1. Molecular identification of the forensically important greenbottle fly species Lucilia caesar and L. illustris (family Calliphoridae) Sonet, Gontran 1, Desmyter, Stijn 2, Braet, Yves 2, Jordaens, Kurt 1 1. Joint Experimental Molecular Unit – JEMU, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B1000 Brussels and Royal Museum for Central Africa, Leuvensesteenweg 13, B3080 Tervuren, Belgium; gontran.sonet@naturalsciences.begontran.sonet@naturalsciences.be 2. National Institute of Criminalistics and Criminology – NICC, Vilvoordsesteenweg 100, B1120 Brussels, Belgium INTRODUCTION: Some greenbottle flies (Calliphoridae: Lucilia; Fig. 1) colonise dead bodies and are useful in forensic entomology to estimate the post-mortem interval (PMI). Two of them, Lucilia caesar and L. illustris, are difficult to identify: - Only males can be identified with accuracy using the shape of the hypopygium (Fig. 2). - Sequence divergences between both species range from 0 to 2.9% for COI and from 0 to 5.1% for 16S. The latter value is above the range of intra- and even interspecific p-distances observed in the genus Lucilia (1.2-4.5%). OBJECTIVES: - To discriminate between both species using DNA sequences. MATERIAL & METHODS: - Sequencing 36 additional males of L. caesar and 24 of L. illustris for COI and, for a subset of samples, two other mitochondrial (16S and COII) and two nuclear markers (28S and ITS-2). - Material originated from Europe, an area where both species co-occur. - 95 homologous sequences from Canada, China, Europe, Korea, Singapore and USA were retrieved from GenBank and used for network analyses. Fig. 1 Greenbottle fly, Lucilia caesar. © Krzysztof Szpila, Nicolaus Copernicus University, Torun RESULTS AND DISCUSSION: For each marker, both species shared at least one haplotype/genotype and could therefore not be distinguished from each other (Fig. 3). The apparent differentiation between both species that has been reported previously is probably due to: - a too restricted sampling that did not include specimens with shared haplotypes and - species misidentifications for 16S since one divergent (5.1%) sequence (GQ396697) was identical to GenBank sequences of Chrysomya megacephala and the others were highly divergent from any other Lucilia species (>2.3%). In the forensic practice, distinguishing between both species is only useful if their biological characteristics are consistently different. The accumulation of data about the life cycle and the ecology of these two species will reveal the extent of phenotypic variation present in this group of Lucilia. Acknowledgments: The authors wish to thank the members of the team of DNA and Microtraces analysis of the NICC, J. Amendt, S. Swoboda, B. Vincent, J. Parrott, R. Wall, C. Richard, C. Rowlinson, K. Szpila, E. Pammeels and R. DeBry. More info: Sonet et al., Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic? Forensic Science International, DOI: 10.1016/j.forsciint.2012.08.020 Fig. 3 Haplotype network for L. illustris and L. caesar based on COI, 16S, COII, ITS-2 and 28S. White = L. illustris, grey = L. caesar. Geographical origins are given as follows: BE: Belgium, CA: Canada, CN: China, DE: Germany, FR: France, KR: Korea, PL: Poland, SG: Singapore, UK: United-Kingdom. Voucher numbers or GenBank accession numbers of peculiar samples are indicated. L. illustris L. caesar Fig. 2 Detail of hypopygium. © Krzysztof Szpila, Nicolaus Copernicus University in Torun