A Journal of Conservation Biogeography
Diversity and Distributions, (Diversity Distrib.) (2011) 17, 757–768
BIODIVERSITY RESEARCH
High mitochondrial DNA diversity of an introduced alien carnivore: comparison of feral and ranch American mink Neovison vison in Poland Andrzej Zalewski1, Aleksandra Michalska-Parda2, Mirosław Ratkiewicz3, Michał Kozakiewicz1,2, Magdalena Bartoszewicz4 and Marcin Brzezin´ski2*
1
Mammal Research Institute, Polish Academy of Sciences, 17-230 Białowie_z a, Poland, 2 Department of Ecology, University of Warsaw, Banacha 2, 02-097 Warsaw, Poland, 3 Institute of Biology, University of Białystok, S´wierkowa 20B, 15-950 Białystok, Poland, 4 Szpitalna 2, 66-436 Słon´sk, Poland
ABSTRACT Aim Invasive alien species usually exhibit very high adaptation and rapid
evolution in a new environment, but they often have low levels of genetic diversity (invasive species paradox). Genetic variation and population genetic structure of feral American mink, Neovison vison, in Poland was investigated to explain the invasion paradox and to assess current gene flow. Furthermore, the influence of mink farming on adaptation of the feral population was evaluated by comparing the genetic structure of feral and ranch mink. Location Samples from feral mink were collected in 11 study areas in northern
and central Poland and from ranch mink at 10 farms distributed throughout the country.
Diversity and Distributions
Methods A 373-bp-long mtDNA control region fragment was amplified from 276 feral and 166 ranch mink. Results Overall, 31 haplotypes, belonging to two groups from genetically diverse sources, were detected: 11 only in feral mink, 12 only in ranch mink and eight in both. The genetic differentiation of feral mink from the trapping sites was high, while that among ranch mink from various farms was moderate. There was no significant relationship between genetic and geographic distance. The number of trapping sites where given haplotypes occurred correlated with the number of farms with these haplotypes. The mink from two sites were the most divergent, both from all other feral mink and from ranch mink. Comparison of mtDNA and microsatellite differentiation suggests male-biased dispersal in this species. Main conclusions American mink in Poland exhibit high genetic diversity and
originate from different source populations of their native range. The process of colonization was triggered by numerous escapees from various farms and by immigrants from Belarus. The genetic structure of local feral mink populations was shaped by the founder effect and multiple introductions. The genomic admixture that occurred during mixing of different populations might have increased the fitness of individuals and accelerated the invasiveness of this species. *Correspondence: Marcin Brzezin´ski, Department of Ecology, University of Warsaw, Banacha 2, 02-097 Warsaw, Poland. E-mail:
[email protected]
Keywords Biological invasions, invasive alien species, mink farming, multiple introduction, population genetics, propagule pressure.
The genetic characteristics of introduced populations have a profound impact on their capacity for establishment and range expansion (Tsutsui et al., 2000). Populations founded by a
small number of individuals usually suffer from low genetic variation, which might reduce their ability to adapt to the new conditions that they face (Frankham & Ralls, 1998; Spielman et al., 2004; Dlugosch & Parker, 2008). The high risk of inbreeding, demographic stochasticity and the inverse density-
ª 2011 Blackwell Publishing Ltd
DOI: 10.1111/j.1472-4642.2011.00767.x http://wileyonlinelibrary.com/journal/ddi
INTRODUCTION
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A. Zalewski et al. dependent effect all limit the long-term viability of such populations (Allee, 1931; Nei et al., 1975; Courchamp et al., 1999). However, investigations of the ongoing global process of alien species introductions to novel ranges have shown that the reduced genetic variability of their populations, resulting from the founder effect, population bottlenecks and genetic depletion, does not necessarily lead to colonization defeat (Grapputo et al., 2005; Dlugosch & Parker, 2008; Tepolt et al., 2009). The paradox of successful invading species is that they are likely to be genetically depauperate when compared with their source population (Sax & Brown, 2000; Grapputo et al., 2005). One of the best examples, the muskrat (Ondatra zibethicus), has succeeded in colonizing large areas of Europe despite the small founder population and very low genetic variability, and this shows that genetic variability is not always a necessary prerequisite for ecological success (Zachos et al., 2007). The history of muskrat expansion in Europe is rather exceptional because a large part of the present stock originates from the release of a few founder individuals and subsequent multiple introductions probably did not extensively supply the expanding population (Zachos et al., 2007). Invasions of alien species are rapid evolutionary events in which populations are usually subjected to the founder effect followed by rapid expansion and sequential bottlenecks during colonization of any new area (Clegg et al., 2002; Estoup et al., 2004; Dlugosch & Parker, 2008). According to the steppingstone model, the genetic structure of populations is created by distance and it is expected that population genetic variability will decline across the expanding range (e.g. Rollins et al., 2009). Thus, the adaptation possibilities of local populations in the newly colonized areas may be limited, and the expansion rate should be reduced in subsequently colonized sites (Lambrinos, 2004). Many recent studies have shown, however, that the lower genetic diversity of invasive populations compared with native ones is not always the rule. In many cases, successful invasive populations of alien species were found not to show the genetic signature of population bottlenecks and exhibited high genetic diversity (Kolbe et al., 2004; Genton