Strong reproductive barriers in a narrow hybrid zone of West-Mediterranean green toads (Bufo viridis subgroup) with Plio-Pleistocene divergence

Abstract

Background: One key question in evolutionary biology deals with the mode and rate at which reproductive isolation accumulates during allopatric speciation. Little is known about secondary contacts of recently diverged anuran species. Here we conduct a multi-locus field study to investigate a contact zone between two lineages of green toads with an estimated divergence time of 2.7 My, and report results from preliminary experimental crosses.

Results: The Sicilian endemic Bufo siculus and the Italian mainland-origin B. balearicus form a narrow hybrid zone east of Mt. Etna. Despite bidirectional mtDNA introgression over a ca. 40 km North-South cline, no F1 hybrids could be found, and nuclear genomes display almost no admixture. Populations from each side of the contact zone showed depressed genetic diversity and very strong differentiation (FST = 0.52). Preliminary experimental crosses point to a slightly reduced fitness in F1 hybrids, a strong hybrid breakdown in backcrossed offspring (F1 x parental, with very few reaching metamorphosis) and a complete and early mortality in F2 (F1 x F1).

Conclusion: Genetic patterns at the contact zone are molded by drift and selection. Local effective sizes are reduced by the geography and history of the contact zone, B. balearicus populations being at the front wave of a recent expansion (late Pleistocene). Selection against hybrids likely results from intrinsic genomic causes (disruption of coadapted sets of genes in backcrosses and F2-hybrids), possibly reinforced by local adaptation (the ranges of the two taxa roughly coincide with the borders of semiarid and arid climates). The absence of F1 in the field might be due to premating isolation mechanisms. Our results, show that these lineages have evolved almost complete reproductive isolation after some 2.7 My of divergence, contrasting sharply with evidence from laboratory experiments that some anuran species may still produce viable F1 offspring after > 20 My of divergence.

Figure 1

Geographical overview of the study region (with population genetic clustering). a. Sampling sites across the entire study area with major mtDNA haplotype groups. Orange symbols: B. siculus; green symbols: B. balearicus. Haplotypes from both lineages were detected in three localities (pop. 13 to 15) east of Mt Etna with ratios shown as pie charts. b. Sampling sites of southern Apennine Peninsula, Sicily and two off-coast islands with mtDNA haplotype groups. Also plotted are assignment probabilities based on STRUCTURE analyses for all B. siculus individuals (K = 3, left) and all B. balearicus individuals (K = 3, right). For clusters (balearicus: b1 to b3, siculus: s1 to s3) see text; the dashed line (in b) between localities 14 and 15 refers to the region where the abrupt change for the nuclear markers is observed.

Figure 2

Phylogenetic trees of mitochondrial and nuclear markers. Maximum likelihood trees based on 577 bp of the mitochondrial d-loop (left, a), and of several clones (cl.) obtained from 580 bp of an intron of tropomyosine, situated between exons 5 and 6 (right, b). Specimen number (sometimes several with same haplotype and locality) is followed by locality information and population number (as in Figure 1 and Table 1). Individuals highlighted in colour possess a d-loop haplotype group of one species but tropomyosine alleles from the opposite species.

Figure 3

Genotype-based assignment of Sicilian green toads based on Bayesian cluster analyses. Bar plots from the program Structure based on seven microsatellite markers for green toads coming from Sicily for K = 2. Population 25 represents F₁-hybrids coming from a laboratory cross between one female B. balearicus (pop. 11) with a male B. siculus (pop. 22).

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.

Figure 5

Crosses of green toads from Sicily. a: Cross B. balearicus female × B. siculus male; b: reciprocal cross B. siculus × B. balearicus; c-h F₁-offspring from cross shown in a; c-d: offspring in the age of seven days, showing dead and malformed embryos and tadpoles in comparison with apparently normally developing ones; e: about one-months old normal tadpole (left) in comparison with leucistic "large" tadpole (right); f: in the age of two months (from left to right): retarded tadpole, "giant" leucistic tadpole with developmental arrest, malformed dwarfed tadpole, leucistic tadpole that turned later out to be incapable of metamorphosis, apparently normally metamorphosing tadpole; g: adult, two-year-old F₁-male; h: adult, two-year-old F₁-female. Photographs: M. Stöck.

Figure 6

Backcrosses of F₁ (B. balearicus × B. siculus, Fig. 6a) to parental species of Sicilian green toads. a-d: Wild-caught animals involved in backcrosses. a: B. balearicus female; b: B. balearicus male; c: B. siculus female; d: B. siculus male. e-h: Backcrosses. e: female F₁ (B. balearicus × B. siculus) × male B. balearicus; f: female B. balearicus × male F₁ (B. balearicus × B. siculus); g: female F₁ (B. balearicus × B. siculus) × male B. siculus; female B. siculus × male F₁ (B. balearicus × B. siculus). Photographs: a-d: G.F. Turrisi; e-h: M. Stöck.

Figure 7

Survival and development of backcrosses. a, c to e, g: tadpoles, one week after spawning. a: female B. siculus × male F₁ (B. balearicus × B. siculus) - note dead embryos and malformations; b: postmetamorphic toadlets exhibited size differences among siblings and low survival; c: F₂ from among hybrid crosses, female F₁ (B. balearicus × B. siculus) × male F₁ (B. balearicus × B. siculus) - all tadpoles malformed; d: female F₁ (B. balearicus × B. siculus) × male B. balearicus - most tadpoles malformed; e, f: female B. balearicus × male F₁ (B. balearicus × B. siculus) - note enormous size differences among siblings in the age of one month after spawning (f); g: female F₁ (B. balearicus × B. siculus) × male B. siculus - note most tadpoles show malformations; h: F₁-hybrid from crossing female B. siculus × male B. balearicus (as shown in Figure 6b) in the age of one year. Photographs: M. Stöck.