DNA barcoding of soldierless termites from South America: the Anoplotermes group (Termitidae) Genetic investigations were carried out by JEMU - Joint Experimental Molecular Unit supported by the Belgian Science Policy. Gontran Sonet 1, Zoltán Tamás Nagy 1, Joris Vermeulen 2, Aurélie Dery 3 & Thomas Bourguignon Royal Belgian Institute of Natural Sciences, rue Vautier 29, B-1000 Brussels, Belgium; 2 – Gezondheidszorg en Technologie, Katholieke Hogeschool, Leuven, Belgium; 3 – Sciences Biologiques, Université Libre de Bruxelles, Brussels, Belgium; presenting author's DNA barcoding can be a valuable and easy-to-use tool for taxonomists, especially when identifying specimens for which diagnostic morphological characters are not available (e.g. incomplete specimen, juvenile) or difficult to access (e.g. tiny individual, or internal anatomy). In the case of termites, taxonomy is traditionally based on morphological characters of soldiers and winged imagos. The “Anoplotermes group” (Termitidae) comprises soldierless termites and its taxonomy is mainly based on the worker’s morphology. This group is therefore a challenge for taxonomists and, although it represents ca. 30% of the Neotropical termite species, it is still in need of complete revision (Inward et al. 2007). Genus Morpho species Anoplotermes sensu stricto parvus banksi U AB AK Ø Anoplotermes sensu lato nigripunctatus T E§E§ M AO AD Ø AF Ø C§C§ AR Ø HØHØ IØIØ Y SØSØ AM § AS § K B Q L AV AN AP N AE Gen. nov. manni Grigiotermes metoecus Grigiotermes VØVØ Armitermes minutus Labiotermes labralis DNA barcoding approach DNA was extracted and the barcode region of the mitochondrial COI gene was amplified and sequenced. Sequences were edited, aligned by eye and a neighbour-joining tree was constructed based on K2P distances (see figure below). Here we tested the hypothesis that identification based on morphological characters is consistent with DNA barcoding. Conclusion The DNA barcoding approach of the analyzed specimens appears to be effective and consistent with the morphological analysis. The combination of both strategies is valuable to initiate the study of the Anoplotermes group. Indeed, due to the absence of soldiers, morphology alone can hardly define reliable diagnostic morphological characters. Similarly, DNA barcoding is inapplicable on its own since it lacks a reference library based on a well established taxonomy. Comparison between morphotypes and DNA barcodes 59 sequences ( bp) were obtained representing 24 morpho-species of the “Anoplotermes group” and two outgroups (A. minutus and L. labralis). 20 clusters of similar sequences (0-5% divergence) match unequivocally 20 morpho-species. One morpho-type (AS) is represented by two divergent sequences (10%) and three morpho-types (C, E and AM) share similar sequences. Technical aspects The success rate of initial experiments was surprisingly low (< 50% of PCR amplifications). This was not expected for samples collected recently ( ). A series of optimization allowed the improvement of the protocol. 1 - Sampling issue Extracting DNA from different collections; from isolated body parts of the individuals (legs, head and thorax or abdomen) or after pooling 2-10 specimens from the same colony showed that most recent collections preserved in 96% ethanol (2008 and 2009) provided the best success rates at downstream applications. 2 – Amplification optimisation Testing different PCR parameters (hot start taq, normal taq, touchdown PCR, primers, annealing temperatures, BSA and Mg 2+ concentration) led to the conclusion that additional BSA significantly increased the performance of the sequencing. Morphological analysis All 250 samples were classified into 34 morpho-species (see table below) based on the morphology of workers, particularly, the enteric valve and the mixed segment of the digestive tract. parvus (3, 3) AO (1, 1) metoecus (1, 1) nigripunctatus (1, 1) E (1, 1) T (1, 1) banksi (8, 8) AV (1, 1) Manni (1, 1) E (1, 1) C (1, 1) AM (1, 1) C (1, 1) E (3, 3) E (1, 1) K (4, 3) N (1, 1) AP (1, 1) B (1, 1) U (3, 3) AN (3, 2) AB (1, 1) AS (1, 1) Q (1, 1) AE (2, 2) M (7, 7) L (5, 5) Minutus (1, 1) labralis (1, 1) * * * * * * * * * * * Sampling 250 specimens collected in French Guiana were submitted to both morphological and genetic analysis. Labels on the tree are species names followed by: “(number of sequences, number of specimens represented)”. Ø No sequence obtained; § Equivocal barcode; * Bootstrap support is 99 %. Lines 1-4: coll. 70% eth Lines 5-9: coll. 70% eth Lines 10-13: coll. 96% eth Line 14: DNA size ladder Electropherograms (same position) of the same sample: without (A) or with (B) additional BSA in PCR. A. B. (A) Queen; (B) workers; (C) larvae; (D) alate of Anoplotermes sp. A. B. C. D. Petit Saut Kaw Mountain Nouragues Inward, D J G, Vogler A P, Eggleton P (2007). A comprehensive phylogenetic analysis of termites (Isoptera) illuminates key aspects of their evolutionary biology. Molecular Phylogenetics and Evolution 44,