et al., 2005; Roman & Darling, 2007; Fo¨rster et al., 2009) that was sometimes even higher than that of populations in their native range (Gillis et al., 2009). This may be explained by the fact that many invasive species have been introduced as a large number of individuals and/or on multiple occasions (Geptner & Naumov, 1967; Facon et al., 2008; Da Silva et al., 2010). Propagule pressure (number of introduction events and/or number of individuals introduced) is an emerging explanation for the establishment success of invasive species (Lockwood et al., 2005). Higher levels of genetic diversity in introduced populations compared with single native populations also result from the introduction of individuals from genetically diverse sources within the native range (Genton et al., 2005; Gillis et al., 2009). If introduction occurs at many sites in a novel range from various sources of the native range, the gene flow among multiple introductions creates zones with high genetic variation and potentially highly viable populations (Crawford
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& Whitney, 2010). Such genomic admixture might increase the fitness of individuals and may accelerate the invasiveness of the species (Ellstrand & Schierenbeck, 2000; Keller & Taylor, 2010). Therefore, with high propagule pressure and multiple sources of introduction, the ‘paradox of invasions’ does not exist, and the possibility to manage the invasive species population is reduced. American mink (Neovison vison) is endemic to North America and has been introduced into Europe, Asia and South America. In Europe, this species was introduced for commercial fur farming, which started to develop in the 1920s (Dunstone, 1993). Farms were established in many countries and harboured mink stocks of different native origin. Because of the large-scale and long-term nature of this farming, the number of escapees, which started to breed in the wild and became the founder individuals of feral populations, was high (Bonesi & Palazon, 2007). In many regions, these populations, derived from farm escapees, were highly supplied by ongoing multiple introductions. Unlike the feral mink populations in Western Europe and Fennoscandia, those in Eastern Europe originate not only from farm escapees, but also from thousands of individuals that, since the 1930s, were deliberately released into the wild in the former Soviet Union (Geptner & Naumov, 1967). In Poland, it has been documented that a migration wave from the east crossed the Polish–Belarusian border at the end of the 1970s and has spread west over subsequent years (Ruprecht et al., 1983; Brzezin´ski & Marzec, 2003). The present geographic range of feral mink covers the whole of northern and central Poland and is continuous (Brzezin´ski & Marzec, 2003). However, it seems highly probable that the presence of mink farms has greatly complicated the process of mink expansion in Poland and means that it has not yet been completed. Mink farming developed in Poland much later than in other European countries, with the first farm being opened in 1953 (Lisiecki & Sławon´, 1980). Since this time, observations of single escapees have been reported from various sites, but there is no evidence that local feral populations originating from ranch mink were established in any region of Poland until the beginning of the 1980s. By this time, feral mink invading Poland from Belarus were already present in the north-east of the country and were successfully enlarging their range. In the following years, there was an increasing number of reports of feral mink also living in the west (Bartoszewicz & Zalewski, 2003; Brzezin´ski & Marzec, 2003). Thus, there is still confusion as to whether the invasion from the east is the only process that has led to the colonization of Poland, or if it was supported by the development of local populations originating from Polish farms. A comparison of the genetic diversity of feral populations and ranch mink in Poland, based on microsatellite markers, revealed that these two groups are genetically separate (Michalska-Parda et al., 2009). The feral mink population in Poland exhibits very high genetic diversity and is characterized by a well-pronounced genetic structure and distance-limited gene flow (Zalewski et al., 2010). Moreover, the proportion of ranch
Diversity and Distributions, 17, 757–768, ª 2011 Blackwell Publishing Ltd
Genetic diversity of introduced alien American mink mink in feral populations is positively related to the number of mink kept on farms in areas where sampling was performed, confirming the occurrence of multiple introductions and high propagule pressure in north-west Poland (Zalewski et al., 2010). Ongoing introductions have strongly modified the genetic structure of the feral mink populations and overwhelmed the potential influence of landscape factors affecting gene flow, which underlines the impact of human activity on the gene flow among populations (Zalewski et al., 2010). However, we still do not know how local populations enlarged their range, in which regions of the country expanding populations started to mix and how mink farms have affected feral populations in present and past years. In the present study, the genetic variability and structure of American mink in Poland were investigated by analysing mitochondrial DNA (mtDNA) sequences. Levels of genetic differentiation and contemporary gene flow were assessed among mink from different regions of Poland. In particular, we addressed the following questions: (1) Is there a loss of genetic diversity in (a) feral mink compared with ranch mink and (b) feral mink in the areas believed to be colonized more recently (NW Poland) compared with feral mink in the areas of earlier colonization from the east (NE Poland)? (2) Is there evidence for high ongoing propagule pressure based on the Table 1 Measures of haplotype (h) and nucleotide (p) diversity among 11 study sites of feral and 10 sites of ranch American mink Neovison vison in Poland.
