfed F 1 -males that reached only B. balearicus occupies the north-eastern part of Sicily.
about two-thirds the size of normal individuals. Ten Based on their geographical localization and patterns of
males and ten females were further raised until matur- genetic similarity with mainland Italy, we infer that
ity (Figures 5g-h). In spring 2009, we used one F 1 of these Sicilian B. balearicus populations recently origi-
each sex to produce F 2 -hybrids (F 1 ×F 1 ) and recipro- nated from close-by Calabrian populations. Faunal
cal backcrosses with either B. siculus or B. balearicus exchange across the Strait of Messina [including amphi-
(one new, wild-caught male and one female each, Fig- bians [29]] are well documented for the Upper Pleisto-
ure 6). F 2 -hybrids turned out to be unviable, with all cene [30]. From our genetic analyses, these two species
tadpoles dying a few days after hatching. While 200 nowadays meet at the eastern coast of Sicily, between
out of 328 tadpoles from the backcrosses were still the Mount Etna and the Ionian Sea. We cannot exclude
alive two months after spawning, they presented, a that another contact exists along the North coast
number of developmental abnormalities, including (north-west of Mount Etna), but could not find any cur-
greenish individuals and a bimodal size distribution rently occupied site in this area despite thorough
within thesamecross (Figure7), andsufferedfrom examination.
dramatic mortality at later stages, with two individuals Though very restricted, the documented contact zone
only surviving after metamorphosis. shows signs of past hybridization, with differential intro-
gression patterns depending on markers. Mitochondrial
Discussion alleles show a clear North-South cline, where the fre-
Our study shows that two distinct lineages of green quency of balearicus haplotypes progressively decreases
toads (Bufo viridis subgroup) occur parapatrically in from 94% (pop. 13, Calatabiano) to 0% (pop. 16, Mister-
Sicily. These lineages have diverged some 2.7 My ago bianco) over a distance of ca 40 km. Cytonuclear dise-
[23], a time frame long enough to allow significant dif- quilibrium occurred in individuals from both species,
ferentiation on both mitochondrial and nuclear DNA pointing to a two-way introgression. This presumably
sequences (Figure 2). As our study further shows, this involved symmetric events of hybridization, followed by