Water availability and hydrological connectivity mediate the responses of alpine soil prokaryotes to temperature changes at the watershed scale.

Temperature and water are crucial drivers of soil prokaryotic community structure and function. However, their coupling effects remain many uncertainties, particularly in alpine watersheds experiencing drastic hydrological changes. Here, we investigate how water availability, hydrological connectivity, and temperature affect the alpine soil prokaryotic community at the watershed scale. Six sampling sites were established along an elevation gradient (representing a temperature gradient) in a typical alpine watershed on the Tibetan Plateau, each covering three ecosystem types with distinct water availability (namely, wetlands, grasslands, and transition zones). Using 16S rRNA sequencing and redundancy analysis of 74 soil samples, we found that temperature significantly affected prokaryotic communities in wetlands and transition zones with higher water availability (explained variations = 10.8 % and 11.2 %, respectively; p < 0.05). However, temperature had non-significant effects in water-limited grasslands, where soil moisture was the primary driver (explained variations = 23.3 %, p < 0.01). Moreover, the distance-decay rate of community similarity from upstream to downstream decreased progressively from grasslands to wetlands (slopes = -0.01, -0.007, and -0.005, respectively) due to increasing hydrological connectivity's effects on community assembly. While homogeneous selection was the sole mechanism in grasslands and transition zones, dispersal processes were also important in wetlands' community assembly. Therefore, the increasing precipitation and glacial meltwater due to global warming may enhance hydrological dispersal and watershed connectivity, thus leading to the homogenization of soil prokaryotic communities in alpine watersheds. Future studies should focus on how intensifying hydrological changes alter alpine ecosystem functions via soil prokaryote dispersal.