Despite only emerging in the past decade, white-nose syndrome has become among the most devastating wildlife diseases known. The pathogenic fungus Pseudogymnoascus destructans infects hibernating bats and typically leads to high rates of mortality at hibernacula during winter in North America. We developed a set of genetic markers to better differentiate P. destructans isolates. We designed and successfully characterized these 23 microsatellite markers of P. destructans for use in disease ecology and epidemiology research. We validated these loci with DNA extracted from a collection of P. destructans isolates from the US and Canada, as well as from Europe (the likely introduction source based on currently available data). Genetic diversity calculated for each locus and for the multilocus panel as a whole indicates sufficient allelic diversity to differentiate among and between samples from both Europe and North America. Indices of genetic diversity indicate a loss of allelic diversity that is consistent with the recent introduction and rapid spread of an emerging pathogen.

White-nose syndrome (WNS) in bats is one of the leading conservation crises in North America. Caused by the fungus Pseudogymnoascus (Geomyces) destructans, this disease has decimated bat populations throughout the eastern US and Canada, with recent spread into the Midwest and Pacific Northwest (Lorch et al. 2016). The fungus affects hibernating bats by invading their skin during hibernation, causing a suite of physiological changes that often result in mortality (Verant et al. 2014). The disease differentially affects various bat species, with some, such as the little brown bat (Myotis lucifugus), experiencing up to 99% declines in some hibernacula with regional extinctions possible within several decades (Frick et al. 2010, 2015). Previous genetic studies using relatively few markers have revealed little variation in the fungus in North America and have found limited differentiation in European samples (Ren et al. 2012; Khankhet et al. 2014; Leopardi et al. 2015). Genetic markers that provide sufficient resolution to precisely identify the source population (Puechmaille et al. 2011; Leopardi et al. 2015) of P. destructans or track the spread of the fungus in the North American epizootic zone, are absent. We therefore sought to develop a set of microsatellite markers with sufficient polymorphisms to genotype samples for epidemiological studies of WNS.

Microsatellites 2–6 base pairs (bp) in length were identified in the whole genome sequence of the type isolate P. destructans ATCC MYA-4855 (GenBank accession nos. GL573169–GL575015), excluding mitochondrial DNA, with msatcommander 1.0.8 beta software (Faircloth 2008). We tested the msatcommander output of 127 primer pairs for microsatellites with length polymorphisms among a panel of haploid DNA samples extracted from pure P. destructans colonies from culture medium inoculated with bat skin swabs from throughout the North American WNS epizootic zone and from Europe (Supplementary Table S1). Forward primers were fluorescently labeled for visualization with capillary electrophoresis. The PCR reactions were 25 μL in volume and contained 1.2 units Platinum Taq DNA Polymerase and 1× buffer (Life Technologies, Carlsbad, California, USA), 2 mM MgCl, 0.2 mM deoxynucleotide triphosphates, and 0.2 μM each for forward and reverse primers. Thermal cycling conditions were as follows: 2 min denaturation at 94 C, 40 cycles of 94 C denaturation for 30 s, annealing for 30 s, and 72 C extension for 1 min, followed by a final 72 C extension for 2 min. Annealing temperature varied by primer pair. The PCR products were run on an ABI PRISM 3130 Genetic Analyzer with a LIZ 1200 size standard (ThermoFisher Scientific, Waltham, Massachusetts, USA). Twenty-three markers were selected based on producing the most polymorphisms among the sample panel (Table 1). These markers all came from different contigs throughout the reference genome, indicating a breadth of genomic coverage. Several of the markers were complexes of 2–6 bp repeats, which could be better modeled as a minisatellite with a larger period. Tandem Repeats Finder v. 4.0.9 (Benson 1999), which can find repeats with longer periods and imperfect repeat motifs, was used to analyze these loci and select the most likely repeat unit. Loci excluded from the panel are listed in Supplementary Table S2 for reference. Genetic diversity analyses were conducted with poppr version 2.2.1 (Kamvar et al. 2015).

