D. Pseudoobscura (EPS vs PS)

I performed differential expression analysis by comparing cells in the EPS cluster with cells in PS1, PS2, and PS3. I then took the list of genes from this analysis (EPS-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.

	muller_A	muller_D	muller_E
conserved	54	114	177
recent_conserved	0	0	0
moved_on	7	1	3
gene_death	34	20	24
moved_off	3	8	6
other	14	34	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): 0.00014820920368461394

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

Muller D

Mann-Whitney U (Favor vs Against): 4.247933807388154e-20

Muller E

Mann-Whitney U (Favor vs Against): 4.278607873507591e-40

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 Muller elements D and E.

Muller A

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

bias	conserved	moved_on	gene_death	moved_off
NS	4	0	3	1
PS	0	4	4	0
EPS	50	3	27	2

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

bias	Selection Favor	Selection Against
PS Biased	4	4
Not Biased	57	33

Fisher’s Exact Test: 0.47126741340750544

Muller D

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

bias	conserved	moved_on	gene_death	moved_off
NS	9	0	5	0
PS	23	1	8	6
EPS	82	0	7	2

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

bias	Selection Favor	Selection Against
PS Biased	24	14
Not Biased	91	14

Fisher’s Exact Test: 0.0034996761816397386

Muller E

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

bias	conserved	moved_on	gene_death	moved_off
NS	19	0	2	3
PS	42	2	15	1
EPS	116	1	7	2

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

bias	Selection Favor	Selection Against
PS Biased	44	16
Not Biased	136	14

Fisher’s Exact Test: 0.0020621301227712065