0 10 20 30 40 50Km Sangro Basin Study Area Abruzzo Region boundary Sangro basin Sangro main hydrography Rome Introduction The Eurasian otter ( Lutra lutra ) is an elusive and rare species, still listed as Critically Endangered in Italy, where two small and distinct populations occur in south and south-central Italy [FIG 1 ], geographically and genetically isolated from the other European populations (Randi et al. 2003 ). Non-in- vasive genetic sampling (NGS) was used to gather essential information on otters living in the Sangro basin, part of the northernmost population. This basin was recently re-colo- nized (De Castro and Loy 2007) and plays a critical role for the future expansion of the species northward. To better characterize our small population we compared two difer- ent microsatellite sets (Lut and OT). Aims The main objectives of our study were to assess the den- sity and distribution of otters in the river and to highlight the social structure of the population in terms of spatial and temporal overlap of individual males and females. These are crucial information to assess the status of the Italian ot- ter population and adopt successful conservation measures claimed by the national action plan (Loy et al. 2011 ). Materials and methods A total of 225 non-invasive samples (spraints, hair, jellies) were collected at 62 sampling sites along 116 km of river stretches between 2011 and 2012 [FIG 2A]. To characterize individual ot- ters from non-invasive samples we used a panel of 13 nu- clear microsatellite loci (Lut 453 , Lut604 , Lut 701 , Lut832, Lut833 , Lut902, OT04, OT05 , OT07 , OT14, OT17 , OT19 , OT22 ). We performed 5 multiplex PCRs (Lut 453 , OT05 , OT22; Lut833 , OT19; Lut604 , Lut 701 ; Lut832, Lut902; OT17 , OT07) and 1 simplex (OT04 ) to reduce costs and time spent for analyses. All samples were initially screened at three loci to assess sample quality. Only samples with 50% or more positive screening PCRs were analysed at all loci and for molecular sexing (Mucci and Randi 2007). To reduce allelic dropouts and false allele error rates a mul- tiple tubes approach was used during both screening and subsequent analyses (Taberlet et al. 1996 ). Two loci subsets were compared (Lut – Dallas et al. 2003 ; OT – Huang et al. 2005 ) to highlight any better performance. Parentage analy- sis (Colony software package, Jones and Wang 2009 ) were run to hypothesize kinship within the sampled individuals. AN IMPROVED NON- INVASIVE GENETIC SAMPLING PROTOCOL FOR OTTERS Discussion Density of otters in the peripheral Sangro river populationzis in accordance with previous results for the core otter area in southern Italy (Prigioni et al. 2006 ), thus contradicting previ- ous hypothesis of low species density at the range periphery (Marcelli and Fusillo 2009 ). Our results conirmed the spat ial overlap among related females observed in other Europe- an populations (Kruuk and Moorhouse 1991 ; Kruuk 2006 ), but also ascertained a spatial overlap of related males that was not reported previously. During the same night more than one otter was sampled in the same area. Temporal and spa- tial overlap among individuals could be consistent with the hypothesis that scent-marking may communicate the use of a resources patch on a small-temporal scale (Kruuk 1995). Otters could take advantage from exclusive access to food resource patches within overlapping ranges, avoiding intra- speci ic encounters and hunt ing eforts on partially deplet- ed resources. The otter density found and the large extension of hydro- graphic network suggest that the Sangro basin could likely be able to support a viable otter population, and promote the further expansion of the species northward. References  Dallas, J. F, S. B. Piertney, 2003. Microsatellite primers for Eurasian otter. Molecular Ecology 7: 1247-1251  De Castro G., Loy A., 2007. Un nuovo censimento della lontra (Lutra lutra, Carnivora, Mammalia) nel iume Sangro (Abruzzo): inizia la ricolonizzazione dell’Italia centrale? 68° Convegno Unione Zoologica Italiana, Lecce, 24-27 Settembre 2007. Riassunti: 105.  Huang, C.C., Y.C. Hsu, L.L. Lee, S.H. Li, 2005. Isolation and characterization of tetramicrosatellite DNA markers in the Eurasian otter (Lutra lutra). Molecular Ecology 5: 314-316  Jones O.R, J. Wang, 2010. COLONY: a program for parentage analysis and sibship inferences from multilocus genotype data. Molecular Ecology Resources 10(3): 551-555  Kruuk H., 1995. Wild Otters. Predation and Populations. Oxford University Press, Oxford  Kruuk H. 2006. Otters ecology, behaviour and conservation. 