Transpiration is the dominant component of terrestrial evapotranspiration and is increasing globally. Extensive research has been conducted in the Maimai M8 catchment (New Zealand) and across many sites on streamflow sources and generation processes, yet uncertainty exists surrounding the sources of transpiration, the periods when transpiration and streamflow sources are hydrologically connected or separated and the mechanisms driving this connectivity or separation. We investigated these questions at M8 over a 10-month period and a separate 10-day period in the summer by combining observations of isotope tracing (δ18O and δ2H) of water from xylem, soil, subsurface stormflow, bedrock moisture, and the stream and measurements of stem water potential and soil moisture. Transpiration and streamflow followed seasonal precipitation inputs, with xylem water showing more enriched isotope values than the stream in the summer. In summer, when both fluxes were ecohydrologically separated, potential evapotranspiration (PET) was greater than in winter, and trees relied on a mixture of summer rainfall and deeper bedrock moisture. Stream and xylem water sources were ecohydrologically connected during winter and fed by the same, mixed, subsurface water pools. These findings suggest seasonal shifts between ecohydrological connectivity and separation between streamflow and transpiration sources driven by changes in PET, which influence the age of these hydrological fluxes. Overall, streamflow young water fraction (90%) was greater than transpiration (47%). Our work underscores the importance of understanding how PET influences ecohydrological connectivity and separation between transpiration and streamflow sources and the contribution of bedrock moisture to the transpiration flux in dry periods.