comparison of patterns of genetic variation in ranch and feral mink? (3) Are feral mink introduced from multiple, genetically diverse sources within the native range sources? (4) Is there evidence of contemporary or historical gene flow between the studied populations of feral mink, or of sexbiased dispersal? METHODS Sample collection In total, 442 tissue samples were collected from feral (N = 276) and ranch mink (N = 166; Table 1). Feral mink samples were obtained from live-trapped animals and from individuals killed by hunters and conservationists under legal permits during local eradication programmes in 2000–2007. Mink were sampled at 14 sites in four regions (NW – northwest Poland, CE – central Poland, SC – south central Poland and NE – north-east Poland); however, at three sites located in south central Poland (San River, middle Warta River and Milicz Ponds), no mink were captured (Fig. 1). Tissue samples from feral mink were collected at the following sites: ‘Warta Mouth’ National Park – FNW1, Gwda River – FNW2, Słupia River – FNW3, Wel River – FCE1, lower Narew River – FCE2, N
Region
Site
North-west Poland
FNW1 FNW2 FNW3 FCE1 FCE2 FCE3 FCE4 FNE1 FNE2 FNE3 FNE4
Central Poland
North-east Poland
All feral mink
Nh
Nu
h
p (%)
0.647 0.417 0.778 0.673 0.409 0.786 0.054 0.000 0.733 0.608 0.071 0.824
0.235 0.644 1.246 0.392 0.770 1.411 0.116 0.000 0.234 0.599 0.076 0.942
0.725 0.786 0.356 0.559 0.442 0.327 0.733 0.694 0.764 0.674 0.711 0.808
0.753 0.807 0.096 0.441 0.120 0.441 0.605 0.796 0.606 0.763 0.637 0.852
Feral mink 51 9 10 11 12 8 37 33 6 43 56 276
4 3 4 4 2 4 2 1 3 5 3 19
2 1 1 1 0 2 2 0 1 2 1
Ranch mink North-west Poland
North-east Poland South-west Poland
South-east Poland All ranch mink Total
RNW1 RNW2 RNW3 RNW4 RNE1 RNE2 RSW1 RSW2 RSW3 RSE1
16 8 10 30 20 11 10 30 11 20 166 442
4 4 2 4 2 2 4 8 4 6 21 31
2 1 0 1 0 1 1 4 1 3
N, sample size, Nh, number of haplotypes for control region mtDNA, Nu, number of private haplotypes. See Fig. 1 and the text for the names and locations of the sampling sites.
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A. Zalewski et al. (a)
(b)
Figure 1 Locations of 14 trapping sites of feral American mink Neovison vison (a) and of 10 American mink farms (b) in Poland. See Methods for site descriptions.
middle Vistula River near Warsaw – FCE3, middle Vistula River near Puławy – FCE4, Mazurian Lakeland – FNE1, Romincka Forest – FNE2, Biebrza National Park and surrounding area – FNE3 and Białowie_za Forest – FNE4 (see Table 1 for sample sizes at each site). To compare the genetic diversity of feral mink with that of ranch mink, tissue samples were also collected at 10 mink farms distributed throughout the country. The farms were located in or near the following cities: Kołobrzeg – RNW1, Buszkowy Go´rne – RNW2, Lez´no – RNW3, Nowy Tomys´l – RNW4, Olsztyn – RNE1, Choroszcz – RNE2, Stary Kisielin – RSW1, Pilawa Dolna – RSW2, Czarnowa˛sy – RSW3 and Chorzelo´w – RSE1 (Fig. 1). All tissue samples were placed in 95% ethanol and stored at )20 C prior to DNA extraction. DNA isolation, fragment amplification, and sequence analysis Genomic DNA was extracted from each tissue sample using a Genomic Mini Kit (A&A Biotechnology, Gdynia, Poland) and this was used as the template to amplify a 373-base pair (bp) fragment of mitochondrial control region (CR) with primers LGL283 and ISM015 (Hundertmark et al., 2002). PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, CA, USA) in 5-lL reaction volumes containing 10–20 ng of genomic DNA and RNase-free water, 0.2 lm of each primer and Qiagen Multiplex PCR Master Mix (1 ·). Polymerase chain reaction started with an initial activation step at 95 C for 15 min, followed by 30–35 cycles of denaturation at 94 C for 30 s, annealing at 57 C for 90 s, extension at 72 C for 60 s, and then a final extension at 60 C for 30 min. Direct sequencing of the amplified fragments was
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performed using the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems) according to the manufacturer’s recommendations, using primers LGL283 and ISM015 to sequence both strands. The sequencing products were analysed using an abi prism 3130 capillary automated sequencer (Applied Biosystems). DNA sequences were aligned in BioEdit v 7.0.4 (Hall, 1999) with manual revision. The sequences have been deposited in GenBank with accession nos. JF430903-JF430933. Relationships among haplotypes were represented as a haplotype network obtained by the statistical parsimony method using TCS software (Clement et al., 2000). The net divergence between mtDNA lineages was calculated according to Kumar et al. (1993). Estimates of the number of haplotypes, haplotype diversity (h), nucleotide diversity (p) and the number of segregating sites were calculated using arlequin 3.11 (Excoffier et al., 2005). Population divergence estimates were obtained from UST