Assessment of bias in carbon isotope composition of organic leaf matter due to pre-analysis milling methods.

RATIONALE: Stable isotope analysis of leaf material has many applications including assessment of plant water-use efficiency and palaeoclimatology. To facilitate interpretations of small shifts in the carbon isotope composition (δ13 C) of leaves, accurate and repeatable results are required. Pre-sample homogenisation is essential to ensure a representative sample is analysed, but can also introduce error. METHODS: We investigate how different grinding methods (freezer-milling and ball-milling) affect the carbon content and δ13 C of tree leaves from a wetland in Queensland, Australia, commenting on how increased temperature, sample contamination, sample loss or poor homogenisation may impact results. RESULTS: No alteration of leaf δ13 C is observed due to different milling methods, although there may be a significant increase in %C of samples processed using ball-milling. CONCLUSIONS: We suggest %C variability is possibly due to contamination from abraded plastic vials or insufficient homogenisation during ball-milling, with no significant impact on δ13 C. Overall, we suggest that intermittent ball-milling may be the best solution to reduce costs, preparation time and use of liquid nitrogen, aiming to achieve complete homogenisation using the shortest possible duration of milling.

Closure of the Bering Strait caused Mid-Pleistocene Transition cooling.

The Mid-Pleistocene Transition (MPT) is characterised by cooling and lengthening glacial cycles from 600-1200 ka, thought to be driven by reductions in glacial CO2 in particular from ~900 ka onwards. Reduced high latitude upwelling, a process that retains CO2 within the deep ocean over glacials, could have aided drawdown but has so far not been constrained in either hemisphere over the MPT. Here, we find that reduced nutrient upwelling in the Bering Sea, and North Pacific Intermediate Water expansion, coincided with the MPT and became more persistent at ~900 ka. We propose reduced upwelling was controlled by expanding sea ice and North Pacific Intermediate Water formation, which may have been enhanced by closure of the Bering Strait. The regional extent of North Pacific Intermediate Water across the subarctic northwest Pacific would have contributed to lower atmospheric CO2 and global cooling during the MPT.