D. Pseudoobscura (SP vs EPS)

I performed differential expression analysis by comparing cells in the SP cluster with cells in EPS. I then took the list of genes from this analysis (SP-biased, EPS-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	46	100	141
moved_on	7	0	4
gene_death	34	23	20
moved_off	4	9	7
other	16	34	27

EPS Expression

In Figures 1-3 I look at the proportion of EPS 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 EPS expression for genes with “Selection Against” on Muller elements D and E. 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): 0.06408074884649775

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

Muller D

Mann-Whitney U (Favor vs Against): 5.421007642766501e-16

Muller E

Mann-Whitney U (Favor vs Against): 4.400211255192757e-30

Enrichment of EPS Biased Genes

Next, we looked at contingency tables to test for a relationship between EPS 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). Again 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	5	1	8	0
EPS	1	5	16	2
SP	40	1	10	2

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

bias	Selection Favor	Selection Against
EPS Biased	6	18
Not Biased	47	20

Fisher’s Exact Test: 0.0002062765044224908

Muller D

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

bias	conserved	gene_death	moved_off
NS	17	7	3
EPS	27	14	6
SP	56	2	0

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

bias	Selection Favor	Selection Against
EPS Biased	27	20
Not Biased	73	12

Fisher’s Exact Test: 0.0005337227613258306

Muller E

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

bias	conserved	moved_on	gene_death	moved_off
NS	19	2	3	1
EPS	44	1	14	6
SP	78	1	3	0

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

bias	Selection Favor	Selection Against
EPS Biased	45	20
Not Biased	100	7

Fisher’s Exact Test: 5.007160270119653e-05