No signs of inbreeding despite long-term isolation and habitat fragmentation in the critically endangered Montseny brook newt (Calotriton arnoldi)

Endemic species with restricted geographic ranges potentially suffer the highest risk of extinction. If these species are further fragmented into genetically isolated subpopulations, the risk of extinction is elevated. Habitat fragmentation is generally considered to have negative effects on species survival, despite some evidence for neutral or even positive effects. Typically, non-negative effects are ignored by conservation biology. The Montseny brook newt (Calotriton arnoldi) has one of the smallest distribution ranges of any European amphibian (8 km2) and is considered critically endangered by the International Union for Conservation of Nature. Here we apply molecular markers to analyze its population structure and find that habitat fragmentation owing to a natural barrier has resulted in strong genetic division of populations into two sectors, with no detectable migration between sites. Although effective population size estimates suggest low values for all populations, we found low levels of inbreeding and relatedness between individuals within populations. Moreover, C. arnoldi displays similar levels of genetic diversity to its sister species Calotriton asper, from which it separated around 1.5 million years ago and which has a much larger distribution range. Our extensive study shows that natural habitat fragmentation does not result in negative genetic effects, such as the loss of genetic diversity and inbreeding on an evolutionary timescale. We hypothesize that species in such conditions may evolve strategies (for example, special mating preferences) to mitigate the effects of small population sizes. However, it should be stressed that the influence of natural habitat fragmentation on an evolutionary timescale should not be conflated with anthropogenic habitat loss or degradation when considering conservation strategies

Integrative Phylogeography of Calotriton Newts (Amphibia, Salamandridae), with Special Remarks on the Conservation of the Endangered Montseny Brook Newt (Calotriton arnoldi)

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. © 2013 Valbuena-Ureña et al.This work was funded by Forestal Catalana, S.A., and DAAM of the Generalitat de Catalunya. Miloca and Zoo de Barcelona (PRIC-2011) also helped with the funding of this study.Peer Reviewe

Preliminary genetic structure of the endemic Montseny brook newt, Calotriton arnoldi inferred from mtDNA and nDNA

Trabajo presentado en el I Congreso Ibérico de Sistemática Animal (CISA2012), celebrado en Madrid del 17 al 19 de enero de 2012.The Montseny brook newt Calotriton arnoli, endemic to the Montseny massif (NE Iberian Peninsula), is catalogued as critically endangered by the IUCN. This rare and threatened species inhabits oligotrophic, cold (below 15°C) fast running brooks. Given its small population size and small distribution area, management measures should be considered. Genetic studies are a very useful tool and therefore are widely implemented in many conservation programs. Two mitochondrial (ND4 and Cyt b) and one nuclear (RAG-1) marker from 105 individuals were used to infer the population genetic structure and phylogeography of this species, covering its total distribution range. Both mitochondrial and nuclear markers confirm the differentiation between C. asper and C. arnoldi. Moreover, haplotype networks and analysis of molecular variance suggest that two distinct groups of populations can be differentiated within C. arnoldi. This is in concordance with morphological differentiation, and correlates with its geographical distribution, as the two groups are situated on each side of the Tordera river valley. Glaciations occurred during the lasts periods may have forced the species to retreat into these two isolated areas, with a subsequent extinction of the in-between populations. The limited dispersion capability owing to its totally aquatic behaviour caused the isolation of these two groups of populations, and the consequent independent evolution. Thus, these results strongly justify the management of this species as two independent ESU’s (Evolutionary Significant Units) to guarantee the long-term population viability.Peer reviewe

Characterization of microsatellite loci markers for the critically endangered Montseny brook newt (Calotriton arnoldi)

Fifteen novel tetranucleotide polymorphic microsatellite loci are described and characterized for the critically endangered Montseny brook newt Calotriton arnoldi. Until now little is known about genetic variability and dispersal of this species across its very limited distribution area, mainly represented by two main sectors (eastern and western from the Tordera river valley). The level of genetic diversity of the new loci was assessed in 23 individuals from the eastern sector. Number of alleles per locus ranged from 2 to 8 (mean = 4.7) and observed and expected heterozygosity ranged from 0.087 to 1.000 and 0.162 to 0.814, respectively. Almost all loci were found to be in Hardy-Weinberg equilibrium and were not linked. These polymorphic loci can be used in population structure, gene flow, and population differentiation. The value of such information should be high for successful management and conservation of this potentially highly threatened species. © 2013 Springer Science+Business Media Dordrecht.This research was supported by Miloca and Zoo de Barcelona (PRIC-2011).Peer Reviewe

Phylogeography of the critically endangered Montseny Newt (Calotriton arnoldi) inferred from the microsatellite markers analyses: implications for its conservation

Trabajo presentado en el II Iberian Congress of Biological Systematics (CISA2013), celebrado en Barcelona del 25 al 27 de septiembre de 2013.N

Genètica aplicada a la conservació del tritó del Montseny (Calotriton arnoldi)

Trabajo presentado en las XV Jornades Herpetològiques Catalanes, celebradas en Sant Feliu de Codines del 10 al 12 de octubre de 2012.The Montseny brook newt (Calotriton arnoldi) is one of the most endangered amphibians in Europe. One key aspect for the conservation of this species is to understand its population genetics. To date, all known localities have been analyzed using mitochondrial (Cyt b) and nuclear (RAG-1 and microsatellites) genes in order to investigate its population structure, gene flow and genetic diversity. Results obtained from these studies are surprising. Although this species has an extremely reduced distribution range, the levels of genetic diversity found are similar to other non-endangered urodel species. It has been confirmed that there are two genetically isolated population sectors, eastern and western, being the former one the most diverse. Therefore, it is stated that these two significant units may be conserved separately. Another significant and surprising aspect of this singular species is the strong population genetic structure, even within sectors. Nevertheless, the low levels of effective population size are concerning, and agree to the categorization of this species by the IUCN as critically endangered. The results obtained are important in order to correctly design the conservation management of this Catalan endemism.N

What remains of a 454 run: estimation of success rate of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, Triturus cristatus)

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