http://www.alpha-gruppen.com/artikler/dna_analysis.htm Här fanns denna att hämta tidigare.
Men av någon anledning fungerar inte länken längre. Kanske någon inte gillar innehållet, men för att inte göra läsarna besvika finns den här nedan i stället. Kenneth Erikson 121203
Alpha gruppen http://miljolare.no/aktiviteter/land/areal/la1/innspel/?org=alphagruppen har åtminstone tidigare sorterat under http://miljolare.no/, som i sin tur finansieras av Norska staten. Det var på deras hemsida länken till nedanstående DNA-undersökning tidigare fanns.
På fråga under offentligt informationsmöte om varg av Olof Liberg sommaren 2013 om denna DNA-undersökning förnekar han dess existens. Orsaken sades vara att den ej finns och att det saknas uppgift om författare. Det går att förstå. Vem vill skriva och stå bakom denna DNA-undersökning, längre, när någon från Folket i busken har upptäckt att den innehåller olycklig information. Då tänker jag naturligtvis på det jag markerat i rött, som om det sammanvägs, bör betyda att den Norsk – Svenska vargstammen är illegalt inplanterad.
Tror fasen det att Olof Liberg inte vill kännas vid denna.
Visst var det tråkigt att undersökningen av dessa vargar i Norge inte längre kunde hittas. Och som jag skrivit tidigare, pensionerade rovdjursforskaren Olof Liberg förnekade dess existens med argumentet att det saknades uppgifter om vilka som gjort den. Det är inte svårt att förstå att Liberg reagerade på mina slutsatser när det tydligen hade förbisetts av de som skapade den. Det visar också på att det är troligt att Liberg själv sökt efter den och kanske även påverkat att den försvann från norska Alpha-gruppen.
Nog för att jag själv försökte hitta den på nätet, men jag fick ge upp. Tur då att det finns personer som också såg värde i undersökningens slutsatser och inte gav sig lika snabbt som undertecknad.
Combined use of maternal, paternal and bi-parental genetic markers for the identification of wolf–dog hybrids, av: C Vilà, C Walker, A-K Sundqvist, Ø Flagstad. Accepted 3 August 2002.
Det visar sig att det är Uppsala Universitet ( i samarbete med Lettland, Italien, Finland, Estland och USA) som står bakom den med flera garanterat kända namn och personer för Olof Liberg. Han återfinns faktiskt själv tillsammans med kollegor under referens nr. 51.
Obs! Som jämförelsematerial för hundar har endast hundar från USA använts.
Samma uppgifter återfinns på sid. 23 och 24 i denna rapport från Uppsala Universitet: Conservation Genetics of Wolves and their Relationship with Dogs av Anna-Karin Sundqvist 2008, ISSN 1651-6214.
Jag klistrar in den första rapporten på slutet, efter nedanstående.
DNA-analysen som sannsynliggjorde at de omtalte dyrene var hybrider av Canis lupus lupus og Canis lupus familiaris
PRESENTATION OF DNA ANALYSIS
Direktoratet for naturforvaltning arranged for us to be provided with three samples from Norway for genetic analyses.
Sample A. Blood from a juvenile individual run over and killed in Våler (Østfold) on 1 October 1999.
Sample B. Snow with urine and blood collected 1 March 1999. Assumed to correspond to the alpha female in the wolf pack in Moss (in heat). Preserved frozen.
Sample C. Drop of blood collected from the snow on 9 January 1999. From the injured back foot of the animal. Assumed to correspond to the alpha female in the wolf pack in Moss. Preserved in 95% ethanol.
Information concerning the respective samples given above is as we have been informed through contacts with Terje Bø and Morten Kjørstad at Direktoratet for naturforvaltning and with Rune Bergstrøm and Asmund Fjellbakk at fylkesmannen in Østfold. We will generally refer to the samples by the letters A, B and C.
We have analysed samples A, B and C in relation to our own stock of wolf and dog samples. The latter wolves are from Sweden and Norway (25), Finland (25), Russia (24) and Estonia (23), and have been collected mainly during the last few years, with the exception of some samples from Sweden which are from 1983 and onwards. Our 44 dog samples correspond to pure bred huskies (19), German shepherd dog (7), American eskimo (7), Alaskan malamute (6), elkhound (3), keeshound (1) and kuvasz (1), all from the USA. We assume that members of the same breed sampled on different continents will be sufficiently similar to each other to be useful as reference population in this analysis. Importantly, the critical issue is that they are more similar to each other than to different populations of wolves, which seems to be a reasonable assumption. Dog breeds were chosen based on somewhat morphological similarity with wolves.
Laboratory and data analysis: mitochondrial DNA
A general overview of the characteristics of the genetic markers used in this study is provided in Appendix 1. DNA was isolated using slight variations on phenol-chloroform extraction methods (Sambrook et al. 1989). For sample B, a part of the volume of snow containing the urine and blood had to be thawed and centrifugated for an extended period of time to concentrate the DNA before isolation. For sample C, a small amount of coagulated blood was taken from the ethanol for the extraction.
Amplification of a fragment of about 400 base pairs (bp) from the mitochondrial DNA control region was performed via the polymerase chain reaction (PCR) using universal primers modified from Kocher et al. (1989). Precise primer sequences, PCR conditions and profile are available upon request. PCR products were sequenced using Big Dye Terminator cycle sequencing chemistry on an ABI 377 instrument (Perkin Elmer), following protocols provided by the manufacturer. Sequences were aligned using the program CLUSTAL W (Higgins et al. 1992) and checked by eye. All sequences were compared to each other and to sequences available in GenBank and databases previously developed (based on Ellegren et al. 1996, Okumura et al. 1996, Taberlet et al. 1996, Tsuda et al. 1997, Vilà et al 1997, Pilgrim et al. 1998, Vilà et al. 1999) using the program PAUP* (Swofford 1998).
