N driver within this evolution, then at least some specific visual signals need to be correlated, at a sizable phylogenetic scale, with an underlying chemical defense (see Figure 1).MethodsStudy group and taxon samplingTenthredinidae could be the biggest sawfly family with more than five,500 species described worldwide, covering all continents except Antarctica [54]. Most tenthredinid species are dietary specialists: larvae normally feed on one particular or maybe a few associated plant genera [55]. The majority of your hosts belong to di- and monocotyledonous angiosperms, but numerous species feed on gymnosperms, ferns, horsetails, and in some cases mosses [55]. Normally, tenthredinids have been subdivided in seven, but far more lately in the six subfamilies Allantinae, Blennocampinae, Heterarthrinae, Nematinae, Selandriinae, and Tenthredininae [54]. The sawfly species applied in this study comprise 106 tenthredinid species (Added file 1), with representatives from all subfamilies and 29 of their important tribes. We mainly focused on species for which information on chemical defense traits but no Bay 59-3074 accurate phylogenetic analyses have been accessible, which is specially the case for the Phymatocerini, represented here by 22 exemplars. From ten non-tenthredinid sawfly families, 13 species had been integrated in as outgroups. Sawfly adults have been identified following Benson [56] and Zhelochovtsev Zinovjev [57], and larvae with Lorenz Kraus [48]. Specimens were stored in one hundred ethanol at -20 or -80 , and vouchers are kept at the Royal Belgian Institute of All-natural Sciences (JLB collection; More file 1).DNA extraction, PCR amplification and sequencingTotal genomic DNA was extracted from legs or abdomens of adult sawflies or components of larvae following a normal CTAB protocol. We amplified and sequenced two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome oxidase I (COI), as well as the nuclear 28S ribosomal gene. Cytb was amplified (and the PCR items 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 combination using the reverse primer mod-A2590 (5′-ACT GCT CCT ATT GAT AAT ACA TAA TG-3′; GM, own style). For 28S, the primers 28SF2 (5′-CAC GAG CCG ATA GCG AAC AAG T-3′; GM, personal design) and 28SB2 (5′-CCA AGG CCT CTA ATC ATT CGC T-3′; GM, own design and style) have been made use of. PCR reactions contained 10 mM Tris Cl, 50 mM KCl, 1.5 mM MgCl2, 50 MBoevet al. BMC Evolutionary Biology 2013, 13:198 http:www.biomedcentral.com1471-214813Page 4 ofof each dNTP, 0.four M of each primer, and 0.026 unitsl of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21338362 Taq polymerase (Amersham Bioscience). The PCR applications consisted of an initial denaturation step at 94 for four 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 were followed by a final extension step at 72 for ten min. PCR products were purified employing either alcohol precipitation or the Amersham Bioscience GFX PCR and Gel Band Purification Kit. Cytb was sequenced on an Amersham ALF express automatic sequencer making use of Cy5 labelled primers and the Amersham sequencing kit, and with an annealing temperature of 50 . COI and 28S items have been sequenced applying ABI PRISM BigDye Terminator cycle sequencing kits as well as a Perkin Elmer ABI sequencer in the Vlaams Instituut voor Biotechnologie in Antwerp, Belgium. Sequences have been assembled and checked making use of the base-calling application in the respective sequencers, and th.