O. Merkl szerk.: Folia Entomologica Hungarica 68. (Budapest, 2007)
94 °C (30 sec), 48 °C (60 sec) and 68 °C (90 sec) and a final extension step at 68 °C (10 min). PCR was performed in an Eppendorf Mastercycler in 200 ul tubes. The sequence reactions were performed on an AB 3730XL sequencer at the University of Illinois, Chicago. Statistical analysis - The mitochondrial DNA sequence alignment was performed by Clustal X (THOMPSON et al. 1997) using default setting. Distance analysis was performed by the Neighbor Joining NJ algorithm (SAITU & NEI 1987, STUDIER & KEPPLER 1988) as it is implemented in the MEGA version 3 (KUMAR et al. 2004). The distance matrix was calculated based on the Tamura-Nei substitution model (TAMURA & NEI 1993), while the robustness of the topology was tested by bootstrapping with 1000 repetitions (FELSENSTEIN 1985, 1988). For comparison also the GenBank entry of Xylosandrus mancus (BLANDFORD, 1898) (submitted by NORMARK et al: AF 187143) were taken. RESULTS Morphological comparison - The collected individuals (all females) were compared to individuals deposited at the Coleoptera Collection (Hungarian Natural History Museum, Budapest). All comparison materials originated from Germany, collected between 1953 and 1974. Both the antennái and elytral morphology were similar. Genetical comparison - Five individuals from Hungary represent one haplotype (HT-1), while the three German individuals represent two haplotypes (HT1 and HT-2) on the 397bp fragment of the COI gene (Table 1 ). The outgroup X. mancus showed a sequence divergence of 20.91% to the X. germanus populations (Table 2, Fig. 1). The vast majority of sequence variation observed occur on third codon positions (65 sites, 78.31%) followed by first (13 sites, 15.67%) and second codon positions (5 sites, 6.02%). Both haplotypes found (HT1 and HT2) were submitted to the GenBank under the accession numbers HT-1 EF433438 and HT-2 EF433439. Table 1. Collection sites and haplotypes of investigated Xylosandrus germanus samples Individual/ Haplotype GenBank Country Area Abbreviation Haplotype identifier Hungary Nagymáté HU-1 HT-1 EF433438 HU-2 HT-1 HU-3 HT-1 HU-4 HT-1 HU-5 HT-1 Germany Baden-Württemberg DE-1 HT-1 DE-5 HT-2 EF433439 DE-6 HT-2