Laboratory and data analyses: Y chromosome microsatellite
We made use of a canine Y chromosome microsatellite marker developed by Olivier et al. (1999), with some optimization to allow more specific amplification. The forward primer was labeled with a fluorescent dye and the PCR products run on an ABI 377 instrument (Perkin Elmer), following protocols provided by the manufacturer. PCR primers, conditions and profile, as well as technical details on the protocols are available upon request. Using the program GENESCAN (Perkin Elmer), the electrophoresis running times were correlated to the size of the DNA fragments, and the alleles observed for each microsatellite were subsequently scored using the software GENOTYPER (Perkin Elmer).
Laboratory and data analysis: autosomal microsatellites
Eightteen microsatellites developed for dogs were selected for this study: c2001, c2010, c2017, c2054, c2079, c2088 and c2096 (Francisco et al. 1996), vWF (Shibuya et al. 1994), u213, u250 and u253 (Ostrander et al. 1993), and PEZ01, PEZ03, PEZ05, PEZ06, PEZ08, PEZ12 and PEZ20 (Perkin Elmer, Zoogen). As for the electrophoresis of PCR products, allele identification and scores, we followed the same protocol used for the Y chromosome microsatellites.
To study the likelihood of finding one of the observed genotypes in each one of the populations we used an assignment test (Paetkau et al. 1995, Waser and Strobeck 1998). This analysis calculates the likelihood of finding a certain genotype in each population and assigns the individual to the population for which it has the highest likelihood. If the frequency of allele A in a population is pA, the frequency of homozygote AA for a certain locus would be pA2 and the frequency of a heterozygoteAB would be 2pApB, according to simple Hardy-Weinberg principles. For a multilocus genotype (assuming that loci are not linked, which seems reasonable in this case), the likelihood of finding the genotype in a population will be the product of the frequencies for all alleles at all loci. If the likelihood of finding a certain genotype is significantly higher for one population than for the others, this could suggest that the individual sample belongs to or originated from that population (for example, see Paetkau et al. 1998). Since the likelihood values are typically extremely low, the log likelihood is indicated.
However, a higher likelihood may indicate that a certain genotype is more similar to the genotypes expected in one population than to the genotypes expected in others, but this does not indicate that it originated in the first population. Since we have only been able to genotype a limited number of individuals from each population, we can not expect our sample to represent most of the genotypic variability in the population. To characterize how well an individual genotype fits into the distribution of genotypes that should be expected from each population, we generated 1000 synthetic genotypes taking random alleles for each locus according to their frequency. These synthetic genotypes represent multilocus genotypes that could occur in the observed allelic distributions but may or may not have been sampled. Similarly, we generated populations of 1000 synthetic genotypes of hybrids between dogs and Swedish wolves. In this case, each synthetic genotype contained per locus one allele derived from each one of the parent populations. If the likelihood of the assignment of the target sample is outside the range observed for the 1000 synthetic animals corresponding to one population, we have a statistical basis for rejecting that population as the origin of the target sample.
Since the number of microsatellites successfully scored was different for each target sample, a new set of histograms was constructed for each one. To standardize the histograms of Figure 2, the log likelihood of assignment of the target sample to the wolf population was subtracted from the log likelihoods of all synthetic genotypes. If the assignment for the target sample lies outside the distribution of assignment likelihoods for the synthetic population (or inside the 2.5% margins at each side of the distribution), -that is to say, if the value 0 is included in the distributions shown in the histograms- the hypothesis that the target sample belongs to that population should be rejected.
In order to determine if the female of sample B could be the mother of the young canid (sample A), we compared the alleles found at each microsatellite. Assuming that she is the mother, we can determine what should be the allelic composition of the paternal contribution at some microsatellites. We constructed a synthetic genotype homozygous for those alleles and calculated its assignment likelihood to different populations.
Results and discussion
Mitochondrial DNA (mtDNA) sequences
Sample A and sample B showed the same mtDNA D-loop haplotype. This is the same haplotype that was found in all Swedish and Norwegian wolves sampled from 1983 and onwards by Ellegren et al. (1996). Sample C showed a distinctly different haplotype, which excludes it representing the same individual as sample B as well as excluding it being the mother of sample A. Moreover, the haplotype shown by sample C (D10, according to Vila et al. 1997) has previously only been seen in dogs (e.g. dachshund and flat-coated retriever; Vilà et al. 1997). Consequently, samples A and B are compatible with being from animals that have wolf ancestry in the maternal line, while sample C is compatible with being an animal that is either a dog or has dog ancestry in the maternal line.
Y chromosome microsatellite
The Y chromosome microsatellite was successfully amplified in samples A and C, but not in sample B. A reasonable interpretation is thus that sample B comes from a female. Sample A and sample C had different alleles, suggesting that the sample C(now known to be a male) is not the father of the young of sample A. Importantly, neither the allele of sample A or of sample C has previously been seen among Scandinavian wolves (our unpublished results). However, both alleles have been seen in other wolf populations, as well as in dogs.
An assignment test comparing, on the one hand, the Scandinavian wolf population and, on the other hand, our population of dogs shows that the observed allelic distributions allow for distinguishing between these two groups of animals (Figure 1). In this graphical illustration of the test, all dogs are located in the upper left corner (higher likelihood of being dogs than wolves), whereas the Scandinavian wolves are in the lower right corner.
The plot also includes the target samples. Sample A lies between the distributions of dogs and wolves, a position that would be expected for a wolf-dog hybrid. Sample B appears on the wolf side of the distribution. Finally, sample C, for which mtDNA and Y chromosome microsatellite data suggest dog ancestry, appears in the middle of the group of dogs, supporting the same conclusion.