Table 1

Characteristics of the 23 polymorphic microsatellite loci in the Pseudogymnoascus destructans genome selected for the Pd23 genotyping panel. The reference sequences, belonging to P. destructans 20631-21, were obtained from National Center for Biotechnology Information (NCBI) GenBank and used to identify microsatellite loci, and to design PCR primers to amplify these loci. The number of repeat motif copies (base pairs [bp]) in the reference sequences are listed, as are the size ranges (bp) of the PCR products obtained from analyzing a panel of P. destructans samples obtained from North America and Europe (Supplementary Table S1). F = forward primer sequence; R = reverse primer sequence.

Characteristics of the 23 polymorphic microsatellite loci in the Pseudogymnoascus destructans genome selected for the Pd23 genotyping panel. The reference sequences, belonging to P. destructans 20631-21, were obtained from National Center for Biotechnology Information (NCBI) GenBank and used to identify microsatellite loci, and to design PCR primers to amplify these loci. The number of repeat motif copies (base pairs [bp]) in the reference sequences are listed, as are the size ranges (bp) of the PCR products obtained from analyzing a panel of P. destructans samples obtained from North America and Europe (Supplementary Table S1). F = forward primer sequence; R = reverse primer sequence.
Characteristics of the 23 polymorphic microsatellite loci in the Pseudogymnoascus destructans genome selected for the Pd23 genotyping panel. The reference sequences, belonging to P. destructans 20631-21, were obtained from National Center for Biotechnology Information (NCBI) GenBank and used to identify microsatellite loci, and to design PCR primers to amplify these loci. The number of repeat motif copies (base pairs [bp]) in the reference sequences are listed, as are the size ranges (bp) of the PCR products obtained from analyzing a panel of P. destructans samples obtained from North America and Europe (Supplementary Table S1). F = forward primer sequence; R = reverse primer sequence.

Genetic diversity of each locus in the Pd23 microsatellite genotyping panel, as measured by Simpson's index of genetic diversity, was substantially higher in European than North American samples (Table 2). Overall genetic diversity per locus ranged from 0.05–0.67 (mean 0.42, SD 0.17), with mean diversity across all loci in North American samples of 0.07 (SD 0.11) and in European samples 0.61 (SD 0.27). Genotype diversity was likewise lower in North America than in Europe, though the 95% confidence intervals of both estimates overlapped (Table 3). The 13 European isolates each produced a unique genotype, whereas the 27 North American isolates comprised 15 genotypes. This is in contrast to previous studies, which reported little or no diversity among North American samples in the genetic loci studied (Ren et al. 2012; Khankhet et al. 2014; Leopardi et al. 2015). The standardized index of association (), which is an average measure of linkage between the loci being analyzed, is near zero for both North American isolates (=−0.04) and the European isolates (=0.06). The low indices of linkage indicate a primarily clonal mode of propagation for the fungus, as was determined previously using other markers (Ren et al. 2012). The negative index for North American isolates indicates that the observed variance in genetic differences between individuals was lower than the calculated expected variance. The much higher value of (0.501) for the total P. destructans population suggests overall linkage disequilibrium between the North American and European populations, which is most likely due to sampling bias (a lack of European samples with alleles shared by the North American isolates). Lower genetic diversity and clonal propagation are common characteristics of recently introduced fungal pathogens (Gladieux et al. 2015), suggesting that P. destructans in North America is a recent emergence from a genetically monomorphic introduction.

Table 2

Summary of allelic results for each locus and tallies for all loci of the Pseudogymnoascus destructans Pd23 microsatellite genotyping panel. Tallies are calculated separately for the North American and European P. destructans subpopulations, as well as the total population. Allelic diversity is expressed as Simpson's index of genetic diversity. Diversity in North American isolates is less than in European isolates, consistent with the recent introduction and/or rapid spread of P. destructans to North America.