values (using pairwise distance), which incorporate information on nucleotide differences between mtDNA haplotypes in arlequin. The significance of pairwise UST values was ascertained by 1000 permutations. Principal component analysis (PCA) of pairwise UST data and the PC1 scores for each population was performed using genalex 6.0 (Peakall & Smouse, 2006). The pairwise genetic distances between populations (UST), obtained from analyses of mtDNA, were correlated (using Mantel test with 10,000 permutations) with pairwise FST values obtained from analyses of microsatellite loci reported by Zalewski et al. (2010). To assess the levels of female gene flow among different sampling sites, the migration parameters Nm and M were estimated from the UST (mtDNA) and FST values (microsatellites; Zalewski et al. (2010). Nm = (1)FST)/4 FST and M is
Diversity and Distributions, 17, 757–768, ª 2011 Blackwell Publishing Ltd
Genetic diversity of introduced alien American mink 2Nfm for mtDNA. The isolation by distance (IBD) pattern was determined by comparing genetic differentiation between populations, as measured by pairwise UST/(1)UST), to the logarithm of geographical distance using ibd software (Bohonak, 2002). Analysis of molecular variance (AMOVA; Excoffier et al., 2005) performed using arlequin (with 10,000 permutations) was used to assess structuring within the data, and the 11 feral mink sampling sites were grouped as a single group of populations. To explore patterns of genetic divergence in more detail, the spatial AMOVA procedure was applied using samova ver. 1.0 (Dupanloup et al., 2002). This identified the grouping of feral mink samples that maximized the FCT value based on 10,000 simulated annealing steps for K = 2 to K = 10 partitions of the sampling sites. RESULTS Genetic diversity Comparison of a 373-bp mitochondrial CR DNA fragment amplified from 442 individuals of feral and ranch American mink yielded 31 haplotypes as defined by 25 polymorphic sites (19 transitions, a single transversion and six indels; a single site showed two different mutations in different individuals: C-T transition and C-A transversion). The percentage of variable sites was 6.7%. Nineteen haplotypes were detected in the feral mink, while 20 haplotypes were identified in ranch mink. Among the haplotypes found in ranch mink, 12 were not present in the feral mink. Eight haplotypes were detected in both feral and ranch mink, with Hap1 being the most common (see Appendix S1 in Supporting Information for haplotype
frequencies in all the samples studied). The number of haplotypes varied considerably among trapping sites and farms. Trapping site FNE1 was fixed for a single haplotype (Hap1), while five haplotypes were found at site FNE3 (mean 3.18 haplotypes per site). In ranch mink, the number of haplotypes ranged from 2 to 8 (mean 4.00; Table 1). In almost all feral mink sampling sites (except FNE1 and FCE2), one or two private haplotypes were found (in total, 13 haplotypes occurred exclusively in single feral sampling sites). The frequency (representation) of a given haplotype in the feral mink sampling sites correlated positively with its frequency in mink farms (Pearson correlation, r = 0.824, P < 0.001, n = 31). The haplotypes that were found in ranch mink at most farms were also present in feral mink from the majority of trapping sites. A network was drawn using the statistical parsimony method to identify possible relationships between haplotypes (Fig. 2). Two possible haplotype groups of mink in Poland, for which the net divergence was 0.9%, were distinguished. These two mtDNA groups were present in both the feral and ranch mink. The first group consisted of 26 haplotypes, including Hap1, while the second consisted of only five haplotypes (Hap11, Hap12, Hap21, Hap28 and possibly Hap24). Hap11 was found in both feral (FNW2 site) and ranch mink (RSW2 farm), even though these sites were distant from one another (over 300 km). Another haplotype from this group (Hap12) was found at the FNW3 site only. The haplotypes Hap11 and Hap24 were detected exclusively in the pearl colour breed of ranch mink and Hap28 only in the pastel breed. A large number of feral mink possessed Hap1 or haplotypes that could have been derived from Hap1 (Fig. 2, see also Appendix S1). The average nucleotide (p) and haplotype diversity (h) values for the total sample were estimated at 0.86% ± 0.49 and 0.808 ± 0.015 SE, respectively. For the feral
Figure 2 Haplotype network and the relationships between 31 mtDNA control region haplotypes of American mink Neovison vison found in Poland. The areas of the circles are proportional to the number of mink sharing each haplotype. Black represents the proportion of haplotypes found in feral mink, and white is the proportion found in ranch mink. The smallest black circles without numbers represent haplotypes that were not observed in the sample.