To analyze if the target samples significantly differ from the distribution of expected genotypes for either Scandinavian wolves, dogs or F1 hybrids between these two groups, the histograms in Figure 2 have been constructed. Each histogram reflects the distribution of the log likelihood of assignment to the Scandinavian wolf population of 1000 synthetic hybrids, 1000 synthetic dogs and 1000 synthetic Scandinavian wolves. Figure 2a shows that the genotype of sample A is significantly different from what we would expect for pure dogs or Scandinavian wolves, but is inside the distribution for F1 hybrids. We can reject the hypothesis that sample A originated from either a pure wolf or a pure dog population, but we cannot reject it from having a hybrid origin. Figure 2b indicates that the genotype of sample B is outside the expected distribution for hybrids and dogs, but is inside the distribution expected for Scandinavian wolves. We can reject the hypothesis that sample B has either a hybrid or dog origin, but we cannot reject the hypothesis of Scandinavian wolf origin. Finally, sample C has a genotype that can only be expected from pure dogs. Here, we can reject the hypothesis that sample C has either a pure Scandinavian wolf or a hybrid origin, but we cannot reject the hypothesis of a pure dog origin. The data are summarized in Table 1
A similar analysis shows that none of the target samples can be identified as a wolf immigrant from Finland or Russia (Table 1). For sample A and sample B, we also tested if their likelihood was outside the expected distribution for a F1 hybrid between a dog and an immigrant. In both cases the target samples are outside the distributions and thus this possibility can also be excluded.
The allelic composition of the three target samples is indicated in Table 2. At four loci (C2001, C2017, U253, PEZ06) sample Aand C are not compatible, reasonably excluding the animal of sample C to be the parent of the animal of sample A. For the comparison of sample A and sample B, we saw non-congruence for one marker. This may either suggest an errornous genotyping (given that sample B and A were parent-offspring), or that that the animal of sample B is not the parent of the animal of sample A.
Since the information provided by Direktoratet for Naturforvaltning states that the female from which sample B originated was the mother of the young of sample A, we have made this assumption for determining the alleles that should come from the father (Table 2). The likelihood of a genotype homozygous for these alleles will be the square of the likelihood for the paternal haplotype. In the same way that the origin of each one of the samples was assessed by comparison to synthetic genotypes, the origin of the father of sample A is analyzed in Table 1. The likelihood of obtaining this haplotype from a Scandinavian, Finnish or Russian wolf is very low and outside the distribution expected for each of them. However, the likelihood for the paternal haplotype falls inside the distribution for pure dogs. Consequently, the microsatellite data suggest the young of sample A having one wolf and one dog as parents.
Given the available reference material (wolves from Scandinavia, Finland, Russia and Estonia; dogs from North America), we come to the following conclusions.
• Sample A, stated to be from a young wolf-like animal run over by a vehicle in Østfold on 1 October 1999, has a DNA profile that is compatible with that arising from a hybridization between a wolf and a dog, but is not compatible with that of a pure Scandinavian wolf. It thus seems reasonable to assume that the animal is a wolf-dog hybrid.
• Sample A has a mitochondrial DNA haplotype that suggests it to have a wolf as mother.
• Sample A has a Y chromosome DNA profile that suggests it to not have a Scandinavian wolf as father, unless recent immigration has occured.
• The blood samples B and C, stated to be collected from the snow on 1 March and 9 January 1999, respectively, correspond to two different animals. Most likely, sample Bis a female wolf from the Scandinavian population whereas sample C is a male dog.
• The presumed male dog of sample C is not the father of the young animal of sampleA.
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Figure 1. Assignment log likelihood for dogs and Swedish+Norwegian wolves. The three target samples are also indicated.
Figure 2. Distribution of the assignment likelihood to the Scandinavian wolf population of 1000 synthetic genotypes corresponding to dogs, Scandinavian wolves and F1hybrids between dogs and wolves. The synthetic genotypes are generated taking into consideration the allelic distributions in the parental populations (see text). Since the number of microsatellites successfully amplified for each target sample varied, the distributions for the synthetic populations were repeated considering only the loci that had been successfully amplified for the sample studied. The likelihoods are standardized by subtracting the likelihood calculated for each target sample. Consequently, if the distribution of likelihoods in the synthetic populations does not encompass the value observed for the target sample (corresponds to the value ”0” after standardization), or if this value is inside the 2.5% margins at each side of the distribution, we can conclude that the sample does not fit the expected allelic distribution for the synthetic population. That combination of alleles is uncommon.
Table 2. Microsatellite alleles identified for each target sample. Last column indicates alleles that could be identified as coming from the father of A assuming that B is the mother.
Microsatellite Locus Sample A (991001) Sample B (990301) Sample C (990109) Paternal Alleles for sample A
c2001 149/153 153/153 133/145 149
c2010 225/237 x/x 237/237 x
c2017 258/266 x/x 262/270 x
c2054 148/152 148/148 148/160 152
c2079 275/283 271/271 275/279 x
c2088 131/135 127/135 x/x 131
c2096 95/103 95/95 99/103 103
PEZ01 112/120 120/120 120/120 112
PEZ03 132/138 138/138 132/144 132
PEZ05 96/104 96/96 104/108 104
PEZ06 174/174 174/174 186/190 174
PEZ08 238/238 x/x 234/238 238
PEZ12 272/272 x/x 258/272 272
PEZ20 177/177 177/177 173/177 177
u213 159/162 x/x x/x x
u250 126/138 x/x x/x x
u253 106/112 106/106 108/108 112
VWF 157/157 x/x 157/187 157
Appendix 1. Types of genetic markers used in this study
Mitochondrial DNA sequences
Mitochondrial DNA (mtDNA) is exclusively inherited on the maternal side. For this reason, and because it is easy to amplify, mt DNA has been extensively used in phylogenetic studies. Different regions of the mtDNA molecular evolve at different rates. The control region contains the origin of the replication of the heavy strand of the DNA. It does not code for any protein and any mutation is likely to have no or only a very small effect on the survival of the individual, and can be transmitted to the next generation. As a result, mutations accumulate faster in this region of the mtDNA than in other areas of the genome. In a shorter time some differentiation of DNA sequences can be achieved. Several studies have shown that mitochondrial DNA control region sequences in wolves and dogs can be distinguished in most cases (Okumura et al. 1996, Vilà et al. 1997, Vilà and Wayne 1999). Consequently mtDNA can be used to investigate the maternal ancestry of a certain individual. However, it does not provide information about the overall genetic composition of the individual. For example, if the maternal grand mother is a dog and all other grand parents are wolves, the mtDNA sequence would correspond to one of those in dogs.
