Microtus Duodecimcostatus Research Articles

Disentangling the Effects of Climate and Land Uses on Small Mammals in Agroecosystems of NE Spain

We analyzed the two main drivers (climate and land uses) shaping the composition of small mammal communities at 16 localities situated in the confluence of the Mediterranean and Eurosiberian regions (Barcelona, Spain). The study area represents a land use and land cover gradient showing urbanization and crop intensification in the lowlands and forest encroachment in mountain areas. We identified 2458 small mammal individuals of 12 different species from barn owl (Tyto alba) pellets. Three open-land species (Microtus duodecimcostatus, Crocidura russula, and Mus spretus) and one forest/generalist species (Apodemus sylvaticus) were dominant in the diet, accounting for 93% of prey. In order to disentangle the effects of both main drivers on the small mammal community, we used partial constrained ordination techniques, which allowed us to determine the pure effects (and shared effects) of the environmental factors. Land use predictors explained 33.4% of the variance (mostly crops), followed by 23.4% of the variance explained by the geo-climatic variables (mostly rainfall), and an additional 24.8% of the variance was shared by both groups of predictors, totaling 81.6% of environmental variance. The remaining 18.4% of variance was unexplained by environmental matrices. This pattern was consistent with expected associations of species and biotic influences at small spatial scales and highlighted that the number of species increased from the crops in the lowlands towards the highlands covered by deciduous and coniferous forests.

Cell adhesion and immune response, two main functions altered in the transcriptome of seasonally regressed testes of two mammalian species.

In species with seasonal breeding, male specimens undergo substantial testicular regression during the nonbreeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, nonapoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing, and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK, and TGF-β signaling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the "immune privilege" that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.

Diet of sympatric Barn Owls Tyto alba and Short-eared Owls Asio flammeus in an agricultural landscape in south-east Spain

The diets of the Barn Owl Tyto alba and the Short-eared Owl Asio flammeus have been extensively studied worldwide over the past few years, especially on the Iberian Peninsula. Nevertheless, very few studies have examined the diets and the trophic niche overlap in areas where these two raptor species occur in sympatry. As such, in this study we compared the diets of the Barn Owl and the Short-eared Owl inhabiting agricultural landscapes of the Vega de Granada, south-east Spain, based on pellet analysis. The diets were very similar, as both owls preyed mainly on small mammals, the Algerian Mouse Mus spretus being the prey most commonly found in pellets from both species. Although the diet of the Barn Owl was more diverse than that of the Short-eared Owl, the food niche overlap was very high, thus indicating a low interspecific trophic segregation. Despite the similarities between both diets, the frequency of the Mediterranean Pine Vole Microtus duodecimcostatus was much higher in pellets from the Barn Owl, thus suggesting that the Barn Owl may exert pest control in years when the Mediterranean Pine Vole occurs in high numbers.

Genetic variation at the p53 locus of two ecologically divergent Microtus pine voles: identification of molecular markers for species assignment.

The Lusitanian (Microtus lusitanicus) and the Mediterranean (Microtus duodecimcostatus) pine voles are recently diverged sister species endemic of the Iberian Peninsula that can be identified with ecological and morphological characters, but in areas where the 2 species co-occur, species designation may be difficult. Genetic discrimination between M. lusitanicus and M. duodecimcostatus has not been achieved yet possibly because of their estimated recent split and an evolutionary history that includes inter-species gene flow. Following our previous observations on exons 5-7 of the p53 gene, here we analyze the potential use of the p53 genomic region as a discrimination marker of these species by extending our analyses to several kb upstream and downstream of the p53 gene and characterizing the degree of genetic differentiation in 7 markers within this region. Additionally, we fully sequenced the P53 protein of both species. We observed: (i) generally high differentiation in this region; (ii) M. duodecimcostatus showed in general higher values of nucleotide and haplotype diversities; (iii) the concatenated phylogenetic tree separates the 2 species; (iv) the 2 P53 proteins only differ in 1 amino acid; (v) 4 of the markers, 2 in p53, one in Atp1b2, and another in Wrap53, contain species-specific genetic variation thus allowing a reliable discrimination between specimens from both species, irrespective of sampling location or introgression status. We provide additional data on the putative role of p53 in the evolution of these species and present researchers with a fast and cost-effective resource for M. lusitanicus and M. duodecimcostatus identification.

Mediterranean Pine Vole, Microtus duodecimcostatus: A Paradigm of an Opportunistic Breeder.

Simple SummaryIn temperate zones of the Earth, some mammalian species reproduce seasonally whereas others do it continuously. Other species are summer breeders in the north and winter breeders in the south. Thus, the reproductive pattern seems not to be a species-specific but a population-specific trait. We investigated the reproduction pattern of the Mediterranean pine vole, Microtus duodecimcostatus, in the area around the city of Granada in Southern Spain, and found that individuals living in wastelands reproduce seasonally whereas those living in close poplar plantations (just 8 km apart) reproduce throughout the year, as did voles captured in wastelands and kept in captivity. These animals represent thus a paradigm of an opportunistic breeder as particular individuals stop breeding or not, depending on the environmental conditions they face at any moment. Sexually inactive male voles undergo complete testis inactivation and their sperm production is halted. The immune system in active testes is depressed, a phenomenon known as “immune privilege” that protect germ cells from autoimmune attack. We studied gene activity in active and inactive testes and our results indicate that such an immune privilege is lost in inactive testes, suggesting an important role for this process during testis regression.Most mammalian species of the temperate zones of the Earth reproduce seasonally, existing a non-breeding period in which the gonads of both sexes undergo functional regression. It is widely accepted that photoperiod is the principal environmental cue controlling these seasonal changes, although several exceptions have been described in other mammalian species in which breeding depends on cues such as food or water availability. We studied the circannual reproductive cycle in males of the Mediterranean pine vole, Microtus duodecimcostatus, in the Southeastern Iberian Peninsula. Morphological, hormonal, functional, molecular and transcriptomic analyses were performed. As reported for populations of other species from the same geographic area, male voles captured in wastelands underwent seasonal testis regression in summer whereas, surprisingly, those living either in close poplar plantations or in our animal house reproduced throughout the year, showing that it is the microenvironment of a particular vole subpopulation what determines its reproductive status and that these animals are pure opportunistic, photoperiod-independent breeders. In addition, we show that several molecular pathways, including MAPK, are deregulated and that the testicular “immune privilege” is lost in the inactive testes, providing novel mechanisms linking seasonal testosterone reduction and testis regression.

Erratum to “Palaeoecological context for the extinction of the Neanderthals: A small mammal study of Stratigraphic Unit V of the El Salt site, Alcoi, eastern Spain” [Palaeogeography, Palaeoclimatology, Palaeoecology 530 (2019) 163–175

Abstract El Salt is an important reference site for understanding the extinction of Neanderthal populations in the eastern Iberian Peninsula during MIS 3. In this paper, we describe the small mammal assemblage from Stratigraphic Unit V, the youngest unit with evidence of human presence, based on nearly 1300 specimens. A total of seven rodents (Microtus arvalis, Microtus duodecimcostatus, Microtus cabrerae, Sciurus vulgaris, Arvicola sapidus, Eliomys quercinus and Apodemus sylvaticus), three insectivores (Talpa occidentalis, Crocidura sp., Sorex sp.) and one lagomorph (Oryctolagus cf. cuniculus) were identified. Palaeocological analyses point to drier conditions in this part of the stratigraphic sequence, supporting the hypothesis that an aridification scenario may have played a role in the extinction of the Neanderthal groups inhabiting this region of the Iberian Peninsula.

Nest-boxes for raptors as a biological control system of vole pests: High local success with moderate negative consequences for non-target species

Biological control of rodents in agricultural areas, increasing avian predator abundance by nest-box provisioning, has been proposed during the last decades in several regions around the world as an alternative to the widespread use of anticoagulant rodenticides (AR) to protect crops. However these experiences have often lacked a “Before-After-Control-Impact” (BACI) experimental design providing evidence strong enough about their efficacy reducing vole abundance or crop damages. Furthermore, little is known about possible indirect effects on non-target species.Here we test the effectiveness of providing nest-boxes to common kestrels (Falco tinnunculus) and barn owls (Tyto alba) in reducing the abundance of two different vole species using indirect index methods to estimate rodent abundance in late spring (May). We monitored the abundance of both vole species in a treatment (with nest boxes) and control (without nest boxes) areas during three years in alfalfa fields, using a BACI design. We studied also the efficiency of this control method in three different crops (alfalfa, cereal and fruit-trees) during the last year of the experiment. We compare the results obtained in this study area in NE Spain with those obtained simultaneously in other three experimental areas in NW Spain. Finally we explored potential negative effects of this method on the abundance of several passerine birds present in our research area.Our results showed a clear-cut reduction in the abundance of the two vole species, the common vole (Microtus arvalis) and the mediterranean-pine vole (Microtus duodecimcostatus) in the experimental area after applying the treatment (nest box installation) in alfalfa crops. We also found a significant decrease in the presence of both species of voles on fruit-tree plantations and alfalfa crops but we did not find significant effects in the cereal fields, where voles were in general very scarce. These results suggest higher efficacy of biological control in this study area than in some areas of NW Spain, what could be explained by several non-exclusive hypotheses presented in the discussion.For other species, we found a negative effect of nest-boxes on the abundance of a limited number of common bird species, but also positive effects over other species using poles with nest-boxes as perches.In conclusion, under the conditions of our study area, nest-box provisioning seemed to be an effective technique to reduce vole abundance, but also had some undesired side-effects on non-target species. However, these side-effects affected mainly to common bird species and can thus be considered less relevant than those caused by SGARs, often affecting to endangered rodent predators and that could thus have negative effects over any potential future natural control of vole abundance, potentially increasing future needs of SGAR use in a vicious circle.

Palaeoecological context for the extinction of the Neanderthals: A small mammal study of Stratigraphic Unit V of the El Salt site, Alcoi, eastern Spain

El Salt is emerging as a reference site for the study of the extinction of Neanderthal populations in the Eastern Iberian Peninsula during MIS 3. The small vertebrate assemblage analysed in this work is framed within this general objective and comes from Stratigraphic Unit V, the most recent unit with human presence. Nearly 1300 small mammal remains have been studied in order to reconstruct the palaeoecological conditions of this debated period. A total of seven rodents (Microtus arvalis, Microtus duodecimcostatus, Microtus cabrerae, Sciurus vulgaris, Arvicola sapidus, Eliomys quercinus and Apodemus sylvaticus), three insectivores (Talpa occidentalis, Crocidura sp., Sorex sp.) and one lagomorph (Oryctolagus cf. cuniculus) were identified. Palaeocological analyse points to drier conditions in the upper part (Unit V) of the stratigraphic sequence, supporting the hypothesis that an aridification scenario may have played a role in the disappearance of the Neanderthal groups inhabiting this region of the Iberian Peninsula.

Energetics and thermal adaptation in semifossorial pine-voles Microtus lusitanicus and Microtus duodecimcostatus.

Rodents colonising subterranean environments have developed several morphological, physiological and behaviour traits that promote the success of individuals in such demanding conditions. Resting metabolic rate, thermoregulation capacity and daily energy expenditure were analysed in two semi-fossorial pine-vole species Microtus lusitanicus and Microtus duodecimcostatus inhabiting distinct areas of the Iberian Peninsula. Individuals capture location varied in habitat and soil features, allowing the comparison of energetic parameters with ecological characteristics, that can help explain the use of the subterranean environment and dependence of the burrow system. Results showed that M. duodecimcostatus has lower mass independent resting metabolic rate when compared with M. lusitanicus, which may be a response to environmental features of their habitat, such as dryer soils and lower water availability. Thermal conductance increased with body mass and was dependent on the ambient temperature. No significant differences were observed in the daily energy expenditure, but water economy data demonstrated the influence of the water available in the habitat on the energetics of voles. These species may rely on behavioural adaptations and seasonal use of burrows to cope with thermal challenges of subterranean activity and soil constraints. We found strong evidence that M. lusitanicus is able to use torpor as a response to low ambient temperatures which is a new observation among Arvicolines.

Reproductive isolation between sister species of Iberian pine voles, Microtus duodecimcostatus and M. lusitanicus

Contact zones of recently diverged species provide the ideal opportunity to study the nature of reproductive barriers. The Mediterranean (Microtus duodecimcostatus) and Lusitanian pine voles (M. lusitanicus) are a pair of recently diverged sister species endemic to Western Europe. Recent microsatellite data raised the possibility that hybridisation events, although rare, may still occur in the area of sympatry of both species. Here we evaluated the nature and extent of reproductive isolation between M. duodecimcostatus and M. lusitanicus through a laboratory crossbreeding experiment and two-way mate preference tests on the basis of chemical cues. Crossbreeding of sympatric individuals from the two species was successful and followed Haldane’s rule, revealing hybrid male sterility and reduced fertility of hybrid females. Results from mate preference tests showed males of both pine vole species could discriminate and preferred female conspecifics to heterospecifics or hybrid females on the basis of chemical cues alone. On the contrary, females of both pine vole species did not show a clear preference for conspecific over heterospecific male chemical cues, suggesting females may need additional cues to discriminate between males of the two species. As a whole our results indicate both prezygotic (i.e. species-specific mate recognition systems) and postzygotic barriers (i.e. male hybrid sterility, female reduced fertility and behavioural sterility ― decreased ability to find or attract mates) have evolved, and should make hybridisation of the Iberian pine voles under natural conditions an unlikely event. The role of both species mating preferences and interspecific behavioural interactions in the asymmetric mitochondrial introgression pattern of the Iberian pine voles is discussed.

Detection of Rabbit Hemorrhagic Disease Virus GI.2/RHDV2/b in the Mediterranean Pine Vole ( Microtus duodecimcostatus) and White-Toothed Shrew ( Crocidura russula).

The European wild rabbit ( Oryctolagus cuniculus) is a key prey species on the Iberian Peninsula, and several predator species that are at risk of extinction are dependent on them as prey. A new rabbit hemorrhagic disease (RHD) virus genotype (GI.2/RHDV2/b) emerged in 2010 and posed a threat to wild rabbit populations. During a survey aimed at investigating RHD epidemiology in wild rabbits, GI.2/RHDV2/b was detected by duplex real-time PCR in carcasses of one Mediterranean pine vole ( Microtus duodecimcostatus) and two white-toothed shrews ( Crocidura russula). Laboratory New Zealand white rabbits that were challenged with inocula obtained from the liver of the small mammals died showing RHD lesions, confirming the infectiousness of the isolates. Phylogenetic analysis of the VP60 gene nucleotide sequences showed complete homology between the isolates from the two small mammal species and a high degree of similarity, but not complete homology, to GI.2/RHDV2/b sequences from wild rabbits. The GI.2/RHDV2/b genotype has not been reported in species outside the order Lagomorpha.

Species-specific movement traits and specialization determine the spatial responses of small mammals towards roads

The barrier effect is a pervasive impact of road networks. For many small mammals individual avoidance responses can be the mechanism behind the barrier effect. However, little attention has been paid to which species and road characteristics modulate road avoidance and mortality risk. We measured the strength of the barrier effect imposed by the road on three rodent species with different body sizes and habitat specializations: Southern water vole (Arvicola sapidus), Mediterranean pine vole (Microtus duodecimcostatus) and Algerian mouse (Mus spretus). We analysed the effect of traffic intensity on use of space and direction of movement and the effect of road type (4-lane highway, 2-lane paved road and 1-lane unpaved road) on crossing rates with simulations of roads bisecting each home range. Finally, we estimated annual mortality risk from collision based on individual speed, crossing rates, body length, traffic volume and road width. Individual mobility and ecological preferences were assumed as two hypotheses to explain species’ responses towards roads. The effects were species-specific, with traffic intensity having a negative influence on the use of space and movement directionality only for water voles and with avoidance of paved roads being evident for the Algerian mouse. Road-kill mortality risk was high in pine voles. Habitat specialization, individual mobility and home-range location were identified as important factors of species’ responses towards roads. This study draws attention to the role of behaviour and ecological requirements as well as road characteristics on the strength of the barrier effect imposed by roads for different species.

Los micromamíferos (Lagomorpha, Eulipotyphla y Rodentia) del yacimiento del Pleistoceno Medio de Cuesta de la Bajada (Teruel, España): Estudio sistemático y consideraciones paleoambientales

La asociación de micromamíferos determinada en este trabajo es la siguiente: Lagomorpha: Oryctolagus cuniculus; Eulipotyphla: Crocidura cf. russula, cf. Sorex sp., Neomys sp., Soricidae indet. y Talpa sp.; y Rodentia: Eliomys quercinus, Apodemus cf. sylvaticus, Cricetulus (Allocricetus) bursae, Arvicola aff. sapidus, Microtus (Iberomys) brecciensis y Microtus (Terricola) duodecimcostatus. Es una asociación característica del Pleistoceno Medio. El estadio evolutivo de Cricetulus (A.) bursae, Arvicola aff. sapidus y Microtus (I.) brecciensis le aproximan a las poblaciones de dichas especies de algunos yacimientos de la Península Ibérica del Pleistoceno Medio avanzado, pero no final, lo que es acorde con las dataciones numéricas obtenidas en el yacimiento (243-337 ka) que le sitúan en el MIS 8 o 9. Los micromamíferos indican la predominancia de los espacios abiertos con abundante vegetación herbácea y arbustiva, en los que podría haber también alguna zona arbolada. El clima que indican es de tipo mediterráneo, similar al actual en la zona o quizás algo más benigno y más húmedo.

Heterogeneous road networks have no apparent effect on the genetic structure of small mammal populations

Roads are widely recognized to represent a barrier to individual movements and, conversely, verges can act as potential corridors for the dispersal of many small mammals. Both barrier and corridor effects should generate a clear spatial pattern in genetic structure. Nevertheless, the effect of roads on the genetic structure of small mammal populations still remains unclear. In this study, we examine the barrier effect that different road types (4-lane highway, 2-lane roads and single-lane unpaved roads) may have on the population genetic structure of three species differing in relevant life history traits: southern water vole Arvicola sapidus, the Mediterranean pine vole Microtus duodecimcostatus and the Algerian mouse Mus spretus. We also examine the corridor effect of highway verges on the Mediterranean pine vole and the Algerian mouse. We analysed the population structure through pairwise estimates of FST among subpopulations bisected by roads, identified genetic clusters through Bayesian assignment approaches, and used simple and partial Mantel tests to evaluate the relative barrier or corridor effect of roads. No strong evidences were found for an effect of roads on population structure of these three species. The barrier effect of roads seems to be site-specific and no corridor effect of verges was found for the pine vole and Algerian mouse populations. The lack of consistent results among species and for each road type lead us to believe that the ability of individual dispersers to use those crossing structures or the habitat quality in the highway verges may have a relatively higher influence on gene flow among populations than the presence of crossing structures per se. Further research should include microhabitat analysis and the estimates of species abundance to understand the mechanisms that underlie the genetic structure observed at some sites.

Olfactory receptors and behavioural isolation: a study on Microtus voles

Genetic variation in olfactory receptors may trigger mate choice, suggesting that olfaction has undergone diversifying selection in diverging populations and may contribute to premating reproductive isolation. In the present study, we analysed two olfactory receptor genes as candidate barriers of reproductive isolation between two recently divergent voles: Microtus lusitanicus and Microtus duodecimcostatus. In addition, evolutionary relationships and signs of positive selection were inferred in a European subgenera context, based on 76 samples from 14 species. DNA sequence analysis revealed the presence of shared haplotypes among various Microtus species. Tests of selection detected negatively selected amino acids in the extracellular loops of both olfactory receptors and a majority of negatively selected residues in the transmembrane helices, the most variable regions responsible for the reception of odorants. Our findings suggest that, for several Microtus species, including M. lusitanicus and M. duodecimcostatus, these proteins probably recognise conserved odour cues not related to behavioural isolation.

Pair-bonding behaviour of the sister species Microtus lusitanicus and M. duodecimcostatus

Monogamous mating systems can be inferred by analysis of social behaviour, for example pair-bonding. We studied the Iberian sister species Microtus lusitanicus and Microtus duodecimcostatus. On the basis of morphological, reproductive, and ecological characteristics, but not behavioural studies, these voles are regarded as monogamous species. Pair-bonding behaviour was inferred by use of partner preference tests using chemical stimuli. Two scenarios were considered: in the first we examined whether the member of a breeding pair prefers chemical stimuli from its partner or from a sexually experienced stranger, simulating a widow/widower of the population, while in the second scenario we assessed whether there was a preference for chemical stimuli from its partner or from a sexually naive stranger, mimicking an immigrant individual. Results support a social monogamous mating system for both species and reveal a significant female preference for the male partner, rather than a stranger. Conversely, male preference differed, depending on the sexual status of the female strangers. When the stranger was sexually experienced, a significant preference for its partner was observed, whereas in the presence of a sexually naive female stranger no significant preference for the female partner was revealed. These results suggest rare male extra-pair mating in M. lusitanicus and M. duodecimcostatus.

