Investigating The Genetic Diversity And Ecological Drivers Of Batrachochytrium Dendrobatidis And B. Salamandrivorans Infections In Amphibian Populations

Amphibians are a critical component of both aquatic and terrestrial ecosystems.Despite their ecological significance,they have become the most threatened class of vertebrates,experiencing an unprecedented loss of biodiversity.The decline is accelerated by a multitude of factors,including habitat loss,environmental pollution,climate change and infectious diseases.Among these,chytriomycosis stands out as a particularly devastating infectious disease,attributed to the decline of at least 501 amphibian species globally including 90 species presumed extinctions.The disease is caused by two sister chytrid fungal pathogens,Batrachochytrium dendrobatidis(Bd)and B.salamandrivorans(Bsal).Interestingly,both pathogens are thought to have originated in Asia,where no chytrid-related mortalities have been witnessed in natural habitats.It is hypothesized that Asian amphibians may have evolved immunity to the clinical infections of the chytrid fungal pathogens,but this has not been explored in depth due to limited knowledge of endemic lineages,infection patterns,and relevant ecological factors.This highlights the necessity to investigate the genus Batrachochytrium infections at the putative point of origin of these pathogens.This dissertation aims to address four main objectives:(1)To explore the uncharted genetic diversity and infection patterns of Bd in the Guangxi Zhuang Autonomous Region of south China.(2)To screen Bsal infection as well as evaluate its geographic distribution and niche space in the Guangxi region.(3)To investigate the associations of the pathogen Bd with skin-associated microbiome of Asian amphibian hosts.(4)To analyze the ecological niche correlates between Bd and Bsal,and how climate change would affect their suitable range and habitat suitability in Eurasia including invaded and native areas of both pathogens.From 2019 to 2021,I collected 1088 swab samples from 36 amphibian species across 17 natural locations and 95 skin swabs of amphibian individuals of two species from four captive facilities in the Guangxi region.Firstly,using the internal transcribed spacer(ITS)marker and nested PCR,I conducted a detailed investigation into the genetic diversity of Bd and infection patterns.The result was the discovery of seven new haplotypes,four of which are closely related to the Bd ASIA-1 lineage from South Korea.The most prevalent haplotype(close to Bd ASIA-3 lineage)was found in 11 out of 16 infected amphibian species,highlighting the complexity and extent of genetic diversity of Bd in its putative region of origin.Statistical analyses using generalized linear models revealed that Bd infection prevalence was strongly associated with mean diurnal temperature range and elevation.This finding adds nuance to our understanding of how abiotic factors influence the spread and infection dynamics of the pathogenic Bd,offering new directions for future research and to formulate wildlife conservation strategies.Secondly in my pursuit to explore the presence of Bsal including whether it co-occurs and coinfects with Bd,I found no evidence in support of the presence of this pathogen despite comprehensive screening over period of two years.To understand the potential distribution and niche space of Bsal,I created ecological niche models of Bsal,utilizing existing occurrence records from both Asia and Europe.These models estimated potential suitable habitats for Bsal largely in the northern and southwestern parts of the Guangxi region.Although Bsal was absent in all tested individual samples,the niche models identified 10 study sites as being potentially suitable for this pathogen.Furthermore,out of these 10 sites,Bd was detected at 8.This suggests that Bsal and Bd could possibly co-exist in these habitats,if Bsal was present.Several factors seem to influence the distribution of Bsal,including variations in temperature,local caudate species diversity,elevation,and human population density;however,it is climate-related factors that hold the greatest significance,accounting for a notable 60% contribution.I also investigated the associations of the pathogen Bd(presence/absence and infection intensity)with Asian amphibian skin-associated microbiome.The16 S r RNA and ITS amplicon sequencing were used to characterize skin bacteria and fungi of four amphibian species inhabiting natural habitats in Guangxi region.Statistical analyses showed significant associations of both skin bacterial and fungal community structure and compositions with Bd presence and infection intensity(numbers of Bd sequence reads).I also found that the richness and relative abundance of putative anti-Bd bacteria were correlated with Bd presence and/or infection intensity,and observed that the relative abundance of putative anti-Bd bacteria roughly correspond with changes in both Bd prevalence and mean infection intensity in populations.Notably,the correlations among microbial community on Bd-uninfected frogs were more complex compared to infected frogs,indicating more stable microbial communities;however,the microbial network of Bd-infected frogs showed more putative keystone species(connectors)with larger links between bacteria,suggesting stronger intermodule bacterial communication.These results indicate that the likely mutual effects between Bd infection and skin-associated microbiome,including the interplays between bacteria and fungi might vary with Bd infection of hosts.Lastly,I extended the geographical focus to include Eurasia,examining ecological niche correlates of Bd and Bsal,and evaluating how climate change would affect the dispersal and geographic range of these pathogens.The niche overlap analyses revealed a partial overlap of the two pathogens,with a unique4% non-overlapping niche for Bsal.Bd was found to be more tolerant to temperature fluctuations,while Bsal required more stable and wetter conditions.Future projections indicate the impacts of global climate warming on their dispersal and distributions,particularly under extreme climate scenarios;there is a greater range expansion for Bd than Bsal.This signifies that climate change could exacerbate the threats posed by these pathogens on amphibians,making conservation efforts even more urgent.In conclusion,this dissertation provides a multifaceted view of chytrid pathogen infections in the Guangxi region,an area of significant importance due to its status as the supposed point of origin of the two pathogens and comprehensively evaluated the different impacts of climate change on Bd and Bsal.The research uncovers significant novel genetic diversity of Bd and highlights the complex interplay between host microbiome,climatic factors,and chytrid pathogens.Importantly,these findings have immediate implications for amphibian conservation,both in Asia and globally.Furthermore,this work contributes substantively to the broader understanding of host-pathogenmicrobiome-environment interactions,a critical perspective in the ecology and evolution of fungal pathogens.