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Colliard et al. BMC Evolutionary Biology 2010, 10:232 Page 8 of 16
http://www.biomedcentral.com/1471-2148/10/232

Table 3 Crossing experiments
Cross Cross Female Species/ Male Species/ Nof Ca.% Nof % Remarks Survival at day
type hybrid hybrid available Estimated tadpoles Survival 40 after
tadpoles hatching (2 months (2 months metamorphosis
(at day 7 success after after
after spawning) spawning)
spawning)
1 F 1 Si41 balearicus Si11 siculus 200 > 80 158 79 malformations, 150
some dwarfed
or leucistic
larvae
2 F 1 Si337 balearicus Si334 siculus 100 > 80 98 98 died through N.A.
technical
accident
3 F 1 Si335 siculus Si336 balearicus 100 > 80 93 93 - 4
4 Backcross Si337 balearicus F 1 F 1 (bal. × 100 > 80 84 84 67 (big), 17 2
(F 1 × Cross sic.) (small)
parental 13
species)
5 Backcross Si335 siculus F 1 F 1 (bal. × 100 > 80 91 91 - 0
(F 1 × Cross sic.)
parental 13
species)
6 Backcross F 1 F 1 (bal. × Si334 siculus 100 ca. 50 1 1 - 0
(F 1 × Cross sic.)
parental 11
species)
7 Backcross F 1 F 1 (bal. × Si336 balearicus 28 ca. 30 24 85.7 1 color mutant, 0
(F 1 × Cross sic.) all dead (at
parental 11 day 116 after
species) spawning)
8 F 2 (F 1 × F 1 F 1 (bal. × F 1 F 1 (bal. × 100 ca. 20 0 0 all dead (at 0
F 1 ) Cross sic.) Cross sic.) day 17 after
11 13 spawning)
Control1 Intra- Ky109 turanensis Ky103 turanensis 50* > 80 48 94 - 47
specific
mating
Control2 Intra- Ky100 pewzowi Ky87 pewzowi 50* > 80 47 90 - 45
specific
mating
Cross number, cross type, sexes, species, estimated hatching success, numbers of tadpoles at 7 day and two months after spawning, percentage of survival two
months after spawning, remarks and survival at day 40 after metamorphosis for each cross. *Note, in controls from two related green toad species, only 50
tadpoles were further raised.

backcrossing of fertile F 1 -females with their paternal by the patterns of genetic differentiation: The two popu-
species. Though no F 1 -hybrids were detected in the lations each side of the contact zone (pop. 14 and 15),
field, theoccurrence ofrareand symmetriceventsof though harboring a mix of mitochondrial haplotypes
hybridization was confirmed by a few backcrosses (F 2 or from both lineages, display the highest differentiation
more) identified via STRUCTURE and NEWHYBRIDS value observed in the study area (pairwise F ST = 0.52).
in both B. siculus and B. balearicus populations, as well Populations at the contact zone (clusters b3 and s3) are
as a two-way leak of diagnostic nuclear alleles. actually the ones most differentiated on the first PCA-
However, nuclear introgression was surprisingly low GEN factors (Figure 4).
overall. The transition between tropomyosine alleles Genetic drift certainly plays a role in this strong local
from the two clades was abrupt, occurring at some differentiation. The B. balearicus populations at the con-
point between populations 14 (Giarre) and 15 (Gravina), tact zone represent the front wave of a recent expan-
separated by just 16 km. The same holds for autosomal sion, as evidenced by the drastic decrease in genetic
microsatellites in general, since STRUCTURE assigned diversity from mainland Italy (Hs = 0.74) to the south-
(with 100% probability) all individuals from pop. 14 to ernmost populations (Hs = 0.38 in population 14). Drift
balearicus, and all individuals from pop. 15 to siculus is certainly further amplified by the geographic localiza-
(Figure 3). The sharpness of this transition is underlined tion of the contact zone: The geographical bottleneck