Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data.

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.

The genetic structure of adders (Vipera berus) in Fennoscandia: congruence between different kinds of genetic markers.

In order to elucidate the colonization history of Fennoscandian adders (Vipera berus), the phylogeographical patterns of two nuclear sets of DNA markers (random amplified polymorphic DNA and microsatellite) are compared with that previously obtained from mitochondrial DNA. An eastern and a western lineage within Fennoscandian adders is readily distinguishable using both sets of nuclear markers, corroborating the hypothesis that the lineages stem from separate glacial refugia. Moreover, the same contact zones as were derived from mitochondrial data are clearly identifiable. Both sets of nuclear markers detect a high level of admixture across one zone in northern Finland, with introgression reaching far west into Sweden.

No apparent reduction of gene flow in a hybrid zone between the West and North European karyotypic groups of the common shrew, Sorex araneus.

The common shrew, Sorex araneus, exhibits an unusually high level of karyotypic variation. Populations with identical or similar karyotypes are defined as chromosome races, which are, in turn, grouped into larger evolutionary units, karyotypic groups. Using six microsatellite markers, we investigated the genetic structure of a hybrid zone between the Sidensjö and Abisko chromosome races, representatives of two distinct karyotypic groups believed to have been separated during the last glacial maximum, the West European karyotypic group (western group) and the North European karyotypic group (northern group), respectively. Significant FST values among populations suggest some weak genetic structure. All hierarchical levels show similar levels of genetic differentiation, equivalent to levels of genetic structure in several intraracial studies of common shrew populations from central Europe. Notably, genetic differentiation was of the same order of magnitude between and within karyotypic groups. Although the genetic differentiation was weak, the correlation between genetic and geographical distance was positive and significant, suggesting that the genetic variation observed between populations is a function of geographical distance rather than racial origin. Hence, considerable chromosomal differences do not seem to prevent extensive gene flow.

Applicability of rabbit microsatellite primers for studies of hybridisation between an introduced and a native hare species.

Introduced species may hybridise with relatives in the native fauna or flora and thereby compete for matings and transmit alien DNA. Such interference may contaminate unique genepools, disturb existing ecological balances and may ultimately result in the extinction of the native species. In Sweden, the introduced brown hare (Lepus europaeus Pall.) hybridise with the native mountain hare (L. timidus L.), both relatively common members of the present Swedish fauna. This hybridisation has resulted in the transmission of mitochondrial DNA from the mountain hare to the brown hare, but absence of species differences in karyotype and allozymes have prevented investigations of the amount of nuclear gene flow. More polymorphic genetic markers are needed to analyse evidence of hybridisation in the nuclear genome. The conservation of microsatellite loci across taxa usually enables PCR amplification of microsatellites in closely related species with the same primers. We have used five microsatellite primer pairs, developed for the European wild rabbit (Oryctolagus cuniculus L.) to amplify microsatellites in the two species of hare in Sweden. The obtained allelic variation was used to construct a genetic distance tree based on the amount of shared alleles between all pairs of individuals (shared-allele index). This method offered sufficient differences to arrange all individuals in two groups, one for each species. Identification of individual hybrids based on the number of alleles shared between the species is not possible with these five microsatellite markers.

Microsatellites in the sand lizard (Lacerta agilis): description, variation, inheritance, and applicability.

We developed microsatellite markers for the sand lizard (Lacerta agilis) to enable investigations of the genetic variability within and among populations with a heterogeneous spatial distribution in Sweden. The populations, which could not be characterized by variation in allozymes or mitochondrial DNA, had a substantial level of variability in microsatellite loci. However, the variability in Swedish populations was limited compared to a large, outbred Hungarian population. In the sand lizard, the number of (GT/CA)n repeats was approximately three times higher than that for (CT/GA)n. The number of repeats and the frequency of microsatellites were within the range reported for other species. Three of nine microsatellite loci showed alleles that could not be amplified, which is in agreement with recent reports describing microsatellite "null alleles" as a common occurrence. We discuss the caution which this calls for when calculating paternity probabilities and when estimating between-population allelic differentiation. A potential problem with different mutation rates for alleles within the same locus is discussed.

The occurrence of mountain hare mitochondrial DNA in wild brown hares.

