Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.     

Karyotype evolution of eulipotyphla (insectivora): the genome homology of seven sorex species revealed by comparative chromosome painting and banding data

The genus Sorex is one of the most successful genera of Eulipotyphla. Species of this genus are characterized by a striking chromosome variability including XY1Y2 sex chromosome systems and exceptional chromosomal polymorphisms within and between populations. To study chromosomal evolution of the genus in detail, we performed cross-species chromosome painting of 7 Sorex species with S. granarius and S. araneus whole-chromosome probes and found that the tundra shrew S. tundrensis has the most rearranged karyotype among these. We reconstructed robust phylogeny of the genus Sorex based on revealed conserved chromosomal segments and syntenic associations. About 16 rearrangements led to formation of 2 major Palearctic groups after their divergence from the common ancestor: the S. araneus group (10 fusions and 1 fission) and the S. minutus group (5 fusions). Further chromosomal evolution of the 12 species inside the groups, including 5 previously investigated species, was accompanied by multiple reshuffling events: 39 fusions, 20 centromere shifts and 10 fissions. The rate of chromosomal exchanges upon formation of the genus was close to the average rate for eutherians, but increased during recent (about 6-3 million years ago) speciation within Sorex. We propose that a plausible ancestral Sorex karyotype consists of 56 elements. It underwent 20 chromosome rearrangements from the boreoeutherian ancestor, with 14 chromosomes retaining the conserved state. The set of genus-specific chromosome signatures was drawn from the human (HSA)-shrew comparative map (HSA3/12/22, 8/19/3/21, 2/13, 3/18, 11/17, 12/15 and 1/12/22). The syntenic association HSA4/20, that was previously proposed as a common trait of all Eulipotyphla species, is shown here to be an apomorphic trait of S. araneus.     

CHROMOSOMAL VERSUS MITOCHONDRIAL DNA EVOLUTION: TRACKING THE EVOLUTIONARY HISTORY OF THE SOUTHWESTERN EUROPEAN POPULATIONS OF THE SOREX ARANEUS GROUP (MAMMALIA,INSECTIVORA)

The shrews of the Sorex araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorex granarius is generally considered ancestral to those of Sorex coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well-separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.     

Genetic and karyotypic structure in the shrews of the Sorex araneus group: are they independent?

The species of the common shrew (Sorex araneus) group are morphologically very similar but exhibit high levels of karyotypic variation. Here we used genetic variation at 10 microsatellite markers in a data set of 212 individuals mostly sampled in the western Alps and composed of five karyotypic taxa (Sorex coronatus, Sorex antinorii and the S. araneus chromosome races Cordon, Bretolet and Vaud) to investigate the concordance between genetic and karyotypic structure. Bayesian analysis confirmed the taxonomic status of the three sampled species since individuals consistently grouped according to their taxonomical status. However, introgression can still be detected between S. antinorii and the race Cordon of S. araneus. This observation is consistent with the expected low karyotypic complexity of hybrids between these two taxa. Geographically based cryptic substructure was discovered within S. antinorii, a pattern consistent with the different postglaciation recolonization routes of this species. Additionally, we detected two genetic groups within S. araneus notwithstanding the presence of three chromosome races. This pattern can be explained by the probable hybrid status of the Bretolet race but also suggests a relatively low impact of chromosomal differences on genetic structure compared to historical factors. Finally, we propose that the current data set (available at http://www.unil.ch/dee/page7010_en.html#1) could be used as a reference by those wanting to identify Sorex individuals sampled in the western Alps.     

