Abstract
Sex determination evolves rapidly, often because of turnover of the genes at the top of the pathway. The house fly, Musca domestica, has a multifactorial sex determination system, allowing us to identify the selective forces responsible for the evolutionary turnover of sex determination in action. There is a male determining factor, M, on the Y chromosome (YM), which is probably the ancestral state. An M factor on the third chromosome (IIIM) has reached high frequencies in multiple populations across the world, but the evolutionary forces responsible for the invasion of IIIM are not resolved. To test if the IIIM chromosome invaded because of sex-specific selection pressures, we used mRNA sequencing to determine if isogenic males that differ only in the presence of the YM or IIIM chromosome have different gene expression profiles. We find that more genes are differentially expressed between YM and IIIM males in testis than head, and that genes with male-biased expression are most likely to be differentially expressed between YM and IIIM males. This suggests that male phenotypes, especially those related to male fertility, are more likely to be affected by the male-determining chromosome, supporting the hypothesis that sex-specific selection acts on alleles linked to the male-determining locus driving evolutionary turnover in the sex determination pathway. We additionally find that IIIM males have a “masculinized” gene expression profile, suggesting that the IIIM chromosome has accumulated an excess of male-beneficial alleles because of its male-limited transmission.
1. Introduction
Sex determination (SD) is an essential developmental process responsible for sexually di-morphic phenotypes. It is therefore paradoxical that SD pathways are poorly conserved, with master SD (MSD) genes at the top of the pathway differing between closely related species and even variable within species (Bull, 1983; Wilkins, 1995; Pomiankowski et al., 2004; Beukeboom and Perrin, 2014). The hypotheses to explain the rapid evolution of SD pathways fall into three categories. First, SD evolution may be selectively neutral if MSD turnover is the result of mutational input without phenotypic or fitness consequences (van Doorn, 2014). Second, frequency dependent (sex-ratio) selection could favor a new MSD variant if one sex is below its equilibrium frequency (Eshel, 1975; Bull and Charnov, 1977; Bulmer and Bull, 1982; Werren and Beukeboom, 1998). Third, a new MSD locus can invade a population if the new MSD variant itself or genetically linked alleles confer a fitness benefit (Charlesworth and Charlesworth, 1980; Rice, 1986; Charlesworth, 1991; van Doorn and Kirkpatrick, 2007, 2010). Those fitness effects could be beneficial to both sexes (natural selection), increase the reproductive success of the sex determined by the new MSD variant (sexual selection), or be beneficial to the sex determined by the MSD variant and deleterious to the other sex (sexually antagonistic selection). Sexually antagonistic selection is predicted to be an especially important driver of MSD turnover because linkage to an MSD locus allows the sexually antagonistic allele to be inherited in a sex-limited manner, thereby resolving the inter-sexual conflict (Charlesworth and Charlesworth, 1980; van Doorn and Kirkpatrick, 2007; Roberts et al., 2009; van Doorn and Kirkpatrick, 2010).
The house fly, Musca domestica, is an ideal model for testing hypotheses about the evolution of SD because it has a multifactorial SD system, with male- and female-determining loci segregating in natural populations (Dübendorfer et al., 2002; Hamm et al., 2015). Most relevant to the work presented here is the the fact that the male-determining factor, M, can be located on the Y chromosome (Y M), any of the five autosomes (AM), and even the X chromosome (Hamm et al., 2015). It is unknown whether these M -factors are the same gene in different locations or different genes that have independently assumed the role of an MSD locus (Bopp, 2010). YM is a common arrangement (Hamm et al., 2015), and it is thought to be the ancestral state because it is the genotype found in close relatives of the house fly (Boyes et al., 1964; Boyes and Van Brink, 1965; Dübendorfer et al., 2002). M on the third chromosome (IIIM) is also common, but it is not clear what was responsible for the invasion of the IIIM chromosome (Hamm et al., 2015). Note that when the M factor arrived on chromosome III, this entire chromosome essentially assumed Y -like properties, including restriction to males and reduced recombination (Hamm et al., 2015). Identifying the selective forces responsible for the invasion of IIIM will be a powerful test of the hypotheses to explain SD evolution.