Summary of allelic results for each locus and tallies for all loci of the Pseudogymnoascus destructans Pd23 microsatellite genotyping panel. Tallies are calculated separately for the North American and European P. destructans subpopulations, as well as the total population. Allelic diversity is expressed as Simpson's index of genetic diversity. Diversity in North American isolates is less than in European isolates, consistent with the recent introduction and/or rapid spread of P. destructans to North America.
Summary of allelic results for each locus and tallies for all loci of the Pseudogymnoascus destructans Pd23 microsatellite genotyping panel. Tallies are calculated separately for the North American and European P. destructans subpopulations, as well as the total population. Allelic diversity is expressed as Simpson's index of genetic diversity. Diversity in North American isolates is less than in European isolates, consistent with the recent introduction and/or rapid spread of P. destructans to North America.
Table 3

Genetic diversity of microsatellites in Pseudogymnoascus destructans using the Pd23 panel. Allelic diversity measured using Simpson's index of genetic diversity (λ) and linkage was assessed with the standardized Index of Association (). Genetic diversity is slightly reduced for the North American isolates, consistent with an introduction. The European and North American isolates exhibit near-zero indices of association (linkage equilibrium), consistent with the clonal reproductive lifestyle hypothesized for this prodigious conidia producer. Linkage disequilibrium occurs for the P. destructans population as a whole, likely due to a lack of shared alleles between isolates from the two subpopulations.

Genetic diversity of microsatellites in Pseudogymnoascus destructans using the Pd23 panel. Allelic diversity measured using Simpson's index of genetic diversity (λ) and linkage was assessed with the standardized Index of Association (). Genetic diversity is slightly reduced for the North American isolates, consistent with an introduction. The European and North American isolates exhibit near-zero indices of association (linkage equilibrium), consistent with the clonal reproductive lifestyle hypothesized for this prodigious conidia producer. Linkage disequilibrium occurs for the P. destructans population as a whole, likely due to a lack of shared alleles between isolates from the two subpopulations.
Genetic diversity of microsatellites in Pseudogymnoascus destructans using the Pd23 panel. Allelic diversity measured using Simpson's index of genetic diversity (λ) and linkage was assessed with the standardized Index of Association (). Genetic diversity is slightly reduced for the North American isolates, consistent with an introduction. The European and North American isolates exhibit near-zero indices of association (linkage equilibrium), consistent with the clonal reproductive lifestyle hypothesized for this prodigious conidia producer. Linkage disequilibrium occurs for the P. destructans population as a whole, likely due to a lack of shared alleles between isolates from the two subpopulations.

We tested this set of microsatellite and minisatellite markers on DNA from a near-relative, Pseudogymnoascus sp. 24MN13 (National Center for Biotechnology Information Biosample SAMN03382560; Minnis and Lindner 2013). Seven of the loci did not amplify in this near relative, whereas a maximum of two loci would not amplify in P. destructans samples. This indicates that several loci might be specific to P. destructans, although further study is needed to determine this specificity.

Microsatellite analysis is relatively rapid, inexpensive, and specific. The focus of this study was to find appropriate loci for genetic differentiation of P. destructans, particularly among low-diversity North American isolates. The Pd23 microsatellite genotyping panel fulfills this need, and reflects other observed genetic characteristics of P. destructans that have already been determined, such as clonal propagation. Our sample sizes are low but are sufficient for methods development. We are in the process of applying these markers to a large dataset of isolates. These markers should be useful for identifying and tracking P. destructans, both within the North American epizootic zone and globally as new regions are sampled, without the cost and infrastructure required for other technologies, such as whole-genome sequencing.

We thank Gudrun Wibbelt and Karen Vanderwolf for each providing a P. destructans isolate for our testing panel. Funding was provided by the US Fish and Wildlife Service, the US Geological Survey, Bat Conservation International, and the National Science Foundation-EEID program (DEB-1115895 to JTF).

Supplementary material for this article is online at http://dx.doi.org/10.7589/2016-09-217.

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Supplementary data