3D organization of interphase fibroblast nuclei in two closely related shrew species, Sorex granarius and S. araneus, differed by the structure of chromosome termini

To study 3D organization of fibroblast interphase nuclei in two sibling shrew species, Sorex araneus from Cordon race and S. granarius, FISH with probe to telomeric and rDNA repeats, and immunofluorescence with ANA CREST and antibodies to nucleolus protein B23 were used. Karyotypes of studied species are composed of near identical chromosomal arms and differ by the number of metacentrics and the structure of terminal chromosome regions. The large telomeres containing on the average 218 kbp of telomere repeats characterize the short arms in all of 32 S. granarius acrocentrics. Telomere repeats in them alternate with nbosomal repeats. These regions also contain active NORs. In contrast, active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005, 2007b). We have shown that telomere associations of chromosomes and contacts of a part of telomere clusters with inner nuclear membrane and nucleolus characterize interphase nuclei of both S. granarius and S. araneus. Moreover, the partial colocalization of telomere and ribosomal clusters, and spatial nearness of centomeric and telomeric regions were revealed in the interphase nuclei of S. granarius. Evidently, only those ribosomal clusters that contain a number of active ribosomal genes display connection with nucleolus. The stripping of nucleolus materials during transition of fibroblasts to mitosis and the role of B23 protein in this process has been studied.     

Intraspecies phylogenetic connections in Sorex araneus L.: a southern Baltic subgroup of chromosome races

Results of chromosome G-banding were used to identify two new karyotypic races of the common shrew (Sorex araneus L.) in the European part of Russia. The chromosomal diagnosis of race St. Petersburg included five metacentrics (hk, ip, jl, mq, and nr) and two acrocentrics (g and o) (2Na = 20); the diagnosis of race West Dvina included six metacentrics (gm, hk, ip, jl, no, and qr) (2Na = 18). The phylogenetic significance of the chromosomal markers gm, hk, and ip is considered and the possibility of reticulate evolution in the species S. araneus is discussed.     

Phylogenetic relationships of Caucasian shrew Sorex satunini Ogn. (mammalia) in the superspecies Sorex araneus inferred from the data of karyological analysis and the mtDNA cyt b gene sequencing

Using the data of karyological analysis, the phylogenetic relationships of Caucasian shrew Sorex satunini and the cryptic species of superspecies Sorer araneus were examined. In the population of Sorex satunini from the plain of North Ciscaucasia two deeply radiated cytochrome b genes (A and B) were identified. Genetic distance between haplotype A and B groups constituted 0.0675 +/- 0.008, which is higher than any distance in superspecies S. araneus. Possible introgression of type B haplotypes from the populations of the evolutionary lineage S. subaraneus--S. araneus in Pleistocene and the time of the appearance of the chromosomal polymorphism of S. araneus is discussed. Our results show that the use of only one mitochondrial marker can lead to false conclusions on taxonomic diversity     

Telomeric DNA allocation in chromosomes of common shrew (Sorex araneus, eulipotyphla)

Recently, we displayed an Iberian shrew species (Sorex granarius) with telomere structures unusual for mammals. Long telomeres on the short acrocentric arms contain an average of 213 kb of telomere repeats, whereas the other chromosomal ends have only 3.8 kb (Zhdanova et al., 2005; 2007). However, it is not clear whether these telomeres are typical for all shrew species or only for S. granarius. S. granarius and common shrew Sorex araneus are sibling species. In this study, using modified Q-FISH we demonstrated that telomeres in S. araneus from various chromosomal races distinguished by their number of metacentrics contain 6.8–15.2 kb of telomeric tracts. The S. araneus telomere lengths appear to correspond to telomere lengths in the majority of both shrew species and wild mammals, whereas S. granarius has telomeres with unique or rare structures. Using DNA and RNA high-specific modified probes to telomeric repeats (PNA and LNA), we showed that interstitial telomeric sites in S. araneus chromosomes contain mainly telomeric DNA and that their localization coincide with some evolutionary breakpoints. Interstitial telomeric DNA in S. granarius chromosomes was not revealed. Thus, the distribution of telomeric DNA may be significantly different, even in closely related species whose chromosomes are composed of almost identical chromosomal arms.     

