Galemys Pyrenaicus) Is a Small Endangered Semi-Aquatic Mammal Endemic To
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Mammalian Biology 80 (2015) 505–509 Contents lists available at ScienceDirect Mammalian Biology jou rnal homepage: www.elsevier.com/locate/mambio Original Investigation A new method to identify the endangered Pyrenean desman (Galemys pyrenaicus) and to study its diet, using next generation sequencing from faeces a,b,c,∗ d d c Franc¸ ois Gillet , Marie-Laure Tiouchichine , Maxime Galan , Frédéric Blanc , c b a,e Mélanie Némoz , Stéphane Aulagnier , Johan R. Michaux a Department of Life Sciences, Conservation Genetics Unit, University of Liège, 4000 Liège, Belgium b Comportement et Ecologie de la Faune Sauvage (CEFS), Institut National de la Recherche Agronomique, 24 Chemin de Borde Rouge, Auzeville, CS 52627, 31326 Castanet-Tolosan Cedex, France c Conservatoire d’Espaces Naturels de Midi-Pyrénées, 75 voie du Toec, BP 57611, 31076 Toulouse Cedex 3, France d CBGP (Centre de Biologie et de Gestion des Populations), UMR INRA/IRD/Cirad/Montpellier SupAgro, Campus international de Baillarguet CS 30016, Montferrier-sur-Lez Cedex, France e CIRAD, Agirs Unit, TA C-22/E-Campus international de Baillarguet, 34398 Montpellier Cedex 5, France a r t i c l e i n f o a b s t r a c t Article history: The Pyrenean desman (Galemys pyrenaicus) is a small endangered semi-aquatic mammal endemic to Received 18 April 2015 the Pyrenean Mountains and to the northern half of the Iberian Peninsula whose ecology and biology Accepted 18 August 2015 are still poorly known. The aim of this study was to identify Pyrenean desman faeces and to analyze its Handled by Frank E. Zachos diet from this material using next-generation sequencing methods. We amplified and sequenced a small Available online 24 August 2015 DNA minibarcode (133 bp) of the COI gene in twenty-four putative faeces samples of Pyrenean desman and successfully identified the species in 16 samples. Other identified species were mammals, birds and Keywords: amphibians, evidencing the potential application of our methods to a larger panel of taxa. Diet Faeces In the Pyrenean desman faeces, we were able to identify nineteen prey species with a positive match (more than 98% of identity with a reference sequence) and eleven putative prey species with lower iden- Galemys pyrenaicus Identification tity scores (90–96%). The nineteen species belonged to four orders and eleven families among which Next-generation sequencing Trichoptera and Hydropsychidae were the most frequent, respectively. Future improvements could be obtained by extending the reference DNA sequence collection to reach precise identifications over the Desman’s range and by increasing the sampling to gain a better knowledge of the local diet of this endan- gered species. Such information is of great importance to propose the best management measures for its conservation. © 2015 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. Introduction inevitably impacting its distribution (Nores et al. 2007; Némoz and Bertrand 2008; Gisbert and Garciá-Perea 2014; Charbonnel 2015). The Pyrenean desman (Galemys pyrenaicus) is a small semi- The biology and ecology of this species are still poorly known, aquatic mammal endemic to the Pyrenean Mountains and to the notably because of its elusive behaviour and its primarily noctur- northern half of the Iberian Peninsula. It is endangered and is cur- nal activity (Stone 1987; Bertrand 1994). Its distribution area is not rently considered as vulnerable in the IUCN Red List (Fernandes even definitively established. However, this information is essential et al. 2008) and is legally protected in the four countries encompass- in order to develop the best management strategies for the species ing its distribution area (Andorra, France, Portugal and Spain). It has (Némoz and Bertrand, 2008). been suffering from habitat loss and fragmentation for decades, The nature of trophic interactions is a fundamental topic in ecol- ogy and has aroused the attention of biologists for decades. Diet analyses provide key data for understanding animal ecology, evo- lution and conservation (Shehzad et al. 2012). This knowledge is ∗ Corresponding author at: Department of Life Sciences, Conservation Genet- particularly important when a species is as endangered and elu- ics Unit, University of Liège, Quartier Vallée 1, Chemin de la Vallée 4, 4000 Liège, sive in nature as the Pyrenean desman. This species lives in cold Belgium. Tel.: +32 497210869. and well-oxygenated flowing waters of mountain rivers and is E-mail address: [email protected] (F. Gillet). http://dx.doi.org/10.1016/j.mambio.2015.08.002 1616-5047/© 2015 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. 