Y chromosome microsatellites
Y chromosomes are only present in males of mammals and are always directly inherited from the father. Consequently, any polymorphic genetic marker on this chromosome could allow to follow the paternal lineage in the same way as mtDNA can reveal the maternal lineage. Olivier et al. (1999) were able to isolate some DNA sequences that would amplify only in male dogs and thus would be expected to be in the Y chromosome. One of the sequences reported contained one microsatellite. After constructing primers specific for the amplification of that microsatellite, two PCR products were obtained, both of them variable in male dogs, suggesting that the microsatellite was duplicated inside the Y chromosome. The analysis of the variability in these two copies of the microsatellite can be used to infer paternal lineages. However, as for the mtDNA, the information obtained is only partial (i.e. paternal lineage whereas mtDNA is maternal lineage).
Microsatellites are sequences made up of a single sequence motif, no more than six bases long, that is tandemly repeated (Hancock 1999). Microsatellites are distributed over the entire genome and their high mutation rate makes them exceptionally useful for evolutionary and genetic studies (Bruford and Wayne 1993). This instability is mainly the result of changes in the number of copies of the microsatellite repeat, caused by slipped-strand mispairing errors during DNA replication (Eisen 1999). The large variability of microsatellites makes possible a wide range of applications, from the comparison of populations (i.e. Roy et al. 1994) to the identification of individuals (Balding 1999) and their relatedness (Ellegren 1999). If different wolf populations are more or less isolated we can expect some degree of differentiation in the diversity of alleles observed at multiple microsatellite loci. Especially if the populations have gone through a demographic bottleneck, as seems to be the case for some wolf populations, the differentiation should be accelerated due to random genetic drift (Hartl and Clark 1997). Consequently, the comparison of the genotypes at multiple loci observed for each unknown sample with the distribution of alleles observed for each population could allow us to identify the population most likely to be the source for that individual. The combination of alleles that constitute one genotype can be much more probable in one population than in the others. Compared to mtDNA or Y chromosome sequences, the study of microsatellites can show the existence of admixture or migration independently of the sex of the alien parent. The combination of all three techniques may produce a better picture of the pattern of differentiation and introgression between populations.
Här rapporten enligt uppdatering 140824:
Heredity (2003) 90, 17–24. doi:10.1038/sj.hdy.6800175
Combined use of maternal, paternal and bi-parental genetic markers for the identification of wolf–dog hybrids
- 1Department of Evolutionary Biology, Uppsala University, Norbyvägen 18D, S-75236 Uppsala, Sweden
- 2Kemeri National Park, ‘Meza maja’, Kemeri-Jurmala, LV-2012, Latvia
- 3Laboratory of Parasitology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy
- 4Finnish Game and Fisheries Research Institute, Oulu Game and Fisheries Research, Tutkijantic 2A, FIN-90570 Oulu, Finland
- 5Institute of Zoology and Hydrobiology, Tartu University, EE2400, Vanemuise 46, Estonia
- 6QuestGen Forensics, 1902 E, 8th Street, Davis, CA, 95616, USA
Correspondence: C Vilà, Department of Evolutionary Biology, Uppsala University, Norbyvägen 18D, S-75236 Uppsala, Sweden. E-mail:email@example.com
Received 18 November 2001; Accepted 3 August 2002.
The identification of hybrids is often a subject of primary concern for the development of conservation and management strategies, but can be difficult when the hybridizing species are closely related and do not possess diagnostic genetic markers. However, the combined use of mitochondrial DNA (mtDNA), autosomal and Y chromosome genetic markers may allow the identification of hybrids and of the direction of hybridization. We used these three types of markers to genetically characterize one possible wolf–dog hybrid in the endangered Scandinavian wolf population. We first characterized the variability of mtDNA and Y chromosome markers in Scandinavian wolves as well as in neighboring wolf populations and in dogs. While the mtDNA data suggested that the target sample could correspond to a wolf, its Y chromosome type had not been observed before in Scandinavian wolves. We compared the genotype of the target sample at 18 autosomal microsatellite markers with those expected in pure specimens and in hybrids using assignment tests. The combined results led to the conclusion that the animal was a hybrid between a Scandinavian female wolf and a male dog. This finding confirms that inter-specific hybridization between wolves and dogs can occur in natural wolf populations. A possible correlation between hybridization and wolf population density and disturbance deserves further research.
