618 A. Balestrieri et al.
1995). Since the 1980s, the otter has been translocated (UK: Wayre, 1985; Roche et al., 1995; Spain: Fernández-Morán et al., 2002) or reintroduced in several countries (Sweden: Sjöåsen, 1996; The Netherlands: Koelewijn et al., 2010; France: Geboes et al., 2016), and further projects are under- way (Denmark: Christiansen, 2017; Japan: Murakami, 2017). At the end of the 20th century, the Eurasian otter
became extinct in the northern and central Italian peninsula (Prigioni et al., 2007). On the River Ticino (northern Italy, Piedmont/Lombardy regions), one of the best conserved lowland riverine habitats of northern Italy, the species was declared extinct in 1985 (Cassola, 1986; Prigioni, 1986), after the last reliable signs of its presence had been recorded in 1980 (Galeotti, 1981). The river was later identified as a potential reintroduction area in the Action Plan for the Conservation of European Otters (Macdonald & Mason, 1990), and deemed suitable for otters by a feasibility study (Prigioni, 1995). In the EEP framework, two breeding centres were built and a reintro- duction trialled with the release of one pair of otters (Montanari & Boffino, 2000). Concern about the reintroduction was raised by the
Italian Institute for Wildlife, because analyses of the mito- chondrial DNA of the otters hosted in European breeding centres confirmed that individuals of the Asiatic subspecies Lutra lutra barang had contributed to the gene pool of the EEP population (the so-called genetic B-line; Randi et al., 2001). In addition, although the European River Otter Studbook was established with the aim to increase the gen- etic variability of captive-bred otters, a high level of inbreed- ing was recorded (Randi et al., 2001, 2005). The reintroduction was interrupted soon after the first
release in 1997,mainly because of concerns about the genetic make-up of the captive population. Nonetheless, a second pair of animals was released or escaped in 1998 and a further female with her cub probably escaped before 2000. None of these otterswere equipped with radio transmitters; their fate is thus unknown and the precise number of reintroduced animals is uncertain. Although translocations of large numbers of individuals
can have negligible effects on local populations (Arrendal et al., 2004), and some reintroductions have been successful with relatively few founders (lynx Lynx lynx: Breitenmoser et al., 1998; fisher Martes pennanti: Aubry & Lewis, 2003; Eurasian badger Meles meles: Balestrieri et al., 2018), success can be affected by the number of released animals (Griffith et al., 1989). Small founder populations are vulnerable to both demographic and environmental stochasticity (Gilpin & Soulé, 1986) and thus at risk of local extinction. Nevertheless, since 2007 signs of otter presence such as roadkill and spraints have been recorded regularly around a short stretch (5–7 km) of the Ticino River near the release site (Balestrieri et al., 2016). In 2016–2017 otters were re- corded by camera traps near the release site on three
occasions, and otter signs were found in 16 out of 32 sampling locations (600m long river stretches) surveyed (Smiroldo et al., 2019). Throughout 2018, a total of 77 km of watercourses weremonitored, but only five otter spraints were found, in four surveyed stretches of the river (Tremolada et al., 2020). Compared to the core area of the Italian otter range, marking density around the release site has generally been low (0.15–0.2 vs 3.7 spraints/100 m), suggesting that the local otter population probably consists of few individuals (Prigioni et al., 2006a). The persistence of the species in the release area despite
the small founder population also raised questions about the possible survival of a residual native otter population that was not recorded during the last national census (Prigioni, 1986). To assess the abundance of the otter population on the River Ticino and examine the genetic origin of the rein- troduced population, we analysed DNA from faecal sam- ples. This method has proved to be effective for assessing the minimum number of individuals and haplotypes of elu- sive species such as the Eurasian otter (Dallas et al., 2003; Hung et al., 2004; Kalz et al., 2006; Koelewijn et al., 2010). In addition, we used population viability analysis to deter- mine a posteriori the number of founders and predict the likely fate of the population.
Methods
Genetic analyses During the 2016–2018 monitoring surveys, which covered the entire Italian stretch of the River Ticino, we collected 25 sufficiently fresh otter spraints and preserved them in
ethanol at −20 °C for genetic analyses (Bonesi et al., 2013; Vergara et al., 2014). In addition, using the Wizard Genomic DNA Purification Kit (Promega Italia, Milan, Italy), we extracted genetic material from five tissue samples of otters found as roadkill, to use as controls. Before DNA extraction, spraint samples were placed in
a vacuum to evaporate the remaining ethanol. We extract- ed DNA with the QIAamp DNA Stool Mini Kit (Quiagen S.r.l., Milan, Italy) and performed genotyping using 11 polymorphic autosomal microsatellite loci, chosen among those reported in previous studies (Dallas & Piertney, 1998; Dallas et al., 1999; Huang et al., 2004, 2005; Hájková et al., 2006; Kalz et al., 2006; Bonesi et al., 2013; Lerone et al., 2014; Vergara et al., 2014), based on expected poly- morphisms and size of PCR fragments (Table 1). We split the 11 primer pairs into two multiplex PCRs and labelled them with different fluorescent dyes for distinguishing the amplified fragments. We used the Multiplex PCR Kit (Quiagen S.r.l., Milan, Italy) for PCR amplifications, follow- ing the manufacturer’s recommendations, except for the final volume, which was reduced to 25 μl. We sequenced PCR amplification fragments using the commercial
Oryx, 2022, 56(4), 617–626 © The Author(s), 2021. Published by Cambridge University Press on behalf of Fauna & Flora International doi:10.1017/S0030605321000107
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