The Effect of Hybridization on Dosage Compensation in Member Species of the Anopheles gambiae Species Complex

The Effect of Hybridization on Dosage Compensation in Member Species of the Anopheles gambiae... Dosage compensation has evolved in concert with Y-chromosome degeneration in many taxa that exhibit heterogametic sex chromosomes. Dosage compensation overcomes the biological challenge of a "half dose" of X chromosome gene transcripts in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes and interact with genes expressed on the autosomes. Sex determination and heterogametic sex chromosomes have evolved multiple times in Diptera, and in each case the genetic control of dosage compensation is tightly linked to sex determination. In the Anopheles gambiae species complex (Culicidae), maleness is conferred by the Y-chromosome gene Yob, which despite its conserved role between species is polymorphic in its copy number between them. Previous work demonstrated that male An. gambiae s.s. males exhibit complete dosage compensation in pupal and adult stages. In the present study we have extended this analysis to three sister species in the An. gambiae complex: An. coluzzii, An. arabiensis, and An. quadriannulatus. In addition, we analyzed dosage compensation in bi-directional F1 hybrids © The Author(s) 2018. . Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 between these species to determine if hybridization results in the mis-regulation and disruption of dosage compensation. Our results confirm that dosage compensation operates in the An. gambiae species complex through the hyper-transcription of the male X chromosome. Additionally, dosage compensation in hybrid males does not differ from parental males, indicating that hybridization does not result in the mis-regulation of dosage compensation. Keywords: Anopheles, Dosage Compensation, Hybridization, RNAseq Introduction Sex chromosomes evolved independently from autosomes multiple times within Diptera (Vicoso and Bachtrog, 2015, Bachtrog et al., 2014). While X chromosomes largely retain their original gene content (Vicoso and Charlesworth, 2006), sexually antagonistic mutations that occur on nascent Y-chromosomes suppress recombination with the X (Rice 1996). This leads to Y-chromosome degeneration as male-biased genes move from the X to the Y chromosome or the autosomes or are lost (Parisi et al., 2003), and eventually the formation of heterogametic sex chromosomes (Bachtrog 2013). Old Y-chromosomes harbor few protein coding genes and are repeat-rich, hampering, until recently, their sequencing and characterization (Mahajan and Bachtrog, 2017). As neo-Y chromosomes degenerate, X-linked genes that are not female-biased in their expression must be equalized or compensated for in the heterogametic sex. The evolution of dosage compensation (DC) has occurred in concert with Y- chromosome degeneration in many taxa that exhibit heterogametic sex determination (Disteche 2012, Graves 2015). DC overcomes the biological challenge of a "half dose" of X chromosome gene transcripts (or Z chromosome Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 transcripts in ZZ/ZW systems) in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes. In Drosophila and Anopheles the regulation of DC is closely tied to the sex-determination pathway despite striking differences in sex determination between these genera. In Drosophila females, a double dose of X chromosome-linked signal elements (XSE) initiates female-specific pre-mRNA splicing of sex lethal (Sxl) transcripts. Sxl in turn regulates sex-specific splicing of transformer (tra), which, along with its non-sex-specific co-factor transformer2 (tra2), promotes female-specific splicing of doublesex (dsx) and fruitless (fru) pre-mRNAs. Male and female isoforms of dsx and fru modulate the expression of genes involved in sexually dimorphic morphologies and physiologies. A single dose of XSE fails to initiate the cascade of female- specific splicing and male transcripts are produced (Penalva and Sanchez, 2003). While male and female isoforms of DSX share the same DNA-binding domain, they differ in their C-terminal domains that confer sex-specific gene regulation (Clough et al., 2014). Dsx and fru are highly conserved in insects despite their variety of sex-determination mechanisms (Herpin and Schartl, 2015, Salz and Erickson, 2010, Wilkins 1995). In mosquitoes (Diptera: Culicidae), sex determination is controlled by a M-factor located on the Y chromosome in the subfamily Anophelinae, which has heterogametic sex chromosomes (Krzywinska et al., 2016), or on the homomorphic sex-determining chromosome of the subfamily Culicinae (Hall et al., 2016). Transcription of the M-factor during early embryo development activates a cascade of sex-specific gene splicing that controls male development through sex-specific splicing of dsx and fru. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 In the Anopheles gambiae species complex the M-factor Yob is located within the male- determining locus of the Y chromosome. Yob expression is male-specific, begins within 2.5 hours of embryo oviposition, and occurs prior to dsx splicing by up to six hours (Krzywinska et al., 2016). This indicates that Yob is either a direct or indirect upstream regulator of dsx splicing. Yob is the only protein-coding gene on the Y chromosome that is shared by all members of the An. gambiae species complex (Hall et al., 2016). Yob is polymorphic in copy number between An. gambiae complex species and within An. gambiae s.s. (Hall et al. 2016). An. stephensi, which diverged from the An. gambiae species complex ~30 million years ago, lacks a Yob homolog, indicating a recent origin of Yob in the An. gambiae complex lineage (Hall et al. 2016). Bernardini et al. (2017) were able introgress the An. gambiae Y chromosome into an An. arabiensis genetic background using an F1 x F0 crossing strategy, which allowed them to overcome a severe bottleneck associated with this cross. They found that when isolated, An. gambiae and An. arabiensis Y-chromosomes are interchangeable; the An. gambiae Y chromosome has no effect on An. arabiensis gene expression and its introgression does not cause male sterility. Thus, by itself a heterospecific Y-chromosome does not appear to disrupt dosage compensation in this cross. However, we do not know how dosage compensation is affected in F1 hybrids when the genetic background of the autosomes is not homozygous. DC of the hemizygous X in male Drosophila occurs through the binding of the male- specific lethal (MSL) complex to the X. The MSL complex is comprised of four proteins encoded by the male-specific lethal (msl) genes 1-3 and maleless (mle), all of which cause male inviability when mutated (Penalva and Sanchez, 2003). Sxl mutations, or genes impacting its regulation, result in over-expression of genes on the X chromosome. This results in female lethality during embryogenesis (Erickson and Quintero, 2007, Lucchesi and Skripsky, 1981, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Gergen 1987, Hilfiker and Amrien, 1995) likely due to the mis-regulation of DC (Biedler and Tu, 2016, Cline 1978). Like Drosophila, An. stephensi and An. gambiae s.s exhibit complete DC through the hyper-transcription of the X chromosome (Jing et al., 2015, Rose et al., 2016). Ectopic injections of Yob mRNA into An. gambiae embryos causes female lethality due to abnormal X chromosome transcription, but has no effect on males. Additionally, knockdown of Yob in male An. gambiae embryos results in 100% mortality (Krzywinska et al., 2016). Krzywinska et al (2016) suggested that these results implicate Yob as an upstream regulator of DC in Anopheles gambiae due to the interconnected roles of sex-determination genes and DC regulatory pathways in other insects. Orthologs of the five Drosophila genes that encode the MSL complex are present in the An. gambiae transcriptome. However, Anopheles msl-1 and msl-2 are highly diverged from their Drosophila orthologs (Rose et al., 2016), which may indicate that they may have different functions in Anopheles. Studies in Drosophila initially implicated defects in DC regulation in hybrid male lethality (Chatterjee et al., 2007, Rodriguez et al., 2007, Bachtrog 2008) under a model where MSL proteins and their binding sites evolved on the D. melanogaster lineage in a way that a heterospecific mix of MSL complex proteins in F1 D. melanogaster - D. simulans hybrid males did not effectively regulate DC. Barbash (2010) subsequently refuted this hypothesis through a genetic screen of mle and msl mutant lines. The An. gambiae species complex harbors the most important vectors of human malaria and has recently emerged as a model system for the study of speciation genetics (Neafsey et al., 2015). However, little is known about dosage compensation regulation in Anopheles: sex determination differs drastically between Drosophila and Anopheles, our knowledge of the link Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 between Yob expression and DC regulation is incomplete, and msl-1 and msl-2 are highly diverged in sequence, and perhaps function, between Drosophila and Anopheles. In the present study, we explored whether DC regulation has diverged between members of An. gambiae species complex, and is mis-regulated in their hybrids, through an analysis of genome-wide gene expression in three sister species: An. coluzzii, An. arabiensis, and An. quadriannulatus, as