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Permafrost thaw in the Arctic is mobilizing old carbon (C) from soils to aquatic ecosystems and the atmosphere. Little is known, however, about the assimilation of old C by aquatic food webs in Arctic watersheds. Here, we used C isotopes (d13C, D14C) to quantify C assimilation by biota across 12 streams in arctic Alaska. Streams spanned watersheds with varying permafrost hydrology, from ice-poor bedrock to ice-rich loess (that is, yedoma). We measured isotopic content of (1) C sources including dissolved organic C (DOC), dissolved inorganic C (DIC), and soil C, and (2) stream biota, including benthic biofilm and macroinvertebrates, and resident fish species (Arctic Grayling (Thymallus arcticus) and Dolly Varden (Salvelinus malma)). Findings document the assimilation of old C by stream biota, with depleted D14C values observed at multiple trophic levels, including benthic biofilm (14C ages = 5255 to 265 years before present (y BP)), macroinverte- brates (4490 y BP to modern), and fish (3195 y BP to modern). Mixing model results indicate that DOC and DIC contribute to benthic biofilm composition, with relative contributions differing across streams draining ice-poor and ice-rich terrain. DOC originates primarily from old terrestrial C sources, including deep peat horizons (39–47%; 530 y BP) and near-surface permafrost (12–19%; 5490 y BP). DOC also accounts for approximately half of fish isotopic composition. Analyses suggest that as the contribution of old C to fish increases, fish growth and nutritional status decline. We anticipate increases in old DOC delivery to streams under projected warming, which may further alter food web function in Arctic watersheds.