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Colliard et al. BMC Evolutionary Biology 2010, 10:232 Page 2 of 16
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Under natural conditions, however, reproductive isola- sequences show two highly homogeneous and strongly
tion could arise much earlier than detected in the distinct clades (Figure 2), corresponding to B. balearicus
laboratory. In frogs, as in many other taxa, “surveys of and B. siculus. The tropomyosine tree displays a clear
natural hybrid zones (...) in the field are needed to com- geographic pattern: the balearicus clade includes all
plement laboratory-based studies to establish the signifi- individuals from mainland Italy (populations 1 to 8, Fig-
cance and strength of specific barriers in nature” [7]. ure 1, Table 1) and north-eastern Sicily, southwards to
Little is known about secondary contact in allopatrically population 14 (east coast), while the siculus clade
diverged lineages of anurans, where reproductive isola- includes individuals from western and southern Sicilian
tion may quickly arise as a result of reinforcement [8], populations, from population 15 (East coast) south-west-
in addition to genetic drift and local adaptation. Extant wards. This pattern points to a very narrow contact
studies of contact zones in anurans have mostly focused zone separating populations 14 and 15, between the
on hybrid fitness [9,10] or on mechanisms of pre- or Mount Etna and the Ionian coast (Figure 1b).
post-mating isolation [9-15]. Such studies classically The mtDNA clades also show a clear geographic sig-
relied on allozymes [e.g. [9,11-13]] or (more recently) nal, with however, some overlap. Populations from
nuclear and mitochondrial DNA markers [e.g. mainland Italy (pop. 1 to 8) and north-eastern Sicily
[10,14,15]], but often lack any molecular-based estimates (pop. 9 to 12) present only balearicus haplotypes, and
of divergence times, which are at best inferred from populations from western and southern Sicily (pop. 16
geological information. Some phylogeographic studies to 24) only siculus, but haplotypes from both clades are
include molecular-based estimates of divergence time [e. found in populations 13 to 15, around the contact zone
g. [16-20]], but very few have combined such estimates identified with tropomyosine.
with multi-locus transect approaches to infer the time These phylogenetic trees also provide evidence for
required to reach reproductive isolation in natural con- past hybridization, as revealed by cytonuclear disequili-
texts [e.g. [8,21,22]]. bria (see highlighted individuals in Figure 2): one indivi-
The current study focuses on Palearctic green toads dual from pop. 14 possesses balearicus tropomyosine
[Bufo viridis subgroup, [18]]. After range-wide phylogeo- alleles but a siculus mtDNA-haplotype, while three indi-
graphic analyses, secondary contact zones of clades were viduals from pop. 15 present siculus tropomyosine
predicted [18,23], in which possible hybridization can be alleles but balearicus mtDNA haplotypes.
examined using fast evolving molecular markers. To do These patterns of mitochondrial distribution were
this, we recently developed microsatellites for two West- widely confirmed by larger-scale mitotyping (Figure 1).
Mediterranean species [24]: B. balearicus (Boettger 1880; All populations on the Apennine Peninsula (pop. 1 to 8)
Peninsular Italy, north-eastern Sicily, Corsica, Sardinia, and four populations (pop. 9 to 12) from the North-East
Balearic Islands), and B. siculus [[23]; endemic to Sicily, of Sicily presented only B. balearicus haplotypes. All
Figure 1]. Using a Bayesian-coalescence approach populations from western and southern Sicily (pop. 16
(mtDNA control region and 16 S rRNA), divergence time to 22) and the two islands off the coast of western Sicily
for the two species was estimated to late Pliocene (2.7 (pop. 23, 24) presented only B. siculus haplotypes. In the
My), with a range from the early Pliocene (4.9 My) to three populations east of Mount Etna (pop. 13 to 15),
Pleistocene (1.1 My) [23]. A single record of Italian main- both B. balearicus and B. siculus haplotypes were pre-
land-origin B. balearicus in north-eastern Sicily [18] sug- sent, with a marked north-south cline (Table 2): The
gests their recent (late Pleistocene) invasion into Sicily, frequency of balearicus haplotypes declined from
where they may secondarily meet the endemic B. siculus. 93.75% in Calatabiano (pop. 13) to 68% in Giarre (pop.
In this work, we combined mitochondrial and nuclear 14) and 50% in Gravina (pop. 15), down to 0% in Mis-
intronic sequences with multilocus microsatellite mar- terbianco (pop. 16).
kers to examine (i) whether B. siculus and B. balearicus
meet each other in north-eastern Sicily, (ii) if so, Autosomal microsatellites and population-genetics
whether these two closely related species hybridize, and analyses
(iii) in such a case, what are the patterns of hybridiza- There was no evidence for allelic dropout from any
tion. In parallel, we conducted limited and preliminary locus in any population. Null alleles at low frequencies
experimental crosses to help interpreting field data. were detected (and corrections performed) in one popu-
lation each for loci C203 (pop. 14) and D105 (pop. 13),
Results and in two populations each for loci C218 (pop. 6, 22),
Nuclear and mitochondrial DNA sequences and C223 (pop. 18, 21) and D5 (pop. 17, 18). Tests for link-
mitotyping age disequilibrium between loci (after sequential Bonfer-
Both of the phylogenetic trees built from mitochondrial roni corrections) revealed four significant combinations
(D-loop) and nuclear (Tropomyosine intron) DNA (Bcal μ10 × C203 for pop. 9, D5 × Bcal μ10 for pop. 15,