Mechanisms of chromosomal evolution in three European species of the Sorex araneus-arcticus group (Insectivora: Soricidae)

Presented are the R-banding patterns of the karyotypes of three European species of the Sorex araneus-arcticus group (Insectivora: Soricidae). The eight species of this Holarctic complex are characterized by sharing a male chromosomal set of XY1Y2 elements. Robertsonian and tandem translocations are common in this complex, at the population level as well as at the species level. The rough morphology of the karyotypes looks similar between all the species presently described. An R-banding technique RHG, RBG) has allowed us to make a comparative analysis of the chromosomal similarities in three species, namely S. araneus, S. coronatus and S. granarius. The data provide evidence that Robertsonian and tandem translocations, accompaniei in some cases by centromeric shifts, are the main, if not the only, mechanisms of chromosomal evolution in this grou. It appears that S. granarius presents a karotype which is most similar to the hypotheticar ancestral type from which the chromosomar sets of two other European species might be derived.     

Chromosomal rearrangements do not seem to affect the gene flow in hybrid zones between karyotypic races of the common shrew (Sorex araneus)

Chromosomal rearrangements are proposed to promote genetic differentiation between chromosomally differentiated taxa and therefore promote speciation. Due to their remarkable karyotypic polymorphism, the shrews of the Sorex araneus group were used to investigate the impact of chromosomal rearrangements on gene flow. Five intraspecific chromosomal hybrid zones characterized by different levels of karyotypic complexity were studied using 16 microsatellites markers. We observed low levels of genetic differentiation even in the hybrid zones with the highest karyotypic complexity. No evidence of restricted gene flow between differently rearranged chromosomes was observed. Contrary to what was observed at the interspecific level, the effect of chromosomal rearrangements on gene flow was undetectable within the S. araneus species.     

Restricted gene flow at specific parts of the shrew genome in chromosomal hybrid zones

The species and races of the shrews of the Sorex araneus group exhibit a broad range of chromosomal polymorphisms. European taxa of this group are parapatric and form contact or hybrid zones that span an extraordinary variety of situations, ranging from absolute genetic isolation to almost free gene flow. This variety seems to depend for a large part on the chromosome composition of populations, which are primarily differentiated by various Robertsonian fusions of a subset of acrocentric chromosomes. Previous studies suggested that chromosomal rearrangements play a causative role in the speciation process. In such models, gene flow should be more restricted for markers on chromosomes involved in rearrangements than on chromosomes common in both parent species. In the present study, we address the possibility of such differential gene flow in the context of two genetically very similar but karyotypically different hybrid zones between species of the S. araneus group using microsatellite loci mapped to the chromosome arm level. Interspecific genetic structure across rearranged chromosomes was in general larger than across common chromosomes. However, the difference between the two classes of chromosomes was only significant in the hybrid zone where the complexity of hybrids is expected to be larger. These differences did not distinguish populations within species. Therefore, the rearranged chromosomes appear to affect the reproductive barrier between karyotypic species, although the strength of this effect depends on the complexity of the hybrids produced.     

A hybrid zone with coincident clines for autosomal and sex-specific markers in the Sorex araneus group

In hybrid zones, endogenous counter-selection of hybrids is usually first expressed as reduced fertility or viability in hybrids of the heterogametic sex, a mechanism known as Haldane's rule. This phenomenon often leads to a differential of gene flow between sex-linked markers. Here, we address the possibility of a differential gene flow for Y chromosome, mtDNA and autosomal markers across the hybrid zone between the genetically and chromosomally well-differentiated species Sorex antinorii and Sorex araneus race Vaud. Intermarker comparison clearly revealed coincidental centre and very abrupt clines for all three types of markers. The overall level of genetic differentiation between the two species must be strong enough to hinder asymmetric introgression. Cyto-nuclear mismatches were also observed in the centre of hybrid zone. The significantly lower number of mismatches observed in males than in females possibly results from Y chromosome-mtDNA interactions. Results are compared with those previously reported in another hybrid zone between S. antinori and S. araneus race Cordon.     