2ed. Oxford University Press, Oxford  Loy A., Boitani L., Bonesi L., Canu A., Di Croce A., Fiorentino P. L., Genovesi P., Mattei L., Panzacchi M., Prigioni C., Randi E., Reggiani G., 2010. The Italian action plan for the endangered Eurasian otter Lutra lutra. Hystrix It. J. Mamm. (n.s.) 21(1) : 19-33  Mucci, N, E. Randi, 2007. Sex identiication of Eurasian otter (Lutra lutra) non-invasive DNA samples using ZFX/ZFY sequences. Conservation Genetics 8: 1479-1482  Prigioni C., Remonti L., Balestrieri A., Sgrosso S., Priore G., Mucci N., Randi E., 2006. Estimation of European otter (Lutra lutra) population size by fecal DNA typing in Southern Italy. Journal of Mammalogy 87(5):855-858  Randi E., F. Davoli, M. Pierpaoli, C. Pertoldi, A.B. Madsen, V. Loeschcke, 2003. Genetic structure in otter (Lutra lutra) populations in Europe: implications for conservation. Animal Conservation 6: 93-100  Taberlet P., S. Griin, B. Goossens, S. Questiau, V. Manceau, N. Escaravage, L.P. Waits, J. Bouvet , 1996. Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Research 26: 3189–3194 1 DEPARTMENT OF ENVIRONMENTAL BIOLOGY - UNIVERSITÀ ROMATRE, I-00146 ROMA, ITALY 2 ISPRA, I-40064, OZZANO DELL’EMILIA, ITALY 3 ENVIRONMETRICS LABORATORY - UNIVERSITÀ DEL MOLISE, I-86090 PESCHE, ITALY LAURA LERONE 1 , CHIARA MENGONI 2 , GIUSEPPE MARIA CARPANETO 1 , ETTORE RANDI 2 , ANNA LOY 3 laura.lerone gmail.com 0 10 20 30 40 50Km 62 sampled marking sites A 0 10 20 30 40 50Km B 2 ♀? 4♀ 8♂ 14 OTTERS Results Despite the low DNA quality and quantity that usually char- acterizes otter non-invasive samples, we successfully geno- typed 32% of samples (N= 72 ), assessing the presence of at least 14 otters, 8 males and 4 females and 2 likely females. Considering extremes locations of successful sampling sites ( 64 km) we obtained a density of 0. 17 otters/km [FIG 2 B]. Subset comparison resulted in slight diferences among Lut and OT microsatellites [FIG 3 ], despite OT loci were more polymor- phic [FIG 4 ] and then more informative for our small otter population. The critical values of theoretical probability of identity among unrelated (PID) and sibling (PIDsibs) individ- uals were 0 .001 (six loci) and 0 .002 ( 13 loci) respectively. Geno- typed samples belonged to 64km of the total sampled river stretches [FIG 2 B]. During the 2011 sampling season (June- October) we obtained a mean density of 0.17otters/km. The spatial distribution of capture and recaptures of indi- vidual otters highlighted a wide both intra and intersexual spatial and temporal overlap among individuals. Parentage analysis disclosed the presence of 5 potential full-sibs famil- iar clusters [TAB 1 ]. The familiar clusters are strictly related to the overlap diagram [FIG 5], as related females and males show the highest degrees of spatial and temporal overlap. FIG 1. The two major otter nuclei in south and south-central italy. On the right the study area: Sangro Basin FIG 3. No signiicant diferences arose comparing Lut and OT loci for positive PCR, false alleles and allelic dropout rates FIG 2. A. 62 sampling stations along 116 km river stretches; B. successfully genotyped samples form 64 km river stretches LUT 453 LUT 604 LUT 701 LUT 832 LUT 833 LUT 902 OT 04 OT 05 OT 07 OT 14 OT 17 OT 19 OT 22 125 129 202 188 147 147 176 175 200 120 145 211 148 131 206 196 155 151 204 210 163 179 204 124 153 215 152 157 223 227 164 DARKER COLORS CORRESPOND TO HIGHER ALLELE SIZES FIG 4. 13 nuclear microsatellites’ panel used. All loci but one (Lut 453) resulted polymorphic with a number of alleles per locus ranging between 2 and 4 FIG 5. Familiar clusters spatial distribution on an ideally stretched Sangro river. Circles represent individuals captured only once FULLSIBS FAMILY PROB (INC) PROB (EXC.) MEMBER 1 MEMBER 2 MEMBER 3 MEMBER 4 1 0.1573 0.1572 F1 F1 F5 F6 2 0.9994 0.9665 M1 F2 M2 3 1.0000 1.0000 F3 4 0.9734 0.9734 M3 M4 M6 M8 5 0.9396 0.9396 M5 M7 TAB 1. Five potential Full-Sibs families inferred by Colony software package Acknowledgements  Arturo Leone (latestuggine.it) for the graphic design. M6 M5 M7 M8 M4 M3 M3 64 +700 Km F3 M2 M1 F2 F5 F6 F4 F1 F2 Km33 +145 Km97 +845 2011 2012 Estuary at Km120 +500 Spring at Km0 01 5 10Km Wild Musteloid Conference — Oxford, UK, March 18-21, 2013 View publication stats View publication stats