Our data supported both of our initial hypotheses. They demonstrated that genetic diversity of grey squirrels around Perugia is very low following the severe bottleneck imposed when a few individuals were introduced from Piedmont, an area where genetic diversity was already low (Signorile et al. in review). It was not known prior to our work that the Perugia population had its origins in Piedmont; the double effect of low founder sizes, combined with founders from a region that had low diversity already, means diversity in Perugia is very low. Our results also showed that range expansion in Perugia has so far been very slow—comparisons to spread rates for other European populations are made below.
Genetic diversity and spread
There is limited consensus on the intrinsic physiological and ecological characteristics of good invaders, and the relationship between intrinsic characteristics of species and invasion may be complex and of limited predictive use (Kolar and Lodge 2001; Crawford and Whitney 2010). Instead, research often indicates that invasion success depends on the size of introduction events (Veltman et al. 1996; Kolar and Lodge 2001; Cassey et al. 2004; Lockwood et al. 2005; Sol et al. 2007; Dlugosch and Parker 2008; LeRoux and Wieczorek 2009; Hardesty et al. 2012). Genetic bottlenecks at the time of introduction can cause low genetic diversity, inbreeding depression and reduced ability to adapt to the new environment, limiting the probability of successful invasion (Frankham et al. 2002). When effective population size is low, stochastic loss of genetic variation is more likely and drift can overcome selection on evolutionary pathways. This can have a long term impact on fitness and population dynamics (Lammi et al. 1999; Reed and Frankham 2003; Reed et al. 2007; Roman and Darling 2007). Conversely, many introduced individuals or multiple introductions can lead to much genetic variation, which limits inbreeding depression, promotes adaptation, and facilitates invasion. These ideas and evidence supporting them are reviewed by Sakai et al. (2001), Lee (2002), and Dlugosch and Parker (2008). The invasiveness demonstrated by grey squirrels around Perugia and elsewhere in Italy therefore may be unexpected if expectations are based on the work cited above. The success of the invasion in spite of the genetic disadvantage of the population demonstrates the overall ability of the species as an invader in Europe. Further supporting this viewpoint, by compiling records on squirrel introductions worldwide, Bertolino (2009) estimated that Sciurus populations can be established from one introduced pair with 50 % success rate.
Although the population in Perugia has established and is spreading, the spread is slow in comparison with other populations in Europe. Low genetic diversity is the most probable explanation for the slowness—although squirrels can invade even when genetically poor, the rate of invasion may be decreased. Average spread rate in Piedmont was estimated to be about 0.6 km/year. For Northumberland, England it was 8.25 km/year. For East Anglia, England, it was 5.7 km/year, and for Northern Ireland it was 1.92 km/year (Signorile et al. in review). Earlier studies (Williamson and Brown 1986; Tangney and Montgomery 1995; O’Teangana et al. 2000) estimated 7.7 km/year in East Anglia and 1.94 km/year in Ireland, similar values. These are all markedly higher than our spread rate estimate for Perugia, 0.29 km/year. Bertolino and Genovesi (2003) also show results that indicate a higher expansion rate in Piedmont than in Perugia. The spread rates listed here are consistent with the hypothetical explanation that genetic diversity is negatively correlated with population spread rate (Signorile et al. in review). Although spread rate in Perugia is slow, ecological factors appear to be entirely in favor of rapid spread, in the sense that the largely unfragmented forests surrounding Perugia are dominated by large-seeded deciduous trees, and are an ideal habitat for fast population growth for an arboreal species native to the forests of the Eastern USA were oaks, walnuts, hickory and beeches are abundant (Koprowski 1994). Densities measured at MMA1 and FON1 were high—most densities previously measured in other European woodlands have been approximately in the range 1–5 individuals per hectare (Lurz et al. 2001).
There are at least two alternative hypothetical explanations for the slow spread in Perugia that can be compared in future work to the genetic explanation we suggest here. First, “lag phases” of slow expansion are often observed in the initial stages of an invasion (Crooks 2005). The Perugia expansion may accelerate after some years of initial slow expansion. Second, it was suggested by a referee of this study that fragmentation may speed population range expansion in some circumstances (With 2002). Hence slow expansion in Perugia may have been caused by the unfragmented nature of the Perugia forest instead of happening in spite of it, according to this hypothesis. One possible mechanism for such an effect would be if fragmentation forces individual squirrels to undergo larger dispersal events in order to bridge gaps between fragments. But the modelling and empirical results reviewed by With (2002) seem to suggest that greater habitat coverage is usually or always associated with faster range expansion. Another possible mechanism is that some degree of fragmentation is actually beneficial to grey squirrel ecology. Koprowski (2005) found a negative relationship between grey squirrel density and fragment size, and Fisher and Merriam (2000) found higher grey squirrel densities in fragmented landscapes than in continuous forest. However, as mentioned above, densities of grey squirrels in the Perugia region were quite high, casting doubt, in our view, on the hypothesis that lack of fragmentation is what has slowed expansion in the Perugia area. Nevertheless, landscape effects on spread rate involve a complex interaction of species demography, dispersal behaviour, ability to detect distant fragments, and precise landscape layout; further evaluation of landscape effects on spread rate and comparison to genetic effects will be an interesting topic for future research.