Canis lupus, Canis familiaris, hybridization, Y chromosome, mtDNA, microsatellites
Hybridization is a natural process that can lead to speciation. It is also an undesirable issue threatening the genetic integrity of endangered species (Arnold, 1997). Detecting the degree or extent of hybridization between species is thus important for evolutionary studies of speciation processes, as well as for conservation biology studies of species potentially in genetic peril. Moreover, being able to detect individual cases of hybridization may be important from a management perspective. Studies on hybridizing species and populations have increasingly sought to use genetic markers that are unique for each taxon (Saetre et al, 2001), in some cases combined with morphological characters (Beaumont et al, 2001). Also, hybrid populations have been compared to pure populations to infer the degree of gene flow (Reich et al, 1999;Madrigal et al, 2001). However, given that hybridization is most likely between closely related taxa, in many cases differentiation between hybridizing populations may be primarily in the form of allele frequency differences rather than the frequent occurrence of private alleles. Identifying individual hybrids in such cases may be particularly problematic. The issue of potential hybridization between wolves (Canis lupus) and dogs (C. familiaris) represents an example of this situation.
Hybridization can occur between many species of the canid family (Gray, 1954; Lehman et al, 1991; Mercureet al, 1993; Roy et al, 1996; Wayne and Brown, 2001) and sometimes threatens the survival of endangered canid species or populations (Nowak, 1979; Wayne and Jenks, 1991; Gottelli et al, 1994; Roy et al, 1994). The close relationship between wolves and dogs, a consequence of their recent divergence (Vilà et al, 1997), suggests that hybridization between these species could be especially common since reproductive isolation may not be completely developed. Wolves coexist with dogs across most of their range.
Wolf populations in Eurasia have become increasingly fragmented during the last centuries (Mech, 1970;Wayne et al, 1992). Their numbers have dramatically decreased and in most areas of Europe only small populations survive in close contact with increasing numbers of humans and domestic dogs (Promberger and Schröder, 1992). It is under these conditions that hybridization between wolves and dogs is most likely to occur (Boitani, 1983; Bibikov, 1988; Blanco et al, 1992). Boitani (1984) hypothesized that the recovery of wolf populations in Italy could have been the result of hybridi-zation with dogs, and Butler (1994) suggested that European wolf populations could be composed mainly of hybrids.
Despite these concerns, a recent review of genetic evidence has suggested that wolf–dog hybridization may not be a threat even in small, endangered wolf populations near human settlements (Vilà and Wayne, 1999). Specifically, the analysis of mitochondrial DNA (mtDNA) suggests that hybridization between wolves and dogs is uncommon, that is, there is no clear evidence of introgression of dog mtDNA into wolf populations, except a few cases in an east European wolf population (Randi et al, 2000). However, this infrequent presence of dog mtDNA haplotypes in wolves only implies that offspring of crosses between female dogs and male wolves are uncommon or do not back-cross into wolf populations. The use of mtDNA cannot provide any information about introgression of hybrids of crosses between a male dog and a female wolf. However, pairs composed of a female wolf and a male dog have been observed in Russia, Israel, Italy and Spain (Ryabov, 1985; Randi et al, 1993; Vilà and Wayne, 1999; however, see Randi et al, 2000) and some recent studies involving nuclear markers have shown that hybridization occasionally occurs in the wild (Andersone et al, 2002; Randi and Lucchini, 2002). More detailed genetic studies using a variety of genetic markers and in different populations are thus necessary to conclusively address the issue of wolf–dog hybridization and to understand its directionality and frequency of occurrence. As a result of the current fragmentation of the wolf distribution range into more or less small patches (Promberger and Schröder, 1992), the detection of these inter-specific crosses may be especially troublesome in areas where the arrival of wolves from other populations – likely to be genetically differentiated to some degree – may occur.
Hybridization with dogs could potentially be expected for Scandinavian (Swedish+Norwegian) wolves. This wolf population, presumed extinct during the 1970s, was founded by a very small number of individuals in the early 1980s (Wabakken et al, 2001), and by the winter 2001–2002 was about 92–107 animals (Aronsonet al, 2002). In 1999, a presumed juvenile wolf was found road-killed in southern Norway, close to Oslo. The uncommon morphology of the animal gave rise to questions about its possible hybrid origin. In this study we combined the use of mtDNA, autosomal and Y chromosome markers to analyze the identity of this juvenile canid and we attempt to genetically characterize it as either a pure Scandinavian wolf, a migrant from Finland or Russia, a domestic dog, or a first-generation hybrid between any of these groups.
Materials and methods
The study focused on two samples from the county of Østfold in southern Norway: sample A was blood from a juvenile individual killed by a car in October 1999 and sample B constituted snow with urine and blood collected at the end of the previous winter, in March 1999. Sample A is derived from the suspected hybrid, while sample B was assumed to correspond to the alpha female in estrus from a wolf pack close to the site where sample A was killed. In the winter of 1998/99, when sample B was collected, she was in estrus but snow tracking suggested that she was not yet paired to a male. Apparently, she was the only wolf in the area. However, during spring 1999 she was sighted with a male wolf and in the summer a litter of at least four pups was detected (Terje Bø, personal communication). As far as is known, this was the first time that this female was breeding.
Samples A and B were analyzed together with DNA samples extracted from muscular tissue of wolves from Scandinavia collected after 1980 (n=25), Finland (n=23), northwest Russia (n=24) Latvia (n=8) and Estonia (n=23), as well as of 44 domestic dogs. The dog samples correspond to pure-bred Huskies, Eskimo dogs, Akita, Elkhound, Wolfspitz, Great Pyrenees, Kuvasz and German Shepherd dogs. Although the dog samples originated from the USA, we assume that members of the same breeds in different continents will still be more similar to each other than to different populations of wolves. A separate set of 38 male pure-bred Scandinavian dogs from diverse breeds was also genotyped for Y chromosome markers.
DNA was isolated using variations on phenol–chloroform extraction methods (Sambrook et al, 1989). For sample B, snow containing urine and blood was centrifuged for over 30 min to concentrate cells before attempting DNA isolation.