It is possible that AM chromosomes invaded house fly populations because of selection on phenotypic effects of either the autosomal M loci themselves or alleles linked to M - factors (Franco et al., 1982; Tomita and Wada, 1989; Kozielska et al., 2006; Feldmeyer et al., 2008). Strong linkage to AM is expected for alleles on the same autosome because recombination is low or non-existent in house fly males (Hiroyoshi, 1961; Hamm et al., 2015), but see Feldmeyer et al. (2010). Phenotypic effects of M variants include splicing and expression differences of genes downstream in the SD pathway between Y M and AM males (Schmidt et al., 1997; Hediger et al., 2004; Siegenthaler et al., 2009). In addition, AM chromosomes form stable latitudinal clines on multiple continents (Franco et al., 1982; Tomita and Wada, 1989; Hamm et al., 2005; Kozielska et al., 2008), and seasonality in temperature is somewhat predictive of their distribution (Feldmeyer et al., 2008). Furthermore, in laboratory experiments, IIIM males out-competed Y M males for female mates; the IIIM chromosome increased in frequency over generations in population cages; and IIIM males had higher rates of emergence from pupae than Y M males (Hamm et al., 2009). The most specific phenotype that has been linked to AM is insecticide resistance (Kerr, 1960, 1961, 1970; Denholm et al., 1983; Kence and Kence, 1992), but insecticide resistance alone cannot entirely explain the invasion of AM chromosomes (Shono and Scott, 1990; Hamm et al., 2005).
To test whether sex-specific selection pressures could be responsible for the invasion of the IIIM chromosome, we used high throughput mRNA sequencing (mRNA-seq) to compare gene expression profiles between nearly isogenic YM and IIIM males that only differ in their M -bearing chromosome. These contrasts are essentially a comparison between flies with the ancestral Y chromosome (YM) and individuals with a recently evolved “neo-Y” (IIIM). The gene expression differences we detected were the result of both differentiation of cis regulatory regions between the IIIM and “standard” third chromosome and trans effects of the IIIM and/or Y M chromosome(s) on expression throughout the genome. We found that genes responsible for male phenotypes are more likely to be differentially expressed between YM and IIIM males, suggesting that YM and IIIM males have phenotypic differences that would be differentially affected by male-specific selection pressures. This supports the hypothesis that sexual or sexually antagonistic selection drives evolutionary turnover at the top of SD pathways.
2. Materials and Methods
2.1. Strains
We compared gene expression between two nearly isogenic house fly strains that differ only in the chromosome carrying M. The first, Cornell susceptible (CS), is an inbred, lab adapted strain with XX males that are heterozygous for a IIIM chromosome and a standard third chromosome that lacks an M factor (X/X; IIIM /IIICS) (Scott et al., 1996; Hamm et al., 2005) (Figure 1A). CS females are XX and homozygous for the standard third chromosome (X/X; IIICS/IIICS). We created a strain with Y M males that has the X chromosome and all standard autosomes from the CS strain. To do so, we used a backcrossing approach to move the Y chromosome from the genome strain (aabys) onto the CS background (Figure 1B), creating the strain CS-aabys-Y (CSaY). CSaY males are XY and homozygous for the standard CS third chromosome (X/Y ; IIICS/IIICS). The aabys strain has a recessive phenotypic marker on each of the five autosomes (Wagoner, 1967; Tomita and Wada, 1989). To confirm that the aabys autosomes had been purged from the CSaY genome, we crossed CSaY flies to aabys and observed only wild-type progeny. CS and CSaY males are nearly isogenic, differing only in that CS males are XX and heterozygous for the IIIM and standard IIICS chromosomes, and CSaY males are XY and homozygous for the standard IIICS chromosome (Figure 1). Females are genetically identical between strains.
