perennially ice-covered lakes that host benthic microbial ecosystems are present in many regions of Antarctica. Lake Untersee is an ultra-oligotrophic lake that is substantially different from any other lakes on the continent as it does not develop a seasonal moat and therefore shares similarities to sub-glacial lakes where they are sealed to the atmosphere. Here, we determine the source of major solutes and carbon to Lake Untersee, evaluate the carbon cycling and assess the metabolic functioning of microbial mats using an isotope geochemistry approach. The findings suggest that the glacial meltwater recharging the closed-basin and well-sealed Lake Untersee largely determines the major solute chemistry of the oxic water column with plagioclase and alumino-silicate weathering contributing < 5% of the Ca 2+-na + solutes to the lake. the tic concentration in the lake is very low and is sourced from melting of glacial ice and direct release of occluded co 2 gases into the water column. The comparison of δ 13 c tic of the oxic lake waters with the δ 13 c in the top microbial mat layer show no fractionation due to non-discriminating photosynthetic fixation of HCO 3 in the high pH and carbon-starved water. the 14 C results indicate that phototrophs are also fixing respired CO 2 from heterotrophic metabolism of the underlying microbial mats layers. The findings provide insights into the development of collaboration in carbon partitioning within the microbial mats to support their growth in a carbon-starved ecosystem. Numerous perennially ice-covered lakes have been inventoried in Antarctica, including in the McMurdo Dry Valleys (MDV), Bunger Hills, Vestfold Hills, Schirmacher Oasis, and Soya Coast 1,2. These lakes have varied chemistries as a result of source water and Holocene history of the lakes, but many are oligotrophic and support benthic cyanobacterial mats and heterotrophic bacterial communities 3-5. Primary productivity is often limited by light attenuation through the ice-cover and nutrient availability (e.g., C and P), however, summer moating and streams provide seasonal recharge of nutrients to the lakes 4,6. Analysis of carbon isotopes (e.g., δ 13 C, 14 C) can provide insights about the source of carbon and transformations of organic matter as photosynthesis and remineralization control the isotopic composition of most organic matter. For example, carbon isotope geochemistry of dissolved inorganic carbon (δ 13 C DIC) and organic carbon (δ 13 C DOC) have been used to trace carbon sources and cycling in the MDV lake ecosystem 3,7-9. Lake Untersee is a 169-m deep ultra-oligotrophic lake that is substantially different from other lakes in Antarctica 10,11. It is recharged by subaqueous melting of glacial ice and subglacial meltwater, and the lake remains ice-covered with no open water along the margin (summer moating) that would provide access to nutrients, CO 2 and enhanced sunlight 12-14. In the absence of large metazoans, photosynthetic microbial mats cover the floor of the lake from just below the ice cover ...

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