Background: Tropical rainforest streams harbour extraordinarily high amphibian diversity, yet the mechanisms enabling multiple species to coexist within these structurally heterogeneous yet spatially confined habitats remain incompletely understood. Stream microhabitat structure — encompassing variation in substrate type, water depth, current velocity, perch height, riparian vegetation complexity, and humidity gradients — is hypothesised to create a mosaic of ecological niches that facilitates species coexistence through niche partitioning, thereby reducing interspecific competition below the levels that would lead to competitive exclusion.

Objectives: This study was designed to: (1) characterise the microhabitat preferences of six co-occurring amphibian species in three tropical rainforest streams; (2) quantify niche overlap between species pairs using Pianka's overlap index; (3) determine which microhabitat variables most strongly predict species-specific habitat use through multivariate analysis; and (4) assess the relative contribution of spatial, temporal, and resource-use dimensions to overall niche partitioning within the assemblage.

Methods: Visual encounter surveys were conducted monthly from June 2025 to June 2026 along three 100-metre stream transects in the Eastern Ghats tropical moist deciduous forest zone of Andhra Pradesh, India. For each amphibian individual detected, seven microhabitat variables were recorded: substrate type, water depth, current velocity, perch height above water, distance from stream edge, canopy cover, and ambient relative humidity. A total of 1,284 individuals across six species — Nyctibatrachus major (Boulenger, 1882), Indirana semipalmata (Boulenger, 1882), Micrixalus saxicola (Jerdon, 1854), Nyctibatrachus jog (Boulenger, 1882), Fejervarya teraiensis (Dutta & Manamendra-Arachchi, 1996), and Uperodon systoma (Schneider, 1799) — were recorded over the study period. Non-metric multidimensional scaling (NMDS), permutational MANOVA (PERMANOVA), and Pianka's overlap index were applied to quantify niche differentiation.

Results: All six species exhibited significant microhabitat preferences (p<0.001 by PERMANOVA), with substrate type, perch height, and current velocity emerging as the strongest discriminators of species-specific habitat use. Pianka's overlap index between species pairs ranged from 0.12 (N. major vs. U. systoma) to 0.74 (N. major vs. N. jog), with a mean overlap of 0.41 (±0.18 SD). Null model analysis (EcoSim 7.0) confirmed that mean pairwise niche overlap was significantly lower than expected by chance (observed mean 0.41 vs. simulated mean 0.68, p=0.003), providing evidence of non-random niche partitioning. Temporal (diel) and acoustic (calling frequency and microsite) dimensions provided additional axes of differentiation between the two most spatially overlapping species pairs.