Genetics

The rresearches in the field of genetics of the European mink are very limited and they need to be urgently completed, especially in the context of the rapidly extinction of the species and the disappearance of its numerous populations, among others, in France, Belarus and Russia. The rapidly disappearing genetic resources will largely never be studied and described, which is an irreversible loss from the cognitive and practical point of view – the meagre data on intra- and inter-population genetic diversity significantly impair the efficacy of the implemented activities for restitution of the European mink, especially in the context of reconstruction and conservation breeding, as well as the reintroduction of the species.

The ppioneer genetic studies on the European mink concerned the cytogenetics of the species and were conducted in the former USSR. To date, works by Volobuev & Ternovsky, Volobuev et al., Graphodatsky et al., Graphodatsky et al. and Graphodatsky & Radjabli are the primary sources of information on the M. lutreola karyotype.

The diploidal number of the chromosomes of the European mink is 38, which is typical for many species of the Mustelidae family – for more than 60% of species of this group 2n = 38. Among the representatives of the Mustela genus the diploid number of chromosome ranges from 38 to 44. Significantly, in the case of the American mink 2n = 30, which, additionally, on the cytogenetic basis, indicates a relatively low degree of its evolutionary relationship with the European mink.

The chromosomal set of the M. lutreola consists of five pairs of metacentric chromosomes, two pairs of subtelocentric chromosomes, five pairs of submetacentric chromosomes and seven pairs of telocentric chromosomes. The chromosome X is submetacentric, while the chromosome Y is metacentric. As with other representatives of the Mustelidae family, the chromosome Y is the smallest chromosome. The basic number of arms of autosomes (FNa) is 58, indicating significant similarity between chromosome morphology between the American mink and the Siberian weasel (both species have the same diploid number of chromosomes and equal FNa). The pattern of karyotype of the European mink has not yet been established.

The characteristics of the G and C bands of chromosomes of the European mink is presented by Graphodatsky et al. and Graphodatsky et al.. The pattern of AgNOR bands (nucleolus organizer regions), obtained by the method of silvering the secondary constriction of chromosomes was also described. A detailed comparison of the pattern of G bands of chromosomes of the European mink and the American mink, the least weasel Mustela nivalis, the mountain weasel Mustela altaica, the Japanese marten Martes melampus, the European badger Meles meles and the striped polecat Ictonyx striatus was presented by Graphodatsky et al.. Both in terms of the pattern of G bands and the chromosome number and morphology, the European mink shows many similarities with the Siberian weasel.

The genome of the European mink has not been sequenced yet, and 158 nucleotide sequences (DNA and RNA) have been deposited in GenBank for this species. This number includes 61 fragments of the sequence of the mitochondrial genome, 30 of them representing haplotypes of the cytb gene (26 for fragments of length 337 to 504 bp and four for the complete gene sequence of 1,140 bp) and 23 haplotypes for the control region (357 up to 990 bp). By comparison, the number of records of the nucleotide sequences of M. putorius and M. putorius furo deposited in GenBank is 357,607 and of N. vison – 19,550.

Due to its high cytogenetic similarity and proven close phylogenetic relationships, the size of the nuclear genome of the European mink should be estimated on the basis of the sequenced genome of the ferret (MusPutFur1.0, GenBank accession code: NC_020638.1), as about 2 411 million bp, the content GC pairs as about 42%, and the number of genes as about 27.3 thousand.

The best known genetic markers of the European mink are the microsatellite nuclear sequences (Short Tandem Repeats – STRs, Simple Sequence Repeats – SSRs), which are sequential patterns of DNA consisting of several nucleotides and repeating in tandems. They are used in phylogenetic studies, in the analysis of interpopulation genetic variation and internal genetic structure, in detection of evolutionary events in phylogenesis of the European mink, in the phylogeographic reconstructions, and also potentially in identification of inter-species M. lutreola × M. putorius hybrids.

