Hi @smith13mr,
What this error is telling us is that there is no grouping factor for the script to perform statistical testing. Based on your metadata file you are feeding it there are no replicates, rather every item under SampleTreatment is unique. That means the script cannot calculate averages, variance etc. You were able to calculate each sample's alpha diversity index, but now you're asking it to compare between groups of which there is none.
You'll have to expand on your metadata file columns to include categories.
For example (and I'm totally making this up, you'll make it the way it makes sense for your experiment)
Sample: A or B
Concentrations: 10 or 50
Chemical: urea or NH4 or control