Hybrid zone analysis confirms cryptic species of banded newt and does not support competitive displacement since secondary contact.

When two putatively cryptic species meet in nature, hybrid zone analysis can be used to estimate the extent of gene flow between them. Two recently recognized cryptic species of banded newt (genus Ommatotriton) are suspected to meet in parapatry in Anatolia, but a formal hybrid zone analysis has never been conducted. We sample populations throughout the range, with a focus on the supposed contact zone, and genotype them for 31 nuclear DNA SNP markers and mtDNA. We determine the degree of genetic admixture, introgression, and niche overlap. We reveal an extremely narrow hybrid zone, suggesting strong selection against hybrids, in line with species status. The hybrid zone does not appear to be positioned at an ecological barrier, and there is significant niche overlap. Therefore, the hybrid zone is best classified as a tension zone, maintained by intrinsic selection against hybrids. While the two banded newt species can evidently backcross, we see negligible introgression and the pattern is symmetric, which we interpret as supporting the fact that the hybrid zone has been practically stationary since its origin (while extensive hybrid zone movement has been suggested in other newt genera in the region). Our study illustrates the use of hybrid zone analysis to test cryptic species status.

Genomic analysis reveals complex population structure within the smooth newt, Lissotriton vulgaris, in Central Europe.

Species with wide-range distributions usually display high genetic variation. This variation can be partly explained by historical lineages that were temporally isolated from each other and are back into secondary reproductive contact, and partly by local adaptations. The smooth newt (Lissotriton vulgaris) is one of the most widely distributed amphibians species across Eurasia and forms a species complex with a partially overlapping distribution and morphology. In the present study, we explored the population genomic structure of smooth newt lineages in the Carpathian Basin (CB) relying on single-nucleotide polymorphisms. Our dataset included new and previously published data to study the secondary contact zone between lineages in the CB and also tested for the barrier effect of rivers to gene flow between these lineages. We confirmed the presence of the South L. v. vulgaris Lineage distributed in Transdanubia and we provided new distribution records of L. v. ampelensis inhabiting the eastern territories of the CB. High genetic diversity of smooth newts was observed, especially in the North Hungarian Mountains and at the interfluves of the main rivers in the South with four distinct lineages of L. v. vulgaris and one lineage of L. v. ampelensis showing a low level of admixture with the spatially closest L. v. vulgaris lineage. Moreover, admixture detected at the interfluve of the main rivers (i.e. Danube and Tisza) suggested a secondary contact zone in the area. Finally, we found that the river Danube has a very weak effect on population divergence, while the river Tisza is a geographical barrier limiting gene flow between smooth newt lineages. As the range boundaries of L. v. vulgaris and L. v. ampelensis in the CB coincide with the river Tisza, our study underpins the influence of rivers in lineage diversification.

Genetic traces of hybrid zone movement across a fragmented habitat.

Theoretical and empirical studies suggest that the structure and position of hybrid zones can change over time. Evidence for moving hybrid zones has been directly inferred by repeated sampling over time, or indirectly through the detection of genetic footprints left by the receding species and the resulting asymmetric patterns of introgression across markers. We here investigate a hybrid zone formed by two subspecies of the Iberian golden-striped salamander, Chioglossa lusitanica, using a panel of 35 nuclear loci (31 SNPs and 4 allozymes) and one mitochondrial locus in a transect in central Portugal. We found concordant and coincident clines for most of the nuclear loci (n = 22, 63%), defining a narrow hybrid zone of ca. 6 km wide, with the centre positioned ca. 15 km south of the Mondego River. Asymmetric introgression was observed at another 14 loci. Their clines are displaced towards the north, with positions located either close to the Mondego River (n = 6) or further northwards (n = 8). We interpret these profiles as genetic traces of the southward displacement of C. lusitanica lusitanica by C. l. longipes over the wider Mondego River valley. We noted the absence of significant linkage disequilibrium, and we inferred low levels of effective selection per locus against hybrids, suggesting that introgression in the area of species replacement occurred under a neutral diffusion process. A species distribution model suggests that the C. lusitanica hybrid zone coincides with a narrow corridor of fragmented habitat. From the position of the displaced clines, we infer that patches of locally suitable habitat trapped some genetic variants that became disassociated from the southward moving hybrid zone. This study highlights the influence of habitat availability on hybrid zone movement.

Next-generation phylogeography of the banded newts (Ommatotriton): A phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure.