The names of microsatellite markers of the European mink consist of a unique numbers preceded by abbreviation Mlut. Cabria et al. identified eight unique microsatellite loci of M. lutreola: Mlut04 (GenBank accession code: EF093582, the repetitive motif GT)16,, five identified alleles), Mlut08 (GenBank accession code: EF093583, the repetitive motif (GT)12, four identified alleles Mlut15 (GenBank accession code: EF093585, repetitive motif (GT)14, five identified alleles), Mlut20 (GenBank accession code: EF093587, (GT)18, eight identified alleles), Mlut25 (GenBank accession code: EF093588, repetitive motif (GT)15, Mlut27 (GenBank Accession Code: EF093589, Repetitive Motif (GT)8NN(GT)14, two identified alleles), Mlut32 (GenBank accession code: EF093590, repetitive motif (GT)59), eight identified alleles), Mlut35 (GenBank accession code: EF093591, repetitive motif (GT)15NNNN(GT)4NN(GT)7, four identified alleles).

Microsatellite markers of European mink have been successfully amplified in other species of the Mustelidae family, among others Mustela eversmannii, Mustela furo, Mustela sibirica, Mustela nivalis, Neovison vison, Martes martes and Martes foina. This demonstrates the possibility of using markers STR of M. lutreola in studies on genomes of other representatives of the Mustelidae family. In turn, Peltier & Lodé, Michaux et al., Lodé et al. and Cabria et al. positively assessed the possibility of using starter sequences developed for amplification of microsatellite sequences in the genome of the American mink (MTV02, Mvi20, Mvi22, Mvi72, Mvi75, Mvi389, Mvi1843, Mvi054, Mvi111), European polecat (PutFK1) and the stoat (Mer09, Mer22, Mer41) for studies on population genetics, phylogenetics and phylogeography of the European mink.

Besides the polymorphism of neutral genetic markers of the European mink, differentiation of allozymes and the drb gene from the family of genes of the major histocompatibility complex (MHC) class II were analysed. Of 36 allozyme loci of the European mink analyzed by Lodé et al. two allelic forms were determined only for four of them (the gene for carboxylesterase / EC 3.1.1.1 – est-2, the gene for NADP-dependent cytosolic malate dehydrogenase / EC 1.1.1.40-me-1, the gene for malate dehydrogenase MDH / EC 1.1.1.37 – mdh-1 and the gene for non-specific protein). All other loci in the European mink were monomorphic, while for parallelly analyzed samples from the European polecat, nine polymorphic loci were found. For the drb gene Becker et al. described nine allelic forms. Nishita et al. conducted the phylogenetic analyses based on the sequence of exon of the second drb gene, demonstrating the evolutionary closeness of this sequence in M. sibirica, M. itasi and M. lutreola and its inter-species polymorphism, as an indirect proof of the effects of stabilizing selection.

The complete mitochondrial genome of the European mink was sequenced de novo in 2017 – the length of with a gained nucleotide sequence is 16 523 bp. The comparison of the recognized sequence of mitogenome of the M. lutreola with the complete sequences of 24 species of the Mustelidae family of mitochondrial genomes deposited in the GenBank conducted in the in BLAST program showed a similarity between 86-99%. The phylogenetic tree made on the basis if the recognized sequence of mtDNA was characterized by the high affinity of the European mink with the European polecat and the ferret, and its explicit presence in the clade including also M. eversmanni, M. nigripes, M. sibirica and M. itatsi. Comparison of the sequences of the mitochondrial genome of the European mink and the European polecat (GenBank accession code: KT693383.1) showed a discrepancy of 158 single-nucleotide differences.

Level of ssimilarity (Max Identity parameter) between the complete sequence of the mitogenome of the European mink and the complete sequences of the mitochondrial genome of selected species of the Mustelidae family (source: developed by means of the BLAST program)

It is worth mentioning here that genetic tests of the differential identification of the European mink have already been developed. The method developed by López-Giráldez et al. is based on the amplification of the species-specific, nuclear microsatellite sequence Mel08, according to the procedure described by Domingo-Roura. At the stage of evaluation of the length of the amplification products, M. lutreola and M. putorius (221 bp product for both species) can be distinguished from N. vison (436 bp), whereas the use of digestion with restriction enzyme AciI can distinguish the European mink from the European polecat. The great advantage of the method is its simplicity and low cost of its performance, while its applicatory value is highlighted by the possibility of identifying species that often coexist and belong to the same ecological guild in Europe (land-and-water predatory mammals) yet require a totally different approach (control and eradication of the alien and invasive population of the N. vison vs. urgent and careful conservative efforts of the M. lutreola). Furthermore, collecting of the genetic material for the above described test may be non-invasive, by using hair traps for sampling hair with hair follicles.

