Rust fungi are obligate parasites of plants with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry-spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilised mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.

Partially nested hierarchical sampling

Location level: Picea abies cones with aecia were collected from 7 different locations in Sweden, Norway, and Finland. At each location 30 cones were collected, from which one scale with aecia per cone and one aecium per scale were randomly sampled. At tree level: a more extensive sampling was made at the seed orchard in Ålbrunna (Sweden), where 100 cones with aecia were sampled from 6 different trees at a distance of ca. 20 m to 600 m from each other. From each cone, one scale with aecia and one aecium per scale were randomly sampled. At cone level: 10 cones with aecia were randomly sampled from two locations in Sweden (a-1 and a-4) and split longitudinally to select 10 scales across each cone, from which 10 aecia per scale were randomly sampled (100 aecia per cone). At scale level: 3 individual scales with aecia from cones from two different locations in Sweden (a-1 and a-4) were thoroughly sampled (ca. 40 aecia per scale).

Single aecium genotyping

DNA was extracted from each aecium following the protocol of Capador et al., (2018). The samples were genotyped with 8 polymorphic microsatellites markers; Tha9, Tha61, Tha91, Tha92, Tha96, Tha105, Tha136, and Tha137, microsatellite amplification and scoring was performed as formerly described (Capador et al., 2018) except for a subset of samples which were analysed with DreamTaq (ThermoFisher) instead of PIR00 (Sigma).

The dataset is in Genalex format