Irwise ITS distances have been utilised as a measure of genetic distance
Irwise ITS distances were used as a measure of genetic distance between fungal haplotypes and significance was assessed by , random permutations.Because S.amabilis and T.sp.each cultivated a single distinct fungal haplotype, A and B respectively, the AMOVA was also performed just after excluding these two species and as a result only containing Trachymyrmex species cultivating extra than a single species of symbiont.To validate the AMOVA final results, we constructed a contingency table with columns representing ant species and rows fungal ITS haplotypes, and each cell containing the observed variety of antfungal combination, so that feasible patterns of specificity of randomness (independence) could be assessed with Fisher’s exact test as implemented in R .We performed the same two tests as within the AMOVAs by initial contemplating the entire data set and following that only the data for the four ant species (T.zeteki, T.sp T.cornetzi sp T.cornetzi sp and T.cornetzi sp) that cultivated far more than a single fungal haplotype.Enzyme activity measurementsUpon collection, the fungus gardens were instantly measured for enzyme activity ahead of any meals items wereDe Fine Licht and Boomsma BMC Evolutionary Biology , www.biomedcentral.comPage ofadministered.Visible ants, larvae, pupae and eggs had been removed ahead of total proteins were extracted by grinding mg fresh garden material using a sterile pestle in .mL Eppendorf tubes containing l mM Tris pH .Extracts were centrifuged at , g for min plus the supernatants containing crude total protein extracts have been right away utilized in enzyme activity assays.Enzyme activity was assayed with AzurineCrosslinked (AZCL) polysaccharides as previously described .Briefly, an agarose medium of agarose, mM phosphoric acid, mM acetic acid and mM boric acid was heated until the agarose was melted and then cooled to when .weightvolume AZCL substrate was added as well as the medium poured into Petri dishes.Wells had been created in the solidified agarose plates using a reduce off pipette tip to offer a continual diameter of mm ahead of l of enzyme supernatant was placed in every effectively.The plates have been incubated for hours at plus the area of your blue halo surrounding the effectively was photographed and measured applying the software plan ImageJ ver…AZCLpolysaccharides are hugely purified polysaccharides, that are dyed with azurineblue and crosslinked to kind a water insoluble chromogenic substrate assay (AZCL, Megazyme.Enzymes present in the protein extracts diffuse into the assay media and within the event of a optimistic reaction the hydrolysis of AZCLpolysaccharides releases dyed watersoluble fragments at a rate that is Indirubin-3-monoxime chemical information proportional to enzyme activity .Measuring the area of bluecoloration around the assay plates is therefore a quantitative measure of enzyme activity against the polysaccharide substrate used .AZCL plate assays don’t supply absolute enzyme activities and are much less sensitive than laboratorybased photometric assays standardized to protein content.On the other hand, field measurements have high reproducibility and are appropriate for largerscale comparisons of enzyme activity spectra with all-natural, instead of laboratory, substrates .We employed unique AZCLpolysaccharides PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21324549/ to test for enzyme activity that cleave the polysaccharide chain of stored starch and proteins inside the plant cells and also the pectins, celluloses and xylans related with all the plant cell walls (Table).Evaluation of variance (ANOVA) with kind of enzyme, cultivar haplotype, ant species, and their interaction.