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The co-occurrence of larvae of congeneric chironomid species is common in natural stream assemblages, and raises the problem of finding mechanisms to explain the co-existence of species with similar ecological requirements. In this contribution, we explored the co-occurrence of chironomid larvae belonging to congeneric species within four genera of chironomids: Cricotopus, Eukiefferiella, Orthocladius and Rheocricotopus (with 2, 7, 2 and 4 species, respectively) in the headwaters of a small Mediterranean calcareous stream. Due to the intrinsic, natural spatial and temporal variability in these habitats, we studied three different sites at two different seasons within the annual hydrological cycle (spring vs summer samples), and each microhabitat unit was surveyed with an effort proportional to its cover at the sampling site. The Outlying Median Index method was used to distinguish the niches of the different chironomid species in relation to the hydraulic features and substrates within each site. Our results showed that hydraulic features helped to improve discrimination between the niches of five congeneric species of Eukiefferiella, and the same occurred within the species belonging to Cricotopus and Orthocladius, although niche overlap was high in general. One Rheocricotopus species was associated to algal substrate. Schoener’s co-occurrence at the microhabitat scale was low among the congeneric species found in our study, suggesting exclusion mechanisms. Flow regime, substrate type and shifts in environmental conditions may favor the settlement of some species to the detriment of other congeneric ones, but other mechanisms such as lottery competition, diffusion competition or predation should be considered in further studies.