Profound genetic divergence and asymmetric parental genome contributions as hallmarks of hybrid speciation in polyploid toads.

The evolutionary causes and consequences of allopolyploidization, an exceptional pathway to instant hybrid speciation, are poorly investigated in animals. In particular, when and why hybrid polyploids versus diploids are produced, and constraints on sources of paternal and maternal ancestors, remain underexplored. Using the Palearctic green toad radiation (including bisexually reproducing species of three ploidy levels) as model, we generate a range-wide multi-locus phylogeny of 15 taxa and present four new insights: (i) at least five (up to seven) distinct allotriploid and allotetraploid taxa have evolved in the Pleistocene; (ii) all maternal and paternal ancestors of hybrid polyploids stem from two deeply diverged nuclear clades (6 Mya, 3.1-9.6 Mya), with distinctly greater divergence than the parental species of diploid hybrids found at secondary contact zones; (iii) allotriploid taxa possess two conspecific genomes and a deeply diverged allospecific one, suggesting that genomic imbalance and divergence are causal for their partly clonal reproductive mode; (iv) maternal versus paternal genome contributions exhibit asymmetry, with the maternal nuclear (and mitochondrial) genome of polyploids always coming from the same clade, and the paternal genome from the other. We compare our findings with similar patterns in diploid/polyploid vertebrates, and suggest deep ancestral divergence as a precondition for successful allopolyploidization

Phylogeography of Aegean green toads (Bufo viridis subgroup): continental hybrid swarm vs. insular diversification with discovery of a new island endemic

BACKGROUND: Debated aspects in speciation research concern the amount of gene flow between incipient species under secondary contact and the modes by which post-zygotic isolation accumulates. Secondary contact zones of allopatric lineages, involving varying levels of divergence, provide natural settings for comparative studies, for which the Aegean (Eastern Mediterranean) geography offers unique scenarios. In Palearctic green toads (Bufo viridis subgroup or Bufotes), Plio-Pleistocene (~ 2.6 Mya) diverged species show a sharp transition without contemporary gene flow, while younger lineages, diverged in the Lower-Pleistocene (~ 1.9 Mya), admix over tens of kilometers. Here, we conducted a fine-scale multilocus phylogeographic analysis of continental and insular green toads from the Aegean, where a third pair of taxa, involving Mid-Pleistocene diverged (~ 1.5 Mya) mitochondrial lineages, earlier tentatively named viridis and variabilis, (co-)occurs. RESULTS: We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids. CONCLUSIONS: The genetic signatures of insular Aegean toads appear governed by bathymetry and Quaternary sea level changes, resulting in long-term isolation (Central Cyclades: Naxos) and recent land-bridges (coastal islands). Conversely, Crete has been isolated since the end of the Messinian salinity crisis (5.3 My) and Cretan populations thus likely result from human-mediated colonization, at least since Antiquity, from Peloponnese and Anatolia. Comparisons of green toad hybrid zones support the idea that post-zygotic hybrid incompatibilities accumulate gradually over the genome. In this radiation, only one million years of divergence separate a scenario of complete reproductive isolation, from a secondary contact resulting in near panmixia

Genomic Evidence for Cryptic Speciation in Tree Frogs From the Apennine Peninsula, With Description of Hyla perrini sp. nov

Despite increasing appreciation of the speciation continuum, delimiting and describing new species is a major yet necessary challenge of modern phylogeography to help optimize conservation efforts. In amphibians, the lack of phenotypic differences between closely-related taxa, their complex, sometimes unresolved phylogenetic relationships, and their potential to hybridize all act to blur taxonomic boundaries. Here we implement a multi-disciplinary approach to evaluate the nature of two deeply-diverged mitochondrial lineages previously documented in Italian tree frogs (Hyla intermedia s. l.), distributed north and south of the Northern Apennine Mountains. Based on evidence from mitochondrial phylogenetics, nuclear phylogenomics, hybrid zone population genomics, niche modeling analyses, and biometric assessments, we propose that these lineages be considered distinct, cryptic species. Both mitochondrial and nuclear data affirm that they belong to two monophyletic clades of Pliocene divergence (~3.5 My), only admixing over a relatively narrow contact zone restricted to the southeast of the Po Plain (50–100 km). These characteristics are comparable to similarly-studied parapatric amphibians bearing a specific status. Inferred from their current geographic distribution, the two Italian tree frogs feature distinct ecological niches (<15% of niche overlap), raising questions regarding potential adaptive components contributing to their incipient speciation. However, we found no diagnostic morphological and bioacoustic differences between them. This system illustrates the speciation continuum of Western-Palearctic tree frogs and identifies additional cryptic lineages of similar divergence to be treated as separate species (H. cf. meridionalis). We recommend combined approaches using genomic data as applied here for the future taxonomic assessment of cryptic diversity in alloparapatric radiations of terrestrial vertebrates, especially in controversial taxa. Finally, we formally described the northern Italian tree frogs as a new species, Hyla perrini sp. nov