We are confident that the strains are isogenic except for the M -bearing chromosome because there is very little evidence for recombination in male house flies with an XY genotype (Hiroyoshi, 1961; Hamm et al., 2015). However, if there were minimal recombination between the CS and aabys chromosomes in our crossing scheme, the majority of autosomal alleles in the CSaY strain would still have originated from the CS genotype, with very little contribution from aabys autosomes.
2.2. Samples and mRNA-seq
CS and CSaY flies were kept at 25°C with a 12:12 hour light:dark cycle. Larvae were reared in media made with 1.8 L water, 500 g calf manna (Manna Pro, St. Louis, MO), 120 g bird and reptile litter wood chips (Northeastern Products, Warrensburg, NY), 60 g dry active baker’s yeast (MP Biomedical Solon, OH), and 1.21 kg wheat bran (Cargill Animal Nutrition, Minneapolis, MN), as described previously (Hamm et al., 2009).
We sampled two types of tissue from CS and CSaY males and females: head and gonad. All dissections were performed on living, non-anesthetized 4–6 day old unmated adult flies. Heads were separated from males and females, homogenized in TRIzol reagent (Life Technologies) using a motorized grinder, and RNA was extracted on QIAGEN RNeasy columns following the manufacturer’s instructions including a genomic DNA (gDNA) elimination step. Testes were dissected from males, and ovaries were dissected from females in Ringer’s solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris-Cl in ddH2O). Ovary and testis samples were dissolved in TRIzol and RNA was extracted on QIAGEN RNeasy columns with gDNA elimination. Three biological replicates of CS (IIIM) male heads, CSaY (Y M) male heads, CS testes, and CSaY testes were collected; one sample was collected for each of the four female dissections (CS head, CSaY head, CS ovary, and CSaY ovary).
Barcoded mRNA-seq libraries were prepared using the Illumina TruSeq kit following the manufacturer’s instructions. The samples were run on 2 lanes of an Illumina HiSeq2500 at the Cornell Medical School Genomics Resources Core Facility. One lane had the eight head samples, and the other lane had the eight gonad (testis and ovary) samples. We generated 101 base pair single-end reads, and the sequencing reads were processed using Casava 1.8.2.
2.3. mRNA-seq data analysis
Illumina mRNA-seq reads were aligned to house fly genome assembly v2.0.2 and annotation release 100 (Scott et al., 2014) using TopHat2 v2.0.8b (Kim et al., 2013) and Bowtie v2.1.0.0 (Langmead et al., 2009) with the default parameters. We tested for differential expression between males and females and between Y M and IIIM males using the Cuffdiff2 program in the Cufflinks v2.2.1 package (Trapnell et al., 2013) with the default parameters and a false discovery rate (FDR) of 0.05. In comparisons between male and female expression levels, we treated all 6 male samples as biological replicates and did the same for both female samples.
We used expression level estimates from Cuffdiff2 (Fragments Per Kilobase of transcript per Million mapped reads, FPKM) to calculate correlations of expression levels between our experimental samples (Figure S1). The correlations between testis and ovary expression are lowest, which is expected because they are dramatically different tissues. The correlations between male and female head samples are substantially higher than between testis and ovary, but still lower than the correlations within sexes. The two ovary samples are more highly correlated than any of the pairwise comparisons between CS and CSaY testis samples (Figure S1), most likely because CS and CSaY females are genetically identical (Figure 1).
2.4. Gene ontology and chromosomal assignment of house fly genes
We used the predicted Drosophila melanogaster orthologs (Scott et al., 2014) to infer the functions of house fly genes. Gene ontology (GO) annotations of house fly genes were determined using Blast2GO (Conesa et al., 2005; Götz et al., 2008) as described previously (Scott et al., 2014).
