Computational protocol: Species-specific PCR to describe local-scale distributions of four cryptic species in the Penicillium chrysogenum complex☆

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Protocol publication

[…] Eleven representative Penicillium spp. isolates and type isolates, including some not previously present in the analysis by , were sequenced for each of the following four loci; benA, trpC, crt1 and ITS. These isolates included P. chrysogenum isolate strains NRRL-792 (P. rubens), NRRL-834, NRRL-841, CBS-118871, CBS-101347, CBS-306.48 and CBS-302.67, and additionally, closely related ancestors of the Chrysogenum complex: Penicillium confertum (CBS-171.87), Penicillium mononematosum (CBS-172.87) and P. flavigenum (CBS-419.89 and CBS-110409). Alignments for the four loci were produced in MEGA 5 () for a total of 217 isolate DNA sequences including the 11 sequenced isolates and a previous set of P. chrysogenum sequences from (n = 206, available from GenBank). Neighbour-joining phylogenetic trees based on maximum likelihood distances for each locus were produced and isolates were assigned to the four species as described by . These species assignments were used for the species phylogeny technique developed by and applied using *BEAST and Beauti, parts of the BEAST package (). The *BEAST technique allows co-estimation of species phylogeny and population parameters using a mixture of coalescent and Yule processes (). Thirty-seven isolates were selected for BEAST analysis, including the 11 newly sequenced isolates and 26 isolates from . These isolates, sequenced for the four loci, were loaded into Beauti and used to generate an XML file of 1 000 000 generations for preliminary test runs. Using Tracer v1.5 to assess the runs, the XML was optimised in Beauti for a final run of 100 000 000 generations with parameters logged every 1000 generations. The HKY substitution model with empirical bases and invariant sites was implemented. To calculate divergence dates from the node heights, a molecular clock with fixed mean substitution rate of 3 × 10−9 per yr was used, as previously described for fungi of the class Eurotiomycetes (). TreeAnotator was used to generate the maximum clade credibility tree after discarding the first 10 % of generations as burn-in and Tracer v1.5 was used to assess convergence. […]

Pipeline specifications

Software tools MEGA, BEAST
Application Phylogenetics
Organisms Penicillium chrysogenum, Penicillium rubens
Chemicals Penicillins