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0 0.282 0.202 0.240 0.741 0.553 0.237 0.354 0.820 0.262 0.146 0.309 0.354 0.417 0.214 0.194 0.127 0.233 0.098 0 0.057 0.232 0.742 0.479 0.201 0.234 0.903 0.284 0.073 0.237 0.294 0.320 0.130 0.114 0.074 )0.020 0.135 0 0.046 0.727 0.351 0.117 0.128 0.817 0.112 )0.019 0.155 0.247 0.248 0.017 0.052 0.053 0.003 0.094 0 0.726 0.651 0.282 0.264 0.736 0.071 0.090 0.365 0.365 0.470 0.220 0.168 0.227 0.153 0.268 0 0.925 0.858 0.705 0.955 0.756 0.720 0.873 0.771 0.870 0.808 0.782 0.641 0.710 0.694
FCE4
0 0.521 0.356 0.964 0.610 0.424 0.309 0.367 0.336 0.267 0.344 0.103 0.363 0.194
FNE1
0 0.242 0.936 0.361 0.113 0.070 0.141 0.119 0.084 0.034 0.009 0.121 0.016
FNE2
0 0.718 0.185 0.119 0.254 0.329 0.309 0.122 0.183 0.147 0.155 0.240
FNE3
0 0.665 0.844 0.942 0.860 0.937 0.864 0.873 0.755 0.861 0.811
FNE4
0 0.102 0.413 0.417 0.493 0.200 0.225 0.222 0.214 0.260
0 0.169 0.244 0.275 0.001 0.042 0.030 )0.027 0.056
RNW2
0 0.115 )0.054 0.094 0.030 0.010 0.156 0.083
RNW3
0 0.105 0.208 )0.015 0.088 0.260 0.195
RNW4
0 0.186 0.058 0.055 0.249 0.157
RNE1
Significant values are in bold (P < 0.05, no Bonferroni correction was applied). See Fig. 1 and the text for the names and locations of the sampling sites.
0 0.158 0.265 0.221 0.301 0.715 0.588 0.251 0.338 0.895 0.364 0.041 0.320 0.351 0.415 0.215 0.220 0.131 0.150 0.126
FCE3
0 0.460 0.511 0.367 0.343 0.566 0.819 0.334 0.238 0.390 0.899 0.556 0.356 0.196 0.292 0.230 0.277 0.241 0.185 0.330 0.253
FCE2
FNW1 FNW2 FNW3 FCE1 FCE2 FCE3 FCE4 FNE1 FNE2 FNE3 FNE4 RNW1 RNW2 RNW3 RNW4 RNE1 RNE2 RSW1 RSW2 RSW3 RSE1
FCE1
RNW1
FNW3
FNW1
Site
FNW2
Ranch mink
Feral mink
Table 2 Pairwise UST values between samples taken from feral and ranch American mink Neovison vison in Poland.
0 0.027 )0.006 0.031 0.043
RNE2
0 )0.023 0.073 0.063
RSW1
0 0.044 0.016
RSW2
0 0.106
RSW3
0
RSE1
A. Zalewski et al.
Diversity and Distributions, 17, 757–768, ª 2011 Blackwell Publishing Ltd
Genetic diversity of introduced alien American mink mink sampling sites, p ranged from zero to 1.411% (mean 0.52%) and h from zero to 0.786 (mean 0.47; Table 1). In the ranch mink farms sampled, p ranged from 0.096 to 0.807% (mean 0.54%) and h from 0.356 to 0.786 (mean 0.61; Table 1). There were no significant differences between the feral and ranch mink samples with respect to the number of haplotypes, and the haplotype and nucleotide diversity values (Mann– Whitney test, P > 0.05 in all cases). Genetic structure Pairwise UST values were highly variable between the feral mink sites and ranged from 0.056 to 0.956 (mean 0.490), while the corresponding probabilities indicated significant differentiation in 93% of the sampling site pairs (P < 0.05, not Bonferroni corrected) (Table 2). Especially high UST values were obtained when the sites FNE4 and FCE4 were compared with the other sites (range 0.71–0.96), indicating great genetic differentiation between mink at these two sites and all others, even those located in the same region. The average genetic differentiation among mink farm samples was moderate and highly significant (UST = 0.126, P < 0.001), and a significant (P < 0.05) subdivision was found between 49% of pairs (range 0.000–0.494; Table 2). Genetic divergence among feral mink samples was significantly larger than for ranch mink (UST = 0.490 vs. 0.126, Mann–Whitney test, U = 1770, P < 0.001). The comparison between feral and ranch mink revealed great genetic divergence (average UST = 0.319, P < 0.001). This value was still high (UST = 0.212, P < 0,001) even after excluding the most divergent sampling sites (FNE4, FCE4). The correlation between the logarithm of geographic distance and the pairwise UST/(1)UST) values among feral mink samples was not significant and showed a lack of IBD (r2 = 0.065, P > 0.05). There was no correlation between the pairwise UST values obtained from mtDNA and FST values for microsatellites (Zalewski et al., 2010) (Mantel test, r = 0.19, P = 0.23; Fig. 3). The migration parameters Nm and M deduced from the average UST value for mtDNA from the feral sampling sites were 0.26 and 0.52, respectively (the range for M was 0.02–8.40 mink exchanged between sites per
Figure 4 Principal component analysis (PCA) of American mink samples examined for mtDNA variation. PC1 and PC2 – the first and second axes of the PCA. Black circles represent feral mink, and white circles represent ranch mink.
generation). The average Nm and M for the samples collected at the mink farms were 1.73 and 3.46, respectively (M ranged from 0.51 to infinity). The first and second axes of the PCA (PC1 and PC2) performed on the whole data set (21 sampling sites) explained 32.9% and 22.6% of the total variability, respectively. This analysis showed that mink trapped at the sampling sites FCE4 and FNE4 were the most divergent from mink from all other sites and from farms (Fig. 4). Geographical structuring among feral mink in Poland was supported by the results of amova, where all sampling sites were treated as a single group of populations (UST = 0.692, P < 0.001). samova was then used to identify the subdivision most likely to explain the observed genetic structure. According to the samova, the differentiation among the studied feral mink populations was best explained assuming three groups: (1) FNE4; (2) FCE4; and (3) all other samples. The percentage of variation was the highest among groups of populations at 61.86%, while among populations within groups, it was 15.37% and within populations, it was 22.78% (UCT = 0.619, P < 0.05; UST = 0.772, P < 0.001; USC = 0.403, P < 0.001, respectively). DISCUSSION
Figure 3 Correlation between pairwise UST and FST values for feral American mink samples obtained from the analysis of mtDNA sequences (this study) and microsatellite markers (Zalewski et al., 2010).
