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Abstract Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival. Graphic abstract

Globalization has facilitated the worldwide movement and introduction of pathogens, but epizoological reconstructions of these invasions are often hindered by limited sampling and insufficient genetic resolution among isolates. Pseudogymnoascus destructans , a fungal pathogen causing the epizootic of white-nose syndrome in North American bats, has exhibited few genetic polymorphisms in previous studies, presenting challenges for both epizoological tracking of the spread of this fungus and for determining its evolutionary history. We used single nucleotide polymorphisms (SNPs) from whole-genome sequencing and microsatellites to construct high-resolution phylogenies of P. destructans . Shallow genetic diversity and the lack of geographic structuring among North American isolates support a recent introduction followed by expansion via clonal reproduction across the epizootic zone. Moreover, the genetic relationships of isolates within North America suggest widespread mixing and long-distance movement of the fungus. Genetic diversity among isolates of P. destructans from Europe was substantially higher than in those from North America. However, genetic distance between the North American isolates and any given European isolate was similar to the distance between the individual European isolates. In contrast, the isolates we examined from Asia were highly divergent from both European and North American isolates. Although the definitive source for introduction of the North American population has not been conclusively identified, our data support the origin of the North American invasion by P. destructans from Europe rather than Asia. IMPORTANCE This phylogenetic study of the bat white-nose syndrome agent, P. destructans , uses genomics to elucidate evolutionary relationships among populations of the fungal pathogen to understand the epizoology of this biological invasion. We analyze hypervariable and abundant genetic characters (microsatellites and genomic SNPs, respectively) to reveal previously uncharacterized diversity among populations of the pathogen from North America and Eurasia. We present new evidence supporting recent introduction of the fungus to North America from a diverse Eurasian population, with limited increase in genetic variation in North America since that introduction.

A Cylindrocarpon species with up to 10 microm wide, straight and predominantly 3-septate macroconidia, subglobose to ovoidal microconidia and chlamydospores, is described as Cyl. pauciseptatum. It is most similar to Cyl. austrodestructans but no chlamydospores and microconidia are formed in the latter. Similar macroconidia also occur in Cyl. theobromicola, which forms oval to ellipsoidal microconidia at least sparsely and has slightly curved macroconidia, and Cyl. destructans var. crassum, which forms abundant 1-celled microconidia. DNA sequence data of the internal transcribed spacer regions 1 and 2 plus the 5.8S rDNA and the partial beta-tubulin gene were used for phylogenetic inferences. Cylindrocarpon pauciseptatum and Cyl. macrodidymum are monophyletic and are closely related to other species of Cylindrocarpon sensu stricto including members of the Cyl. destructans (teleomorph, Neonectria radicicola) species complex, which accommodates Cyl. liriodendri (teleomorph, Neon. liriodendri), Cyl. destructans var. crassum and Cyl. austrodestructans (teleomorph, Neonectria austroradicicola comb. nov.). Cylindrocarpon theobromicola is distantly related to species of Cylindrocarpon sensu stricto or Neonectria sensu stricto. It clustered among cylindrocarpon-like species with curved macroconidia, of which some belong to the Neon. mammoidea group. Relatively voluminous cells in sporodochial conidiophores of Cyl. theobromicola resembled those described for Campylocarpon, which is closely related to members of the Neon. mammoidea group including Cyl. theobromicola. Cylindrocarpon pauciseptatum has been isolated from roots of Vitis spp. in South-eastern Europe (Slovenia) as well as New Zealand, where it also occurs on roots of Erica melanthera.

Four Cylindrocarpon or Cylindrocarpon-like taxa isolated from asymptomatic or diseased Vitis vinifera plants in nurseries and vineyards of South Africa, New Zealand, Australia, and France were morphologically and phylogenetically compared with other Neonectria/Cylindrocarpon taxa. Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial -tubulin gene introns and exons were used for phylogenetic inference. Neonectria/ Cylindrocarpon species clustered in mainly three groups. One monophyletic group consisted of three subclades comprising (i) members of the Neonectria radicicola/Cylindrocarpon destructans complex, which contained strains isolated from grapevines in South Africa, New Zealand, and France; (ii) a Neonectria/Cylindrocarpon species isolated from grapevines in South Africa, Canada (Ontario), Australia (Tasmania), and New Zealand, described here as Cylindrocarpon macrodidymum; and (iii) an assemblage of species closely related to strains identified as Cylindrocarpon cylindroides, the type species of Cylindrocarpon. This monophyletic group excluded two other groups, which comprised (i) members of the Neonectria mammoidea complex, with anamorphs characterised by curved macroconidia, violet or purple pigments in cultures of most of its members, and lack of microconidia and chlamydospores; and (ii) two undescribed Cylindrocarpon-like species, both from grapevines in South Africa. The latter two clades formed a paraphyletic group in LSU rDNA analysis but were supported as a monophyletic group in ITS and -tubulin gene analysis. Strains of the Neonectria radicicola/Cylindrocarpon destructans complex isolated from grapevines matched C. destructans in morphology and DNA sequences. Cylindrocarpon macrodidymum formed micro- and macroconidia, but rarely formed chlamydospores. Its mostly 3-septate macroconidia were more or less straight, minutely widening towards the tip, and had an apical cell slightly bent to one side. Its teleomorph, Neonectria macrodidyma, was obtained in mating experiments, and was characterised by smooth to finely warted ascospores, smooth to finely warted perithecia, and moderately sized angular to subglobose cells in the outer region of the perithecial wall. The other two undescribed Cylindrocarpon-like species mentioned above were characterised by mostly 3-5-septate, curved macroconidia, and by the lack of microconidia. Both species differed from members of the Neonectria mammoidea group by brownish colonies and by brownish hyphal strands formed in the aerial mycelium. For these species a new genus, Campylocarpon gen. nov., is proposed. It comprises the new species Campylocarpon fasciculare and Campylocarpon pseudofasciculare, respectively. Inoculation of 6-mo-old potted grapevine rootstocks (cv. Ramsey) with selected isolates of Cylindrocarpon destructans, Neonectria macrodidyma, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare resulted in a reduced root and shoot mass of inoculated plants and appearance of symptoms typical of black foot disease.