text: These two pathways, with the same effect on RuBisCO, evolved a number of times independently – indeed, C alone arose 62 times in 18 different plant families.  A number of pre-adaptations seem to have paved the way for C, leading to its clustering in certain clades: it has most frequently developed in plants that already had features such as extensive vascular bundle sheath tissue. Whole-genome and individual gene duplication are also associated with C evolution. Many potential evolutionary pathways resulting in the  phenotype are possible and have been characterised using Bayesian inference, confirming that non-photosynthetic adaptations often provide evolutionary stepping stones for the further evolution of .
  The C construction is most famously used by a subset of grasses, while CAM is employed by many succulents and cacti. The trait appears to have emerged during the Oligocene, around ; however, they did not become ecologically significant until the Miocene, . Remarkably, some charcoalified fossils preserve tissue organised into the Kranz anatomy, with intact bundle sheath cells, allowing the presence C metabolism to be identified without doubt at this time. Isotopic markers are used to deduce their distribution and significance.
  C plants preferentially use the lighter of two isotopes of carbon in the atmosphere, C, which is more readily involved in the chemical pathways involved in its fixation. Because C metabolism involves a further chemical step, this effect is accentuated. Plant material can be analysed to deduce the ratio of the heavier C to C. This ratio is denoted . C plants are on average around 14‰ (parts per thousand) lighter than the atmospheric ratio, while C plants are about 28‰ lighter. The  of CAM plants depends on the percentage of carbon fixed at night relative to what is fixed in the day, being closer to C plants if they fix most carbon in the day and closer to C plants if they fix all their carbon at night.
  It is troublesome procuring original fossil material in sufficient quantity to analyse the grass itself, but fortunately there is a good proxy: horses. Horses were globally widespread in the period of interest, and browsed almost exclusively on grasses. There's an old phrase in isotope palæontology, "you are what you eat (plus a little bit)" – this refers to the fact that organisms reflect the isotopic composition of whatever they eat, plus a small adjustment factor. There is a good record of horse teeth throughout the globe, and their  has been measured. The record shows a sharp negative inflection around , during the Messinian, and this is interpreted as the rise of C plants on a global scale.