Applicability of mitochondrial DNA for the identification of Arvicolid species from faecal samples: a case study from the threatened Cabrera’s vole

Molecular Ecology Resources (Impact Factor: 7.43). 02/2011; 11(2):409 - 414. DOI: 10.1111/j.1755-0998.2010.02939.x

ABSTRACT Arvicolid mitochondrial genomes evolve faster than in any other mammalian lineage. The genetic diversity exhibited by these rodents contrasts sharply with their phenotypic homogeneity. Furthermore, faecal droppings from Arvicolid rodents of similar body size are almost undistinguishable on the basis of pellet morphology and content. In this study, we advantaged from their high genetic diversity vs. phenotypic homogeneity to document the applicability of mtDNA extraction from vole droppings for latter identification of such via a rapid and efficient nested PCR-based technique using the threatened Microtus cabrerae as a model species. We sequenced the mitochondrial control region from 75 individuals belonging to 11 species of Arvicolinae from Spain, Portugal, Greece and Italy, and an additional 19 sequences from ten Microtus species from other countries were downloaded from Genbank. Based on these control region sequences, we successfully designed and applied a nested PCR for M. cabrerae-specific and arvicolid-generic mtDNA markers to differentiate Cabrera’s vole faecal samples among other species of the Arvicolinae subfamily. Although this study used Cabrera’s vole as a model species, similar techniques based on mtDNA sequences may find a broader applicability for noninvasive genetic conservation of vole species and their populations.

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    ABSTRACT: Species identification through noninvasive sampling is increasingly used in animal conservation genetics, given that it obviates the need to handle free-living individuals. Noninvasive sampling is particularly valuable for elusive and small species such as rodents. Although rodents are not usually assumed to be the most obvious target for conservation, of the 21 species or near-species present in Iberia, three are considered endangered and declining, while several others are poorly studied. Here, we develop a genetic tool for identifying all rodent species in Iberia by noninvasive genetic sampling. To achieve this purpose, we selected one mitochondrial gene [cytochrome b (cyt-b)] and one nuclear gene [interphotoreceptor retinoid-binding protein (IRBP)], which we first sequenced using tissue samples. Both genes allow for the phylogenetic distinction of all species except the sibling species Microtus lusitanicus and Microtus duodecimcostatus. Overall, cyt-b showed higher resolution than IRBP, revealing a clear barcoding gap. To allow these markers to be applied to noninvasive samples, we selected a short highly diagnostic fragment from each gene, which we used to obtain sequences from faeces and bones from owl pellets. Amplification success for the cyt-b and IRBP fragment was 85% and 43% in faecal and 88% and 64% in owl-pellet DNA extractions, respectively. The method allows the unambiguous identification of the great majority of Iberian rodent species from noninvasive samples, with application in studies of distribution, spatial ecology and population dynamics, and for conservation.
    Molecular Ecology Resources 10/2012; · 7.43 Impact Factor
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    ABSTRACT: A cost-effective, reliable and efficient method of obtaining DNA samples is essential in large-scale genetic analyses. This study examines the possibility of using a threatened vole species, Microtus cabrerae, as a model for the collection and preservation of faecal samples for subsequent DNA extraction with a protocol based on the HotSHOT technique. Through the examination of the probability of multi-copies (mitochondrial) and single copy (microsatellite) loci amplification (including the genotype error) and of the DNA yield (estimated by real-time qPCR), the new protocol was compared with both the frequently employed methods that successfully use ethanol to preserve faecal samples and with commercial kit-based DNA extraction. The single-tube HotSHOT-based protocol is a user-friendly, non-polluting, time-saving and inexpensive method of faeces sample collection, preservation and PCR-quality gDNA preparation. This technique therefore provides researchers with a new approach that can be employed in high-throughput, noninvasive genetic analyses of wild animal populations.
    European Journal of Wildlife Research · 1.36 Impact Factor
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    ABSTRACT: Capturing wild animals can be time consuming and difficult or even impractical. Noninvasive sampling is potentially a cost-effective and efficient means to monitor wild animals, thereby avoiding the need of capture and disturb species in the wild. On the basis of the morphological and genetic analyses of owl pellet contents, a so far undetected European snow vole (Chionomys nivalis) population was discovered in the Sierra Segura mountain range (Southern Spain). The mtDNA sequence from the newly discovered haplotype was compared with sequences from vole populations of the Sierra Nevada and Sierra Peñalara mountain ranges (Spain) and from Churwalden (Switzerland). The nine recovered haplotypes clustered in four distinct lineages according to their geographical origin. The vole sequence from the Sierra Segura owl pellet belonged to a new haplotype, constituting a new lineage. The evolutionary divergence between sequences from the Sierra Segura and other Spanish populations was higher than that among other Spanish haplotypes. The new snow vole haplotype from this new locality duplicates the number of occurrence sites of this critically endangered species in Southern Spain, which is of great interest for further conservation and management plans of the European snow vole in the most southwestern area of its entire distribution range.
    Mitochondrial DNA 03/2013; · 1.71 Impact Factor


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May 31, 2014