Beta-Tubulin

Genomic DNA was isolated from fungal mycelium grown on the agar plates following the protocol of Lee & Taylor (1990 ) or the UltraClean™ Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc., Solana Beach, CA, USA). All isolates were sequenced with five genomic loci. The primers ITS5 or ITS1 and ITS4 (White et al. 1990 ) were used to amplify the internal transcribed spacers areas as well as the 5.8S rRNA gene (ITS) of the nrDNA operon. Part of the actin gene (ACT) was amplified using the primer set ACT-512F and ACT-783R (Carbone & Kohn 1999 ) and part of the translation elongation factor 1-a gene (EF) using the primer set EF1-728F and EF1-986R (Carbone & Kohn 1999 ). The primer set CAL-228F and CAL-737R (Carbone & Kohn 1999 ) was used to amplify part of the calmodulin gene (CAL) whereas the primer set CylH3F and CylH3R (Crous et al. 2004c ) was used to amplify part of the histone H3 gene (HIS). Additional degenerate primers were developed from sequences obtained from GenBank as alternative forward and reverse primers for some of the loci during the course of the study (Table 2); however, these were rarely used but based on their degenerate design could be of use to the broader scientific community. The protocols and conditions outlined by Groenewald et al. (2005 (link)) were followed for standard amplification and subsequent sequencing of the loci.
Sequences of Septoria provencialis (isolate CPC 12226) were used as outgroup based on availability and phylogenetic relationship with Cercospora (Crous et al. 2004b , 2006b (link)). The Cercospora sequences were assembled and added to the outgroup sequences using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002 ), and manual adjustments for improvement were made by eye where necessary. Gaps present in the ingroup taxa and longer than 10 characters were coded as a single event for all analyses (see TreeBASE).
Neighbour-joining analyses using the HKY85 substitution model were applied to each data partition individually to check the stability and robustness of each species clade under each data set using PAUP v. 4.0b10 (Swofford 2003 ) (data not shown, discussed under the species notes where applicable). Alignment gaps were treated as missing data and all characters were unordered and of equal weight. Any ties were broken randomly when encountered. The robustness of the trees obtained was evaluated by 1 000 bootstrap replications (Hillis & Bull 1993 ).
MrModeltest v. 2.2 (Nylander 2004 ) was used to determine the best nucleotide substitution model settings for each data partition. Based on the results of the MrModeltest, a model-optimised phylogenetic re-construction was performed for the aligned combined data set to determine species relationships using MrBayes v. 3.2.0 (Ronquist & Huelsenbeck 2003 (link)). The heating parameter was set at 0.3 and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies came below 0.05. Trees were saved each 1 000 generations and the resulting phylogenetic tree was printed with Geneious v. 5.5.4 (Drummond et al. 2011 ). New sequences generated in this study were deposited in NCBI’s GenBank nucleotide database (www.ncbi.nlm.nih.gov; Table 1) and the alignment and phylogenetic tree in TreeBASE (www.treebase.org).
Isolates of Cercospora sp. Q were screened with five more loci to test whether additional loci could distinguish cryptic taxa within this species. This species was selected based on the intraspecific variation present in Fig. 2 (part 5) and also the range of host species and countries represented. The primer set GDF1 and GDR1 (Guerber et al. 2003 (link)) was used to amplify part of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, primer set NMS1 and NMS2 (Li et al. 1994 (link)) for part of the mitochondrial small subunit rRNA gene and part of the chitin synthase (CHS) gene was amplified using the primers CHS-79F and CHS-354R (Carbone & Kohn 1999 ). Part of the gene encoding for a mini-chromosome maintenance protein (MCM7) was amplified using primers Mcm7-709for, Mcm7-1348rev, Mcm7-1447rev (Schmitt et al. 2009 (link)) and part of the beta-tubulin gene using mainly the primers T1, Bt2b and TUB3Rd (see Table 2 for references).