The house fly genomic scaffolds have not formally been assigned to chromosomes, but homologies have been inferred between house fly chromosomes and the five major chromosome arms of Drosophila, also known as Muller elements A–E (Foster et al., 1981; Weller and Foster, 1993). Additionally, the house fly X chromosome is most likely homologous to the Drosophila dot chromosome (Muller element F, or D. melanogaster chromosome 4) (Vicoso and Bachtrog, 2013). We therefore assigned house fly genes that are conserved as one-to-one orthologs with D. melanogaster (Scott et al., 2014) to house fly chromosomes based on the Muller element mapping of the D. melanogaster orthologs. We also assigned house fly scaffolds to chromosomes based on the Muller element mapping of the majority of D. melanogaster orthologs on each scaffold.
3. Results
3.1. Genes on the house fly third chromosome are more likely to be differentially expressed between YM and IIIM males than genes on other autosomes
We used mRNA-seq to compare gene expression in heads and gonads of house fly males and females of a Y M strain (CSaY) and a IIIM strain (CS). Males of the IIIM strain are XX and heterozygous for the IIIM chromosome and a standard third chromosome without M (Figure 1A). Males of the Y M strain are XY (with the same X as the IIIM strain) and homozygous for the standard third chromosome found in the IIIM strain (Figure 1B). The rest of the genome is isogenic, and females of the two strains are genetically identical (Figure 1).
We detected 873 and 1338 genes that are differentially expressed between Y M and IIIM males in heads or testes, respectively (Table 1, Figure S2, Supplementary Data). Genes on the house fly third chromosome are more likely than genes on other autosomes to be differentially expressed between Y M and IIIM males (Figure 2). Approximately 30% of the differentially expressed genes are predicted to be on the third chromosome, which is greater than the fraction assigned to any of the other four autosomes (14.8–20.6%). X-linked genes also trend towards an excess that are differentially expressed between Y M and IIIM males, but we do not have power to detect statistically significant deviations from the expectation because only 56 X-linked genes are expressed in head and 52 X-linked genes are expressed in testis.
3.2. More differential expression between YM and IIIM males in testis than in head, but a common set of genes co-regulated in both tissues
A higher fraction of genes is differentially expressed in testes between Y M and IIIM males than in heads (Table 1; P < 10−16 in Fisher’s exact test, FET), suggesting that genes involved in male fertility phenotypes are more affected by the M -bearing chromosome. In fact, the fraction of genes differentially expressed between the testes of YM and IIIM males is nearly as large as the fraction differentially expressed between male and female heads (Table 1). When we restrict the analysis to only genes expressed in both heads and gonads, we still observe more genes differentially expressed in testes than heads between Y M and IIIM males (Table S1).
If the probability that a gene is differentially expressed between Y M and IIIM male heads is independent of the probability that the gene is differentially expressed in testes, we expect <1% of genes to be differentially expressed in both head and testis. We find that 176 genes (2.12%) are differentially expressed between Y M and IIIM males in both head and testis, which is significantly greater than the expectation (P < 10−25, FET). In contrast, there is not a significant excess of genes differentially expressed between males and females (i.e., “sex-biased”) in both head and gonad—we expect 9.41% of genes to have sex-biased expression in both head and gonad (Table S1), and we observe that 809 genes (9.27%) are sex-biased in both tissue samples (P = 0.655, FET). These results suggest that there are genes under common regulatory control by the M -bearing chromosome in both male head and testis, but there is no such sex-specific regulation in common between head and gonad.
3.3. Genes that are differentially expressed between YM and IIIM males are more likely to have male-biased expression
Genes whose expression is significantly higher in males than females are said to have “malebiased” expression, and genes that are up-regulated in females have “female-biased” expression. Genes with male-biased expression in head are more likely to be differentially expressed between Y M and IIIM male heads than genes with either female-biased or unbiased expression (Figure 3A). Similarly, genes that are up-regulated in testis relative to ovary (“testis-biased”) are more likely to be differentially expressed between Y M and IIIM testes than genes with “ovary-biased” or unbiased expression in gonad (Figure 3B). In addition, 14.8% of genes that are up-regulated in IIIM male heads have male-biased expression, whereas <2% of genes that are up-regulated in Y M male heads have male-biased expression (P < 10−10, FET). This suggests that IIIM male heads have a “masculinized” expression profile relative to Y M heads.