This study of American mink in Poland is the first on this species to be based on mtDNA polymorphism. It is also one of the few studies to show the genetic consequences of biological invasion of an alien carnivore mediated by humans. The most common haplotype, Hap1, occurred with high or moderate frequencies in ranch mink from all farms studied and in almost all feral populations. The feral and ranch populations possessed haplotypes located in various regions on the haplotype network, without a discernable pattern, and possible two mtDNA groups were identified. The number of feral populations with a given haplotype was positively correlated with the number of farms with the same haplotype. We demonstrated that feral and ranch mink populations had strikingly similar patterns of genetic variation, as revealed by
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A. Zalewski et al. haplotype and nucleotide diversity. However, the genetic differentiation among feral mink populations, as measured by UST, was very high, while that among different farms was moderate. This may result from colonization from different sources, the restricted gene flow among feral mink and genetic drift. Genetic variation Theoretically, low genetic variability should be expected a priori in alien invasive species because of reduced heterozygosity and the loss of rare alleles, which result from the founder effect and population bottlenecks (Allendorf & Lundquist, 2003; Grapputo et al., 2005). Indeed, in some successful invaders (e.g. muskrat Ondatra zibethicus), the complete lack of variation in typically highly polymorphic markers was recorded (Zachos et al., 2007). The success of invasive species despite low genetic diversity has been referred to as a ‘genetic paradox’ (Allendorf & Lundquist, 2003). Contrary to this expectation, our results showed high levels of mitochondrial DNA variability, not only in ranch mink, but also in feral populations. We found 31 haplotypes, 19 of which occurred in feral mink. There is a dearth of information in the literature about American mink mtDNA diversity in both the native and introduced ranges, and so there are no similar studies to compare our results. However, in studies of other carnivores performed on a similar scale, the mtDNA haplotype diversity in their native ranges was considerably lower: nine haplotypes in a 335-bp fragment in red fox Vulpes vulpes (Kirschning et al., 2007); eight in a 350-bp fragment in pine marten Martes martes (Pertoldi et al., 2008); and 13 in a 555-bp fragment in black bear Ursus americanus (Van Den Bussche et al., 2009). Twenty-four haplotypes were recorded in three European populations of polecat Mustela putorius (Pertoldi et al., 2006) and 76 in four subspecies of raccoon Procyon lotor (Cullingham et al., 2008), but the analysed mtDNA region in these species was much longer than in our study: 2000-bp and 1400-bp fragments of CR, respectively. On the other hand, some introduced carnivores had a low number of haplotypes, e.g. in the raccoon dog Nyctereutes procyonoides in Europe, nine haplotypes were found in a 599-bp fragment (Pitra et al., 2010). Although this comparison gives only a rough index of genetic diversity in carnivores, it convincingly shows that American mink introduced into Europe has high genetic diversity and is not as genetically depauperate as might be expected. The high genetic diversity of invasive mink in Poland may be a result of two non-mutually exclusive reasons: (1) the large number of founding individuals colonizing new areas, (2) releases from multiple source populations at various sites and admixture after expansion and enlargement of their geographical range. Indeed, between the 1930s and 1960s, large numbers of American mink (at least 20,000) were released in Russia and Belarus (Geptner & Naumov, 1967), and the species started to colonize Poland in the 1970s (Brzezin´ski & Marzec, 2003). Such a high number of introduced individuals may have
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reduced the negative influence of the founding effect. The origin of Polish feral mink from multiple introductions from various sources is supported by the high number of private haplotypes and possible presence of two separate mtDNA groups. The number of haplotypes at a single sampling site varied between 1 and 5, and at least one private haplotype was recorded in almost all sites, at high frequency in some cases. For example, haplotypes that were frequent but private to the local population were identified in feral mink from sites FCE1 (Hap15), FCE4 (Hap18) and FNE4 (Hap7). Similarly, high genetic diversity caused by multiple introductions from various sources has been demonstrated for other invasive animals (Kolbe et al., 2004; Vidal et al., 2010) and plants (Genton et al., 2005; Marrs et al., 2008). We found some evidence that two mtDNA groups of the American mink could be present in Poland, both in feral and in ranch mink. They may have come from genetically diverse sources within the native range. This hypothesis can be, however, fully tested after studying phylogeographic data from North America. Interestingly, the two haplotypes (Hap11 and Hap12) from the second group were found in the NW region only, where mink farms are most numerous (Zalewski et al., 2010). Thus, it is highly probable that the rapid increase in the number of mink farms and the size of breeding stocks in NW Poland over the last decade has resulted in the introduction of haplotypes from a second group (in sites FNW2 and FNW3) to this region. Ranch mink in Europe are derived from the crossbreeding of different local North American subspecies (Dunstone, 1993), which has led to high levels of genetic variability. Since the 1920s, when the American mink was first introduced to European farms, the genetic pool of ranch mink has changed considerably owing to the process of human selection (e.g. for large body size or pelt colour). Therefore, the past mink escapees, which possibly gave rise to the first feral populations, and the present escapees that supply existing populations have both contributed to the large genetic variability observed. This could