Computational protocol: Convergent evolution of highly reduced fruiting bodies in Pezizomycotina suggests key adaptations to the bee habitat

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Protocol publication

[…] For two strains of Eremascus fertilis and two strains of Bettsia alvei (Table ), genomic DNA was obtained by picking up ascocarps and mycelium from isolate cultures and grinding them inside a 1.5 ml Eppendorf tube. DNA was then isolated using the Qiagen DNeasy Plant Mini Kit (Hilden, Germany) using the standard protocol and eluted in two separate 50 μl fractions to avoid over-dilution.The LSU and SSU regions were each amplified by PCR. Primers LR0R and LR7 [] were used to amplify 1.4 kb of LSU, and primers NS1 and NS4 [] were used to amplify 1.1 kb of SSU. PCR reactions were prepared in 50 μl volumes containing 29.8 μl of sterile deionized water, 5 μl of Taq polymerase reaction buffer (Sigma®), 1.0 μl 10 mM dNTP, 3.0 μl 25 mM MgCl2, 0.2 μl Taq DNA polymerase (Sigma®), 5.0 μl each 10 μM primer and 1 μl of genomic DNA template. PCR was performed on a Biometra® thermocycler (Whatman) under the following conditions: for LSU: step 1) 1 min at 95 °C, 2) 1 min at 94 °C, 3) 30 sec at 51 °C, 4) 1 min at 72 °C, 5) return to step 2 34 times, 6) final step of 10 min at 72 °C; and for SSU: step 1) 1 min at 95 °C, 2) 1 min at 94 °C, 3) 30 sec at 51 °C, 4) 1 min at 72 °C, 5) 1 min at 94 °C, 6) 30 sec at 53 °C, 7) 65 sec at 72 °C, 8) return to step 2 34 times, 9) final step of 10 min at 72 °C. Samples were kept at 4 °C until electrophoresis was performed on 1 % agarose TAE gels and visualized with EZvision One® (Amresco). PCR products were cleaned using the Qiaquick® PCR purification kit (Qiagen) and were sent to Eurofins MWG Operon AG (Ebersberg, Germany) for sequencing. In addition to the amplification primers, LSU was sequenced with primers LR3R and LR5 [], and SSU with primers NS2 [] and SR7R (http://sites.biology.duke.edu/fungi/mycolab/primers.htm). Sequences were assembled using BioEdit [].For each region, a data matrix that included sequences from two isolates of Eremascus fertilis, two isolates of Bettsia alvei, and 63 other ascomycetes (Table ) was assembled and manually aligned in MEGA5 []. Taxon sampling was focused on genera with reduced fruiting bodies. Many of the sequences came from James et al. [] and were downloaded from the AFToL website (aftol.org), while those from other studies [, ] came from GenBank. The LSU and SSU datasets were exported in NEXUS format and were combined in a single data file in PAUP* v. 4.0.10b []. The combined file was deposited in the Dryad Digital Repository, and can be accessed at http://dx.doi.org/http://dx.doi.org/10.5061/dryad.6s80j. This file was analyzed by maximum parsimony in PAUP*: using a random addition sequence and TBR swapping, 1000 heuristic replicates were performed, saving no more than ten best trees per replicate, followed by a final search of the saved trees. The file was also bootstrapped (2000 replicates) using the same search parameters except that only 10 heuristic replicates were performed per bootstrap replicate. A Bayesian analysis of the combined file was also performed using the program MrBayes v. 3.2 []. Based on the Akaike Information Criterion, the GTR + I + Γ model of DNA sequence evolution was selected as the best-fit model using the program Modeltest v. 3.06 []. A Markov chain Monte Carlo (MCMC) analysis was then run for 2,000,000 generations under the default settings, which was twice the number needed to keep the standard deviation of split frequencies below 0.01. Following Schoch et al. [], Saccharomycotina was used as the outgroup for Pezizomycotina. Published single and multi-gene phylogenies [, , ] were followed in naming the major clades shown in Fig. .Fig. 3 […]

Pipeline specifications

Software tools BioEdit, MEGA, MrBayes, ModelTest-NG
Application Phylogenetics
Organisms Apis mellifera