3.4. Functional annotations of genes that are differentially expressed between YM and IIIM males
We tested for GO categories that are over-represented amongst genes differentially expressed both between Y M and IIIM males and between males and females (Supplementary Data). We found that nearly half (49.7%) of genes that are differentially expressed between Y M and IIIM male heads are annotated with the functional category “catalytic activity”, whereas only 43% of genes not differentially expressed have that GO annotation (P < 0.05 in FET corrected for multiple tests). Over 10% of the genes with the catalytic activity annotation that are up-regulated in IIIM male head are predicted to encode proteins involved in a metabolic process, including metabolism of organic acids, amino acids, and lipids. Among those genes, 15 are annotated as cytochrome P450 (CYP450) genes, and four of those also have male-biased expression in head (Table S4). CYP450s collectively carry out a wide range of chemical reactions including metabolism of endogenous (e.g., steroid hormones) and exogenous (e.g., xenobiotics) compounds (Scott, 2008). All 15 differentially expressed CYP450s are up-regulated in IIIM males, and no CYP450 genes are up-regulated in Y M males. Five of the CYP450s can be assigned to the third chromosome (Table S4), suggesting that cis regulatory sequences controlling the expression of CYP450s have diverged between IIIM and the standard third chromosome. However, five of the CYP450s can be assigned to other autosomes (the remaining 5 cannot be assigned to a chromosome), demonstrating that divergence of trans factors between IIIM and the standard third chromosome are also responsible for differential expression of CYP450s between Y M and IIIM males. The 15 CYP450s represent a range of different clans (2, 3 and 4) and families (4, 28, 304, 313, 438 and 3073). However, an excess of CYP450s from clan 4 are up-regulated in IIIM male head (χ2 = 4.19, P = 0.041), and thus over-expression of CYP450s is not random.
Genes that are annotated as encoding proteins located in extracellular regions are over-represented amongst genes with testis-biased expression (15.0% of genes with testis-biased expression; 9.9% of genes not differentially expressed between testis and ovary; P < 10−3 in FET corrected for multiple tests) and amongst genes that are differentially expressed between Y M and IIIM testes (13.9% of differentially expressed genes; 8.1% of non-differentially expressed genes; P < 10−4 in FET corrected for multiple tests). In addition, 3.1% of the genes differentially expressed between Y M and IIIM testes are predicted to encode carbohydrate binding proteins (compared to 1.4% of non-differentially expressed genes; P < 0.05 in FET corrected for multiple tests), and 7.2% of differentially expressed genes are predicted to encode structural molecules (compared to 3.7% of non-differentially expressed genes; P < 10−3 in FET corrected for multiple tests). Three of those structural molecules are predicted to be β-tubulin proteins encoded by genes that are up-regulated in the testis of Y M males relative to IIIM, and two of those genes also have testis-biased expression. The D. melanogaster genome encodes a testis-specific β-tubulin paralog that is essential for spermatogenesis (Kemphues et al., 1982; Hoyle and Raff, 1990), suggesting that at least one of the β-tubulin genes that is up-regulated in Y M testis may be important for sperm development.
Four genes that are differentially expressed between Y M and IIIM testes are homologs of the D. melanogaster Y-linked fertility factors kl-2, kl-3, and kl-5 (Goldstein et al., 1982; Gepner and Hays, 1993; Carvalho et al., 2000, 2001). These proteins encode components of the dynein heavy chain, which is necessary for flagellar activity of sperm. All four of the predicted dynein heavy chain genes that are differentially expressed between Y M and IIIM testes are autosomal in house fly. Three of these genes have testis-biased expression—two of those are up-regulated in IIIM testis, while the third is up-regulated in Y M testis. The fourth gene is up-regulated in IIIM testis, but it is not differentially expressed between testis and ovary. Two additional genes encoding components of other dynein chains have testis-biased expression and are up-regulated in IIIM testis relative to Y M testis.
