Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach S.-R.VESTERLUND,* J. SORVARI† and A. VASEM € AGI‡§ *Department of Biology, Section of Ecology, University of Turku, Turku FI-20014, Finland, †Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, Kuopio FI-70211, Finland, ‡Department of Biology, Division of Genetics and Physiology, University of Turku, Turku FI-20014, Finland, §Department of Aquaculture, Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, Tartu 51014, Estonia Abstract The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high-quality DNA which makes them less suitable for analysis of low-quality or older samples. We modified the PCR–RFLP protocol for an efficient and cost-effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of sam- ples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subge- nus and is especially useful for fast species identification from degraded samples. Keywords: Bombus, COI, conservation, degraded DNA, PCR–RFLP, species identification Received 22 November 2012; revision received 2 September 2013; accepted 6 September 2013 Introduction Bumblebees (Bombus spp) are important pollinators of natural flowering plants and they are also widely used to pollinate various commercial crops (Goulson 2010). The recent decline of the bumblebees and local extinc- tions in several species worldwide (Fitzpatrick et al. 2007; Goulson 2008) have raised a need for precise iden- tification tools for conservation and management. This is especially important for cryptic taxa, such as the B. luco- rum complex (B. lucorum, B. magnus and B. cryptarum) of the Holarctic subgenus Bombus sensu stricto (s. str.), as it is extremely hard to reliably identify species within this group by using only morphological characters such as coloration (Carolan et al. 2012). As one particular species of the subgenus, B. terrestris, is widely used for pollina- tion, it is very important to be able to distinguish the commercially used species from closely related species such as those belonging to the B. lucorum complex. This is especially relevant in areas where B. terrestris does not naturally occur, for example in most of Finland (Pekkarinen & Kaarnama 1994) and the northern parts of Sweden, as commercial strains of B. terrestris can out-compete native species (Chittka et al. 2004; Ings et al. 2006) and transmit pathogens to local pollinators (Goka et al. 2001; Colla et al. 2006; Murray et al. 2013). Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based methods are rapid, cheap and reliable tools to identify insect species when morphological characters are not sufficient or the sam- ples are of bad quality. Murray et al. (2008) developed recently a PCR–RFLP method for distinguishing four bumblebee species (B. lucorum, B. terrestris, B. magnus and B. cryptarum) in cryptic European taxa of the subge- nus Bombus s. str. Their approach uses a partial mito- chondrial COI sequence (1064 bp) and primers originally developed for Apis mellifera. However, this method is not optimal for degraded DNA because then the amplifi- cation of long DNA fragments often fails. Degraded DNA is often gained when the samples are very old or otherwise damaged, for example, by suboptimal storage conditions, such as when using various trapping mate- rial, where the samples may have been in salt water and detergent for several weeks. Correspondence: Salla-Riikka Vesterlund, Fax: +358-2-3336550; E-mail: salla.vesterlund@utu.fi © 2013 John Wiley & Sons Ltd Molecular Ecology Resources (2013) doi: 10.1111/1755-0998.12168