GENOME EVOLUTION IN GREEN TOADS

Speciation, the evolutionary process by which one species splits into two, is characterized by the évolution of reproductive isolation between diverging lineages allowing the maintenance of their genetic and phenotypic distinctiveness. Reproductive isolating mechanisms are assumed to accumulate gradually as a function of molecular genetic distance and therefore to correlate with time divergence. If reproductive isolation is incomplète, secondary contact between closely related allopatric lineages may lead to hybridization and introgression that challenge speciation process. We have compared the degree of hybridization in two secondary contact zones between lineages from the same radiation with différent divergence times. In contact zone between lineages of more recent divergence, advanced degrees of hybridization suggest that no major reproductive barriers limit the gene flow whereas in contact zone between lineages of deeper divergence, restricted signs of past hybridization suggest that lineages have evolved almost complété reproductive isolation. Hybridization may also create genetic diversity by the formation of entirely new species, through allopolyploidization for instance. Newly formed allopolyploid, with a complété set of chromosomes from each parental species merged in a common nucleus, will undergo an evolutionary trajectory of instability where genomic reorganization is widespread and re- diploidization necessary to obtain adapted polyploids. The duplication of sex- determining genes represents a challenge for the formation of polyploid animais that often display déviations from sexual and/or meiotic reproduction. We have highlighted that six independent events of allopolyploidization were ail unidirectional in terms of ancestry with two diploid forms representing the maternai ancestors and another diploid lineage representing the paternal ancestor. We have also identified rearrangements events occurring only within the paternal subgenomes of allopolyploid toads and exclusively affected the linkage group determining sex. We have finally described for the first time the reproductive mode of a bisexually reproducing triploid green toad where meiotic processes differ between sexes and females display clonal and sexual reproduction simultaneously. -- La spéciation, le processus évolutif par lequel une espèce se divise en deux, est caractérisé par l'évolution de l'isolement reproductif entre les lignées permettant le maintien de leur distinction génétique et phénotypique. On suppose que les mécanismes d'isolement reproductif s'accumulent progressivement en fonction de la distance génétique et donc, en fonction du temps depuis la divergence. Si l'isolement reproductif est incomplet, le contact secondaire entre des lignées allopatriques apparentées, peut entraîner de l'hybridation. Nous avons comparé le degré d'hybridation dans deux zones de contact secondaire entre des lignées de la même radiation possédant des temps différents de divergence. Dans la zone de contact entre les lignées qui se sont séparées récemment, le fort degré d'hybridation suggère qu'aucune barrière reproductive majeure ne limite le flux de gènes alors qu'entre les lignées de divergence plus profonde, des signes restreints d'hybridation passée suggèrent qu'elles ont presque atteint l'isolement reproductif définitif. L'hybridation peut également créer de la diversité génétique en formant de nouvelles espèces notamment par allopolyploïdisation. Un allopolyploïde possède donc un jeu de chromosomes de chaque espèce parentale, fusionné au sein d'un noyau commun. Il peut subir une période évolutive d'instabilité où il va être réorganisé génomiquement. La duplication des gènes déterminant le sexe représente un défi pour la formation d'animaux polyploïdes qui affichent souvent des déviations de la reproduction sexuée ou de la méiose. Nous avons découvert six événements indépendants d'allopolyploïdisation chez les crapauds verts et tous étaient unidirectionnels en termes d'ascendance avec deux formes diploïdes représentant les ancêtres maternels et une autre lignée diploïde représentant l'ancêtre paternel. Nous avons également identifié des événements de réorganisation génomique dans les sous-génomes paternels de crapauds allopolyploïdes ayant lieu uniquement dans le groupe de liaison déterminant le sexe. Nous avons enfin décrit pour la première fois le mode de reproduction d'un crapaud vert triploïde où les processus de la méiose diffèrent entre les sexes et les femelles effectuent simultanément reproduction clonale et sexuelle

Thirteen polymorphic microsatellite markers for the European green toad Bufo viridis viridis, a declining amphibian species.

We report 13 new polymorphic microsatellite markers for the European green toad Bufo viridis viridis (B. viridis subgroup), a declining amphibian from Central, Southeastern and Eastern Europe. Diversity at these loci estimated for 19 individuals ranged from two to ten alleles. Most of these primers also cross-amplify in related West-Mediterranean green toad species (Bufo balearicus, B. siculus and B. boulengeri). These microsatellites will be useful for conservation genetics of threatened Bufo viridis viridis populations and evolutionary studies of green toad taxa in secondary contact to examine hybridization

Development and cross-amplification of thirty microsatellite loci in five diploid and polyploid Central Asian species of Palearctic green toads (Bufo viridis subgroup)

We report 30 polymorphic microsatellite markers for five species of Palearctic green toads (Bufo viridis subgroup): 23 in the diploid B. latastii, 19 in diploid B. turanensis, 20 in diploid B. shaartusiensis, 27 in tetraploid B. pewzowi, and 30 in triploid B. baturae. Genetic diversity at these loci, measured for 10-18 individuals per species, ranged from 2 to 19 alleles. These microsatellite loci will be useful for conservation plans (genetic diversity, population structure, evolutionary units), inheritance patterns, and evolution of green toads

Homologous sex chromosomes in three deeply divergent anuran species.

Comparative genomic studies are revealing that, in sharp contrast with the strong stability found in birds and mammals, sex determination mechanisms are surprisingly labile in cold-blooded vertebrates, with frequent transitions between different pairs of sex chromosomes. It was recently suggested that, in context of this high turnover, some chromosome pairs might be more likely than others to be co-opted as sex chromosomes. Empirical support, however, is still very limited. Here we show that sex-linked markers from three highly divergent groups of anurans map to Xenopus tropicalis scaffold 1, a large part of which is homologous to the avian sex chromosome. Accordingly, the bird sex determination gene DMRT1, known to play a key role in sex differentiation across many animal lineages, is sex linked in all three groups. Our data provide strong support for the idea that some chromosome pairs are more likely than others to be co-opted as sex chromosomes because they harbor key genes from the sex determination pathway

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination

Fifteen shades of green: The evolution of Bufotes toads revisited.

The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (<6My), above which species barely admix and eventually evolve different mating calls (6-10My), or can successfully cross-breed through allopolyploidization (>15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians