Genome-wide relaxation of selection and the evolution of the island syndrome in Orkney voles.

Island populations often experience different ecological and demographic conditions than their counterparts on the continent, resulting in divergent evolutionary forces affecting their genomes. Random genetic drift and selection both may leave their imprints on island populations, although the relative impact depends strongly on the specific conditions. Here we address their contributions to the island syndrome in a rodent with an unusually clear history of isolation. Common voles (Microtus arvalis) were introduced by humans on the Orkney archipelago north of Scotland >5000 years ago and rapidly evolved to exceptionally large size. Our analyses show that the genomes of Orkney voles were dominated by genetic drift, with extremely low diversity, variable Tajima's D, and very high divergence from continental conspecifics. Increased d N/d S ratios over a wide range of genes in Orkney voles indicated genome-wide relaxation of purifying selection. We found evidence of hard sweeps on key genes of the lipid metabolism pathway only in continental voles. The marked increase of body size in Orkney-a typical phenomenon of the island syndrome-may thus be associated to the relaxation of positive selection on genes related to this pathway. On the other hand, a hard sweep on immune genes of Orkney voles likely reflects the divergent ecological conditions and possibly the history of human introduction. The long-term isolated Orkney voles show that adaptive changes may still impact the evolutionary trajectories of such populations despite the pervasive consequences of genetic drift at the genome level.

Did the creeping vole sex chromosomes evolve through a cascade of adaptive responses to a selfish x chromosome?

The creeping vole Microtus oregoni exhibits remarkably transformed sex chromosome biology, with complete chromosome drive/drag, X-Y fusions, sex reversed X complements, biased X inactivation, and X chromosome degradation. Beginning with a selfish X chromosome, I propose a series of adaptations leading to this system, each compensating for deleterious consequences of the preceding adaptation: (1) YY embryonic inviability favored evolution of a selfish feminizing X chromosome; (2) the consequent Y chromosome transmission disadvantage favored X-Y fusion ("XP "); (3) Xist-based silencing of Y-derived XP genes favored a second X-Y fusion ("XM "); (4) X chromosome dosage-related costs in XP XM males favored the evolution of XM loss during spermatogenesis; (5) X chromosomal dosage-related costs in XM 0 females favored the evolution of XM drive during oogenesis; and (6) degradation of the non-recombining XP favored the evolution of biased X chromosome inactivation. I discuss recurrent rodent sex chromosome transformation, and selfish genes as a constructive force in evolution.

Comparing the development of cortex-wide gene expression patterns between two species in a common reference frame.

Advances in sequencing techniques have made comparative studies of gene expression a current focus for understanding evolutionary and developmental processes. However, insights into the spatial expression of genes have been limited by a lack of robust methodology. To overcome this obstacle, we developed methods and software tools for quantifying and comparing tissue-wide spatial patterns of gene expression within and between species. Here, we compare cortex-wide expression of RZRß and Id2 mRNA across early postnatal development in mice and voles. We show that patterns of RZRß expression in neocortical layer 4 are highly conserved between species but develop rapidly in voles and much more gradually in mice, who show a marked expansion in the relative size of the putative primary visual area across the first postnatal week. Patterns of Id2 expression, by contrast, emerge in a dynamic and layer-specific sequence that is consistent between the two species. We suggest that these differences in the development of neocortical patterning reflect the independent evolution of brains, bodies, and sensory systems in the 35 million years since their last common ancestor.

Bank vole genomics links determinate and indeterminate growth of teeth.

BACKGROUND: Continuously growing teeth are an important innovation in mammalian evolution, yet genetic regulation of continuous growth by stem cells remains incompletely understood. Dental stem cells responsible for tooth crown growth are lost at the onset of tooth root formation. Genetic signaling that initiates this loss is difficult to study with the ever-growing incisor and rooted molars of mice, the most common mammalian dental model species, because signals for root formation overlap with signals that pattern tooth size and shape (i.e., cusp patterns). Bank and prairie voles (Cricetidae, Rodentia, Glires) have evolved rooted and unrooted molars while retaining similar size and shape, providing alternative models for studying roots. RESULTS: We assembled a de novo genome of Myodes glareolus, a vole with high-crowned, rooted molars, and performed genomic and transcriptomic analyses in a broad phylogenetic context of Glires (rodents and lagomorphs) to assess differential selection and evolution in tooth forming genes. Bulk transcriptomics comparisons of embryonic molar development between bank voles and mice demonstrated overall conservation of gene expression levels, with species-specific differences corresponding to the accelerated and more extensive patterning of the vole molar. We leverage convergent evolution of unrooted molars across the clade to examine changes that may underlie the evolution of unrooted molars. We identified 15 dental genes with changing synteny relationships and six dental genes undergoing positive selection across Glires, two of which were undergoing positive selection in species with unrooted molars, Dspp and Aqp1. Decreased expression of both genes in prairie voles with unrooted molars compared to bank voles supports the presence of positive selection and may underlie differences in root formation. CONCLUSIONS: Our results support ongoing evolution of dental genes across Glires and identify candidate genes for mechanistic studies of root formation. Comparative research using the bank vole as a model species can reveal the complex evolutionary background of convergent evolution for ever-growing molars.

A single amino acid residue in bank vole prion protein drives permissiveness to Nor98/atypical scrapie and the emergence of multiple strain variants.

Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein (PrPC) into infectious aggregates (PrPSc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrPSc. The transmissibility of prions depends on the PrPC sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.

Interplay between vertebrate adaptive immunity and bacterial infectivity genes: Bank vole MHC versus Borrelia afzelii OspC.

Coevolution of parasites with their hosts may lead to balancing selection on genes involved in determining the specificity of host-parasite interactions, but examples of such specific interactions in wild vertebrates are scarce. Here, we investigated whether the polymorphic outer surface protein C (OspC), used by the Lyme disease agent, Borrelia afzelii, to manipulate vertebrate host innate immunity, interacts with polymorphic major histocompatibility genes (MHC), while concurrently eliciting a strong antibody response, in one of its main hosts in Europe, the bank vole. We found signals of balancing selection acting on OspC, resulting in little differentiation in OspC variant frequencies between years. Neither MHC alleles nor their inferred functional groupings (supertypes) significantly predicted the specificity of infection with strains carrying different OspC variants. However, we found that MHC alleles, but not supertypes, significantly predicted the level of IgG antibodies against two common OspC variants among seropositive individuals. Our results thus indicate that MHC alleles differ in their ability to induce antibody responses against specific OspC variants, which may contribute to selection of OspC polymorphism by the vole immune system.

The expression profile of 79 genes from 107 viruses revealed new insights into disease susceptibility in rats, mice, and muskrats.

The coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread over the world, resulting in a global severe pneumonia pandemic. Both the cell receptor angiotensin-converting enzyme 2 (ACE2) and the breakdown of S protein by transmembrane serine protease 2 (TMPRSS2) are required by SARS-CoV-2 to enter the host cells. Similarly, the expression level of viral receptor genes in various organs determines the likelihood of viral infection. Several animal species have been found to be infected by the SARS-CoV-2, such as minks, posing an enormous threat to humans. Because the mice and rats were closely related to human and the fact that rats and mice have a risk of infection by SARS-CoV-2 with specific variants, we investigated the expression patterns of 79 receptor genes from 107 viruses, including SARS-CoV-2, in 14 organs of the rat and mouse, and 5 organs of the muskrat, to find the most likely host organs to become infected with certain viruses. The findings of this study are anticipated to aid in prevention of zoonotic infections spread by rats, mice, muskrats, and other rodents.

A single protective polymorphism in the prion protein blocks cross-species prion replication in cultured cells.

The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species-matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti-prion small molecules, and the inability of BVPrP-adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross-species prion replication in cultured cells and that a single amino acid change can override the prion-permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.

Phospholipid cofactor solubilization inhibits formation of native prions.

Cofactor molecules are required to generate infectious mammalian prions in vitro. Mouse and hamster prions appear to have different cofactor preferences: Whereas both mouse and hamster prions can use phosphatidylethanolamine (PE) as a prion cofactor, only hamster prions can also use single-stranded RNA as an alternative cofactor. Here, we investigated the effect of detergent solubilization on rodent prion formation in vitro. We discovered that detergents that can solubilize PE (n-octylglucoside, n-octylgalactoside, and CHAPS) inhibit mouse prion formation in serial protein misfolding cyclic amplification (sPMCA) reactions using bank vole brain homogenate substrate, whereas detergents that are unable to solubilize PE (Triton X-100 and IPEGAL) have no effect. For all three PE-solubilizing detergents, inhibition of RML mouse prion formation was only observed above the critical micellar concentration (CMC). Two other mouse prion strains, Me7 and 301C, were also inhibited by the three PE-solubilizing detergents but not by Triton X-100 or IPEGAL. In contrast, none of the detergents inhibited hamster prion formation in parallel sPMCA reactions using the same bank vole brain homogenate substrate. In reconstituted sPMCA reactions using purified substrates, n-octylglucoside inhibited hamster prion formation when immunopurified bank vole PrPC substrate was supplemented with brain phospholipid but not with RNA. Interestingly, phospholipid cofactor solubilization had no effect in sPMCA reactions using bacterially expressed recombinant PrP substrate, indicating that the inhibitory effect of solubilization requires PrPC post-translational modifications. Overall, these in vitro results show that the ability of PE to facilitate the formation of native but not recombinant prions requires phospholipid bilayer integrity, suggesting that membrane structure may play an important role in prion formation in vivo.

Characterization of repetitive DNA on the genome of the marsh rat Holochilus nanus (Cricetidae: Sigmodontinae).

Repetitive DNA are sequences repeated hundreds or thousands of times and an abundant part of eukaryotic genomes. SatDNA represents the majority of the repetitive sequences, followed by transposable elements. The species Holochilus nanus (HNA) belongs to the rodent tribe Oryzomyini, the most taxonomically diverse of Sigmodontinae subfamily. Cytogenetic studies on Oryzomyini reflect such diversity by revealing an exceptional range of karyotype variability. However, little is known about the repetitive DNA content and its involvement in chromosomal diversification of these species. In the search for a more detailed understanding about the composition of repetitive DNA on the genome of HNA and other species of Oryzomyini, we employed a combination of bioinformatic, cytogenetic and molecular techniques to characterize the repetitive DNA content of these species. RepeatExplorer analysis showed that almost half of repetitive content of HNA genome are composed by Long Terminal Repeats and a less significant portion are composed by Short Interspersed Nuclear Elements and Long Interspersed Nuclear Elements. RepeatMasker showed that more than 30% of HNA genome are composed by repetitive sequences, with two main waves of repetitive element insertion. It was also possible to identify a satellite DNA sequence present in the centromeric region of Oryzomyini species, and a repetitive sequence enriched on the long arm of HNA X chromosome. Also, comparative analysis between HNA genome with and without B chromosome did not evidence any repeat element enriched on the supernumerary, suggesting that B chromosome of HNA is composed by a fraction of repeats from all the genome.

Ancient DNA of narrow-headed vole reveal common features of the Late Pleistocene population dynamics in cold-adapted small mammals.

The narrow-headed vole, collared lemming and common vole were the most abundant small mammal species across the Eurasian Late Pleistocene steppe-tundra environment. Previous ancient DNA studies of the collared lemming and common vole have revealed dynamic population histories shaped by climatic fluctuations. To investigate the extent to which species with similar adaptations share common evolutionary histories, we generated a dataset comprised the mitochondrial genomes of 139 ancient and 6 modern narrow-headed voles from several sites across Europe and northwestern Asia covering approximately the last 100 thousand years (kyr). We inferred Bayesian time-aware phylogenies using 11 radiocarbon-dated samples to calibrate the molecular clock. Divergence of the main mtDNA lineages across the three species occurred during marine isotope stages (MIS) 7 and MIS 5, suggesting a common response of species adapted to open habitat during interglacials. We identified several time-structured mtDNA lineages in European narrow-headed vole, suggesting lineage turnover. The timing of some of these turnovers was synchronous across the three species, allowing us to identify the main drivers of the Late Pleistocene dynamics of steppe- and cold-adapted species.

Diversity and Evolution of Highly Repetitive DNA Sequences Constituting Chromosome Site-Specific Heterochromatin in Two Gerbillinae Species.

Constitutive heterochromatin, consisting of repetitive sequences, diverges very rapidly; therefore, its nucleotide sequences and chromosomal distributions are often largely different, even between closely related species. The chromosome C-banding patterns of two Gerbillinae species, Meriones unguiculatus and Gerbillus perpallidus, vary greatly, even though they belong to the same subfamily. To understand the evolution of C-positive heterochromatin in these species, we isolated highly repetitive sequences, determined their nucleotide sequences, and characterized them using chromosomal and filter hybridization. We obtained a centromeric repeat (MUN-HaeIII) and a chromosome 13-specific repeat (MUN-EcoRI) from M. unguiculatus. We also isolated a centromeric/pericentromeric repeat (GPE-MBD) and an interspersed-type repeat that was predominantly amplified in the X and Y chromosomes (GPE-EcoRI) from G. perpallidus. GPE-MBD was found to contain a 17-bp motif that is essential for binding to the centromere-associated protein CENP-B. This indicates that it may play a role in the formation of a specified structure and/or function of centromeres. The nucleotide sequences of the three sequence families, except GPE-EcoRI, were conserved only in Gerbillinae. GPE-EcoRI was derived from the long interspersed nuclear elements 1 retrotransposon and showed sequence homology throughout Muridae and Cricetidae species, indicating that the repeat sequence occurred at least in the common ancestor of Muridae and Cricetidae. Due to a lack of assembly data of highly repetitive sequences constituting heterochromatin in whole-genome sequences of vertebrate species published to date, the knowledge obtained in this study provides useful information for a deep understanding of the evolution of repetitive sequences in not only rodents but also in mammals.

Seasonal expressions of COX-1, COX-2, and EP4 in the scent glands of muskrats (Ondatra zibethicus).

Prostaglandins (PGs) serve as signaling molecules that regulate various physiological processes, including inflammation, immune response, blood clotting, and reproduction. The aim of this study was to investigate the immunolocalizations and expression patterns of prostaglandin-E2 (PGE2), cyclooxygenase (COX)-1, and COX-2, as well as its receptor subtypes 4 (EP4) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and nonbreeding periods. There were significant seasonal differences in the scent glandular mass, with higher values in the breeding season and relatively low in the nonbreeding season. PGE2, EP4, COX-1, and COX-2 have been immunolocalized in the scent glandular and epithelial cells in both breeding and nonbreeding seasons, whereas no immunostaining was observed in the interstitial cells. The protein and mRNA expression levels of EP4, COX-1, and COX-2 were higher in the scent glands of the breeding season than those of the nonbreeding season. The mean mRNA levels of EP4, COX-1, and COX-2 were positively correlated with the scent glandular weights. The circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and PGE2, as well as scent glandular PGE2 and dihydrotestosterone (DHT) concentrations, were also significantly higher in the breeding season. In addition, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to fatty carboxylic monocarboxylic acid, steroidogenic-related pathways, and prostanoid metabolic processes. These findings suggested that prostaglandin-E2 might play an essential autocrine or paracrine role in regulating seasonal changes in the scent glandular functions of the muskrats.

Early-life immune expression profiles predict later-life health and fitness in a wild rodent.

Individuals differ in the nature of the immune responses they produce, affecting disease susceptibility and ultimately health and fitness. These differences have been hypothesized to have an origin in events experienced early in life that then affect trajectories of immune development and responsiveness. Here, we investigate how early-life immune expression profiles influence life history outcomes in a natural population of field voles, Microtus agrestis, in which we are able to monitor variation between and within individuals through time by repeat sampling of individually marked animals. We analysed the co-expression of 20 immune genes in early life to create a correlation network consisting of three main clusters, one of which (containing Gata3, Il10 and Il17) was associated with later-life reproductive success and susceptibility to chronic bacterial (Bartonella) infection. More detailed analyses supported associations between early-life expression of Il17 and reproductive success later in life, and of Il10 expression early in life and later infection with Bartonella. We also found significant association between an Il17 genotype and the early-life expression of Il10. Our results demonstrate that immune expression profiles can be manifested during early life with effects that persist through adulthood and that shape the variability among individuals in susceptibility to infection and fitness widely seen in natural populations.

Bayesian Morphological Clock versus Parsimony: An Insight into the Relationships and Dispersal Events of Postvacuum Cricetidae (Rodentia, Mammalia).

Establishing an evolutionary timeline is fundamental for tackling a great variety of topics in evolutionary biology, including the reconstruction of patterns of historical biogeography, coevolution, and diversification. However, the tree of life is pruned by extinction and molecular data cannot be gathered for extinct lineages. Until recently methodological challenges have prevented the application of tip-dating Bayesian approaches in morphology-based fossil-only data sets. Herein, we present a morphological data set for a group of cricetid rodents to which we apply an array of methods fairly new in paleontology that can be used by paleontologists for the analysis of entirely extinct clades. We compare the tree topologies obtained by traditional parsimony, time-calibrated, and noncalibrated Bayesian inference phylogenetic approaches and calculate stratigraphic congruence indices for each. Bayesian tip-dated clock methods outperform parsimony in the case of our data set, which includes highly homoplastic morphological characters. Regardless, all three topologies support the monophyly of Megacricetodontinae, Democricetodontinae, and Cricetodontinae. Dispersal and speciation events inferred through Bayesian Binary Markov chain Monte Carlo and biodiversity analyses provide evidence for a correlation between biogeographic events, climatic changes, and diversification in cricetids. [Bayesian tip-dating; Cricetidae; Miocene; morphological clock; paleobiodiversity; paleobiogeography; paleoecology; parsimony; STRAP.].

Genetic footprints of a rapid and large-scale range expansion: the case of cyclic common vole in Spain.

In the Anthropocene, many species are rapidly shifting their ranges in response to human-driven habitat modifications. Studying patterns and genetic signatures of range shifts helps to understand how species cope with environmental disturbances and predict future shifts in the face of global environmental change. We investigated the genetic signature of a contemporary wide-range expansion observed in the Iberian common vole Microtus arvalis asturianus shortly after a colonization event. We used mtDNA and microsatellite data to investigate patterns of genetic diversity, structure, demography, and gene flow across 57 localities covering the historical range of the species and the newly colonized area. The results showed a genetic footprint more compatible with a true range expansion (i.e. the colonization of previously unoccupied areas), than with a model of "colonization from within" (i.e. local expansions from small, unnoticed populations). Genetic diversity measures indicated that the source population was likely located at the NE of the historical range, with a declining gradient of genetic diversity towards the more recently invaded areas. At the expansion front, we observed the greatest gene flow and smallest pairwise differences between nearby localities. Both natural landscape features (rivers) and recent anthropogenic barriers (roads, railways) explained a large proportion of genetic variance among populations and had a significant impact on the colonization pathways used by voles.

Different waves of postglacial recolonisation and genomic structure of bank vole populations in NE Poland.

Previous studies indicated that in some species phylogeographic patterns obtained in the analysis of nuclear and mitochondrial DNA (mtDNA) markers can be different. Such mitonuclear discordance can have important evolutionary and ecological consequences. In the present study, we aimed to check whether there was any discordance between mtDNA and nuclear DNA in the bank vole population in the contact zone of its two mtDNA lineages. We analysed the population genetic structure of bank voles using genome-wide genetic data (SNPs) and diversity of sequenced heart transcriptomes obtained from selected individuals from three populations inhabiting areas outside the contact zone. The SNP genetic structure of the populations confirmed the presence of at least two genetic clusters, and such division was concordant with the patterns obtained in the analysis of other genetic markers and functional genes. However, genome-wide SNP analyses revealed the more detailed structure of the studied population, consistent with more than two bank vole recolonisation waves, as recognised previously in the study area. We did not find any significant differences between individuals representing two separate mtDNA lineages of the species in functional genes coding for protein-forming complexes, which are involved in the process of cell respiration in mitochondria. We concluded that the contemporary genetic structure of the populations and the width of the contact zone were shaped by climatic and environmental factors rather than by genetic barriers. The studied populations were likely isolated in separate Last Glacial Maximum refugia for insufficient amount of time to develop significant genetic differentiation.

Constitutive heterochromatin propagation contributes to the X chromosome inactivation.

Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6 kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.

Numerous insertions of mitochondrial DNA in the genome of the northern mole vole, Ellobius talpinus.

BACKGROUND: Ellobius talpinus is a subterranean rodent representing an attractive model in population ecology studies due to its highly special lifestyle and sociality. In such studies, mitochondrial DNA (mtDNA) is widely used. However, if nuclear copies of mtDNA, aka NUMTs, are present, they may co-amplify with the target mtDNA fragment, generating misleading results. The aim of this study was to determine whether NUMTs are present in E. talpinus. METHODS AND RESULTS: PCR amplification of the putative mtDNA CytB-D-loop fragment using 'universal' primers from 56 E. talpinus samples produced multiple double peaks in 90% of the sequencing chromatograms. To reveal NUMTs, molecular cloning and sequencing of PCR products of three specimens was conducted, followed by phylogenetic analysis. The pseudogene nature of three out of the seven detected haplotypes was confirmed by their basal positions in relation to other Ellobius haplotypes in the phylogenetic tree. Additionally, 'haplotype B' was basal in relation to other E. talpinus haplotypes and found present in very distant sampling sites. BLASTN search revealed 195 NUMTs in the E. talpinus nuclear genome, including fragments of all four PCR amplified pseudogenes. Although the majority of the NUMTs studied were short, the entire mtDNA had copies in the nuclear genome. The most numerous NUMTs were found for rrnL, COXI, and D-loop. CONCLUSIONS: Numerous NUMTs are present in E. talpinus and can be difficult to discriminate against mtDNA sequences. Thus, in future population or phylogenetic studies in E. talpinus, the possibility of cryptic NUMTs amplification should always be taken into account.