Abstract
Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.
Highlights
Because species are the basic unit of many fields in biology [1,2], accurate species identification is an absolute prerequisite for studies focusing on agronomy [3,4] human health [5], conservation biology [2,6], ecology and evolution [7]
GenBank searches using the keyword ‘cytb’ yielded 15,121 sequences corresponding to 1,476 rodent species, whereas ‘c oxidase I (COI)’ retrieved only 2,857 sequences corresponding to 503 rodent species
Only 42% of these COI sequences were identified at the genus or species level, the remaining sequences, labeled as ‘Rodentia sp.’, were not included for assessment
Summary
Because species are the basic unit of many fields in biology [1,2], accurate species identification is an absolute prerequisite for studies focusing on agronomy [3,4] human health [5], conservation biology [2,6], ecology and evolution [7]. Disease reservoirs or endangered species are considered, fast and accurate species identification is required for an ever increasing number of animal and plant samples [8]. Classical approaches to identification have traditionally been based on morphological criteria and/or morphometric analyses, often requiring the input of taxonomic experts. Complicating the issue further, precise species identification based solely on morphological criteria can be extremely complex. Identification based on morphology alone is often impossible either due to poorly preserved specimens, or to the difficulties associated with identifying non-invasive samples such as feces, bones in bird’s pellets, shed skin, etc., and to incomplete or degraded museum specimens. Illegally traded products from endangered species are often processed to such an extent that they are useless for forensic investigations based on morphological criteria [8][14]
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