Finally, there are numerous predicted RNAs in the house fly genome annotation that have no identifiable homology to any known RNAs or proteins (Scott et al., 2014). We identified six of these uncharacterized RNAs that both have testis-biased expression and are differentially expressed between Y M and IIIM testes (three up-regulated in Y M, three up-regulated in IIIM). Reproductive proteins are known to evolve rapidly (Swanson and Vacquier, 2002; Clark et al., 2006; Meisel, 2011), suggesting that these testis-biased genes may be evolving so fast that their homologs are undetectable. On the other hand, there are multiple examples of testis-expressed lineage-specific de novo protein-coding genes in Drosophila (Levine et al., 2006; Begun et al., 2007; Reinhardt et al., 2013; Zhao et al., 2014); the testis-biased house fly genes without identifiable homologs may have similarly arisen de novo along the house fly lineage. However, we were unable to detect long open reading frames in the transcripts, suggesting that these may be non-protein-coding RNAs with testis-biased expression.
4. Discussion
4.1. Differential expression between YM and IIIM males is driven by both cis and trans effects
We compared gene expression in head and testis between Y M and IIIM males. Y M males are homozygous for a standard third chromosome that does not have M, whereas IIIM males are heterozygous for a IIIM chromosome and a standard third chromosome (Figure 1). Differences in the expression levels of autosomal genes between Y M and IIIM males could be the result of: 1) divergence of cis-regulatory sequences between the IIIM and standard third chromosomes that affect the expression of genes on the third chromosome; 2) divergence of trans-factors between IIIM and the standard third chromosome that differentially regulate gene expression throughout the genome; 3) downstream effects of the first two processes that lead to further differential expression.
The two strains also differ in the genotype of their sex chromosomes; Y M males are XY, whereas IIIM males are XX (Figure 1). The house fly Y chromosome is highly heterochromatic and does not harbor any known genes other than M (Boyes et al., 1964; Hediger et al., 1998; Dübendorfer et al., 2002). It is clear that the Y chromosome does not contain any genes necessary for male fertility or viability because XX males are fertile and viable. The X chromosome is also highly heterochromatic and probably homologous to the Drosophila dot chromosome (Hediger et al., 1998; Vicoso and Bachtrog, 2013). The heterochromatic Drosophila Y chromosome can affect the expression of autosomal genes (Lemos et al., 2008, 2010; Sackton et al., 2011; Zhou et al., 2012), suggesting that the house fly X and Y chromosomes could have trans regulatory effects on autosomal gene expression (although none yet have been reported).
A higher fraction of third chromosome genes are differentially expressed between Y M and IIIM house fly males than genes on any other autosome (Figure 2). Therefore, divergence of cis-regulatory sequences between IIIM and the standard third chromosome are at least partially responsible for the expression differences between Y M and IIIM males. However, ∼70% of the genes differentially expressed between Y M and IIIM males map to one of the other four autosomes, suggesting that the majority of expression differences are the result of trans effects of the X, Y, and third chromosomes along with further downstream effects.
4.2. Reproductive and male phenotypes are more likely to be affected by M variation
Reproductive traits are more sexually dimorphic than non-reproductive traits, and reproductive traits also tend to evolve faster, possibly as a result of sexual selection (Eberhard, 1985). A similar faster evolution of gene expression in reproductive tissues has been observed across many taxa (Khaitovich et al., 2005; Zhang et al., 2007; Brawand et al., 2011), and increased variation within species for sex-biased gene expression often accompanies elevated expression divergence (Meiklejohn et al., 2003; Ayroles et al., 2009). Consistent with these patterns, more genes are differentially expressed between Y M and IIIM males in testis than head (Table 1, Figure S2).