well as reciprocal F1 hybrids derived from An. coluzzii x An. arabiensis and An. coluzzi x An. quadriannulatus crosses. Materials and Methods Mosquito Rearing Mosquitoes were derived from the SUA2La (An. coluzzii), SANGQUA (aka SANGWE, An. quadriannulatus), and DONGOLA (An. arabiensis) strains, or from bi-directional crosses between An. coluzzii and An. arabiensis, as well as An. coluzzii and An. quadriannulatus. Cross directions are indicated by female x male. Mosquitoes were reared in 12 hr light / 12 hr dark cycles at 25 ˚C and 70-80% relative humidity with access to 5% sucrose solution. Colonies were blood fed via an artificial membrane with warmed, defibrinated sheep blood (Hemostat Laboratories, Dixon, CA). Adults were provided wetted filter paper during the period of 48-72 hr post-feeding for oviposition. Larvae were fed Tetra brand TetraColor Tropical Crisps (fish food) ad libitum until pupation. Mosquitoes were collected 12 hr post-pupation and sexed prior to RNA extraction. Bi-directional crosses were performed by sexing 50-100 male and female pupae from the respective parental colonies prior to mixing them in adult cages. Rearing conditions of F1 larvae and collection of F1 pupae were performed as described above. RNA Isolation and Sequencing Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Mosquitoes were sexed 12 hr post-pupation. Total RNA extractions were performed on five pupae per replicate using a Qiagen RNeasy Mini kit (Qiagen, Redwood City, CA). RNA samples were quantified using a NanoDrop spectrophotometer and their quality was assessed using an Agilent Bioanalyzer. Equimolar amounts of two to four extraction products (10 or 20 total mosquito pupae) were combined to form a single biological replicate. Pooled RNA samples were again run on the Agilent Bioanalyzer and NanoDrop spectrophotometer prior to library prep and sequencing. Library prep was performed using an Illumina TruSeq RNA library prep kit (Illumina Inc., San Diego, CA). Two biological replicates for each strain, cross, and sex (28 libraries total) were sequenced on seven lanes of Illumina HiSeq 2500 machine using 125 bp single-end chemistry. Raw RNA sequencing reads are available on the NCBI Sequence Read Archive at project accession number PRJNAXXXXXX. Pseudo-Genome Construction The first step of our analysis was to construct an An. gambiae-based pseudo-genome for each parental species. By incorporating SNPs from each species into the AgamP4 genome, we were able to use the AgamP4 coordinates and gene set as a common framework to quantify transcript abundance. FastQC package version 0.11.4 (Babraham Bioinformatics) was used to examine read quality. Illumina TruSeq adapters were removed using Trimmomatic version 0.30 (Bolger et al., 2014), while simultaneously soft-clipping the reads from both 5' and 3' ends to an average phred quality score of 20, with no single bp in a four bp window below a phred quality score of 10. Only reads ≥ 50 bp were retained for alignment and subsequent analyses. Trimmed RNAseq data from the three species were initially aligned to their respective, species-specific genomes, which were obtained from VectorBase.org (AcolM1 for An. coluzzii, AaraD1 for An. arabiensis, and AquaS1 for An. quadriannulatus, Giraldo-Calderon et al., 2015). Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Initial alignments were performed using the splice-aware alignment software STAR version 2.4.2a using default parameters with the exception of the option "-- outFilterMismatchNoverLmax 0.1" (Dobin et al., 2013). Next, an index was created for each alignment, which annotated all splice junctions found during the first alignment round. This index informed the second alignment with STAR (as above), which re-aligned the reads around splice junctions. Picard tools version 2.8.1 (http://broadinstitute.github.io/picard/) was used to mark and remove PCR duplicates. BAM alignments from each of the three species (two biological replicates of males and females) were then merged using Picard MergeBamAlignment, and SAMtools/BCFtools version 1.3 (Li et al., 2009) was used to call variants within each merged alignment using the BCFtools "call" tool. This process was informed by a prior mutation rate of 0.02. Next, GATK was used to filter variant call files for only bi-allelic SNPs. The vcfutils.pl varFilter function, a component of the BCFtools package, was used to filter SNPs using default parameters with the exception of not filtering for strand bias, as this can be expected in RNAseq data. Filtered SNPs were converted to the genomic coordinates of the An. gambiae s.s. PEST strain AgamP4 genome (Holt et al., 2002) using the Picard Tools (github.com/broadinstitute/picard ) LiftoverVCF function and a chain file to match base-pair coordinates between genomes. Chain files were created from pair-wise multiple-alignment files between the An. coluzzii, An. arabiensis, and An. quadriannulatus genomes and the An. gambiae s.s. PEST reference originally published by Fontaine et al. (2015), which were downloaded from VectorBase.org. Once SNPs were converted to AgamP4 coordinates, they were incorporated into the AgamP4 genome using the GATK FastaAlternateReferenceMaker to create a pseudo-genome for each species using the AgamP4 backbone/coordinates. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Next, we again performed a STAR 2-pass alignment (as above) for each parental library using the species-specific, AgamP4-baesed pseudo-genome as a reference. SNPs were called using the methods described above, and were then incorporated into the previous pseudo-genome reference to further populate the AgamP4-based pseudo-genome of each species with variation from that strain. Subsequently, the AgamP4-based pseudo-genomes were used as a reference to create a cDNA pseudo-genome for each species using the generate_transcripts function of rSeq version 0.2.2 (Jiang et al., 2009, Salzman et al., 2011). Gene transcript coordinates were derived from the AgamP4.4 gene set (VectroBase.org). Quantification of Gene Expression Parental species libraries (An. coluzzii, An. arabiensis, and An. quadriannulatus) were aligned to their respective cDNA pseudo-genomes using the --very-sensitive mode of Bowtie 2 version 2.2.9 with default parameters (Langmead and Salzberg, 2012). Only uniquely mapped reads were retained to calculate transcript abundances. RNAseq reads from F1 hybrids were aligned to diploid pseudo-genomes comprised of the AgamP4.4 based cDNA pseudo-genomes of each parental species. rSeq version 0.2.2 (Jiang et al., 2009, Salzman et al., 2011) was used to create cDNA genomes from the AgamP4 based pseudo-genomes of each parental species (as above). The maternal and paternal cDNA genomes for each hybrid were then merged into a diploid genome using the ASE-TIGAR program (Nariai et al., 2016, http://nagasakilab.csml.org/ase-tigar/), which concatenates the cDNA genomes of the maternal and paternal strains into one genome file. RNAseq reads from F1 hybrids were assessed for quality and trimmed using FastQC and Trimmomatic as described above. Reads were mapped to the diploid, bi-parental cDNA pseudo-genomes for the respective cross using the --very-sensitive mode Bowtie 2 version 2.2.9 with default parameters (Langmead and Salzberg, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 2012). F1 hybrid reads were allowed to align up to 100 times across the diploid reference genome. ASE-TIGAR was used to identify the most likely parental genome of origin and location of each transcript. Thus, only the highest probability map position of each transcript was quantified. Total transcript abundance (maternal + paternal expression in F1 hybrids) was used for the subsequent analysis of dosage compensation. Analysis of Dosage Compensation Transcript abundances were standardized by converting them to RPKMs (reads per kilo- base of transcript per million reads) in R (www.cran.r-project.org, R Core Team, 2013). Two methods were used to assess dosage compensation in species and F1 hybrid samples. First, the ratio of median expression of X-linked and autosomal genes in each sample was determined. This was performed by dividing the RPKM value of each X-linked gene by the median RPKM expression level of all autosomal loci. The "boot" package in R (Davison and Hinkley, 1997, Cantly and Ripley, 2015) was used to calculate 95% confidence intervals of the median of this distribution for each sample using 10,000 replicates. X to autosomal (X:A) median gene expression was analyzed at increasing minimum RPKM values between zero and 10. In order to be included in the analysis at a specific RPKM threshold, a gene had to exceed the specified RPKM level in all samples in a species comparison (ex. An. coluzzii, An. arabiensis, and their hybrids). Because of this, the number of genes included in this analysis differs for An. coluzzi between the An. coluzzi - An. arabiensis and the An. coluzzi - An. quadriannulatus species comparisons. An analysis of X:A median gene expression without RPKM cut-off (RPKM ≥ 0.0) would also include transcriptionally inactive genes which would bias the analyses (Kharchenko et al., 2011). Additionally, work in Drosophila has shown that genes with low expression have a lower degree of dosage compensation (McAnally and Yampolsky, 2010). Thus, by analyzing Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 X:A expression ratios at increasing minimum RPKM thresholds (RPKM > 