Among the worst part of it all is that Gpuccio’s argument directly depends on how much variation is sampled in the alignment. That’s a giant problem right there. If technically 10 out of the 20 different possible amino acid states are allowed at some position in the protein, but only 5 of these are among the variation known from species that have had the gene sequenced, that’s massively going to impact the FI you calculate as you go through each aligned site in the protein and there are strictly allowed variants for each position not actually present in existing variation.
This idea that we would expect all the allowed variation to actually be present in the form of sequences from each different species is just ridiculous. Particularly when we consider that some states might just have lower fitness and therefore be less likely to be present, yet would still strictly represent functional states, and therefore affect the probability of evolving the sequence.
The method is therefore extremely vulnerable to taxon sampling, and completely ignores the effect of fitness. It treats each site as black or white, with variants not seen in the taxon sampling assumed to be nonfunctional, which will inflate the FI. Besides the fact that it also assumes that the taxon sampling yields an exhaustive sampling of allowed variants.
And then there’s the fact that saturation of synonymous sites tells you nothing about how exhaustive the sampling has been of multi-residue mutations for amino acids.
Amazingly I’ve explained all this on this same website now four years ago. I got crickets back, like we get crickets now. In the latest FI thread I also linked an article co-authored by Swamidass where they prove with simulations that the sampled variations you can expect to be present in some taxon sampling used for your alignment, to “fantastically understimate” the real number of functional sequences.
It’s stupid. It’s just stupid. I cannot get over that there are people who buy it. They must be having trouble actually understanding what is going on. But, we’ve been over all this before so many times. No response.