Who is who? High Resolution Melting analysis to discern between hare species using non-invasive sampling
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TECHNICAL REVIEW
Who is who? High Resolution Melting analysis to discern between hare species using non‑invasive sampling Maria Buglione1 · Simona Petrelli1 · Tommaso Notomista1 · Gabriele de Filippo2 · Romano Gregorio3 · Domenico Fulgione1 Received: 2 October 2019 / Accepted: 15 May 2020 © Springer Nature B.V. 2020
Abstract Identification of the species is a crucial step in many ecological studies. Sometimes, this could become a challenge, because of animal elusive behavior, low density population or for sympatric species leaving similar signs that are impossible to discriminate based only on their morphology. Here, we set up non-invasive molecular method to discern between the Italian hare (Lepus corsicanus) and the European hare (Lepus europaeus) using High Resolution Melting assay on fecal DNA, for the first time on these species. The Italian hare is endemic of the Central-Southern Italy and Sicily and it is classified vulnerable by the International Union for Conservation of Nature. Our procedure could be a useful tool to help conservation and management strategies, mainly in areas where the Italian hare and the European hare live in sympatry. The 9.5% of the peninsular range distribution of the European hare is took up by the range of the Italian hare. Our workflow allows sure species discrimination, rapidly and inexpensively (in one day at least 36 samples could be processed at costs of about 259 euros, including both DNA extractions and HRM run), also when large numbers of samples have to be processed. Moreover, our method could be widely applicable to other Lepus and/or mammalian species with similar concerns, by small adjustments to the protocol and its further validation, focusing on primes and corresponding HRM annealing temperature. Keywords Lepus corsicanus · Lepus europaeus · High resolution melting · Species assignment
Introduction Species identification of a collected sample is the first stage required in all ecological studies, mainly because it is fundamental for the understanding of the presence, abundance and distribution of a species. Indeed, this knowledge represents the key step for studying of population ecology, its interactions with the biotic and abiotic components of the ecosystem or for animal management strategies (Palomares et al. 2002). For low population density and elusive animals, species assignment based only on morphological features of * Domenico Fulgione [email protected] 1
Department of Biology, University of Naples Federico II, Via Cinthia 26, 80126 Naples, Italy
2
Istituto Di Gestione Della Fauna (IGF), Via M. da Caravaggio 143, 80126 Naples, Italy
3
Ente Parco Nazionale del Cilento, Vallo di Diano e Alburni, Via F. Palumbo 18, 84078 Vallo della Lucania, Salerno, Italy
individual and their traces could be problematic and could lead to misinterpretation of the data (Hansen and Jacobsen 1999; Edgar et al. 2004; Reisser et al. 2008). Moreover, methods based on the capture and manipulation of animals introduce logistical and ethical questions, especially wh
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