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0) we were able to identify the minimum RPKM level that removes bias during subsequent gene expression analyses. A comparison of median X:A gene expression ratios between males and females shows if genes on the male hemizygous X chromosome is hyper-expressed to match the level of X chromosome expression in females. In females we expect X:A expression ratios to be at or near one. If the X chromosome is hyper-expressed in males, the X:A expression ratio should be similar to that of females. If dosage compensation is absent, the X:A expression ratio of males should be half that of females. X:A expression ratios in males and females were also compared to the expression ratio of the second and third chromosomes (2:3). We expect the 2:3 expression ratio to be equal in both sexes. Thus, 2:3 median expression serves as an internal standard with which to compare X:A median expression. The 2:3 expression ratios were calculated by correcting the RPKM value of each chromosome 2 gene by the median RPKM of all chromosome 3 genes. The 2:3 expression ratios were also analyzed at increasing minimum RPKM thresholds (RPKM > 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0). The 95% confidence intervals of the median of the expression ratio distribution was calculated using the "boot" package in R using 10,000 replicates (Davison and Hinkley, 1997, Cantly and Ripley, 2015). A Kruskal-Wallis test was used to test for significant differences between the median of the X:A expression ratio distribution between each sample (Kruskal and Wallis, 1952). We used an ANOVA to test for significant differences in the mean of the X:A expression ratio distribution within and between each sample in a species comparison. The same tests were performed to analyze significant differences between the distributions of 2:3 expression ratios between samples. Where appropriate, a Tukey's post-hoc test (Tukey 1949) was Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 performed to test for significant differences in the means of pair-wise comparisons, and a Dunn's test (Dunn 1964) was used to test for significant differences in the medians of pair-wise comparisons. Additionally, we calculated male to female (M:F) expression ratios of X-linked and autosomal genes. Genes were only included in this analysis if they exceeded a RPKM of 10.0 across all samples in a comparison (see results). The expectation of this approach is that the M:F ratio of X-linked and the M:F ratio of autosomal genes will not differ if dosage compensation is operating effectively. M:F expression ratios were calculated by first calculating the mean RPKM expression of each gene between the two biological replicates for each sample. Then, the male to female expression ratio was calculated for each gene within a species or F1 hybrid. Medians and 95% confidence intervals were calculated for X-linked and autosomal distributions separately. The 95% confidence intervals of the median of each M:F expression ratio distribution were calculated as described above. An ANOVA was used to test for significant differences between the means of the X-linked, M:F expression ratio distribution and the autosomal M:F expression ratio distribution within each species and F1 hybrid. Similarly, a Kruskal-Wallis test was used to test for significant differences between the medians of the X-linked, M:F expression ratio distribution and the autosomal M:F expression ratio distribution within each species and F1 hybrid. Results RNA Sequencing and Alignment The sequencing effort yielded an average of 67.23 million reads per sample, though this number is skewed by one sample (COLZ x QUAD Female 2), which had 248.18 million reads (Supplementary Tables S1, S2). If this sample is excluded, the mean number of reads per sample Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 is 60.53 million, ranging from 51.73 to 75.01 million. The mean mapping efficiency of parental libraries to their respective, species-specific reference genomes was 85.5% (min. 80.9%, max 88.7%, Supplementary Table S1). The mapping efficiency of parental species libraries increased when aligned to their respective AgamP4-based pseudo-genomes (mean 97.3%, min. 95.2%, max 99.1%, Supplementary Table S1). The mapping efficiency of all libraries to their respective cDNA pseudo-genomes was reduced in comparison to either DNA genome alignment (mean 63.8%, min. 62.3%, max 65.2%, Supplementary Table S1). However, this reduction was uniform across species and may result from an incomplete AgamP4 gene annotation. F1 hybrid RNAseq reads were aligned to the respective bi-parental cDNA pseudo-genome for that cross. After filtering BAM alignments with ASE-Tigar, the