Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach

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 samples 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 subgenus and is especially useful for fast species identification from degraded samples.

Recognition and identification of bumblebee species in the Bombus lucorum-complex (Hymenoptera, Apidae) – A review and outlook

“…Therefore Murray et al (2008) provided a protocol which was successfully applied by Waters et al (2011). An enhanced version was published recently (Vesterlund et al 2014). This more time consuming approach works well with smaller COI fragments and hence is better suited for degraded DNA.…”

“…Bertsch et al (2004) carefully outlined the distribution of the species. Additional data comes from the recent COIbased studies (Murray et al 2008, Anagnostopoulos 2009, Bertsch 2009, Waters et al 2011, Vesterlund et al 2014 and from the distribution maps on bumblebees in the Atlas Hymenoptera project (Rasmont and Pauly 2010, Rasmont and Iserbyt 2010-2013. In these works the distribution, especially of B. cryptarum and B. magnus, appears fragmentary.…”

Recognition and identification of species in theBombus lucorum-complex – A review and outlook

“…Therefore Murray et al (2008) provided a protocol which was successfully applied in Waters et al (2011). An enhanced version was published recently (Vesterlund et al 2014). This more time consuming approach works well with smaller COI fragments and hence is better suited for degraded DNA.…”

“…Bertsch et al (2004) carefully outlined the distribution of the species. Additional data comes from the recent COI-based studies (Murray et al 2008, Anagnostopoulos 2009, Bertsch 2009, Waters et al 2011, Vesterlund et al 2014 and from the distribution maps on bumblebees in the Atlas Hymenoptera project (Rasmont and Pauly 2010, Rasmont and Iserbyt 2010-2013. In these works the distribution, especially of B. cryptarum and B. magnus, appears fragmentary.…”

Status and monitoring of the buff-tailed bumblebee <i>Bombus terrestris</i> Linnaeus (Hymenoptera: Apidae) in Southern Finland

“…The first B. terrestris was found in year 1993 from Southern Finland (Pekkarinen & Kaarnama 1994). This study reports new observations using transect line trapping data and some sporadic samples that were identified using a modified polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP)-method for degraded DNA (Vesterlund et al 2014). Appropriate trapping methods for monitoring B. terrestris are also discussed.…”