Computational protocol: Knoxdaviesia proteae is not the only Knoxdaviesia-symbiont of Protea repens

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Protocol publication

[…] During November 2012 and January 2013, infructescences were sampled from two Protea repens populations in the Western Cape Province of South Africa () in order to isolate K. proteae individuals as part of a previous study (, ). In the Gouritz population (34.2062°S 21.6812°E), 220 infructescences from the current and 220 from the previous flowering seasons were sampled from 11 different P. repens trees (). The site at Franschhoek (33.9044°S 19.1566°E) had been burnt in 2007, and was sampled just after the new P. repens recruits had flowered for the first time. Some P. repens trees at this site (ca 15-yr-old) had escaped the fire and were included in our surveys. At this site, 20 infructescences were collected from 11 plots (20 m diam) in the burnt area and 19 plots in the unburnt area (). Since the initial aim of the sampling was to collect K. proteae, only one Knoxdaviesia isolate was maintained per infructescence to prevent repeated isolation of the same individual. Because the sexual morphs of both K proteae and K. capensis species are indistinguishable under ×30 magnification (), fungal isolations were made from randomly selected sporing structures. Knoxdaviesia isolation methods and culturing techniques were as given in . Isolates were identified by sequencing the ITS regions of the rDNA () as detailed by .Statistical analyses were conducted in R v. 3.1.0 (). The number of fungal isolates obtained from infructescences at each sampling site (Gouritz or Franschhoek) and for each subdivision (flowering season or burnt/unburnt area) was recorded and tested for normality with Shapiro-Wilk’s W test. Subsequently, a Mann-Whitney U test for independent groups and a Pearson’s Chi-square test was applied to test for significant differences between the numbers of isolates obtained from each infructescence age class (Gouritz population) and between the burnt and unburnt sampling plots (Franschhoek). These tests were chosen because the Mann-Whitney U test takes into account only the number of positive hits (i.e. the presence of the fungus) whereas the Chi-square test also includes the total number of observations (i.e. number of infructescences sampled) ().A Maximum Likelihood (ML) phylogenetic tree was constructed in order to illustrate the difference between the species identified in this study. MAFFT 7 () was used to align the ITS sequences of a subset of the isolated individuals to those of previously characterized species of Gondwanamycetaceae obtained from GenBank®. The ML tree was computed in MEGA6 () under the Tamura-Nei substitution model () and reliability was calculated with 1 000 bootstrap replications. […]

Pipeline specifications

Software tools MAFFT, MEGA
Application Phylogenetics