If interspecific hybrids are fertile and backcross to either parental species, transmission of mitochondrial DNA over the species barrier can occur. To investigate if such transmission has occurred between the brown hare Lepus europeus Pall and the mountain hare L. timidus L. in Scandinavia, an analysis of genetic variation in mitochondrial DNA from 36 hares, collected from 15 localities, was performed. Sequence divergence of mtDNA between species was estimated at 8 +/- 1% (SD). Intraspecific mtDNA sequence divergence varied between 0.09 and 0.38% in brown hares and 0.10 and 1.44% in mountain hares. In six out of 18 brown hares examined, two different haplotypes of mountain hare origin were detected, demonstrating a transmission of mtDNA haplotypes from mountain hares to brown hares. The results indicate that interspecific hybridization between the two species occurs in wild populations.

Distribution and genetic heterogeneity of Puumala virus in Sweden.

Small mammals trapped in Sweden were analysed for specific antibody responses against three hantavirus serotypes and for the presence of viral antigen. To determine the genetic identity of viral RNA in lungs of seropositive bank voles (Clethrionomys glareolus), polymerase chain reactions and subsequent partial sequencing of both the M and S segments were employed. The sequences obtained were all identified as Puumala (PUU) virus, with a high degree of heterogeneity between the different geographical localities. Alignment of nucleotide and deduced amino acid sequences, together with phylogenetic analysis, showed that PUU viruses circulating in central Sweden were distinct from those in the northern region. The localization of the two distinct PUU virus genotypes was shown to correlate with the postglacial recolonization of Sweden by bank voles.

DNA fingerprinting of captive breeding pairs of lesser white-fronted geese (Anser erythropus) with unknown pedigrees.

For a number of decades, the lesser white-fronted goose (Anser erythropus) has been almost-absent from the Fennoscandian fauna and has a current population size of only about 60 breeding pairs, with fewer than 10 pairs in Sweden. During the period 1981-1991 more than 200 young have been reintroduced in northern Sweden. However, the origin and possible relatedness of lesser white-fronted individuals were unknown when the breeding program started. We have used DNA fingerprinting to assess the similarity of 18 individuals, i.e., the entire captive population used for breeding in 1991 and about 60% of the captive population used in 1981-1991. Minisatellite probe 33.15 provided an index for an average similarity of 0.39 between the mates of the 12 breeding pairs used for producing offspring for reintroduction. This is a higher similarity than in natural populations of birds in general but lower than in populations that have passed through serious population bottlenecks. Individuals originating from different breeders are more dissimilar than those from the same breeder. However, the close relationships (similarity, 0.5-0.6) found in a group of five individuals from different breeders show that selecting individuals from different breeding groups is not sufficient to prevent mating between closely related individuals.

Differential male genetic success determines gene flow in an experimentally manipulated mouse population.

Sexual selection arises when genetically different males show heritable differences in reproductive success. Mouse mating behaviour involves both male competition and female choice. In this paper we show that introduced Y-linked DNA markers spread more extensively through a natural population than do genes inherited matrilineally. Differences in mating success between the sexes and among individual males may alter the pattern and rate of gene flow in natural populations. Another interesting possibility is that the success of the introduced Y chromosome may be attributable to so-called 'selfish' traits, such as sex-linked meiotic drive or intra-uterine competition. However, this study provides little unequivocal evidence to support this view. Differential success of introduced versus resident males may have implications for the reintroduction of endangered mammals into residual wild populations.

Colonization history of north European field voles (Microtus agrestis) revealed by mitochondrial DNA.

The genetic structure of field vole (Microtus agrestis) populations from northern Europe was examined by restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) in 150 individuals from 67 localities. A total of 83 haplotypes was observed, most of which were rare and highly localized geographically. Overall nucleotide diversity was high (1.34%), but showed a tendency to decrease with higher latitude. Two major mtDNA lineages differing by 2% in nucleotide sequence were identified. A southern mtDNA lineage was observed in field voles from Britain, Denmark and southern and central Sweden, whereas voles from Finland and northern Sweden belonged to a northern lineage. The strict phylogeographic pattern suggests that the present population genetic structure in field voles reflects glacial history: the two groups are derived from different glacial refugia, and recolonized Fennoscandia from two directions. A 150-200-km-wide secondary contact zone between the two mtDNA groups was found in northern Sweden. Distinct phylogeographic substructuring was observed within both major mtDNA groups.

Paternity determination in the adder (Vipera berus)--DNA fingerprinting or random amplified polymorphic DNA?