Amplification of a 350 base pairs (bp) fragment of the mtDNA control region I was performed via the polymerase chain reaction (PCR) using primers Thr-L 15926 and DL-H 16340 (modified from Kocher et al, 1989). PCR conditions and profile were as described inVilà et al (1999). PCR products were sequenced using Big Dye Terminator cycle sequencing chemistry on an ABI 377 instrument (Perkin Elmer), following protocols provided by the manufacturer. Sequences were aligned using the program CLUSTAL W (Higgins et al, 1992) and checked by eye. All sequences were compared to each other and to sequences available in GenBank and databases previously developed (based on Ellegren et al, 1996; Okumura et al, 1996; Taberlet et al, 1996; Tsudaet al, 1997; Vilà et al, 1997,1999, Pilgrim et al, 1998;Randi et al, 2000), using the program PAUP*4.0b8 (Swofford, 1998).
A total of eighteen autosomal microsatellites developed for dogs were selected for this study: c2001, c2010, c2017, c2054, c2079, c2088 and c2096 (Francisco et al, 1996), vWF (Shibuya et al, 1994), u213, u250 and u253 (Ostrander et al, 1993), and PEZ01, PEZ03, PEZ05, PEZ06, PEZ08, PEZ12 and PEZ20 (Perkin Elmer, Zoogen; see dog genome map athttp://www.fhcrc.org/science/dog_genome/dog.html). In addition, one highly polymorphic Y chromosome microsatellite, MS41B (Sundqvist et al, 2001), was analyzed. This marker was only genotyped in the additional set of 38 pure-bred male dogs and the target samples, and the results were compared to results published by Sundqvist et al (2001) for north European wolves. PCR products, including one fluorescently labeled primer, were run on an ABI 377 instrument (Perkin Elmer) following protocols provided by the manufacturer. PCR primers, conditions and profile were essentially as in the original reports. The alleles observed for each microsatellite were sized and scored using the software Genescan 3.1 and Genotyper 2.1 (Perkin Elmer). Owing to the small amount and low quality of DNA extracted from sample B only a limited number of microsatellite amplifications could be successfully performed for this individual.
To study the likelihood of finding one of the observed autosomal genotypes in each one of the reference populations, we used an assignment test (Paetkau et al, 1995,1998; Waser and Strobeck, 1998). This calculates the log likelihood of finding a certain genotype combination in each population and assigns the individual to the population for which it has the highest likelihood. From the moderate number of genotypes gathered from each population (n=23–44), we cannot expect the samples to represent most of the variability in the populations, although the allele frequencies should be well represented. To characterize how well an individual genotype did fit into the distribution of genotypes expected from each population, we generated 1000 synthetic genotypes taking random alleles for each locus according to their frequency. Similarly, we generated populations of 1000 synthetic genotypes of hybrids between dogs and Scandinavian wolves, and between dogs and wolves from neighboring populations (see Thulin, 2000). In these cases, the synthetic genotypes contained one allele derived from each of the two parent populations at each locus. We then calculated the likelihood of assignment to the Scandinavian wolf population. If the likelihood of assignment of a target sample was outside the range observed for the 1000 synthetic genotype combinations, we assumed that the sample did not belong to this population. To standardize the likelihood estimates, the log likelihood of assignment of the target sample to the wolf population was subtracted from the log likelihoods of the synthetic genotypes. After standardizing, the likelihood for the target sample becomes zero. If the value zero lies outside the distribution of assignment likelihoods for the synthetic population (or inside the 2.5% margins at each side of the distribution), the hypothesis that the target sample belongs to that population should be rejected. Since the number of microsatellites successfully scored was different for each target sample, the analyses were redone for each of the target samples including only the loci successfully amplified.
As a complement to the assignment test, we also used a model-based genetic mixture analysis developed byPritchard et al (2000), which is implemented in the program Structure (available athttp://www.stats.ox.ac.uk/~pritch/software.html). This program is based on a Bayesian approach and we used it to identify two groups (K=2) in a sample composed of Scandinavian wolves and domestic dogs. Besides this initial classification of each individual sample, we used Structure to estimate the probability that each sample represented an immigrant or had a parent or grandparent that was an immigrant.
Assuming that the female of sample B is the mother of sample A (see below), we deduced the composition of paternally contributed alleles. We constructed a synthetic genotype homozygous for those alleles and calculated its assignment likelihood to different populations. Thus, the likelihood for the paternal haplotype is the square root of the likelihood for the synthetic homozygous individual.
Mitochondrial DNA sequences
The Scandinavian wolf population is fixed for a mtDNA haplotype H1 (Ellegren et al, 1996). This variant is also the most common in neighboring populations, present in about 65% of north European wolves, although it is not fixed in any of them (Table 1). Four different haplotypes were observed in Estonia and Finland, and five in Russia. Haplotype H1 has not been reported in domestic dogs (Okumura et al, 1996; Tsuda et al, 1997; Vilà et al, 1997; and complete GenBank searches). Both samples A and B were found to carry the H1 mtDNA haplotype. We thus conclude that the suspected hybrid was either a pure wolf or represented a hybrid with wolf ancestry in the maternal line. However, the geographical origin of this ancestry cannot be revealed by the mtDNA data.
Y chromosome microsatellite
Table 2 shows the alleles observed in one Y chromosome microsatellite (MS41-B) in male wolves from northern Europe and in 38 male dogs. Nine alleles have been observed in wolves: eight of them in the Baltic States (Estonia and Latvia), six in Russia and four in Finland. A total of eight alleles were observed in our sample of domestic dogs, including the two alleles found in Scandinavian wolves and almost all of the alleles observed in other wolf populations.
Table 2 – Y chromosome microsatellite alleles (locus MS41-B) observed in male wolves from northern Europe (data from Sundqvist et al, 2001), pure-bred dogs, and in the target sample A.
