Carbon isotope trends across a century of herbarium specimens suggest CO2 fertilization of C4 grasses

Increasing atmospheric CO2 is changing the dynamics of tropical savanna vegetation. C3 trees and grasses are known to experience CO2 fertilization, whereas responses to CO2 by C4 grasses are more ambiguous. Here, we sample stable carbon isotope trends in herbarium collections of South African C4 and C3 grasses to reconstruct 13C discrimination. We found that C3 grasses showed no trends in 13C discrimination over the past century but that C4 grasses increased their 13C discrimination through time, especially since 1950. These changes were most strongly linked to changes in atmospheric CO2 rather than to trends in rainfall climatology or temperature. Combined with previously published evidence that grass biomass has increased in C4-dominated savannas, these trends suggest that increasing water use efficiency due to CO2 fertilization may be changing C4 plant-water relations. CO2 fertilization of C4 grasses may thus be a neglected pathway for anthropogenic global change in tropical savanna ..., Specimen collection. Samples for analysis were collected from herbaria at Skukuza Biological Reference Collection in Kruger National Park and the South African National Biodiversity Institute (SANBI). In total, we sampled 344 grass specimens of four species: two C4 (Digitaria eriantha [N = 121], Hyparrhenia hirta [N = 56]) and two C3 species (Pentameris pallida [N = 84], Koeleria capensis [N = 73]). To minimize variation based on underlying physiology, both C4 species were chosen to have the same C4 photosynthetic subtype (using a nicotimamide-adenine dinucleotide phosphate-depending malic enzyme, commonly referred to as the NADP-ME subtype), with species selected depending on herbarium specimen availability. Sample collection dates spanned 110 years (1890s-2000s), with a mode in the early to mid 1900s (Table S2).
Each herbarium specimen is associated with a quarter-degree square, commonly used to geolocate specimens. Most of our samples, especially pre-1950, only have locations accurat..., , # Stable isotope analysis for South African herbarium grass specimens

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Carbon and Nitrogen isotope analysis values and accompanying environmental data values for 334 herbarium grass samples across a 115 year timescale.

## Description of the data and file structure

This dataset contains the carbon isotope data and accompanying environmental variables of 334 herbarium specimens sampled from the herbarium grass collections of the South African National Biodiversity Institute (SANBI) and the Kruger National Park (KNP) Skukuza Biological Reference Collection.
The data set includes data for two C3 photosynthetic grass species (*Pentameris pallida*, *Koeleria capensis*) and two C4 photosynthetic grass species (*Hyparrhenia hirta*, *Digitaria eriantha*).

Each specimen has its corresponding herbarium collection identificators listed, its species and photosynthetic pathway identified, its isotope analysis values, and a set of annual environmental variables such as atmospheric CO2 concentr...