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Two transects reveal remarkable variation in gene flow on opposite ends of a European toad hybrid zone


Speciation entails a reduction in gene flow between lineages. The rates at which genomic regions become isolated varies across space and time. Barrier markers are linked to putative genes involved in (processes of) reproductive isolation, and, when observed over two transects, indicate species-wide processes. In contrast, transect-specific putative barrier markers suggest local processes. We studied two widely separated transects along the 900 km hybrid zone between Bufo bufo and B. spinosus, in northern and southern France, for ~1200 RADseq markers. We used genomic and geographic cline analyses to identify barrier markers based on their restricted introgression, and found that some markers are transect-specific, while others are shared between transects. Twenty-six barrier markers were shared across both transects, of which some are clustered in the same chromosomal region, suggesting that their associated genes are involved in reduced gene flow across the entire hybrid zone. Transect-specific barrier markers were twice as numerous in the southern than in the northern transect, suggesting that the overall barrier effect is weaker in northern France. We hypothesize that this is consistent with a longer period of secondary contact in southern France. The smaller number of introgressed genes in the northern transect shows considerably more gene flow towards the southern (B. spinosus) than the northern species (B. bufo). We hypothesize that hybrid zone movement in northern France and hybrid zone stability in southern France explain this pattern. The Bufo hybrid zone provides an excellent opportunity to separate a general barrier effect from localized gene flow-reducing conditions.

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Fig. 1: Overview of the Bufo hybrid zone and genetic data presented in this study.
Fig. 2: Markers identified as β or α outlier by Bayesian genomic cline (BGC) analysis, also stand out in their geographic cline when comparing the parameters ‘center’ and ‘width’ (HZAR).
Fig. 3: Geographic cline graphs coloured by Bayesian Genomic Cline (BGC) analysis outlier identification.
Fig. 4: Alignment of RAD markers to the B. bufo genome for each transect.

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Supplementary figures, tables, and text can be downloaded from the online article version. Raw data used in this study are available on GenBank under NCBI BioProject PRJNA954537. Data assembly, validation, and analysis code and input files can be found on Dryad:


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We thank the reviewers and editor for their comments, which helped improve the manuscript. We thank Frido Welker, Tara Luckau, and the members of the Butlin, Allentoft, Richards, and Bossdorf laboratories for support and discussion. IvR was supported by the ‘Nederlandse Organisatie voor Wetenschappelijk Onderzoek’ (NWO Open Programme 824.14.014). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 655487. Part of this project was carried out by IvR at the Shaffer laboratory in Los Angeles, at the University of California. This study trip has been sponsored by the Leiden University Fund / Swaantje Mondt Fonds (D7102). MR was funded by the Hasselblad Foundation Grant to Female Scientists, a grant from the Swedish Research Council Formas (grant number: 2019-00882), from the Swedish Research Council Vetenskapsrådet (grant number: 2021-05243), and by additional grants from Swedish Research Councils (Formas and VR) to the Centre for Marine Evolutionary Biology at the University of Gothenburg. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.

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IvR, JWA, BS, and BW designed the study. IvR and JWA collected samples. IvR performed the laboratory work and data assembly with contributions from GB, EMM, PS, and ET. IvR analyzed and interpreted the data with contributions from BS, BW, JWA, MR, and PS. IvR, BW, and BS wrote the manuscript with input from all authors.

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Correspondence to I. van Riemsdijk.

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van Riemsdijk, I., Arntzen, J.W., Bucciarelli, G.M. et al. Two transects reveal remarkable variation in gene flow on opposite ends of a European toad hybrid zone. Heredity 131, 15–24 (2023).

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