Clarification of strata argument for adoinis2

[cazzlewazzle89]

Hi all,

As the title suggests, I'm hoping for some clarification on the strata argument in adonis2. I have paired samples from the faecal microbiome of mice, one is a 'total' faecal sample, the other is an extract of bacterial extracellular vesicles from the same sample. I also have two treatment groups.

I am trying to see whether Treatment impacts the extracellular vesicle-producing component of the microbiome, which using the paired 'total' faecal sample to control for the fact that the microbiome will differ between individual mice. Should I use MouseID as the strata argument for PERMANOVA?

A standard two way PERMANOVA gives me no significant effect of Treatment

adonis2(formula = bray_dist_paired ~ metadata_paired$Source * metadata_paired$Treatment)
                                                 Df SumOfSqs      R2       F Pr(>F)    
metadata_paired$Source                            1   4.7516 0.43150 26.7180  0.001 ***
metadata_paired$Treatment                         1   0.2809 0.02551  1.5795  0.142    
metadata_paired$Source:metadata_paired$Treatment  1   0.2882 0.02618  1.6208  0.134    
Residual                                         32   5.6910 0.51681                   
Total                                            35  11.0117 1.00000                   
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ‘ ’ 1

But adding the strata argument makes it significant

adonis2(formula = bray_dist_paired ~ metadata_paired$Source * metadata_paired$Treatment, strata = metadata_paired$MouseID)
                                                 Df SumOfSqs      R2       F Pr(>F)    
metadata_paired$Source                            1   4.7516 0.43150 26.7180  0.001 ***
metadata_paired$Treatment                         1   0.2809 0.02551  1.5795  0.001 ***
metadata_paired$Source:metadata_paired$Treatment  1   0.2882 0.02618  1.6208  0.275    
Residual                                         32   5.6910 0.51681                   
Total                                            35  11.0117 1.00000                   
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ‘ ’ 1

I am unsure which method is correct. The R2 value doesn't change, so I think I'm just unsure of what strata actually does.

Thanks in advance for any advice, Calum