D. Pseudoobscura (SP vs PS)

I performed differential expression analysis by comparing cells in the SP cluster with cells in PS1, PS2, and PS3. I then took the list of genes from this analysis (SP-biased, PS-biased, Not Significant) and filtered out the orthologs. The number of genes analyzed per Muller element and class are in Table 1.

Table 1. Number of genes per Muller Element.

index	muller_A	muller_D	muller_E
conserved	80	151	218
moved_on	7	1	4
gene_death	31	21	27
moved_off	4	9	6
other	18	43	29

PS Expression

In Figures 1-3 I look at the proportion of PS1, PS2, and P3 cells that expressed genes in the different movement classes. The number of genes in the moved_on and moved_off classes are very low (< 10) which makes interpretation difficult. I group genes into “Selection Favor” and “Selection Against” for all statistical analyses. Here I compare the distribution of the proportion of cells using a Mann-Whitney U test. We see an significant increase in PS expression for genes with “Selection Against” on all Muller elements. However, the number of genes in the two classes is very different so I am worried about biases.

Muller A

Mann-Whitney U (Favor vs Against): 4.4927665471754445e-11

**Figure 1. Gene movement on/off of Muller element A.**

Muller D

Mann-Whitney U (Favor vs Against): 6.832147521615045e-31

Muller E

Mann-Whitney U (Favor vs Against): 1.1474515219576066e-51

Enrichment of PS Biased Genes

Next, we looked at contingency tables to test for a relationship between PS1, PS2, and PS3 biased expression and selection against. Again, cell counts are very low for some combinations (Table 2,4,6) so I combined counts to make statistical analysis possible (Table 3,5,7). There is evidence of a enrichment of genes that underwent “Selection Against” for all tested Muller elements.

Muller A

Table 2. Number of genes with differential expression on Muller A.

bias	conserved	moved_on	gene_death	moved_off
NS	0	1	7	0
PS	1	4	7	1
SP	79	2	17	3

Table 3. Number of genes with selection on Muller A.

bias	Selection Favor	Selection Against
PS Biased	5	8
Not Biased	82	27

Fisher’s Exact Test: 0.009540858896492865

Muller D

Table 4. Number of genes with differential expression on Muller D.

bias	conserved	moved_on	gene_death	moved_off
NS	12	0	2	1
PS	31	1	16	7
SP	108	0	3	1

Table 5. Number of genes with selection on Muller D.

bias	Selection Favor	Selection Against
PS Biased	32	23
Not Biased	120	7

Fisher’s Exact Test: 9.485216138214068e-09

Muller E

Table 6. Number of genes with differential expression on Muller E.

bias	conserved	moved_on	gene_death	moved_off
NS	16	0	3	1
PS	50	3	17	5
SP	152	1	7	0

Table 7. Number of genes with selection on Muller E.

bias	Selection Favor	Selection Against
PS Biased	53	22
Not Biased	169	11

Fisher’s Exact Test: 2.068291103314802e-06