Another non-invasive method of identification of the European mink, also differentiating it from the European polecat and the American mink, was developed by Gómez-Moliner et al.. The proposed protocol is based on the nested PCR of the fragment of the sequence of the mitochondrial control region (loop D) and on digestion of the obtained amplicons (length 240 bp) with a mixture of restriction enzymes – RsaI and MspI. In result of the agarose electrophoresis of the products of digestion two haplotypes characteristic to the M. lutreola, haplotypes characteristic to the M. putorius and haplotype characteristic to the N. vison can be identified. The great advantage of this method is that it was designed for gaining small amounts of degraded DNA from faecal samples.

Studies in the area of population genetics of the M. lutreola are focused on defining genetic diversity between preserved populations of the species. Analyses of the intra-species genetic structure of the European mink showed a relatively high genetic diversity of the species, especially in comparison with other taxa of the Mustelidae. However, this diversity is not homogeneous, and various populations show significantly different levels of genetic diversity. The studies of Cabria et al., based on 11 microsatellite loci, have identified three genetically distinguishable populations – the north-eastern (inhabiting the Volga and the Dvina basin), the western (inhabiting south-western part of France and northern & western parts of Spain) and the south-eastern (inhabiting the Danube Delta in Romania). The established values of parameters evaluating the genetic diversity indicate the higher genetic diversity in the north-eastern population, slightly lower in the Romanian population and significantly lower in the western population.

Similar results were obtained by Michaux et al.. In this study the inter-population analysis of genetic diversity of the European mink was based on the complete sequence of the D loop of the mitochondrial genome. The results obtained by the abovementioned team identified 15 haplotypes for the examined fragment of DNA in the Russian-Belarussian population (18 individuals examined), 4 haplotypes in the Romanian population (34 individuals examined) and only one haplotype for the French-Spanish population (124 individuals examined). The nucleotide diversity (π) and haplotype diversity (h) were respectively: for the Russian-Belarussian population – 0.0120 ± 0.0014 and 0.939 ± 0.058, for the Romanian population – 0.0012 ± 0.0003 and 0.469 ± 0.088, for the French-Spanish population – 0 for both indicators.

Indicators of genetic variety of the Northeastern (NE), Southeastern (SE) and Western (W) populations of the European mink, based on 11 microsatellite loci, calculated by Cabria et al.

N – number of examined individuals, NA – number of alleles, PA – number of private alleles, % PA – percentage of private alleles in total number of alleles), A – allelic diversity, HO – observed heterozygosity, HE – expected heterozygosity, FIS – inbreeding coefficient

Studies conducted by Lodé, Davidson et al. and Cabria led to the similar conclusion as far as the genetic diversity of the three abovementioned populations of the European mink is concerned. The cause of high genetic homogeneity of populations living in France and Spain is attributed to the “bottleneck effect” and of the “founder’s effect” that occurred (one or several times) in the relatively recent past, as well as to the limited gene flow.

Although the number of studies on the genetics of the European mink is relatively small, the extremely important applicatory aspect of almost all of the research currently being conducted in this field should be emphasized. Such studies directly contribute to obtaining very valuable practical knowledge about planning of effective protective measures, both ex situ (conservation and recreation breeding) and in situ (reintroduction programs, supplying of the disappearing populations with wild individuals from the outside), conducted ad hoc and as a long-term strategy alike. As such, they are an excellent example of the possibility of the practical use of conservatory genetics. It is worth mentioning here that the studies conducted by Lodé, Davison et al., Peltier & Lodé, Michaux et al., Michaux et al. and Cabria et al. are particularly valuable for the preservation of the species. It also should be emphasized here that only conservatory genetics can provide tools to rescue the species that has been affected by the so-called “extinction vortex”, which, in turn, requires more research initiatives in conservatory genetics of the European mink.