Environment predicts Batrachochytrium dendrobatidis lineage distribution and zones of recombination in South Africa.

The amphibian-infecting chytrid fungus, Batrachochytrium dendrobatidis (Bd), is widespread throughout Africa and is linked to declines of populations and species across the continent. While it is well established that the lineage of Bd encodes traits which determine disease severity, knowledge around how lineages are distributed according to environmental envelope is unclear. We here studied the distribution of Bd in South Africa based on the two lineages found, BdGPL and BdCAPE, in terms of their genome and environmental envelope statistically associated with their distribution. We used Bd surveillance data from published studies, as well as data collected during fieldwork from across South Africa, Lesotho, and eSwatini with samples collected along a transect spanning most of South Africa from Lesotho to the west coast. We utilized lineage-typing qPCR to resolve the spatial distribution of BdGPL and BdCAPE across South Africa and used the resulting surveillance data to create a predictive ecological niche model for Bd lineages in South Africa. Phylogenomic analyses were performed on isolates sourced from across the transect. We show that BdGPL demonstrates a strong isolation by distance suggestive of stepping-stone dispersal, while BdCAPE showed two distinct clusters within their genomic structure that appear geographically and temporally clustered, indicating two separate invasions. Our predictive niche model revealed that the two lineages tended to occur in different ecotypes; BdGPL was associated with lower altitude, arid regions while BdCAPE occurred across cooler, higher altitude environs. Niche predictions identified a zone of lineage contact, where genomics identified inter-lineage recombinants. We argue that this zone of recombination should be prioritized for disease surveillance as it is a potential hotspot for the evolution of variants of amphibian chytrid with novel traits that may be epidemiologically relevant.

Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.