Integrative phylogeography of Calotriton newts (Amphibia, Salamandridae), with special remarks on the conservation of the endangered Montseny brook newt (Calotriton arnoldi)

Abstract

The genus Calotriton includes two species of newts highly adapted to live in cold and fast-flowing mountain springs. The Pyrenean brook newt (Calotriton asper), restricted to the Pyrenean region, and the Montseny brook newt (Calotriton arnoldi), endemic to the Montseny massif and one of the most endangered amphibian species in Europe. In the present manuscript, we use an integrative approach including species distribution modeling (SDM), molecular analyses of mitochondrial and nuclear DNA sequence data and morphology to unravel the historical processes that have contributed to shaping the biogeography and genetic structure of the genus Calotriton, with special emphasis on the conservation of C. arnoldi. The results of the molecular analyses confirm that, despite having originated recently, being ecologically similar and geographically very close, there is no signal of hybridization between C. asper and C. arnoldi. SDM results suggest that tough environmental conditions on mountains tops during glacial periods, together with subsequent warmer periods could have prevented the contact between the two species. Within the critically endangered C. arnoldi, a high genetic structure is revealed despite its extremely small distribution range compared to C. asper. Haplotype networks, AMOVA and SAMOVA analyses suggest that two distinct groups of populations can be clearly differentiated with absence of gene flow. This is in concordance with morphological differentiation and correlates with its geographical distribution, as the two groups are situated on the eastern and western sides of a river valley that acts as a barrier. The genetic and morphological results are highly important for the ongoing conservation program of C. arnoldi and strongly justify the management of this species into at least two independent evolutionary significant units (eastern and western sectors) to guarantee the long-term population viability.

Figure 1. The study area in the NE Iberian Peninsula showing the distribution of the genus Calotriton.

Circles indicate populations of C. asper included in the molecular analyses; triangles correspond to additional populations included in the species distribution modeling (SDM). All localities of C. arnoldi are included in the molecular analyses. Locality codes correspond to names on Table 1. Localities of C. arnoldi represented do not correspond to the exact geographic locations intentionally due to conservation reasons.

Figure 2. Map showing the geographical distribution of Cyt b DNA haplotypes.

(A) Pie diagram size indicates number of individuals. (B) Statistical parsimony networks showing cytochrome b haplotypes found for Calotriton arnoldi (left) and C. asper (right); circle size are proportional to haplotype abundance, straight lines and black dots reflect mutations and unsampled or extinct haplotype.

Figure 3. Map showing the geographical distribution of RAG-1 DNA haplotypes.

(A) Pie diagram size indicates number of individuals. (B) Statistical parsimony network showing nuclear haplotypes found for Calotriton genus; circle sizes are proportional to haplotype abundance, straight lines and black dots reflect mutations and unsampled or extinct haplotype.

Figure 4. Predicted distribution models of Calotriton asper.

(A) Present distribution using climate and topographic variables, landcover and lithology. Last Glacial Maximum based on (B) CCSM model and (C) MIROC model, and (D) Last Interglacial distributions using climate variables and lithology. Warmer colors represent areas of high habitat suitability. Red marks and arrows indicate the Montseny Mountain Natural Park.

Figure 5. Discriminant canonical analysis scatterplot of males and females of Calotriton arnoldi from eastern and western sectors.

Red squares and circles indicate eastern males and females, and blue squares and circles show western males and females, respectively. Black points are the mean values for each group.

Figure 6. Characteristic patterns of coloration of newts belonging to the two sectors of Calotriton arnoldi range.

(A) Adult female from the eastern populations showing yellow spots found in 78.8% of the individuals, and (B) adult male from the western populations with the whitish margin of the snout.