C at position 98 and T at position 253 were common characters in all the strains of P. coccineus (including MUCL 38420) and in
the Chinese strains of P. sanguineus (including CIRM-BRFM 542). C/G substitution at positions 152 and 206 was specific to the East Asian strains of Pycnoporus, and T/C substitution (at position 56) was specific to the Australian strains of Pycnoporus. The phylogenetic trees inferred from ITS1-5.8S-ITS2 and β-tubulin gene sequences (Figs 1 and 2) clearly differentiated the group of P. cinnabarinus strains from the group of P. puniceus strains (100% bootstrap support). The group of the P. coccineus strains from Australia (including strain MUCL 38420), the P. sanguineus strains from China (including CIRM-BRFM 542 of unknown origin) with the Japanese strain of P. coccineus, selleck chemicals and the strain of P. coccineus Palbociclib from the Solomon Islands (positioned alone), formed a well supported clade (84% bootstrap value with ITS). Due to the high similarity of their ITS sequences, the strains of P. sanguineus from Madagascar, Vietnam, New Caledonia, French Guiana and Venezuela could not be distinguished phylogenetically. β-Tubulin molecular data might be of slightly more help than ITS data to disclose genetic polymorphism within these P. sanguineus strains with two groups, although weakly supported (Fig. 2). In
this study, the functional lac3-1 gene, which protein products showed high variability in enzymatic activity between the species of Pycnoporus (Uzan et al., 2010), was targeted to infer the phylogenetic relationships within the genus Pycnoporus, out and especially within the P. sanguineus and P. coccineus species. PCR amplification resulted in laccase F2-R8 products of about 1640 bp. Comparison
between gene and predicted cDNA fragment sequences showed that the corresponding partial coding regions were interrupted by eight introns. A positional homology among these introns could be observed. It is noteworthy that the eight intron lengths were strictly similar for the East Asian strains of Pycnoporus on the one hand, and for the Australian strains on the other (data not shown). The nine exons corresponded to sequences of 1182 nucleotides. The 36 deduced partial proteins (corresponding to about 75–80% of the full length protein) displayed sequence similarity ranging from 87.6% to 99.7%. The 36 laccase sequences from Pycnoporus strains were aligned in 1185 nucleotide positions after hand-refining (see File S3). These regions of the laccase gene had 33% variable positions among the strains of Pycnoporus studied. Informative nucleotide site variations were localized in the conserved copper-binding domains, especially domains II and III with T/C substitution specific to the East Asian strains of Pycnoporus. Phylogenetic construction of our worldwide sample of Pycnoporus lac3-1 sequences led to distinct groups that were correlated with the geographic origin of the strains (Fig. 3).