also indicate ongoing multiple invasions from several genetically divergent source populations, especially in NW Poland. History of colonization and genetic structure The field data suggest that NE Poland was colonized by invasive mink from Belarus (Brzezin´ski & Marzec, 2003). If the genetic diversity of mink in Poland was shaped by a colonization wave from Belarus, we might expect – according to the stepping-stone model – that genetic diversity would decrease in the areas subsequently colonized by mink, and IBD should be observed (e.g. Herborg et al., 2007). However, we did not record decreasing diversity from the east (initial colonization area) to the west (area colonized later), and the relationship between genetic and geographic distances was not significant. The lack of IBD may have resulted from genetic drift, which is a driving force affecting the genetic relationships between populations. However, the relatively short time interval since the first feral mink populations were established
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Genetic diversity of introduced alien American mink in Poland may rather suggest that they developed from founder animals with different genetic characteristics. Generally, the hydrological system of the Polish lowlands, and particularly of the lakelands, which predominate in the north of the country, is well developed and has enhanced the establishment and dispersion of American mink. The isolation of any local feral mink population seems to be unlikely, which implies that the genetic diversity among these populations can be explained mainly by their different origin. However, the lack of geographic barriers should lead to homogenization, and therefore, the question of why local populations are so different is still open. It could be attributed to the short time that has passed since the beginning of colonization (only 20–30 years) or, more probably, to the current high propagule pressure with the large number of mink escapees from various farms that breed genetically different stocks. The feral mink from sites FNE4 and FCE4 were the most genetically divergent from those sampled at all other sites, and they were different from each other. PCA classified them as separate groups, and samova revealed that the majority of the variance was attributable to differences among FNE4, FCE4 and all other sites. These results are consistent with those of microsatellite analysis in which site FCE4 was assigned to one cluster, separate from all other sites (Zalewski et al., 2010). Interestingly, 27% of mink captured at site FCE4 had a pelt colour other than the standard (M. Brzezin´ski, unpublished data). The populations of American mink at site FCE4 most probably originated from individuals that escaped (or were released) from mink farms and are likely to have been founded by a very small number of females. It is much more difficult to explain why mink from FNE4, which is located on the eastern periphery of Poland, close to the Belarusian border, are so genetically distinct. The feral population here is probably the oldest in Poland: wild-living mink were first observed at site FNE4 in 1972, whereas they were not identified at FCE4 until 1995. However, this does not exclude the possibility that escapees from local farms have supplied the feral mink population at FNE4 (unfortunately, no data on the location of mink farms in the 1970s are available). Taken together, these data suggest that the most likely scenario for the colonization of Poland by American mink was the creation of feral populations from farm escapees that were mixed with a wave of invasive mink from Eastern Europe.
from microsatellite data was 2.66 mink per generation, whereas using mtDNA data, this value was only 0.36 (calculated using average UST). This result indicates that the dispersal rate of males was about sevenfold than that of females. Sex-biased dispersal might explain the very high genetic divergence among feral mink populations with respect to mtDNA compared with microsatellite loci (Zalewski et al., 2010). Female dispersal may be less effective than that of males. Long-distance male dispersal homogenizes the structure of populations of mink founded by individuals of different origin. This is probably why a very clear pattern of mink differentiation in relation to geographical distance was identified when microsatellite data were analyzed (Zalewski et al., 2010), but the same was not seen when mtDNA was examined. It is likely that the founder effect has affected genetic structure of mink populations, both in the past and present (by ongoing propagule pressure), but the longdistance dispersal ability of males has shaped both the population genetic diversity and its structure. The discrepancy between mtDNA and microsatellite divergence could also result from the nature and mode of inheritance. In effect, the effective population size for mtDNA is fourfold lower than for microsatellite loci, allowing genetic drift. CONCLUSIONS The introduction pathway and population genetic structure of invasive species play an important role in the invasion success. The adaptation to a novel environment requires genetic variation. Therefore, multiple introductions from various sources circumvent the harmful effect of small population size and increase the probability of successful invasion. Our results indicate that the colonization of Poland by American mink was a complex process triggered not only by immigrants from Belarus but also by numerous escapees from various farms. These data also provide evidence that alien species introductions are not always characterized by the loss of genetic diversity (invasive species paradox). Multiple introductions that shaped the genetic structure of feral mink in Poland and formed high levels of haplotype and nucleotide diversity might have increased the fitness of individuals, the viability of the population and the invasiveness of the species. Further studies are required to elucidate the mechanisms underlying the potentially increased fitness of individuals and accelerated invasiveness of alien species after the mixing of genetically different populations.
