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Background  One of the central issues in ecology is the question what allows sympatric occurrence of closely related species in the same general area? The non-biting midges  Chironomus riparius  and  C. piger , interbreeding in the laboratory, have been shown to coexist frequently despite of their close relatedness, similar ecology and high morphological similarity.    Methodology/Principal Findings  In order to investigate factors shaping niche partitioning of these cryptic sister species, we explored the actual degree of reproductive isolation in the field. Congruent results from nuclear microsatellite and mitochondrial haplotype analyses indicated complete absence of interspecific gene-flow. Autocorrelation analysis showed a non-random spatial distribution of the two species. Though not dispersal limited at the scale of the study area, the sister species occurred less often than expected at the same site, indicating past or present competition. Correlation and multiple regression analyses suggested the repartition of the available habitat along water chemistry gradients (nitrite, conductivity, CaCO 3 ), ultimately governed by differences in summer precipitation regime.    Conclusions  We show that these morphologically cryptic sister species partition their niches due to a certain degree of ecological distinctness and total reproductive isolation in the field. The coexistence of these species provides a suitable model system for the investigation of factors shaping the distribution of closely related, cryptic species.

Reproductive Isolation and Ecological Niche Partition among Larvae of the Morphologically Cryptic Sister Species Chironomus riparius and C. piger