1. Introduction
Invasive alien species (IAS) are one of the major drivers of biodiversity loss [1]. These IAS can reduce or promote the extinction of native populations worldwide through different ecological processes [2], including interspecific competition [3], predation [4], transmission of infectious diseases [5], passing on helminths causing parasitological illness [6], and changes in ecosystems [7].
One of the aims of the EU 2020 biodiversity strategy is to study IAS. In particular, the EU Regulation 1143/2014, on the prevention and management of the introduction and spread of IAS, makes this recommendation. One of the best-known examples of competitive exclusion of a native species by an alien species is the replacement of the Eurasian red squirrel (Sciurus vulgaris) by the Eastern grey squirrel (Sciurus carolinensis) in Great Britain, Ireland, and Italy [8,9,10].
The grey squirrel belongs to the order of Rodentia, family Sciuridae, and is native to eastern North America; this medium-sized squirrel lives mainly in trees and has no sexual dimorphism, neither in body size nor in coat colour [11]. The grey squirrel is one of the most invasive species in Europe and worldwide; accordingly, it has been included among the 100 World’s Worst IAS by the International Union for the Conservation of Nature [12]. Consequently, trading and breeding of the grey squirrel is currently prohibited, even if they are still imported and traded illegally in several countries [13]. The grey squirrel initially expanded its range in several states of the USA and Canada [11]; from there, it was introduced to South Africa [11], Great Britain [8], and Italy [10]. Eradication and sterilization studies have recently been carried out in several Italian regions, including Piedmont [14], Lombardy [10,15], and Umbria [16]. The dynamics of how the initial invasion of grey squirrels took place in Umbria remain unclear. Seven grey squirrels were purchased in 1999 and placed in large cages inside a zoological garden near Perugia [16]. The squirrels managed to escape from their cages in the early 2000s [16], and expanded their range over at least 50 km2, in a natural area close to Perugia connected to nearby Apennines woodlands. The LIFE BIO/IT204 U-SAVEREDS Project was set up for the conservation of the European red squirrel in Umbria [17], at least in part owing to the threat imposed by invasive grey squirrels.
The negative interaction between the two species has been attributed to competition for food resources and habitats, thus reducing breeding and recruitment [3,18], but it also due to the chronic stress induced by co-existing in the same habitat [19], and to interspecific competition leading to behavioural changes [20].
Eastern grey squirrels can have two breeding seasons; generally, most breeding occurs in December-February and May-June [11,21,22]. Consequently, after 44 days of gestation [21,23,24], the females deliver a spring litter (or first litter) and/or a summer litter (or second litter) [21]. The lactation period may last as much as 70 days [23]; the young are weaned at 8-10 weeks old [21], and sexual maturity occurs at about 10-12 months [21].
In male grey squirrels, spermatogenesis generally peaks during the female breeding season [11,21]. Nevertheless, male squirrels undergo periods of sexual regression and redevelopment [25]. There is disagreement as to whether the regression is synchronous throughout the population, or if it is an individual phenomenon. In the same country, Britain, both phenomena were described: sexually regressed males were found during all months of the year [26], but a seasonal cycle of sexual regression, in the autumn, and redevelopment was also described [25]. Similar findings were described in USA [27], but with a greater synchronization of the male reproductive cycle [21,28].
The growth rate of a population is affected by annual fecundity: the product of the number of weaned young per litter and the number of litters produced per year by females [3]. Fecundity rates can be estimated by counting the number of embryos and uterine scars if carcasses of females are available [29,30,31]. Uterine scars form at embryo implantation sites in the utero and can last up to nine months after parturition [29,30,31]. Two successive pregnancies can be also identified because uterine scars have a different colour intensity [15,32].
Studies of the reproductive cycle of an IAS can establish parameters on population dynamics, growth rates, viability, and ultimately allow more precise estimates of how quickly populations of invasive species will spread in their non-native range. These demographic parameters can predict the management effort required (i.e., number of animals to be culled) to achieve population reduction successfully or to estimate future spread rates and demographic trends in population dynamics models [33,34]. Bearing this in mind, the reproductive cycle and fecundity of the Umbrian grey squirrel population was studied by investigating uterine scar evaluation and litter production through the analysis of female and male genital tract morphometric and morphological seasonal changes.
