I am comparing fish communities at 2 sites (upstream vs downstream) with data collected in two seasons (wet and dry) over several years (2017-2022), with data from the first pair of wet and dry seasons representing the period before a treatment and subsequent data representing periods after the treatment. During each season I sampled each site four times, and recorded abundance of each fish species from each site. I did not conduct sampling during the last dry season due to resource constraints. The community compositions of the two sites over different seasons and periods are visualised in the NMDS biplots.
I am trying to do further analysis using PERMANOVA to look for any spatial-temporal changes in the fish commnities, mainly if the two groups are becoming more similar in the years following the treatment. As there are samples with no fishes recorded I have to remove those samples from the dataset, which means I have only three instead of four replicates in some of the site x season x year groups.
My question is, does it still make sense to use PERMANOVA if I have unequal sample size among groups, given the number of replicates from each are small (3-4)? I am planning to run the test separately for wet and dry seasons, but that means I still need to do two-way (site x year) PERMANOVAs for each of the seasons.
I learned from some of the online discussions that unequal sample size would be a problem for two (or more)-way PERMANOVAs and the problem of unequal sample size would be more prominent when the sample sizes are small. Would be grateful for any comments or insight on this. Thanks a tonne!