Multilevel subdivision in the <Emphasis Type="Italic">araneus</Emphasis> species group of the genus <Emphasis Type="Italic">Sorex</Emphasis>: 1. Chromosomal differentiation

This review summarizes the available data on multilevel differentiation in the araneus species group of the genus Sorex (Eulipotyphla, Mammalia). Communication 1 gives basic information about the biology and morphological and karyotypic differentiation of some species belonging to this genus, as well as about the subdivision of one of the species, the common shrew (Sorex araneus), into intraspecific chromosomal races. The list of the 75 races known so far is included (26 of them dwell in Russia).     

Comparative analysis of karyotypes in European shrew species. I. The sibling species Sorex araneus and S. gemellus: Q-bands, G-bands, and position of NORs

The karyotypes of two closely related species of the genus Sorex (Mammalia, Insectivora) were compared with each other by G- and Q-banding techniques and by Ag-AS staining (GOODPASTURE and BLOOM, 1975). By comparing the G-banded karyotypes, it could be ascertained that the basic differences in karyotype between the two species lie in three pericentric inversions, three paracentric inversions, and one reciprocal translocation. This is in near agreement with FORD and HAMERTON (1970), who assumed that both species differ by three pericentric inversions and one tandem translocation. Furthermore, the karyotype of S. araneus (race C) presented by HALKKA et al. (1974) has been compared with the S. araneus of the present report. Considering the species with respect to karyotypic evolution, it is supposed that S. araneus and S. gemellus derive from a common ancestor.     

Morphological stasis and phylogenetic relationships in Tadpole shrimps, Triops (Crustacea: Notostraca)

The tadpole shrimp (Triops) is a well-known 'living fossil' whose fundamental morphology has been unchanged for over 170 million years. Thus, tadpole shrimps are suitable subjects for the study of morphological stasis. We were able to obtain samples of three species of Triops (T. granarius, T longicaudatus and T. cancriformis) from four regions in Japan. Taxonomic species were identified by diagnostic morphology. We inferred phylogenetic relationships between individual samples using mitochondrial 16S rRNA. Carapace shapes were compared among populations using shape coordinate methods. The phylogeny inferred from mtDNA shows that T. granarius is phylogenetically more similar to T. longicaudatus than to T. cancriformis. mtDNA sequences did not differ among the populations of T granarius. However, there were two distinct phylogenetic species within T. longicaudatus. In spite of the similarity in fundamental morphological characteristics among Triops species, mtDNA sequences of Triops showed marked differences among the four phylogenetic species. Among the populations of T. granarius , the carapace shape of the Fukuoka population was significantly different from those of other populations of T. granarius. The carapace shape of the Kagawa population of T. longicaudatus was more similar to those of the Shizuoka and Kagawa populations of T. granarius. The shape of the carapace of T. cancriformis was significantly different from those of T. granarius and T longicaudatus. Thus, taxonomic species, phylogenetic species and populations with similar carapace morphology did not correspond with each other. The present results indicate that most of the morphological change did not occur at the time of speciation (lineage separation) and that morphological stases are important evolutionary patterns, but they are not species-level properties.     

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.      

Using a Bayesian method to assign individuals to karyotypic taxa in shrew hybrid zones

Individuals sampled in hybrid zones are usually analysed according to their sampling locality, morphology, behaviour or karyotype. But the increasing availability of genetic information more and more favours its use for individual sorting purposes and numerous assignment methods based on the genetic composition of individuals have been developed. The shrews of the Sorex araneus group offer good opportunities to test the genetic assignment on individuals identified by their karyotype. Here we explored the potential and efficiency of a Bayesian assignment method combined or not with a reference dataset to study admixture and individual assignment in the difficult context of two hybrid zones between karyotypic species of the Sorex araneus group. As a whole, we assigned more than 80% of the individuals to their respective karyotypic categories (i.e. 'pure' species or hybrids). This assignment level is comparable to what was obtained for the same species away from hybrid zones. Additionally, we showed that the assignment result for several individuals was strongly affected by the inclusion or not of a reference dataset. This highlights the importance of such comparisons when analysing hybrid zones. Finally, differences between the admixture levels detected in both hybrid zones support the hypothesis of an impact of chromosomal rearrangements on gene flow.     