Somatic and germline SD in house fly are under the same genetic control (Hilfiker-Kleiner et al., 1994), so the exaggerated differences in expression between Y M and IIIM testes relative to heads cannot be attributed to differences in the SD pathway between gonad and head. We also find that genes with male-biased expression are more likely to be differentially expressed between Y M and IIIM males (Figure 3). Genes with male-biased expression are more likely to perform sex-specific functions (Connallon and Clark, 2011), suggesting that genes that are differentially expressed between Y M and IIIM males disproportionately affect male phenotypes.
4.3. Evaluating the role of sex-specific selection in MSD turnover
Many models of SD evolution predict that a new MSD locus will invade a population if it is genetically linked to an allele with a beneficial, sexually selected, or sexually antagonistic fitness effect (Charlesworth and Charlesworth, 1980; Rice, 1987; Charlesworth, 1991, 1996; Rice, 1996; van Doorn and Kirkpatrick, 2007, 2010). Alternatively, evolutionary turnover of MSD loci could be the result of neutral drift in a highly labile system (van Doorn, 2014).
The differential expression between Y M and IIIM males is consistent with a model in which the IIIM chromosome invaded populations because it harbors alleles with male-specific beneficial effects. For example, the expression of genes that are likely to perform male-specific functions—especially in male fertility—are more likely to be affected by the IIIM chromosome (Table 1; Figure 3), and those male-specific phenotypic differences could have been targets of sex-specific selection pressures. In addition, IIIM heads have a masculinized expression profile relative to Y M heads (see Results), suggesting that the male-limited transmission of the IIIM chromosome favored the accumulation of alleles with male-beneficial fitness effects (Rice, 1984). Previous work found that IIIM males outperformed Y M males in multiple laboratory fitness assays (Hamm et al., 2009), providing additional support for the accumulation of male-beneficial alleles on the IIIM chromosome. However, despite the apparent selective advantage of the IIIM chromosome, it surprisingly does not appear to be expanding rapidly (Hamm et al., 2015), suggesting that the fitness benefits of IIIM are environment-specific (Feldmeyer et al., 2008).
Our data do not allow us to distinguish between two possible orders of events in the invasion of the IIIM chromosome. In the first scenario, male-beneficial alleles on the third chromosome could have driven the initial invasion of IIIM (van Doorn and Kirkpatrick, 2007). In the second scenario, beneficial alleles could have accumulated on the IIIM chromosome after it acquired an M -locus because male-limited inheritance promotes the fixation of male-beneficial alleles (Rice, 1984, 1987). These scenarios are not mutually exclusive. Regardless of the sequence of events, we have provided evidence that the house fly multifactorial male-determining system is associated with phenotypic differences that likely have male-specific fitness effects, which could explain the invasion of the IIIM chromosome under sexual or sexually antagonistic selection.
