Tadpole Doctor: a UK-based citizen science project that identifies novel Perkinsea clades closely related to candidate amphibian pathogens.

Parasites of the Perkinsea lineage infect both unicellular eukaryotic and metazoan hosts. The Pathogenic Perkinsea Clade (PPC) has been associated with Severe Perkinsea Infection (SPI) and mortality events in wild tadpole populations across the USA and in captive European Tree Frogs (Hyla arborea) in the UK. To investigate the spread of this emergent disease and identify additional putative parasites, we launched a UK-based citizen science project called 'Tadpole Doctor'. Volunteer participants collected tadpoles, sediment samples from ponds suspected to contain tadpoles, or alternatively, sediment from ponds where there was evidence of recent loss of a tadpole population. In addition to the UK samples, tadpoles with SPI symptoms were collected in Alaska and Florida, along with asymptomatic tadpoles in the Czech Republic. To identify traces of PPC and related lineages, we developed a semi-targeted long-read small-subunit (SSU) - Internal Transcribed Spacer (ITS) rDNA sequencing approach, revealing an unprecedented molecular diversity of the wider Perkinsea clade NAG01, where PPC branches in SSU rDNA phylogenies. Seven groups of sequences other than PPC were found associated to tadpole livers, potentially reflecting novel parasitic infections. We also identified three sister groups to the PPC clade, among which one group was found in a tadpole, and another was frequently encountered in environmental DNA metabarcoding surveys. Deeper characterization of this diversity may identify new threats to amphibians. Overall, these data illustrate how citizen-based research can be useful for assessing organismal distribution patterns, enabling further targeted studies, while also raising public awareness of the vulnerability of amphibians to infectious disease.