Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Selection and Gene Duplication Associated With High-Elevation Diversification in Pristimantis, the Largest Terrestrial Vertebrate Genus.

The genus Pristimantis diversified in the tropical Andes mountains and is the most speciose genus of terrestrial vertebrates. Pristimantis are notable among frogs in that they thrive at high elevations (>2,000 m) and are direct developers without a tadpole stage. Despite their ecological significance, little is known about the genetic and physiological traits enabling their success. We conducted transcriptomic analysis on seven Pristimantis species sampled across elevations in the Ecuadorean Andes to explore three hypotheses for their success: (i) unique genes are under selection relative to all other frogs, (ii) common selection occurs across all direct developers, or (iii) common selection occurs across all high-elevation frog clades. Comparative analysis with 34 frog species revealed unique positive selection in Pristimantis genes related to aerobic respiration, hemostasis, signaling, cellular transportation of proteins and ions, and immunity. Additionally, we detected positive selection across all direct developers for genes associated with oxygenase activity and metal ion binding. While many genes under selection in Pristimantis were not positively selected in other high-elevation frog species, we identified some shared genes and pathways linked to lipid metabolism, innate immunity, and cellular redox processes. We observed more positive selection in duplicated- versus single-copy genes, while relaxed purifying selection was prevalent in single-copy genes. Notably, copy number of an innate immunity complement gene was positively correlated with Pristimantis species elevation. Our findings contribute novel insights into the genetic basis of adaptation in Pristimantis and provide a foundation for future studies on the evolutionary mechanisms leading to direct development and coping with high elevations.