average mapping efficiency for F1 hybrid libraries was 64.3% (min. 62.3%, max. 66.7%, Supplementary Table S2). Thus, the mapping efficiency of F1 hybrid and parental libraries to the cDNA pseudo-genomes was equivalent. Dosage compensation in An. coluzzii, An. arabiensis, and An. quadriannulatus The analysis of dosage compensation can be biased when genes with very low expression are included. To overcome this challenge we calculated X:A and chromosome 2:3 gene expression ratios at increasing minimum RPKM thresholds from zero to 10. The number of genes included in the An. coluzzii - An. arabiensis and An. coluzzii - An. quadriannulatus comparison datasets do not differ substantially at any RPKM threshold (Supplementary Table S3). The medians of the X:A and 2:3 distributions were tested for significant differences at each RPKM threshold. X:A median expression ratios approach 1.0 for male and female An. coluzzii, An. arabiensis, and An. quadriannulatus as the RPKM threshold increases (Supplementary Tables Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 S4, S5), and at RPKM > 10, all 95% confidence intervals of the median exression ratios include 1.0 (Figure 1, Table 1). The same is true for 2:3 median expression ratios for males and females of all parental species (Figure 1, Supplementary Tables S8, S9). This result demonstrates that at RPKM > 10, 2:3 median expression ratios serve as an appropriate control with which to compare X:A expression in the effort to determine if dosage compensation is active in males. We tested for significant differences between the medians of the X:A and 2:3 expression ratio distributions in males and females of each parental species and found no significant differences at a minimum RPKM > 10.0 (X p-value > 0.05, Supplementary Tables S8, S9). This result was confirmed through pair-wise tests for differences between of X:A and 2:3 median expression ratios between males and females, with in each parental species. X:A an 2:3 median expression ratios did not differ between any pair-wise comparisons of males and females at RPKM > 2.0 (Supplementary Tables S10, S11). Another way to explore whether dosage compensation is operating is by comparing directly the median male to female expression ratios of X-linked and autosomal genes. Due to the under-representation of male-biased on the X-chromosome (Magnusson et al., 2011, Magnusson et al., 2012), we expect the male to female expression ratios of X-linked genes to be female-biased, but nonetheless close to one. Based on the findings of the chromosome ratio analyses (above), only genes with a minimum RPKM > 10.0 across all samples in a species comparison were included in this analysis. The median M:F expression ratios ranged between 0.89 and 0.99 for X-linked genes in parental species, and between 0.91 and 1.05 for autosomal loci (Table 2, Figure 2). An ANOVA found no significant difference between the means of the X-linked and autosomal distributions, however, a Dunn's test found significant differences between the medians of the distributions Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 (Table 2). Despite these differences, all values are close to 1.0. In each species, the median of the M:F expression ratio distribution was lower for X-linked genes vs. autosomal genes (Figure 2), indicating, as expected, a higher proportion of male-biased genes expressed on the autosomes compared to the X chromosome. An additional explanation for this pattern (though not mutually exclusive) is a higher proportion of female-biased genes on the X chromosome relative to the autosomes. However, previous studies found that genes with female-biased expression in An. gambiae are randomly distributed amongst the X chromosome and autosomes (Magnusson et al., 2012). These results confirm that dosage compensation is acting to balance X chromosome and autosomal gene expression in hemizygous males of An. coluzzi, An. arabiensis, and An. quadriannulatus, and that X chromosome dosage is equivalent between males of these species. Dosage compensation in Anopheles gambiae complex hybrids Like parental species, X:A and 2:3 median expression ratios approach 1.0 for male and female F1 hybrids as the RPKM threshold increases regardless of the parental species or direction of the cross (Supplementary Tables S4, S5). At RPKM > 10 the 95% confidence intervals of the X:A median expression ratios include 1.0 for all hybrids (Figure 1, Table 1). The same is true for 2:3 median expression ratios (Supplementary Tables S6, S7). The X:A median expression ratio of bi-directional hybrids between An. coluzzii and An. arabiensis do not differ significantly from parental species at RPKM > 0.2 (Supplementary Table 4) or at RPKM > 0.5 for 2:3 