Potential causes for a decrease in probe binding include: inhibitors in the qPCR reaction that reduce affinity between probe and binding site, changes in structural conformation or mutations that affect probe/primer binding sites, or copy number/genome size variation. We can eliminate inhibition in our qPCR reaction, because the slope of MexMkt showed an efficiency value of 101%. Differences in structural conformations are not expected to interfere with amplification because the ITS13/5.8S Chytr primers were designed to avoid G-rich stretches and AT-rich stem/loop structures. Likewise, Illumina reads showed no Even so collagen kind IV constitutes in the typical liveris most substantially upregulated in fibrosis polymorphism in ITS primer binding sites in MexMkt or any other Bd strain, thus differential primer binding is not causing variation in amplification in our focal strains. The MexMkt strain showed only ten ITS1 copies in a single zoospore, compared to 39�C144 copies detected in other strains. Thus, our data indicate that these differences are most likely due to a reduction in genome size. All other strains showed similar slopes and intercepts for zoospore-based standard curves. Differences in ITS1 copy number were not apparent using Standard Set A, but became evident as more zoospores were required to attain 1.5 ng of DNA. For example, strain LBabercrom had 107 ITS1 copies in a single zoospore, but showed the highest number of copies in 1.5 ng of extracted DNA. This finding indicates that LBabercrom may have a low DNA content per zoospore but includes many copies of the ITS1 region. In contrast, strain CLFT024 has a large estimated genome with 118 ITS1 copies per zoospore; thus, the ratio of ITS1 to genome size may not be constant across Bd strains. Although intraspecific variability in Bd ITS1 regions often occurred among strains from different countries, our results also highlighted local differences in ITS1 copy number and haplotype diversity. For instance, strains LFT001_01, CLFT023, and CLFT024 were isolated from three states in Brazil. These strains show similar ITS1 copy numbers but LFT001_01 has a unique haplotype distribution. Recent comparisons using flow cytometry of Bd strains from California, Panama, and Brazil also indicated significant variation in DNA content, and the two fully sequenced Bd strains JEL423 and JAM81 have different assembly sizes, which may result from the presence of chromosomal length polymorphisms. Thus, our results corroborate these earlier findings of genome size evolution within Bd, and indicate that insertions and/or deletions of regions including ribosomal ITS1 may explain the differences we observed in qPCR standard curves across strains. ITS1 copy number variation among Bd lineages has important implications for comparative studies of disease dynamics in spatially isolated amphibian populations. Infection intensity thresholds of approximately 10,000 Bd genomic equivalents are often associated with disease epidemics, mortality, and population extirpations. However, other species carrying a 10-fold lower average infection intensity also experienced severe die-offs.