Among the two target samples, the Y chromosome microsatellite was successfully amplified in sample A only, confirming that this came from a male and supporting the notion that sample B was a female. The allele identified (2 2 2) was not found in Scandinavian wolves, but has been seen in other North European wolf populations and in dogs. Thus, this result does not discriminate between a wolf or a dog as the father of sample A. However, it suggests that the father was not a Scandinavian wolf.
An assignment test comparing wolves from the Scandinavian population and dogs clearly shows that the allelic distributions allow for distinguishing between them (Figure 1; all dogs are located above the dia-gonal, indicating a higher likelihood of being dogs than wolves, whereas all wolves are below the diagonal).Figure 1 also includes the target samples. Sample A lies between the distributions of dogs and wolves, a position that would be expected for a wolf–dog hybrid. Sample B appears at the limit of the distribution of wolves. This sample has the highest likelihood, among all animals, of assignment to the Scandinavian wolf population; this extreme position is likely to be a consequence of the low number of microsatellites successfully scored for this individual (11) because of the low quality of the sample (drop of blood in snow). Its likelihood of being a wolf is clearly higher than the likelihood of being a dog.
Log likelihood of assignment for dogs (open triangles) and Scandinavian wolves (black circles). The log likelihoods for the two target samples (A, B) are also indicated.
To analyze if the target samples differed significantly from the distribution of expected haplotypes for either Scandinavian wolves, dogs or F1 hybrids, we studied the distribution of the log likelihood of assignment to the Scandinavian wolf population of three groups: 1000 synthetic hybrids, 1000 synthetic dogs and 1000 synthetic Scandinavian wolves. Figure 2 (left) shows that the genotype combination of sample A is significantly different from that expected for pure dogs or wolves, but is inside the distribution for F1 hybrids.Figure 2 (right) indicates that the genotype of sample B is outside the expected distribution for hybrids or dogs, but inside the distribution expected for Scandinavian wolves. A similar analysis shows that none of the target samples can be identified as a wolf immigrant from Finland or Russia (Table 3). We also tested if the assignment likelihoods of samples A and B were outside the expected distribution for an F1 hybrid between a dog and an immigrant. The target samples were outside the distributions in both cases and thus this possibility could be excluded as well (analyses not shown).
Distribution of the log likelihood of assignment to the Scandinavian wolf population of 1000 synthetic genotypes corresponding to dogs, Scandinavian wolves and F1 hybrids between dogs and wolves. Values are standardized by subtracting the log likelihood calculated for each target sample. If the value 0 (corresponding to the target sample) is outside the distribution, we can conclude that the genotype of the target sample is unlikely to occur in the dog, wolf or hybrid population.
Table 3 – Proportion (P) of 1000 synthetic genotypes in which the likelihood of assignment to the respective wolf population is lower than the likelihood of assignment observed for the target samples (A, B and for the synthetic father, see text).
The allelic composition of the two target samples is indicated in Table 4. For 10 out of 11 loci for which genotyping was successful for both samples A and B, sample B is compatible with being the parent of sample A. However, one locus (c2079) excludes this possibility: sample A is heterozygote for alleles 275 and 283, whereas B is homozygote for allele 271. We consider a technical artifact to be the most likely explanation for this non-congruence and that sample B is indeed parent to sample A. The quality and quantity of the DNA extracted from the thawed snow (sample B) might have been so low that allelic dropout has occurred. Allelic dropout, the accidental lack of amplification of one allele, is more common in samples of poor quality (Taberlet et al, 1999). This idea lends support from the fact that seven loci failed to amplify for sample B and possibly also from the fact that 10 out of 11 (91%) of the amplifying loci appeared homozygous. The average observed heterozygosity for all Scandinavian wolves for the 18 microsatellite markers was 0.65 (SD=0.16) and, consequently, for 11 loci typed for sample B we would expect to have around seven heterozygous loci. Unfortunately, the small amount of DNA obtained for sample B did not allow for further amplifications that may have confirmed allelic dropout.
In order to further test if sample B could correspond to the mother of A, we tried to assess how likely is to find a wolf in Scandinavia that is as similar to A as sample B is. We generated 10 000 simulated genotypes for the 11 microsatellite loci successfully amplified in B using the allele frequencies observed in the Scandinavian wolf population. For each locus, we assume that dropout could lead to the amplification by PCR of only one of the alleles, resulting in a false homozygote if the locus was heterozygote. We used a frequency of allelic dropout of 18%, as observed by Lucchini et al (2002), for autosomal microsatellites on other low-quality samples of wolves (scats). In spite of the extremely low genetic diversity that characterizes the Scandinavian wolf population (Ellegren et al, 1996; Ellegren, 1999), over 90.5% of the simulated genotypes could be excluded as possible parents of A at two or more loci. Sample B, instead, shows incompatibility at only one locus. The evidence that the genotype of B – in spite of the mismatch at locus c2079 ( Table 4) – is highly similar to A, together with the evidence provided by field observations suggesting that it could have been the only wolf in the area during the winter before A was born, supports the notion that B could be the mother of A.
Making the tentative assumption that sample B represents the mother of sample A, we determined the paternally contributed allele at 13 loci (Table 4). Two of the alleles found in A and assumed to come from the father (allele 131 at locus c2088 and 104 at PEZ05) have not been observed before in the Scandinavian wolf population, and other alleles are present in very low frequencies. These alleles were present in wolves from other populations and in dogs. As above, the origin of the paternal haplotype was assessed by comparison with synthetic genotypes (Table 3). The likelihood of obtaining this haplotype from the Scandinavian, Finnish or Russian wolf population is extremely low and outside their expected distribution. Also, this haplotype is not expected from a hybrid between a Scandinavian wolf and a domestic dog. However, the likelihood for the paternal haplotype falls inside the distribution for pure dogs.
