N driver in this evolution, then a minimum of some distinct visual signals must be correlated, at a large phylogenetic scale, with an underlying chemical defense (see Figure 1).MethodsStudy group and taxon samplingTenthredinidae could be the biggest sawfly family with over 5,500 species described worldwide, covering all continents except Antarctica [54]. Most tenthredinid species are dietary specialists: larvae generally feed on a single or maybe a few related plant genera [55]. The majority on the hosts belong to di- and monocotyledonous angiosperms, but many species feed on gymnosperms, ferns, horsetails, and in some cases mosses [55]. Generally, tenthredinids have already been subdivided in seven, but extra recently within the six subfamilies Allantinae, Blennocampinae, Heterarthrinae, Nematinae, Selandriinae, and Tenthredininae [54]. The sawfly species utilized within this study comprise 106 tenthredinid species (Added file 1), with representatives from all subfamilies and 29 of their big tribes. We mostly focused on species for which information on chemical defense traits but no correct phylogenetic analyses had been offered, which is specially the case for the Phymatocerini, represented here by 22 exemplars. From ten non-tenthredinid sawfly households, 13 species have been integrated in as outgroups. Sawfly adults were identified following Benson [56] and Zhelochovtsev Zinovjev [57], and larvae with Lorenz Kraus [48]. Specimens have been stored in 100 ethanol at -20 or -80 , and vouchers are kept in the Royal Belgian Institute of Organic Sciences (JLB collection; Added file 1).DNA extraction, PCR amplification and sequencingTotal genomic DNA was extracted from legs or abdomens of adult sawflies or parts of larvae following a typical CTAB protocol. We amplified and sequenced two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome oxidase I (COI), along with the nuclear 28S ribosomal gene. Cytb was amplified (plus the PCR products sequenced in each directions) working with primers CB-J-10933 and CB-N-11367 [58]. For CoI, we utilised a modified version from the Simon et al. [58] primer C1-J-1718 (5′-GGA GGA TTT GGA AAT TGA TTA ATT CC-3′) in mixture using the reverse primer mod-A2590 (5′-ACT GCT CCT ATT GAT AAT ACA TAA TG-3′; GM, own design and style). For 28S, the primers 28SF2 (5′-CAC GAG CCG ATA GCG AAC AAG T-3′; GM, own design and style) and 28SB2 (5′-CCA AGG CCT CTA ATC ATT CGC T-3′; GM, own style) were applied. PCR reactions contained ten mM Tris Cl, 50 mM KCl, 1.five mM MgCl2, 50 MBoevet al. BMC Evolutionary Biology 2013, 13:198 http:www.biomedcentral.com1471-214813Page 4 ofof every single dNTP, 0.4 M of every single primer, and 0.026 unitsl of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21338362 Taq polymerase (Amersham Bioscience). The PCR programs consisted of an initial denaturation step at 94 for 4 min, followed by 30 cycles of 94 for 60s, annealing at 52 (Cytb and COI) or 54 (28S) for 60s, and extension at 72 for 2 min. The cycles had been followed by a final extension step at 72 for ten min. PCR solutions were purified making use of either alcohol precipitation or the Amersham Bioscience GFX PCR and Gel Band Purification Kit. Cytb was sequenced on an Amersham ALF express automatic sequencer employing Cy5 labelled primers along with the Amersham sequencing kit, and with an annealing temperature of 50 . COI and 28S merchandise were sequenced making use of ABI PRISM BigDye Terminator cycle sequencing kits in addition to a Perkin Elmer ABI sequencer at the Vlaams Instituut voor Biotechnologie in Antwerp, Belgium. Sequences had been assembled and checked making use of the base-calling (-)-Calyculin A web software program of your respective sequencers, and th.