Sex-biased dispersal Male-biased dispersal has been observed in many carnivores (e.g. Wilson et al., 2000; Blundell et al., 2002; Proctor et al., 2004) and has been accepted as a general rule for mammals (Greenwood, 1980). Strong male-biased dispersal in the American mink has been directly and indirectly confirmed by several studies (Mitchell, 1961; Gerell, 1970; Melero et al., 2008; Zalewski et al., 2009). Comparison of genetic differentiation estimated using mtDNA (this study) and microsatellite DNA (Zalewski et al., 2010) also suggests much higher gene flow among males than females. The number of migrants calculated
ACKNOWLEDGEMENTS We thank A.B., G.B., M.M., M.M., M.K. and staff of ‘Warta Mouth’ National Park for help with trapping mink, and local farms, hunters and conservationists for providing mink tissue samples. We are grateful to M.S´. for excellent laboratory work and to J.G. for English correction and useful critical comments on the manuscript. We are also grateful to F.E.Z. and two other anonymous referees for helpful comments. Trapping and handling procedures were approved by the Polish Ethical Commission for Research on Animals. This study was financed
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A. Zalewski et al. by the University of Warsaw and University of Białystok and partly by the Ministry of Science and Higher Education (grant no: N N304 221835). The project was supported by a Marie Curie European Reintegration Grant within the 7th European Community Framework Programme to A.Z. REFERENCES Allee, W. (1931) Animal aggregations: a study in general sociology. University of Chicago, Chicago. Allendorf, F.W. & Lundquist, L.L. (2003) Introduction: population biology, evolution, and control of invasive species. Conservation Biology, 17, 24–30. Bartoszewicz, M. & Zalewski, A. (2003) American mink, Mustela vison diet and predation on waterfowl in the Słon´sk Reserve, western Poland. Folia Zoologica, 52, 225–238. Blundell, G.M., Bendavid, M., Groves, P., Bowyer, R.T. & Geffen, E. (2002) Characteristics of sex-biased dispersal and gene flow in coastal river otters: implications for natural recolonization of extirpated populations. Molecular Ecology, 11, 289–303. Bohonak, A.J. (2002) IBD (isolation by distance): a program for analyses of isolation by distance. Journal of Heredity, 93, 153–154. Bonesi, L. & Palazon, S. (2007) The American mink in Europe: status, impacts, and control. Biological Conservation, 134, 470–483. Brzezin´ski, M. & Marzec, M. (2003) The origin, dispersal and distribution of the American mink Mustela vison in Poland. Acta Theriologica, 48, 505–514. Clegg, S.M., Degnan, S.M., Kikkawa, J., Moritz, C., Estoup, A. & Owens, I.P.F. (2002) Genetic consequences of sequential founder events by an island-colonizing bird. Proceedings of the National Academy of Sciences of the United States of America, 99, 8127–8132. Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657–1659. Courchamp, F., Clutton-Brock, T. & Grenfell, B. (1999) Inverse density dependence and the Allee effect. Trends in Ecology Evolution, 14, 405–410. Crawford, K.M. & Whitney, K.D. (2010) Population genetic diversity influences colonization success. Molecular Ecology, 19, 1253–1263. Cullingham, C.I., Kyle, C.J., Pond, B.A. & White, B.N. (2008) Genetic structure of raccoons in Eastern North America based on mtDNA: implications for subspecies designation and rabies disease dynamics. Canadian Journal of Zoology, 86, 947–958. Da Silva, A.G., Eberhard, J.R., Wright, T.F., Avery, M.L. & Russello, M.A. (2010) Genetic evidence for high propagule pressure and long-distance dispersal in monk parakeet (Myiopsitta monachus) invasive populations. Molecular Ecology, 19, 3336–3350. Dlugosch, K.M. & Parker, I.M. (2008) Founding events in species invasions: genetic variation, adaptive evolution, and
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A. Zalewski et al. genetic monomorphism in a highly successful introduced species: the case of the muskrat (Ondatra zibethicus) in Europe. Mammalian Biology, 72, 123–126. Zalewski, A., Piertney, S.B., Zalewska, H. & Lambin, X. (2009) Landscape barriers reduce gene flow in an invasive carnivore: geographical and local genetic structure of American mink in Scotland. Molecular Ecology, 18, 1601– 1615. Zalewski, A., Michalska-Parda, A., Bartoszewicz, M., Kozakiewicz, M. & Brzezin´ski, M. (2010) Multiple introductions determine the genetic structure of an invasive species population: American mink Neovison vison in Poland. Biological Conservation, 143, 1355–1363. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article:
Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. BIOSKETCHES All authors are interested in the ecology and genetics of terrestrial vertebrates. They are currently conducting research on invasive mammals’ ecology with a focus on processes and patterns underlying the dynamics, adaptation to the new condition and genetics of invasive species. Author contributions: Tissue samples from feral mink were collected by A.Z., M.B4. and M.B2., from ranch mink by A.M.-P. Laboratory work was carried out by A.M-P. and M.R.; A.Z. and M.R. analysed the data; A.Z., M.B.2, M.R. and M.K led the writing.
Appendix S1 Haplotype frequencies. Editor: David Richardson As a service to our authors and readers, this journal provides supporting information supplied by the authors.
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