Phylogenetic divergence between species may weaken both negative and positive biotic interactions. Whether, as a result, divergent species aggregate or segregate remains unclear. These interactions differ among lineages, and without immigration, the effects of interactions might be overridden by dispersal limitation. We studied changes in co-occurrence across 5 years for 1770 pairs of grassland species drawn primarily from four major lineages (families), reducing dispersal limitation by seed addition. Across all lineages, changes in co-occurrence were almost unrelated to phylogenetic divergence, partly reflecting opposing relationships within two groups of families: Fabaceae/Poaceae, where more divergent species segregated, versus Asteraceae/Apiaceae, where they aggregated. Dispersal limitation through lack of immigration existed, without eliminating divergence effects. We conclude that species assembly differed between major lineages, with spatial aggregation in Asteraceae/Apiaceae possibly reflecting limiting similarity, whereas segregation in Fabaceae/Poaceae may reflect mutualist incompatibility or asymmetric competition. These processes could feedback on within-lineage diversification.