Genetic identification of Iberian rodent species using both mitochondrial and nuclear loci: application to noninvasive sampling

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.

p53 gene discriminates two ecologically divergent sister species of pine voles

“…For p53 haplotypes, the TPM2uf model was chosen with uniform rates of variable sites with the following estimated nucleotide frequencies: A = 0.1947, C = 0.2537, G = 0.2938 and T = 0.2578; and substitution rate matrix: (AC) = 1495.6642, (AG) = 8493.0230, (AT) = 1495.6642, (CG) = 1.0000, (CT) = 8492.0230 and (GT) = 1.0000. Because a regional partition of cytb haplotypes according to introgression status was observed in M. lusitanicus (Bastos-Silveira et al, 2012;Barbosa et al, 2013), we reconstructed the phylogenetic tree of M. lusitanicus and M. duodecimcostatus using all the cytb sequences available in GenBank and assigned each specimen to its place of origin to investigate any connection between the geographical distribution of p53 and cytb haplotypes. A total of 129 cytb haplotypes were retrieved from all the published sequences of each species, 77 haplotypes from 79 individuals of M. lusitanicus and 52 from 55 specimens of M. duodecimcostatus (accession numbers in Supplementary Table S2; Jaarola et al, 2004;Tougard et al, 2008;Bastos-Silveira et al, 2012;Barbosa et al, 2013).…”

“…Because a regional partition of cytb haplotypes according to introgression status was observed in M. lusitanicus (Bastos-Silveira et al, 2012;Barbosa et al, 2013), we reconstructed the phylogenetic tree of M. lusitanicus and M. duodecimcostatus using all the cytb sequences available in GenBank and assigned each specimen to its place of origin to investigate any connection between the geographical distribution of p53 and cytb haplotypes. A total of 129 cytb haplotypes were retrieved from all the published sequences of each species, 77 haplotypes from 79 individuals of M. lusitanicus and 52 from 55 specimens of M. duodecimcostatus (accession numbers in Supplementary Table S2; Jaarola et al, 2004;Tougard et al, 2008;Bastos-Silveira et al, 2012;Barbosa et al, 2013). For the construction of the cytb phylogenetic tree the TIM3 substitution model was chosen with a proportion of invariable sites and a gamma-distributed rate variation across sites (TIM3+I+G).…”

“…The separation in two regional groups of populations for M. lusitanicus is also apparent in the reconstructed p53 discriminates two ecologically divergent pine voles AS Quina et al cytb phylogenetic gene tree (Figure 2). Two lineages with high posterior probabilities were observed, one that corresponded to M. lusitanicus of the northern regional group and the other to a cluster comprised of M. duodecimcostatus and introgressed individuals of M. lusitanicus (Bastos-Silveira et al, 2012; Barbosa et al, 2013). Notably, these introgressed M. lusitanicus were also subdivided with high posterior probability in the two regional groups separated by the Douro river, suggesting that regional separation occurred after secondary contact between M. lusitanicus and M. duodecimcostatus.…”

“…The two existing population-level studies on the genetic diversity of M. lusitanicus and M. duodecimcostatus in the Iberian Peninsula have found evidence for a past introgressive hybridization from M. duodecimcustatus to M. lusitanicus of the mitochondrial cytochrome b (cytb) gene (Bastos-Silveira et al, 2012), and possibly of the nuclear gene IRBP (interphotoreceptor retinoid-binding protein) given the impossibility to discriminate individuals from the two species when using this marker (Barbosa et al, 2013). Here we performed a population-genetic analysis involving 96 individuals (52 M. lusitanicus and 44 M. duodecimcostatus) sampled in the Iberian Peninsula, and the p53 DNA sequence encoding part of the DNAbinding region of P53, from the 3′ end of exon 5 to the 5′ extreme of exon 7, which includes residue 174.…”

“…Here we performed a population-genetic analysis involving 96 individuals (52 M. lusitanicus and 44 M. duodecimcostatus) sampled in the Iberian Peninsula, and the p53 DNA sequence encoding part of the DNAbinding region of P53, from the 3′ end of exon 5 to the 5′ extreme of exon 7, which includes residue 174. We used specimens captured both in allopatric and sympatric areas where introgressed animals have been observed (Jaarola et al, 2004;Tougard et al, 2008;Bastos-Silveira et al, 2012;Barbosa et al, 2013;Rodriguez-Prieto et al, 2014), to ultimately evaluate whether genes involved in the differentiation of species separated recently carry signature changes reflecting speciation more accurately than neutral genes (Ting et al, 2000). Variability of the obtained p53-coding sequences of M. lusitanicus and M. duodecimcostatus was further compared with the published sequences from other Microtus species, rodents and mammals.…”