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High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

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Bus M. M. , Zmihorski M. , Romanowski J. , Balciauskiene L. , Cichocki J. , Balciauskas L.

tom 59

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Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

Hill forest dweller: a new cryptic species of Rhinolophus in the ‘pusillus Group’ (Chiroptera: Rhinolophidae) from Thailand and Lao PDR

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Soisook P. , Karapan S. , Srikrachang M. , Dejtaradol A. , Nualcharoen K. , Bumrungsri S. , Lin Oo S. S. , Aung M. M. , Bates P. J. J. , Harutyunyan M. , Bus M. M. , Bogdanowicz W.

Acta Chiropterologica

2016

tom 18

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A new species of Rhinolophus in the pusillus group is described from Ratchaburi, Kamphaeng Phet and Loei Provinces where it was found in evergreen forest at elevations ranging from 550 to 1,320 m a.s.l. It is distinguished from R. shortridgei and other similar species in the same group by its broad, parallel-sided sella, which is squared-off at the tip, relatively large body size with a forearm length of 42.2–44.1 mm, and bulbous rostral swellings. The echolocation frequency from hand-held individuals is 84.1–93.0 kHz. Bayesian analyses of a 654 bp of cytochrome oxidase subunit I (DNA barcode), and an 878 bp fragment of cytochrome b also support differences at the species level. Three specimens from Loaung Namtha, Lao PDR are referred to this new species based on DNA barcodes. Based on distinctive DNA barcodes and craniodental morphology, the taxon refulgens, is here regarded as a separate species from R. lepidus. Morphological comparisons between similar species are discussed and notes on ecology included.