Design and testing of an agricultural implement for underground application of rodenticide bait.

An agricultural implement for underground application of rodenticide bait to control the Mediterranean pocket gopher (Microtus Duodecimcostatus) in fruit orchards has been designed and tested. The main objective of this research was to design and test the implement by using the finite element method (FEM) and considering a range of loads generated on most commonly used furrow openers in agricultural implements. As a second step, the prototype was tested in the field by analysing the effects of forward speed and application depth on the mechanical behaviour of the implement structure. The FEM was used in the design phase and a prototype was manufactured. The structural strains on the prototype chassis under working conditions were tested by using strain gauges to validate the design phase. Three forward speeds (4.5, 5.5, and 7.0 km/h), three application depths (0.12, 0.15, and 0.17 m), and two types of soil (clayey-silty-loam and clayey-silty-sandy) were considered. The prototype was validated successfully by analysing the information obtained from the strain gauges. The Von Mises stresses indicated a safety coefficient of 1.9 for the most critical load case. Although both forward speed and application depth had a significant effect on the stresses generated on the chassis, the latter parameter critically affected the structural behaviour of the implement. The effects of the application depth on the strains were linear such that strains increased with depth. In contrast, strains remained roughly constant regardless of variation in the forward speed.

The end of the Last Glacial Maximum in the Iberian Peninsula characterized by the small-mammal assemblages

This paper presents a palaeoenvironmental and palaeoclimatic approach to the end of the Last Glacial Maximum (LGM) in the Iberian Peninsula on the basis of the small-mammal assemblages (insectivores, bats and rodents). The LGM is an important period in our climate history defined by the maximum extension of ice sheets between ca. 22 and 19 ka BP. In the Mediterranean region the LGM is characte­rized by humid conditions, which allow for the development of arboreal vegetation. The small-mammal remains described in this study were recovered from four different sites within the Iberian Peninsula: El Mirón cave (Ramales de la Victoria, Cantabria), Valdavara-1 (Becerreá, Lugo), El Portalón (Sierra de Atapuerca, Burgos), and Sala de las Chimeneas (Maltravieso, Cáceres). We found in these sites a non-analogue association represented by species associated with mid-European climatic conditions, such as the voles Chionomys nivalis, Microtus arvalis, Microtus agrestis and Microtus oeconomus, together with species associated with Mediterranean requirements, such as Microtus (Iberomys) cabrerae and Microtus (Terricola) duodecimcostatus. These assemblages reveal that the climate was harsher than today in the sites under study, though not as rigorous as elsewhere in Europe, with mean annual temperatures lower than present and an environment dominated by wet open meadows. All our data have been compared with other environmental and climatic proxies, global isotope curves and pollen data, providing a scenario for the palaeoclimatic and palaeoenvironmental conditions that occurred during the LGM in the Iberian Peninsula.

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

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.

Deep mitochondrial introgression and hybridization among ecologically divergent vole species

The completion of speciation is typically difficult to ascertain in rapidly diverging taxa but the amount of hybridization and gene flow in sympatry or parapatry contains important information about the level of reproductive isolation achieved. Here, we examined the progress in speciation between the Mediterranean (Microtus duodecimcostatus) and the Lusitanian pine vole (M.lusitanicus), which are part of the most rapid radiation of species known in mammals. These two Iberian pine voles are classified as separate species because of differences in morphology and ecology, but relatively many ambiguous individuals can be found in sympatric conditions. Our phylogenetic analyses of rangewide data from the mitochondrial cytochrome b gene (mtDNA) demonstrated high levels of diversity and a basal separation in two parapatric lineages. However, mtDNA affiliation was at odds with morphological classification or geographical distribution of the taxa. In contrast, statistical analyses of microsatellites (nucDNA) showed two clear genetic clusters in allopatry and sympatry generally matching morphological classification. This cytonuclear discordance over a large geographic area suggests historical introgression of mtDNA from M.duodecimcostatus to M.lusitanicus. There was statistical evidence for at least two recent hybrids in the sympatry zone but gene flow is apparently low given clear-cut differences in nucDNA. Our results indicate a relatively advanced speciation process in these Iberian pine voles without fully established reproductive isolation. This situation enables use of combined population genomic and experimental approaches for the separation of patterns and mechanisms in the ongoing explosive diversification of these and other Arvicoline rodents in the future.