Additional support for these results was provided by the model-based method of Pritchard et al (2000). All Scandinavian wolves had a probability of at least 0.95 of being classified as pure wolves (the pro-bability was higher than 0.99 for 92% of the wolves). Similarly, all dogs but one had a probability higher than 0.95 of being genetically identified as pure dogs. The target sample B, in spite of its incomplete genotype, had a probability of 0.998 corresponding to a pure Scandinavian wolf. On the other hand, the corresponding probability for sample A was only 0.264. For this sample, the probability of having one dog as parent was 0.402 and the probability of having it as a grandparent was 0.334. The probability of assignment to the dog population was 0.000. Consequently, sample A was likely to have a hybrid origin (probability=0.402+ 0.334=0.736).
The absence of species-specific genetic markers seemingly makes the identification of hybrids difficult, but the recent development of methods aimed at identifying inter-population migrants based on the initial characterization of allelic distributions in the parent populations (species) offers new means for hybrid identification (Paetkau et al, 1995; Pritchard et al, 2000). In addition, the combined use of autosomal markers and both paternally and maternally inherited markers may allow the direction of hybridization events to be determined. However, such precise knowledge on hybridization has so far not been possible to derive owing to a general lack of polymorphic Y chromosome markers. This study therefore represents one of the first applications of Y chromosome polymorphisms, together with mtDNA and autosomal markers, to study hybridization in nature (cf Evans et al, 2001). The combined use of the markers allowed us to conclude that a hybridization event between dog and wolf had occurred in the endangered Scandinavian wolf population. The direction of hybridization was a male dog paired with a female wolf, the latter coming from the Scandinavian wolf population. Indeed, Vilà and Wayne (1999) suggested that if wolves and dogs would hybridize, the most likely direction is male dog crossing with female wolf. However, the lack of observable effects on the wolf populations led these authors to suggest that survival of hybrid pups could be difficult because dog fathers are less likely to help to raise the offspring and because their integration into wolf packs could be difficult (see also Randi and Lucchini, 2002).
An important consequence from our results is the confirmation, with compelling genetic evidence, that hybridization between wolves and dogs does occasionally occur in the wild and that hybrids can be successfully raised. However, as all 25 Scandinavian wolves included in the study are clearly differentiated from domestic dogs, that is, they do not show signs of recent hybridization, it indicates that hybridization may be an uncommon event. Our results agree with recent studies suggesting that this hybridization occasionally takes place across Europe but may be fairly uncommon (in Bulgaria, Randi et al, 2000; in Latvia, Andersone et al, 2002; in Italy, Randi and Lucchini, 2002; in Spain, Vilà and Llaneza, personal observation).
The generation of synthetic genotypes for both pure specimens and hybrids allowed an intuitive representation of the variability that can be expected in each population group. This method allowed us to infer that the genotype of the target sample A would be very uncommon for pure dogs or Scandinavian wolves. The generation of synthetic genotypes is dependent on a fairly accurate knowledge of the allelic frequencies. The low genetic variability of Scandinavian wolves (Ellegrenet al, 1996; Ellegren, 1999) simplifies the estimation of the allele frequencies, but this can be a harder task for dogs. The strong genetic fragmentation of dogs into breeds may limit the power of hybridization tests like the one we present here. Modern breeding practices imply the almost complete reproductive isolation between breeds, each of them with a small effective population size, leading to fast inter-breed differentiation owing to genetic drift (Lingaas et al, 1996; Zajc et al, 1997; Wilton et al, 1999). The selection of local dogs belonging to the breeds that could be most likely to hybridize could increase the resolution of the test, allowing for an increase in power that could enhance the likelihood of detecting F2 hybrids and backcrosses.
The birth of a litter had been detected in the area where the individual corresponding to sample A was killed. During autumn 1999, five pups were observed. The killed animal was assumed to be one of these pups. Direct observation of the litter had suggested that these animals could be of hybrid origin. The determination of the hybrid status of sample A confirmed the suspicion and led to the management decision to remove its presumed siblings. As a result of the management efforts, two of them were killed by government officials. Another one is believed to have been illegally killed, and the last one is unaccounted for (Terje Bø, personal communication). This action should have reduced the chances of dog genes introgressing into the wolf population.
Further research is necessary in order to confirm if fragmented and low-density wolf populations that coexist with a larger number of domestic dogs are at high risk of hybridization, as suggested (Boitani, 1983;Blanco et al, 1992; Andersone et al, 2002). If this is shown to be the case, actions that could result in the decrease of the density of already threatened wolf populations, or in the disruption of social groups, should be avoided.
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The collection of samples in Russia was supported by the Swiss National Science Foundation SNF31-46691.96 and the Institutional Partnership Programme SNF 7IP051224, and was carried out with the help of Vladimir Bologov from the Central Natural Reserve Zapoviednik. Janis Ozolins, Agris Strazds, Janis Baumanis and other employees of the Latvian Forest Service and volunteer hunters assisted in the collection of Latvian wolves. Jonathan Stone developed the program used to generate synthetic genotypes. Jennifer Seddon and Frank Hailer provided valuable comments on the manuscript. This research has been supported by Direktoratet for Naturforvaltning (Norway), Swedish Environmental Protection Agency, the Swedish Research Council for Agriculture and Forestry, the Swedish Hunting Association, the Nordic Arctic Research Program, and by the Olle Engkvist, Carl Trygger, Oscar and Lili Lamms, and the Sven and Lilly Lawski’s foundations. HE is a Royal Swedish Academy of Sciences Research fellow supported by a grant from the Knut and Alice Wallenberg foundation.