Supplementary Data
mRNA-seq results
Column information:
gene - NCBI gene symbol
protein id - NCBI protein identifier
locus - Coordinates in genomic scaffold
orthocat - Orthology category:
sco: single-copy ortholog
mto1: multiple house fly genes orthologous to 1 D. melanogaster gene
1tom: 1 house fly gene orthologous to many D. melanogaster genes
mtom: multiple house fly genes homologous to multiply D. melanogaster genes
lr: lineage-restricted house fly gene without any identified D. melanogaster orthologs
og - Orthology group (gene family)
dmel - FlyBase identifier of D. melanogaster orthologs
Dmel_ME - Muller element location of D. melanogaster ortholog (only if sco)
Musca_chr - House fly chromosome homolog of Muller element
scaff_ME - Muller element assignment of house fly scaffold (based on majority rule of genes on scaffold)
Musca_scaff - House fly chromosome based on scaff_ME
CS_MaleHead_FPKM - Expression level in CS (IIIM) male head
CSaY_MaleHead_FPKM - Expression level in CSaY (YM) male head
MaleHead_TestStat - Test statistic comparing CS and CSaY male head expression levels
MaleHead_qval - FDR corrected p-value of test statistic comparing CS and CSaY male head expression levels
CS_testis_FPKM - Expression level in CS (IIIM) testis
CSaY_testis_FPKM - Expression level in CSaY (YM) testis
testis_TestStat - Test statistic comparing CS and CSaY testis expression levels
testis_qval - FDR corrected p-value of test statistic comparing CS and CSaY testis expression levels
CS_FemaleHead_FPKM - Expression level in CS female head
CSaY_FemaleHead_FPKM - Expression level in CSaY female head
FemaleHead_TestStat - Test statistic comparing CS and CSaY female head expression levels
FemaleHead_qval - FDR corrected p-value of test statistic comparing CS and CSaY female head expression levels
CS_ovary_FPKM - Expression level in CS ovary
CSaY_ovary_FPKM - Expression level in CSaY ovary
ovary_TestStat - Test statistic comparing CS and CSaY ovary expression levels
ovary_qval - FDR corrected p-value of test statistic comparing CS and CSaY ovary expression levels
female_head_FPKM - Expression level in female head
male_head_FPKM - Expression level in CS male head
head_TestStat - Test statistic comparing female and male head expression levels
head_qval - FDR corrected p-value of test statistic comparing female and male head expression levels
ovary_FPKM - Expression level in ovary
testis_FPKM - Expression level in testis
gonad_TestStat - Test statistic comparing ovary and testis expression levels
gonad_qval - FDR corrected p-value of test statistic comparing ovary and testis expression levels
Gene ontology tests
File information (test vs reference comparisons):
head_female-biased_GO.txt - GO categories that are enriched in genes with female-biased expression in head (foreground) vs genes with non-sex-biased expression in head
head_male-biased_GO.txt - GO categories that are enriched in genes with male-biased expression in head vs genes with non-sex-biased expression in head
male_head_diff_GO.txt - GO categories that are enriched in genes that are differentially expressed between CS and CSaY male heads vs genes that are not differentially expressed between CS and CsaY male heads
male_testis_diff_GO.txt - GO categories that are enriched in genes that are differentially expressed between CS and CSaY testes vs genes that are not differentially expressed between CS and CsaY testes
ovary-biased_unbiased_GO.txt - GO categories that are enriched in genes with ovary-biased expression vs genes with non-sex-biased expression in gonad
testis-biased_unbiased_GO.txt - GO categories that are enriched in genes with testis-biased expression vs genes with non-sex-biased expression in gonad
Column information:
GO-ID - Gene ontology ID
Term - GO term
Category - Cellular Component (C), Biological Process (P), or Molecular Function (F)
FDR - False discovery corrected P-value for enrichment
P-Value - Fisher’s exact test comparing #Test, #Ref, #notAnnotTest, #notAnnotRef
#Test - Count of genes with ontology annotation that are differentially expressed
#Ref - Count of genes with ontology annotation that are not differentially expressed
#notAnnotTest - Count of genes without ontology annotation that are differentially expressed
#notAnnotRef - Count of genes without ontology annotation that are not differentially expressed
Over/Under - Term is enriched (over) or depleted (under) in test data set
Acknowledgments
5. Acknowledgements
Cheryl Leichter, Naveen Galla, and Daniel Chazen assisted with the creation of the CSaY strain, and Amanda Manfredo prepared the mRNA-seq libraries. This work benefited from discussions with members of the Clark lab. We were supported by multistate project S-1030 to JGS, NIH grant R01-GM64590 to AGC and A. Bernardo Carvalho, and start-up funds from the University of Houston to RPM. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.
Footnotes
Data deposition: All data have been deposited in the NCBI Gene Expression Omnibus under accession GSE67065.