We detected no spatial genetic structure in the Perugia population using STRUCTURE; this contrasts with our earlier work in which we found structure at small spatial scales in populations in Piedmont and elsewhere (Signorile et al. in review). Lack of structure is probably due to the recency of the introduction or dispersal within the area or both. Structure would be expected to emerge as the population range expands, mirroring what was found, for instance, in Piedmont.
The method used here for calculating spread rate was quite similar to widely used methods described in Andow et al. (1990), but with a few differences because of the nature of our data. Andow et al. (1990) had several population range maps for their species of study, gathered at intervals over a long period of time. So they were able to compute spread rates over intervals and investigate whether the theoretical expectation held that spread was asymptotically linear. But Andow et al. (1990) selected species for study for which data were especially abundant, whereas we were interested for this paper in an important new population of grey squirrel, for which data were good but more limited than those of Andow et al. (1990). We calculated one spread rate per direction for each possible start date, using 2012 data, rather than extrapolating successive estimates over time to an asymptotic rate. This is an approximation of the asymptotic rate of Andow et al. (1990). We tried calculating spread rates for the successive years 2010–2012, and results were similar to those presented here, but because only 3 years of data were available this was not an improvement, and the simpler method was presented. Our estimated spread rate may be an underestimate if asymptotic rate has not been acheived (e.g., if the Perugia population is in a lag phase of expansion—see above), but because spread rate differences with other European populations are so large, spread rate will have to increase dramatically to approach levels seen elsewhere. Andow et al. (1990) also make a comparison between spread rates calculated from range map data and rates estimated using the formula \( \sqrt {4\alpha D} \), where α is an intrinsic growth rate and D is the diffusion coefficient for the species. But this formula is not suitable for our study because estimates of α and D are not available for the Perugia population or for the other populations to which comparisons of spread rate were made.
Ecological issues
Umbria is covered by extensive and largely unfragmented secondary forests for over 60 % of its territory (Regione Umbria et al. 2009). Therefore, grey squirrels have the potential to spread dramatically. From Umbria, squirrels may well move freely northwards and southwards along the Apennine mountains to occupy the whole Italian peninsula. This is the first time that grey squirrels have occupied an extensive, continuous, forested area in continental Europe. Although grey squirrel spread is currently slow, ecological consequences could be serious in the long term, or in the shorter term if spread rate increases.
Although red and grey squirrels are sympatric at present, it is unlikely the peninsular woodlands will be able to indefinitely support both species. Competitive exclusion mechanisms (Wauters and Gurnell 1999) could already be acting on the red squirrel population; grey squirrels were much more commonly captured in the area than were red squirrels. Exclusion effects could become more evident in the next few years. If this proves to be the case, the presence of the grey squirrel will lead to local and regional extinctions of red squirrels.
Invasion of the grey squirrel may have consequences for the forest ecosystems of central and southern Italy more broadly than disappearance of the red squirrel. Squirrels have an important ecological role as seed dispersers because of their scatter-hoarding behaviour, and mutualistic relationships have evolved between tree species and squirrels (Wall 2001). Further studies would be needed to assess the capacity of the ecosystem to adapt to the replacement of the native red squirrel species with the grey squirrel, a species usually living at higher densities and with potentially subtly different seed hoarding and consumption behaviours. Red and grey squirrels overlap in their food preferences (Wauters et al. 2002), so replacement of red with grey squirrels may or may not lead to any substantive change in forest composition in the longer term. Although grey squirrels in the UK are known to debark and kill forest trees (Rowe and Gill 1985; Kenward and Parish 1986; Dagnall et al. 1998), this behaviour has not been observed in Italy (Signorile and Evans 2006). We have focussed here on the importance of the grey squirrel expansion in Perugia as derived from the threat the grey squirrel represents for S. v. italicus and S. v. meridionalis because that threat is very clear based on past observed interactions between grey and red squirrels elsewhere in Europe, whereas wider effects of the species replacement on southern Italian ecosystems may also be important but require further study.