Technological developments now make it possible to employ many markers for many individuals in a phylogeographic setting, even for taxa with large and complex genomes such as salamanders. The banded newt (genus Ommatotriton) from the Near East has been proposed to contain three species (O. nesterovi, O. ophryticus and O. vittatus) with unclear phylogenetic relationships, apparently limited interspecific gene flow and deep intraspecific geographic mtDNA structure. We use parallel tagged amplicon sequencing to obtain 177 nuclear DNA markers for 35 banded newts sampled throughout the range. We determine population structure (with Bayesian clustering and principal component analysis), interspecific gene flow (by determining the distribution of species-diagnostic alleles) and phylogenetic relationships (by maximum likelihood inference of concatenated sequence data and based on a summary-coalescent approach). We confirm that the three proposed species are genetically distinct. A sister relationship between O. nesterovi and O. ophryticus is suggested. We find evidence for introgression between O. nesterovi and O. ophryticus, but this is geographically limited. Intraspecific structuring is extensive, with the only recognized banded newt subspecies, O. vittatus cilicensis, representing the most distinct lineage below the species level. While mtDNA mostly mirrors the pattern observed in nuclear DNA, all banded newt species show mito-nuclear discordance as well.

Tracing species replacement in Iberian marbled newts.

Secondary contact between closely related species can lead to the formation of hybrid zones, allowing for interspecific gene flow. Hybrid zone movement can take place if one of the species possesses a competitive advantage over the other, ultimately resulting in species replacement. Such hybrid zone displacement is predicted to leave a genomic footprint across the landscape in the form of asymmetric gene flow (or introgression) of selectively neutral alleles from the displaced to the advancing species. Hybrid zone movement has been suggested for marbled newts in the Iberian Peninsula, supported by asymmetric gene flow and a distribution relict (i.e., an enclave) of Triturus marmoratus in the range of T. pygmaeus. We developed a panel of nuclear and mitochondrial SNP markers to test for the presence of a T. marmoratus genomic footprint in the Lisbon peninsula, south of the enclave. We found no additional populations of T. marmoratus. Analysis with the software Structure showed no genetic traces of T. marmoratus in T. pygmaeus. A principal component analysis showed some variation within the local T. pygmaeus, but it is unclear if this represents introgression from T. marmoratus. The results may be explained by (a) species replacement without introgressive hybridization and (b) displacement with hybridization followed by the near-complete erosion of the footprint by purifying selection. We predict that testing for a genomic footprint north of the reported enclave would confirm that species replacement in these marbled newts occurred with hybridization.

Testing an hypothesis of hybrid zone movement for toads in France.

Hybrid zone movement may result in substantial unidirectional introgression of selectively neutral material from the local to the advancing species, leaving a genetic footprint. This genetic footprint is represented by a trail of asymmetric tails and displaced cline centres in the wake of the moving hybrid zone. A peak of admixture linkage disequilibrium is predicted to exist ahead of the centre of the moving hybrid zone. We test these predictions of the movement hypothesis in a hybrid zone between common (Bufo bufo) and spined toads (B. spinosus), using 31 nuclear and one mtDNA SNPs along a transect in the northwest of France. Average effective selection in Bufo hybrids is low and clines vary in shape and centre. A weak pattern of asymmetric introgression is inferred from cline discordance of seven nuclear markers. The dominant direction of gene flow is from B. spinosus to B. bufo and is in support of southward movement of the hybrid zone. Conversely, a peak of admixture linkage disequilibrium north of the hybrid zone suggests northward movement. These contrasting results can be explained by reproductive isolation of the B. spinosus and B. bufo gene pools at the southern (B. spinosus) side of the hybrid zone. The joint occurrence of asymmetric introgression and admixture linkage disequilibrium can also be explained by the combination of low dispersal and random genetic drift due to low effective population sizes.

The Near East as a cradle of biodiversity: A phylogeography of banded newts (genus Ommatotriton) reveals extensive inter- and intraspecific genetic differentiation.

The banded newt (genus Ommatotriton) is widely distributed in the Near East (Anatolia, Caucasus and the Levant) - an understudied region from the perspective of phylogeography. The genus is polytypic, but the number of species included and the phylogenetic relationships between them are not settled. We sequenced two mitochondrial and two nuclear DNA markers throughout the range of Ommatotriton. For mtDNA we constructed phylogenetic trees, estimated divergence times using fossil calibration, and investigated changes in effective population size with Bayesian skyline plots and mismatch analyses. For nuDNA we constructed phylogenetic trees and haplotype networks. Species trees were constructed for all markers and nuDNA only. Species distribution models were projected on current and Last Glacial Maximum climate layers. We confirm the presence of three Ommatotriton species: O. nesterovi, O. ophryticus and O. vittatus. These species are genetically distinct and their most recent common ancestor was dated at ∼25Ma (Oligocene). No evidence of recent gene flow between species was found. The species show deep intraspecific genetic divergence, represented by geographically structured clades, with crown nodes of species dated ∼8-13Ma (Miocene to Early Quaternary); evidence of long-term in situ evolution and survival in multiple glacial refugia. While a species tree based on nuDNA suggested a sister species relationship between O. vittatus and O. ophryticus, when mtDNA was included, phylogenetic relationships were unresolved, and we refrain from accepting a particular phylogenetic hypothesis at this stage. While species distribution models suggest reduced and fragmented ranges during the Last Glacial Maximum, we found no evidence for strong population bottlenecks. We discuss our results in the light of other phylogeographic studies from the Near East. Our study underlines the important role of the Near East in generating and sustaining biodiversity.