Exuviae as a reliable source of DNA for population-genetic analysis of odonates

Genetic analyses are widely used for a variety of ecological research, especially to aid species' conservation programs. Where genetic material is required from rare endangered species it is essential that the samples be collected non-destructively, the ultimate goal should be to develop reliable DNA extraction protocols that may be used with non-invasively collected samples. In this paper, we describe and compare three methods of DNA extraction (DNeasy tissue kit, proteinaseK/TNES and Chelex-100) that use odonate (Coenagrion mercuriale) exuviae as a non-invasive source of genetic material. DNA extracted from exuviae produced consistent genotypes at five polymorphic microsatellite loci for all of the samples processed using the DNeasy tissue kit and proteinase-K/TNES methods and 4 out of the 6 exuviae treated with Chelex-100. Exuviae offer an effective source of genetic material from endangered odonates and also highly mobile species that are too difficult to catch in significant numbers. As such, we expect DNA extracted from exuviae to be widely applied to odonatological genetic research. INTRODUCTION DNA-based genetic analyses undoubtedly play an important role in many species' conservation plans (FRANKHAM et al., 2002; DESALLE & AMATO, 2004). Because of the inherent problems (i.e. time, expense and short-term bias) in directly tracking many species (SLATKIN, 1985), molecular markers offer a rapid and cost-effective method to indirectly estimate the extent of interpopulation dispersal and thus help define management units. With increasing degradation and fragmentation of freshwater habitats, it is perhaps not surprising that many odonate populations/species are under threat from extinction (CORBET, 1999). For odonates, a moleculargenetic appraisal of population connectivity and response to fragmentation would benefit the development of appropriate habitat-restoration/management plans. A reliable DNA extraction protocol is the foundation for any population-genetic investigation. For conservation purposes it is obviously essential that a non-destructive sampling regime is followed. Where possible, however, the ultimate goal for any conservation-genetic research should be the non-invasive isolation of genetic material. WATTS et al. (2001) and LODGE & FREELAND (2003) reported methods to obtain DNA from odonate legs. Although leg samples may be removed from odonates without measurably affecting their fitness (FINCKE & HADRYS, 2001; THOMPSON, unpublished data), this procedure is nonetheless invasive. In our experience, a rapid, large-scale genetic analysis of protected species can be hindered because of the need to acquire licences from several different government agencies. Rather than sampling adults directly, potential sources of genetic material are the shed exuviae that remain on aquatic vegetation after larval emergence. DNA from butterfly and honey bee 'exuviae' has been recovered by FEINSTEN (2004) and GREGORY & RINDERER (2004) respectively. The former study used a DNeasy Tissue Kit (Qiagen Inc.) to extract DNA while the latter authors followed a Chelex-100 protocol (see also WALSH et al., 1991). WATTS et al., (2001) and LODGE & FREELAND (2003) both employed Geneclean kits (Bio101) to extract DNA from odonate legs, although to reduce cost and increase sample throughput we now use a proteinase-K/TNES method (SAMBROOK & RUSSELL, 2001) to extract DNA from odonate leg samples. Here, we report the relative success of 3 simple methods DNeasy, Chelex-100 and proteinase-K/TNES of extracting DNA from odonate (Coenagrion mercuriale) exuviae.