A major challenge in the biological monitoring of stream ecosystems in protected wilderness areas is discerning whether temporal changes in community structure are significantly outside of a reference condition that represents natural or acceptable annual variation in population cycles. Otherwise sites could erroneously be classified as impaired. Long-term datasets are essential for understanding these trends, to ascertain whether any changes in community structure significantly beyond the reference condition are permanent shifts or with time move back to within previous limits. To this end, we searched for long-term (>8 years) quantitative data sets of macroinvertebrate communities in wadeable rivers collected by similar methods and time of year in protected wilderness areas with minimal anthropogenic disturbance. Four geographic areas with datasets that met these criteria in the USA were identified, namely: McLaughlin Nature Reserve in California (1 stream), Great Smoky Mountains National Park in Tennesse-North Carolina (14 streams), Wind River Wilderness Areas in Wyoming (3 streams) and Denali National Park and Preserve in Alaska (6 streams).

Two statistical approaches were applied: Taxonomic Distinctness (TD) to describe changes in diversity over time and non-metric multidimensional scaling (MDS) to describe changes over time in community persistence (Jaccards Index) and community stability (Bray–Curtis Index). Control charts were used to determine if years in MDS plots were significantly outside a reference condition. For Hunting Creek, TD showed three years outside natural variation which could be attributed to severe hydrological events but years outside the natural-variation funnel at sites in other geographical areas were inconsistent and could not be explained by environmental variables. TD identified simulated severe pollutant events which caused the removal of entire invertebrate assemblages but not simulated water temperature shifts.

Within a region, both MDS analyses typically identified similar years as exceeding reference condition variation, illustrating the utility of the approach for identifying wider spatial scale effects that influence more than one stream. MDS responded to both simulated water temperature stress and a pollutant event, and generally outlying years on MDS plots could be explained by environmental variables, particularly higher precipitation. Multivariate control charts successfully identified whether shifts in community structure identified by MDS were significant and whether the shift represented a press disturbance (long-term change) or a pulse disturbance. We consider a combination of TD and MDS with control charts to be a potentially powerful tool for determining years significantly outside of a reference condition variation.