Using Environmental DNA to Map a Contact Zone between Deeply Diverged Frog Mitochondrial Lineages in Southern Ontario

Abstract

Contact zones are natural laboratories that facilitate the study of dynamics between diverging evolutionary lineages in primary (evolved in situ) or secondary (evolved in allopatry) contact; outcomes of contact zone dynamics have implications for understanding species range limits, can clarify questions on species delineation and mechanisms of speciation itself, and may be important in conservation prioritization. Chorus frog populations within Ontario and Quebec, Canada (currently collectively classified as Western Chorus Frogs, Pseudacris triseriata) comprise distinct mitochondrial DNA (mtDNA) lineages (> 10% sequence divergence in cytochrome b) representing Boreal (P. maculata) and Western (P. triseriata) Chorus Frogs. Within Ontario and Quebec, there are two designatable units (DUs), with the Great Lakes-St. Lawrence-Canadian Shield DU classified as Threatened in Canada. This DU approximately encompasses the Boreal Chorus Frog mitotype population. These diverging mitochondrial lineages are in probable secondary contact in Southern Ontario, yet the exact location of contact is uncertain, lying somewhere within a large region spanning ~150km (between Toronto, which is north of Lake Ontario, and the southern shores of Georgian Bay, part of Lake Huron). I used environmental DNA (eDNA) from water sampled from 50 chorus frog breeding ponds to better delineate this contact zone. eDNA facilitates intensive geographic sampling, allows us to identify single wetlands containing breeding assemblages with both haplotypes, and is less invasive than tissue sampling (e.g., toe clipping, buccal swabbing). I surveyed for the presence of each mtDNA lineage using droplet digital PCR with custom primer-probe mitochondrial cytochrome b sets to target each lineage. I used landscape layers to determine the habitat composition surrounding each observation. Ten wetlands in Southern Ontario had both mitochondrial lineages with evidence to support a broader, more interdigitated contact zone than previously thought. Landscape composition and bioclimatic variables did not significantly correlate with mitotype detection, at least at the scale I used. My study sets the stage for future research on implications of hybridization and speciation between these lineages; these findings may also help guide classification and conservation of these populations and refine conservation designations.