The single squirrel seen at distance 7.13 km from the release site (Fig. 2) may have come from a human-mediated dispersal event from the main population centre West of the city, or it may have been part of a natural spread through a green belt north of the city. Future work should include sampling north of the city in the green belt. In addition, the distribution of grey squirrel sightings (Fig. 2) seems to suggest that the motorway RA06 has been a barrier to southward grey squirrel expansion. But because a few squirrels have recently been seen south of the road, the barrier appears to recently have been traversed. The section of the motorway closest to the release point is surface highway, but nearby sections are bridges or tunnels, which provide reduced or no barrier. Faster expansion south may be expected in the next several years now that the motorway barrier has been passed.
Conservation and research priorities
The grey squirrel population around Perugia should be eradicated. We have demonstrated that the population is expanding, albeit slowly, and it seems likely that new releases that increase genetic diversity will also increase spread rate: introgression of new genotypes, even of just a few individuals, could generate novel phenotypes, reduce the genetic load and have an effect on the evolutionary responses of invading populations (Lee 2011). The Perugia grey squirrel population is probably the only grey squirrel population that overlaps with the range of S. v. italicus, and is by far the closest population to the range of S. v. meridionalis. The expansion of the Perugia grey squirrel nucleus is probably the principle conservation threat to these subspecies (see below). New releases and increased spread rate are not unlikely given current enforcement of laws on invasive species. Grey squirrels were included in 2012 in Annex B of European Community Regulation 338/97, and this should prevent further imports from the species’ natural range. However, the grey squirrels released in Perugia and purchased from the pet trade were taken from Piedmont, another Italian location. Grey squirrel live-captures for any purpose other than scientific studies are strictly regulated in Italy by law 157/92. Squirrels in the pet trade circuit can therefore come from other Italian locations only through an initial illegal poaching act, and it cannot be ruled out that this could happen again, especially given lax enforcement. Hence eradication is probably the only way to eliminate the threat posed to the peninsular Italian red squirrel subspecies. Eradication is very likely still possible and practical. For instance, eradication of the Piedmont population of grey squirrels was considered practical in 1997, when that population covered 380 km2 (Bertolino and Genovesi 2003), far more than the <50 km2 covered in the Perugia region. Eradication seems likely to be feasible for at least the next 10 years or longer, since the Piedmont population was established in 1948, and it was still considered practical to eradicate in 1997. But this is not guaranteed, especially if additional genetic diversity is imported to the Perugia population. Rapid action is considered crucial to successful eradication (Genovesi et al. 2010), as the task gets harder with population expansion. In addition, further translocations within the area or into the range of S. v. meridionalis are more likely with a nucleus at Perugia.
Past attempts to eradicate grey squirrel populations in Italy have been thwarted by animal rights activists (Bertolino and Genovesi 2003). If eradication is not possible because of this human dimension, the recent EU Regulation 757/2012 and the Legislative decree 24/12/2012 signed by the Italian government are steps in the right direction—these laws are aimed at forbidding the grey squirrel pet trade, releases in the wild, and import. Citizens should be educated about the ecological risks of buying, moving or releasing exotic species, and enforcement of these regulations should be vigilant.
Broader conservation of the Italian peninsular red squirrel subspecies, beyond the mitigation of the threat posed by grey squirrels, should focus on research because, to our knowledge, little has been published on the subspecies S. v. italicus and S. v. meridionalis regarding many aspects of their range, density, demography, genetics, behaviour, ecology or the threats they face, if any, other than the grey squirrel. Habitat loss and destruction have been found in extensive research to strongly affect S. vulgaris generally (Wauters et al. 1994; Verbeylen et al. 2003, 2009), with sensitivity to habitat fragmentation especially emphasized, for instance, by Koprowski (2005). Mortelliti et al. (2011) argued instead that what matters is habitat loss, not fragmentation per se. However, the vast majority of studies of red squirrel ecology use study locations outside the likely ranges of S. v. italicus and S. v. meridionalis (but see Mortelliti et al. (2011)), and we are aware of no studies that compare and contrast the ecology and demography of these subspecies with other red squirrel populations. Perhaps more importantly, there has been no research on whether the particular sensitivities of S. vulgaris to habitat layout and amount, combined with the past and projected changes of habitat in peninsular Italy, actually represent a real threat to S. v. italicus and S. v. meridionalis in the way that the grey squirrel represents a real and present danger to the subspecies.