Genomic DNA of the isolates was extracted using the method of Damm et al. (2008 (link)). The 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial sequences of the chitin synthase 1 (CHS-1), histone3 (HIS3), actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS-1F (Gardes & Bruns 1993 (link)) + ITS-4 (White et al. 1990 ) or V9G (de Hoog & Gerrits van den Ende 1998 (link)) + ITS-4, GDF1 + GDR1 (Guerber et al. 2003 (link)), CHS-354R + CHS-79F (Carbone & Kohn 1999 ), CYLH3F + CYLH3R (Crous et al. 2004b ), ACT-512F + ACT-783R (Carbone & Kohn 1999 ) and BT2Fd + BT4R (Woudenberg et al. 2009 (link)) or T1 (O’Donnell & Cigelnik 1997 (link)) + Bt-2b (Glass & Donaldson 1995 (link)), respectively. The PCRs were performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California) in a total volume of 12.5 μL. The GAPDH, CHS-1, HIS3, ACT and TUB2 PCR mixture contained 1 μL 20x diluted genomic DNA, 0.2 μM of each primer, 1x PCR buffer (Bioline, Luckenwalde, Germany), 2 mM MgCl₂, 20 μM of each dNTP, 0.7 μL DMSO and 0.25 U Taq DNA polymerase (Bioline). Conditions for PCR of these genes constituted an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C, and a final denaturation step of 7 min at 72 °C, while the ITS PCR was performed as described by Woudenberg et al. (2009 (link)). The DNA sequences generated with forward and reverse primers were used to obtain consensus sequences using Bionumerics v. 4.60 (Applied Maths, St-Marthens-Lathem, Belgium), and the alignment assembled and manually adjusted using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002 ).
To determine whether the six sequence datasets were congruent and combinable, tree topologies of 70 % reciprocal Neighbour-Joining bootstrap with Maximum Likelihood distances (10 000 replicates) with substitution models determined separately for each partition using MrModeltest v. 2.3 (Nylander 2004 ) were compared visually (Mason-Gamer & Kellogg 1996 ). A maximum parsimony analysis was performed on the multilocus alignment (ITS, GAPDH, CHS-1, HIS3, ACT, TUB2) as well as for each gene separately with PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2000 ) using the heuristic search option with 100 random sequence additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Alignment gaps were treated as missing and all characters were unordered and of equal weight. No more than 10 trees of score (length) greater than or equal to 10 were saved in each replicate. The robustness of the trees obtained was evaluated by 10 000 bootstrap replications using the Fast-stepwise addition algorithm (Hillis & Bull 1993 ). Tree length, consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the resulting tree. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v. 3.1.1 (Ronquist & Huelsenbeck 2003 (link)) for the combined sequence datasets. Models of nucleotide substitution for each gene determined by MrModeltest v. 2.3 were included for each gene partition. The analyses of two MCMC chains were run from random trees for 1000 000 generations and sampled every 100 generations. The likelihood score of the two runs were 2 500 and 2 200 and therefore, the first 2 350 (the average of both) trees were discarded as the burn-in phase of the analysis and posterior probabilities determined from the remaining trees. For additional comparison, a Neighbour-Joining analysis was performed on the multigene alignment using PAUP and 1000 bootstrap replications. Sequences derived in this study have been lodged at GenBank, the alignment in TreeBASE (www.treebase.org/treebase-web/home.html), and taxonomic novelties in MycoBank (Crous et al. 2004a ).

Single-cell RNA-sequencing data were obtained from the Complete Gene Expression Map of the C. elegans Nervous System (CeNGEN) [85 (link)]. Using the CeNGEN scRNA-seq dataset, gene expression for each of the genes of interest was extracted from the database with a threshold of 2 (Table S8). All expression values are in transcripts per million (TPM) [94 (link)]. All data collection, processing, normalization, and analysis of the CeNGEN data can be found at https://www.cengen.org/.