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

males showing less contrast in marbled patterns than Distribution
females. Brownish to olive (but barely bright greenish) Figure 1 and this paper.
spots often form light dorsal stripes (not to be confused
with a yellowish pigmented stripe), a character rarely Breeding phenology
found in other green toads of Italy, but which is quite Breeding phenology may be explained by phylogenetic
common in B. boulengeri from North Africa. The new spe- history. Lo Valvo & Giacalone [74], and Sicilia et al. [44]
cies is distinguishable from its geographic neighbor,B. bal- reported differences between Italian mainland (including
earicus (as defined by mtDNA haplotype group, Figure 1, Calabrian, i.e.B. balearicus) and Sicilian green toads: B. sic-
2), which exhibits pinhead-sized red (female B. balearicus) ulus exhibits a much longer, potentially bimodal breeding
or brownish (male B. balearicus) spots around tips of lat- period (January-June and September-November), and
eral glands, by the lack of these spots. Bufo siculus almost high plasticity similar to African B. boulengeri (scarce data
never shows a reddish-orange coloration, characteristic of discussed in [44]), versus a short reproductive period in
many B. balearicus, the only form of the subgroup with spring (February-April) that is typical of B. balearicus.
which it may co-occur on Sicily. In Sicily, the ratio VDT/
ED [vertical diameter of the tympanum (VDT) divided by Conclusion
the diameter of the eye (ED)] is smaller in B. siculus [0.530 Our findings on green toads give the first combined mito-
(max) ≥ 0.381 (mean) ≤ 0.247 (min) (N = 42)] than in chondrial and nuclear sequence evidence for a phylogeo-
allo- or parapatric B. balearicus [0.622 ≥ 0.478 ≤ 0.345 (N graphic connection in terrestrial vertebrates across the
= 31)], but similar to that of African B. boulengeri [0.527 ≥ Strait of Sicily. Given the available literature on substitu-
0.362 ≤ 0.25 (N = 29)]. In life, B. siculus has a dark yellow- tion rates of the anuran d-loop and 16S rRNA, which
ish-golden iris. range conservatively from 1% to 3% to higher rates
[53,77] and from 0.3% to 1% [78,79], respectively, we
Variation argue that the African-Sicilian divergence of green toads
The morphometric variation of topotypic B. siculus with a may be younger (but not older) than estimated by the
focus on sexual dimorphism has been recently studied by present analyses. This points towards a post-Messinian (<
Lo Valvo & Giacalone [40] in 354 males and 312 females. 5.3 My) faunal exchange between Africa and Sicily. This
In other parts of its range, stronger variability in size relationship may have implications for comparative phy-
occurs. logeographic research on other terrestrial animals that co-
occur in North Africa and Sicily. We highlight the neces-
Karyotype sity for similar phylogeographic studies that use more
Diploid, 2n = 22 chromosomes; a mitotic metaphase is molecular markers to provide accurate estimates of the
shown in Figure 5b. potential vicariant events and barriers, as well as the pre-
cise dating of the splits between the lineages involved.
Advertisement calls
An advertisement call from the type locality (Figure 6, Methods
recorded as described [75] at 16°C) shows a green toad Sequencing of mitochondrial and nuclear DNA
trill (similar to B. viridis; reminiscent of a canary trill) con- Genomic DNA was extracted from frozen or ethanol pre-
sisting of a series of up to 75 actively pulsed notes with a served blood, liver, muscle tissue, tail tips (tadpoles) and
constant duration separated by constant internote inter- muscle of vouchers from scientific collections using the
vals (Figure 6b, c). Single notes show a symmetrical Qiagen DNeasy™ kit. We either amplified most of the
amplitude increase and decrease as typical of actively mitochondrial control region (or "d-loop", ~860 bps
pulsed calls in Palearctic green toads. Note series are inter- [28]) or a shorter fragment (577 bps) of the control region
rupted by ("inter-call") intervals of more than 12 s (at using the primer pairs CytbA-L/ControlK-H (PCR: 95°C,
16°C, Figure 6a). Sonagram and oscillogram (Figure 6) 3 min, denaturation; cycle [94°C, 45 s, denaturation;
reveal deviations in pulse structure at the beginning of the 55°C, 45 s, annealing; 72°C, 60 s, extension] 35 times;
call, with increasing frequency and signal intensity, as is 72°C, 5 min, final extension [28]).
typical of the subgroup [75]. The fundamental frequency
of the advertisement call, from the topotypic male shown In those individuals from which we sampled the shorter
in Figure 6, was 1600 Hz. The advertisement calls are sim- control region fragment, an additional 611 bps of mito-
ilar in their note repetition rate ("pulse rate") to and typi- chondrial 16S rRNA were amplified using the primer pairs
cal of other diploid members of this complex [75,76] but 16Sar-L/16Sbr-H (PCR: 95°C, 3 min, denaturation; cycle
deserve further investigation with higher sample size [94°C, 45 s, denaturation; 55°C, 45 s, annealing; 72°C,
across a range of temperatures. 60 s, extension] 35 times; 72°C, 5 min, final extension
[80]).

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