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Reintroduced Eurasian otters in Italy 619


TABLE 1 Number of samples successfully amplified (NS) among the genotyped samples of otter Lutra lutra spraint collected on the Ticino River, Italy, during 2016–2018. The table shows amplification success per locus, number (NA) and size of alleles, and observed and expected heterozygosis (HO and HE, respectively).


Locus OT-14


OT-04 Lut 453 OT-17 Lut 833 Lut 701 Lut 818 OT-19 Lut 435 Lut 715 Lut 902 Mean


NS 8


7 8 6 7 4 5 5 8 6 3 6


Amplification success (%) 100.0


87.5


100.0 75.0 87.5 50.0 62.5 62.5


100.0 75.0 37.5 76.1


NA 1


5 4 3 4 2 4 3 3 1 2 3


Size (base pairs) 129


166, 173, 175, 177, 190 123, 127, 129, 131 147, 151, 172


154, 163, 165, 175 207, 247


140, 156, 160, 170 211, 215, 218 123, 129, 143 203


145, 166 HO


0.00 0.40 0.50 0.40 0.80 0.67 0.50 0.40 0.50 0.00 0.67 0.41


HE


0.00 0.34 0.48 0.34 0.70 0.44 0.66 0.32 0.40 0.00 0.44 0.35


TABLE 2 Accession number and origin of European and Asian otter sequences downloaded from GenBank (National Center for Biotechnology Information, Bethesda, USA).


GenBank accession no. LC094961_Lutra_lutra


FJ236015_Lutra_lutra EF672696_Lutra_lutra LC049955_Lutra_lutra LC049954_Lutra_lutra LC049953_Lutra_lutra LC049952_Lutra_lutra LC049378_Lutra_lutra LC049377_Lutra_lutra MN122838_Lutra_lutra


KY117556_Lutra_sumatrana AY860320_Lutra_lutra_captive_Caramanico


fragment analysis service offered byMacrogen (Seoul, South Korea) and analysed electropherograms using GeneMarker 2.2.0 (SoftGenetics, State College, USA; see Tremolada et al., 2020 for further details). We analysed the mitochondrial DNA control region to


verify the genetic strain and determine the occurrence of hy- bridization with a residual native population. We accessed sequences from European and Asian otters from GenBank (National Center for Biotechnology Information, Bethesda, USA; Table 2) to pinpoint a short region amplifiable in poor and degraded faecal mtDNA and capable of identifying the origin of the genotypes. Using the primers OTTDL3- and OTTDH5- (Mucci


et al., 2010), we amplified mtDNA from seven of the 25 sam- ples. The reaction was performed using the following proto- col: 2 minutes at 94 °C, followed by 40 cycles of three steps (30 seconds at 94 °C, 30 seconds at 55 °C, and 30 seconds at 72 °C), before a final extension of 10 minutes at 72 °C. We carried out Sanger sequencing using the reverse primer OTTDH5. Amplicons were separated on an ABI 3130XL automated sequencer (Applied Biosystems, Waltham,


Origin Unknown


South Korea South Korea Japan Russia


Unknown China China China


Denmark Thailand Italy


References Unpublished


Jang et al. (2009) Ki et al. (2010)


Waku et al. (2016) Waku et al. (2016) Waku et al. (2016) Waku et al. (2016) Waku et al. (2016) Waku et al. (2016) Unpublished


Mohd Salleh et al. (2017) Ketmaier & Bernardini (2005)


USA) and visualized and corrected using Seqscape 5.0 (Applied Biosystems,Waltham, USA). We also included a haplotype (GenBank accession no.


AY860320) from an individual otter hosted in Caramanico Otter Centre, Italy, as it belonged to the same captive-bred group as the reintroduced individuals. Weused the mtDNA extracted from tissues of an individual found as roadkill in the lower valley of the River Ticino in 2007 as a reference sample. We aligned the results and sequences from GenBank


in Bioedit 7.0 (Hall, 1999) and created a neighbour joining phylogenetic tree (1,000 bootstrap resamples) in MEGA 7 (Kumar et al., 2016) to identify the maternal lineage of Italian spraint samples. We used a sequence of the hairy- nosed otter Lutra sumatrana (GenBank accession no. AY860320) to root the tree.


Population viability analysis


We performed the population viability analysis using Vortex 10 (Lacy & Pollak, 2018), the most commonly


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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