506 F. Gillet et al. / Mammalian Biology 80 (2015) 505–509 Table 1 well adapted to the aquatic life. The Pyrenean desman is char- French localities and departments of the 24 faecal samples used in this study. acterized by large webbed hindfeet, a double layered fur, a long tail and a mobile prehensile snout, which make it a specialist in Sample Locality Department finding and feeding on the larvae of benthonic macroinvertebrates 1 Saurat Ariège (Palmeirim and Hoffmann 1983; Richard 1986). The diet of the 2 Saurat Ariège Pyrenean desman has been previously studied either by examining 3 Aulus-Les-Bains Ariège 4 Aulus-Les-Bains Ariège digestive tracts of trapped animals or by analyzing faeces sam- 5 Gourbit Ariège ples (Castién and Gosálbez 1995; Santamarina and Guitian 1988; 6 Gourbit Ariège Santamarina 1993; Trutat 1891; Richard and Vallette Vialard 1969; 7 Nescus Ariège Fernández-salvador et al. 1998; Bertrand 1994; Puisségur 1935). 8 Saint-Paul de Jarrat Ariège 9 Le Bosc Ariège While these methods have provided some useful information, they 10 Le Bosc Ariège are not without flaws. Firstly, the taxonomic resolution based on 11 Aleu Ariège these approaches cannot exceed the family rank (Bertrand 1994). 12 Sentenac de Sérou Ariège Secondly, as the Pyrenean desman is a vulnerable species, trapping 13 Sentenac de Sérou Ariège and killing animals is not appropriate and analyzing faeces is time- 14 Boussenac Ariège 15 Beille Ariège consuming and greatly relies on the experience of the identifier. 16 Beille Ariège Indeed, some mistakes can easily occur when identifying the host 17 Beille Ariège species especially when faeces are not fresh or have been in contact 18 Prats-de-Mollo-La-Preste Pyrénées-Orientales with water. A recent reliable and non-invasive molecular method 19 Audressein Ariège 20 Saint-Michel Pyrénées-Atlantiques was developed to easily distinguish faeces of the Pyrenean desman 21 Agnos Pyrénées-Atlantiques (Gillet et al. 2015) but the scope of targeted species was limited 22 Montségur Ariège to other semi aquatic and ecologically related vertebrate species 23 Ansignan Pyrénées-Orientales namely the white-throated dipper Cinclus cinclus and the water 24 Tramezaïgues Hautes-Pyrénées shrew Neomys fodiens. Therefore, it seems appropriate to propose a more precise and universal method that could clearly identify the et al. 2011): 5 -CCTCTCTATGGGCAGTCGGTGATNNNNNNNACTAT- Pyrenean desman and its prey, at a finer taxonomic resolution than AAAARAAAATYTDAYAAA DGCRTG-3 . The 5 parts of the primers the family rank. were modified by the addition of individual-specific MIDs (Mul- More recently, several diets based on faeces of a broad panel tiplex IDentifiers NNNNNNN), consisting in a short 7 bp sequence of mammal species encompassing herbivores, bats, carnivores, and adaptors required for the emPCR and the Ion Torrent sequenc- and rodents among others, have been successfully analyzed with ing. By using combinations of different forward and reverse MIDs molecular techniques using DNA barcoding (Soininen et al. 2009; sequences, several hundred samples can be multiplexed on the Zeale et al. 2011; Galan et al. 2012; Shehzad et al. 2012; Latinne same sequencing run, and the sequences can be recognized after et al. 2014). The different prey species and plant taxa contained in sequencing where all the PCR products from the different sam- the faeces are identified by amplifying small and highly variable ples are mixed together. In this study, the 24 faecal samples were DNA fragments with universal primers and by using them as bar- multiplexed with samples from other projects. PCR amplification codes. Among the different DNA regions that have been used for this was performed twice for each faecal sample and following the pro- purpose, the mitochondrial cytochrome oxydase 1 (COI) has been cedure detailed in Galan et al. (2012). Briefly, PCRs were carried widely used, notably in invertebrate studies (Meusnier et al. 2008; out in a 10 l reaction volume using 5 l of 2× QIAGEN Multiplex Lees et al. 2010; Virgilio et al. 2010). In addition, the international Kit (Qiagen, Hilden, Germany), 0.5 M of each primer, and 1 l of project Barcode of Life (www.barcoding.si.edu) aims to generate a DNA extract. The PCR conditions consisted in an initial denaturation complete species identification catalogue for all animal kingdom ◦ step at 95 C for 15 min, followed by 40 cycles of denaturation at organisms based on the COI gene. ◦ ◦ ◦ 94 C for 30 s, annealing at 45 C for 45 s, and extension at 72 C for As invertebrates, and particularly macroinvertebrates’ ben- ◦ 30 s, followed by a final extension step at 72 C for 10 min. After thonic larvae, represent the majority of the diet of the Pyrenean PCR pooling (4 l per strong PCR amplification products and 7 l desman, the aim of our study was to use a small COI minibarcode per lighter ones) and size selection by gel excision, the amplicon of 133 bp in order to assess the performance of a DNA barcoding libraries were sequenced by the company Genes Diffusion (Douai, technique, using next-generation sequencing, to (1) identify Pyre- France) on a Ion Torrent PGM system using a Ion 316 Chip version nean desman faeces, as this will greatly help future studies on this 2 (LifeTechnologies).