We performed breeding experiments with adders (Vipera berus) to determine whether multiple matings may result in multiple paternity. DNA fingerprinting of mothers, their offspring, and possible fathers using a polydinucleotide probe [(TG)n] gave a low overall similarity between unrelated individuals (0.18 +/- 0.07; SD) and an average of 17 bands that were male-specific. In no cases were there fewer than seven paternal-specific bands present in the fingerprint of an offspring, enabling us unambiguously to identify the biological father among five males. Multiple paternity was detected in the investigated broods with offspring sired exclusively by the captive males. PCR amplification of random amplified polymorphic DNA (RAPD) using 16 decamer primers gave 76 bands and an average similarity of 0.95 (+/- 0.01) between the males, which were collected at different, geographically well-separated localities. Although there were on average 8.3 (+/- 1.9) bands that differ between males in pairwise comparisons, there were only 1.9 (+/- 1.1) bands per male that are specific for a particular individual. Thus, RAPDs are adequate for paternity determination only in experiments with a low number of males, whereas DNA fingerprinting offers sufficient information to discriminate between large numbers of putative fathers.

Multiple paternity in wild common shrews (Sorex araneus) is confirmed by DNA-fingerprinting.

We have tested for the occurrence of multiple paternity in wild common shrews by karyotypic analysis and DNA-fingerprinting of five wild-caught females and their litters. Karyotypic data suggest that some litters were sired by more than one male, but provide no definitive evidence. By using DNA-fingerprinting, it was possible to establish that two males sired the litter of two females. The present report shows that multiple paternity is not a rare phenomenon in the common shrew and by using DNA-fingerprinting it is possible to assign individual offspring to different male parents even when none of the putative fathers are available for inspection.

Genetic divergence in mitochondrial DNA between the wood mouse (Apodemus sylvaticus) and the yellow necked mouse (A. flavicollis).

Genetic variability and the relationships between clones of mitochondrial DNA (mtDNA) provide data which can be used to construct hypotheses about the biogeographical and postglacial colonization history of Fennoscandian species. We have investigated fragment differences in mtDNA among 24 Wood mice (Apodemus sylvaticus) from 8 localities, and 9 Yellow necked mice (A. flavicollis) from four North European localities. The greatest intraspecific sequence divergence found between mtDNA clones was 1.40% within A. sylvaticus and 1.00% within A. flavicollis. The mean pairwise divergence in samples from a local population (n = 14) of A. sylvaticus was only 0.003%. Interspecific fragment comparison of mtDNA from A. sylvaticus and A. flavicollis give a sequence divergence close to 10%. However, comparison of genetic distances based on mtDNA between the two Apodemus species shows that they are more distantly related than suggested by nuclear distances. The observed discrepancy between distances estimated from mitochondrial and nuclear DNA suggests that mtDNA divergence predated divergence in nuclear genes or that there was an influx of nuclear variation during the speciation process or in connection with the expansion and contractions of populations during interglacial and glacial periods.

Somatic hybridization between anther-derived dihaploid clones of potato (Solanum tuberosum L.) and the identification of hybrid plants by isozyme analysis.

Green mesophyll protoplasts of the dihaploid potato line 198∶2 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 67∶9 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%-4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.

Concordant divergence in proteins and mitochondrial DNA between two vole species in the genus Clethrionomys.

The bank vole (Clethrionomys glareolus) and the northern red-backed vole (C. rutilus) are two closely related species where interspecific crosses result in fertile female but sterile male offspring. Mitochondrial DNA (mtDNA) from C. rutilus has passed the species barrier and is found in C. glareolus from northern Fennoscandia. The present report shows that the genetic distance between the two species, calculated from enzyme data (Nei's D), is 0.64. Isoelectric focusing of muscle proteins resolved around 55 bands, of which each species had 6 or 7 bands not present in the other species. Sequence divergence of mtDNA from the two species is 13.9%. A comparison between protein and mtDNA distances in other species pairs reveals a high correlation between the two measures, indicating that differences in mtDNA between taxa are not random when compared to divergence in protein-coding nuclear genes. The relationship between genetic divergence in proteins and that in mtDNA between Clethrionomys glareolus and C. rutilus is similar to that found in other species pairs. It is also shown that despite large differences on the protein level it is still, in some cases, possible for species pairs to produce fertile hybrid females.

Genetic variability in mitochondrial DNA in a regional population of the great tit (Parus major).

Genetic variation of mitochondrial DNA (mtDNA) in 18 great tits (Parus major) from three neighboring localities in Sweden was investigated with eight tetranucleotide restriction endonucleases. The 18 individuals could be separated into 13 different maternal lineages. The high number of female lineages present in this regional population contrasts with a low level of sequence divergence between the different mtDNA clones, with a mean of 0.19% sequence divergence between all individuals. There was no obvious spatial structuring of mtDNA clones among the three localities. The presence of a high number of different clones with a low degree of sequence divergence could be explained by the effects of a large long-term effective population size, with the mtDNA clones having diverged about 25,000-200,000 years ago.