JoinYourPlayfellows - it was interesting to read what you said about your 2 DDs not wanting anything to do with boys around the age of 3, turning 4 - when I moved to Australia and joined a playgroup here, DS1 was just coming up to 2 and would play with anyone - but there was a small group of girls, aged 3 and 4, who wouldn't let him play teaparties or cooking or any of their games "because he was a boy". He was so disappointed to be pushed away!
Luckily those few girls left at the end of that year, and the next lot were more than happy to all play together with no gender bias at all, thank goodness.
And to whomever asked if boys really do run around in pink dresses - well not all the time, but yes, DS1 has dressed up in pink sparkly fairy costumes at playgroup as well, why not?
In my own experience, we didn't have any gender segregation until we were 9 at school. A group of boys and girls had played together since we started school aged 4/5, pretty much, with no problems - our playground games varied around the themes of Bulldog and kiss-chase (between 7 and 9) - then suddenly when we hit the final year of junior school we suddenly separated out. It was such a strange thing, that we actually got together and talked about it, and made an effort to play together again! But it didn't work, sadly and we remained separated.
OP - IMO, it's a shame your DS and the other little boy weren't invited and I think the mother was a bit daft to assume that they wouldn't like it (if that's what she did), but perhaps she doesn't like little boys herself, or thought that they would be "too rough" and change the play dynamic. I like mixed parties at that age and, in reality, up to the point where they naturally start to separate out - I wouldn't expect that to be before they were at least 7.