Research Article DNA from owl pellet bones uncovers hidden biodiversity RITA GOMES ROCHA 1,2 *, JULIANA JUSTINO 1 , YURI LUIZ REIS LEITE 1 & LEONORA PIRES COSTA 1 1 Departamento de Ci^ encias Biol ogicas, Centro de Ci^ encias Humanas e Naturais, Universidade Federal do Esp  ırito Santo, Av. Fernando Ferrari 514, Goiabeiras, 29075-910, Vit oria, ES, Brazil 2 Departamento de Biologia & CESAM, Universidade de Aveiro, Campus de Santiago, Aveiro 3810-193, Portugal (Received 3 February 2015; accepted 27 March 2015) Owl pellets are a useful tool for inventorying small mammals and a robust complement to traditional trapping methods. Here we assessed if bone samples recovered from Barn owl pellets in the Neotropics represent a viable source of modern DNA. We used the upper incisor of rodents and jaws of marsupials of pellet material for DNA extraction and amplification. Extraction and amplification success depended on the type of bone element used, being much higher from rodent incisors. The amplification success of small (429 bp) and large (801 bp) mitochondrial cytochrome b fragments was relatively similar, suggesting that DNA was not severely degraded, probably because our pellet samples were less than 8 years old. Eleven species of small mammals were identified, from which two are new to the study area: Gracilinanus emiliae and Marmosops pinheiroi. These records represent the southeastern boundary of both species, and extended their geographic distribution 370 km (G. emiliae) and 700 km (M. pinheiroi) to the south. Moreover, two of three detected species of Oecomys occurring in sympatry may represent undescribed species. The use of molecular analysis as a complement to morphological analysis of pellet bones is very effective in species identification, especially considering poorly known and elusive species, such as those reported here. The search for owl pellets during fieldwork should be a common practice among mammalogists in the Neotropics because this material provides a valuable non-invasive source of DNA, often revealing previously undetected species and sometimes uncovering new species. Keywords: bones, Cytochrome b, DNA extraction, marsupials, Neotropics, non-invasive DNA, rodents Introduction The Neotropical region hosts one of the richest mamma- lian fauna of the world (see Patterson & Costa, 2012), and is considered a ‘natural museum’ where new genera and species discoveries have grown at a fast pace over recent years (e.g., Guti errez, Jansa, & Voss, 2010; Pardi~ nas, Lessa, Teta, Salazar-Bravo, & C^ amara, 2014; Percequillo, Weksler, & Costa, 2011; Voss, Lim, Diaz-Nieto, & Jansa, 2013). These new taxa largely consist of small mammals: bats, marsupials and rodents weighing less than 1 kg. Despite these advances, our knowledge on several species is still very scarce, and based on scattered specimens from a few collecting localities (e.g., Brand~ ao, da Rocha, Dantas, & Pascoal, 2014; Rocha, Fonseca, Zhou, Leite, & Costa, 2012; Voss, Fleck, & Jansa, 2009; Voss et al., 2013). This collecting gap prevents a full understanding of Neotropical diversity and its associated historical phy- logeographic patterns (Patton, da Silva, & Malcolm, 2000). Some studies evaluated field techniques and suggested methods to improve the effectiveness of Neotropical small mammal surveys (e.g., C aceres, N apoli, & Hannibal, 2011; Lambert, Malcolm, & Zimmerman, 2005; Ribeiro-J unior, Rossi, Miranda, &  Avila-Pires, 2011; Voss & Emmons, 1996), because several species either avoid trapping devi- ces or fail to be detected due to their elusive behaviour and/or low densities. Owl pellets are a useful tool for inventorying small mammals and a robust complement to traditional trapping methods (e.g., Bonvicino & Bezerra, 2003; Rocha, Ferreira, Leite, Fonseca, & Costa, 2011a; Souza, Asfora, Lira, & Ast ua, 2010; Teta, Gonz alez- Fischer, Codesido, & Bilenca, 2010; Torre, Arrizabalaga, & Flaquer, 2004). Barn owls (Tyto alba), for example, are specialized predators that feed mainly on small mammals, and prey bone remains are regurgitated in the form of pel- lets (Bellocq, 2000). Given that the Barn owl is a cosmo- politan species (Sick, 1997), these pellets are often available in several different habitats, thus representing a low-cost, effective tool in detecting small mammals. *Correspondence to: Rita Gomes Rocha. E-mail: rgrocha@ua.pt ISSN 1477-2000 print / 1478-0933 online Ó The Trustees of the Natural History Museum, London 2015. All Rights Reserved. http://dx.doi.org/10.1080/14772000.2015.1044048 Systematics and Biodiversity (2015), 13(4): 403412 Downloaded by [Yuri Leite] at 13:28 21 May 2015