Molecular variability in the commom shrew Sorex araneus L. from European Russia and Siberia inferred from the length polymorphism of DNA regions flanked by short interspersed elements (Inter-SINE PCR) and the relationships between the Moscow and Seliger chromosome races

Genetic exchange among chromosomal races of the common shrew Sorex araneus and the problem of reproductive barriers have been extensively studied by means of such molecular markers as mtDNA, microsatellites, and allozymes. In the present study, the interpopulation and interracial polymorphism in the common shrew was derived, using fingerprints generated by amplified DNA regions flanked by short interspersed repeats (SINEs)-interSINE PCR (IS-PCR). We used primers, complementary to consensus sequences of two short retroposons: mammalian element MIR and the SOR element from the genome of Sorex araneus. Genetic differentiation among eleven populations of the common shrew from eight chromosome races was estimated. The NP and MJ analyses, as well as multidimensional scaling showed that all samples examined grouped into two main clusters, corresponding to European Russia and Siberia. The bootstrap support of the European Russia cluster in the NJ and MP analyses was respectively 76 and 61%. The bootstrap index for the Siberian cluster was 100% in both analyses; the Tomsk race, included into this cluster, was separated with the bootstrap support of NJ/MP 92/95%.     

The occurrence of Centrorhynchus (Acanthocephala) in shrews (Sorex araneus and Sorex minutus) in the United Kingdom

Encysted acanthocephalans belonging to the genus Centrorhynchus were found in the body cavities of Sorex araneus (common shrew) and Sorex minutus (pygmy shrew) from Boxworth, Cambridgeshire, U.K. Fifty percent of the male S. araneus and 67% of the male S. minutus examined were found to be infected, with the mean intensity (+/-SD) being 54.3 +/- 91.3 and 14.7 +/- 18.4, respectively. The species of Centrorhynchus in the shrews may be Centrorhynchus aluconis, which is distributed widely in tawny owls, Strix aluco, in the United Kingdom. Shrews appear to serve as paratenic hosts for C. aluconis.     

Meiotic drive favors Robertsonian metacentric chromosomes in the common shrew (Sorex araneus, Insectivora, mammalia)

Meiotic drive has attracted much interest because it concerns the robustness of Mendelian segregation and its genetic and evolutionary stability. We studied chromosomal meiotic drive in the common shrew (Sorex araneus, Insectivora, Mammalia), which exhibits one of the most remarkable chromosomal polymorphisms within mammalian species. The open question of the evolutionary success of metacentric chromosomes (Robertsonian fusions) versus acrocentrics in the common shrew prompted us to test whether a segregation distortion in favor of metacentrics is present in female and/or male meiosis. Performing crosses under controlled laboratory conditions with animals from natural populations, we found a clear trend toward a segregation distortion in favor of metacentrics during male meiosis, two chromosome combinations (gm and jl) being significantly preferred over their acrocentric homologs. Apart for one Robertsonian fusion (hi), this trend was absent in female meiosis. We propose a model based on recombination events between twin acrocentrics to explain the difference in transmission ratios of the same metacentric in different sexes and unequal drive of particular metacentrics in the same sex. Pooled data for female and male meiosis revealed a trend toward stronger segregation distortion for larger metacentrics. This is partially in agreement with the frequency of metacentrics occurring in natural populations of a chromosome race showing a high degree of chromosomal polymorphism.