Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex

Ceratocystis polonica and C. laricicola are two morphologically similar species that occur on conifers and reside in the Ceratocystis coerulescens species complex. They, however, represent two ecologically distinct entities. C. polonica causes blue stain on Norway spruce (Picea abies) and other spruce species (Picea spp.) in Eurasia and is associated with the bark beetles Ips typographus, I. typographus japonicus, I. amitinus and I. duplicatus. In contrast, C. laricicola lives in a symbiotic relationship with the bark beetles Ips cembrae and I. subelongatus that infest various larch species (Larix spp.). The objective of this study was to consider the phylogenetic relationships of C. polonica and C. laricicola and more specifically to determine the identity of Japanese isolates from both spruce and larch, based on sequences derived from the ITS regions of the rRNA operon, the β-tubulin gene and the HMG box of the MAT-2 gene. Isolates were also compared based on morphology and cultural characteristics. Comparisons of anamorph and teleomorph structures confirmed that C. polonica and C. laricicola are indistinguishable based on morphology. Both species had an optimal growth temperature of 25 °C. However, at temperatures between 31-33°, C. polonica isolates grew slowly or not at all, while C. laricicola isolates grew more actively at these temperatures. Thus, a growth test at 32° can differentiate these species. Phylograms generated using parsimony for the three gene regions were strongly congruent. These showed three distinct clades supported by high bootstrap values. Two of the clades clearly separate C. laricicola from Europe and C. polonica supporting the view that they represent two discrete taxa. A third clade included isolates obtained from galleries of Ips subelongatus on Larix kaempferi in Japan. This fungus clearly represents a discrete taxon that is closely related to, but distinct from C. laricicola which is described here as C.fujiensis sp. nov.