expression ratios (Supplementary Table 6). A Kruskal-Wallis test found no significant differences between median X:A and 2:3 expression ratios within both males and female hybrids at RPKM > 10 (Supplementary Table S8). Additionally, between sex Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 comparisons of median X:A and 2:3 expression ratios in F1 hybrids found no significant differences at RPKM > 10 (Supplementary Table S10). Similar patterns were found in bi-directional hybrids between An. coluzzi and An. quadriannulatus. The median X:A expression ratios of F1 males and females did not differ from parental species at RPKM > 10 (Supplementary Table S5), and median 2:3 expression ratios did not differ above RPKM > 0.5 (Supplementary Table S7). No significant differences were found between median X:A and 2:3 expression ratios within both males and female hybrids at RPKM > 10 (Supplementary Table S9), with on exception (COLZ x QUAD Female 1, Kruskal-Wallis p- value = 0.036). However, this difference is not significant after Bonferroni correction, and this result has no relevant bearing on our dosage compensation analyses. Finally, between sex comparisons of median X:A and 2:3 expression ratios in F1 hybrids found no significant differences above RPKM > 0.5 (Supplementary Table S11). The median M:F expression ratios of ranged between 0.87 and 1.07 for X-linked genes in hybrids, and between 0.90 and 1.11 for autosomal loci (Table 2, Figure 2). Corresponding to the analysis of parental species, an ANOVA found no significant difference between the means of the X-linked and autosomal distributions, but a Dunn's test found significant differences between the medians (Table 2). These results demonstrate that hybridization between An. coluzzii and An. arabiensis, and An. coluzzii and An. quadriannulatus, does not result in the mis-regulation and disruption of dosage compensation in F1 hybrid males. Discussion We analyzed dosage compensation in the closely related species within the Anopheles gambiae complex; An. coluzzii, An. arabiensis, and An. quadriannulatus, as well as in their F1 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 hybrids. Dosage compensation was measured by comparing the ratio of male to female gene expression on the X-chromosome and autosomes, and by comparing X-chromosome vs. autosome median gene expression levels in males and females. By analyzing bi-directional crosses between An. coluzzii - An. arabiensis and An. coluzzii - An. quadriannulatu, we tested if the species from which the Y or X chromosome is inherited affects dosage compensation in male hybrids. Specifically, these comparisons tested if the observed variation in the M locus between these closely related species may impact dosage compensation and if trans-acting factors from the heterozygous autosomes of hybrids affected X chromosome transcription differently depending on which parental species the X chromosome was inherited from. Our results correspond with previous studies that compared X:A and M:F expression ratios in An. stephensi and An. gambiae s.s. to demonstrate that dosage compensation acts in males through the hyper-transcription of the hemizygous X (Jiang et al., 2015, Rose et al., 2016). Jiang et al. (2015) reported median X:A expression ratios in An. stephensi males and females between 0.92 and 0.98 at minimum RPKM thresholds between 0.0 and 4.0, with no significant differences between median X and autosomal expression in males or females above a minimum RPKM > 2.0. In addition, these authors compared expression ratios between An. stephensi X- linked genes and their one-to-one orthologs in Ae. aegypti, a species with homomorphic sex chromosomes. The median RPKM ratio of X-linked An. stephensi genes to their Ae. aegypti orthologs was close to one in both sexes after normalization, indicating full dosage compensation of these genes on the hemizygous X chromosome of An. stephensi males. The evolution of sex determination and heteromorphic sex chromosomes evolved independently between Drosophila and Anopheles mosquitoes. Drosophila and Anopheles differ in their mechanisms of sex-determination (dosage vs. the Yob M-locus), and the mechanisms Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 controlling dosage compensation in Anopheles differ from those in Drosophila. Sxl controls sex- determination in Drosophila, however, the Anopheles gambiae homolog does not have sex- specific transcripts (Saccone et al., 2002), and is not involved in sex determination (Hall et al., 2016). Additionally, while the orthologs of Drosophila MSL complex proteins are transcribed in An. gambiae, they are highly divergent, and their role, or lack thereof, in dosage compensation is unknown (Rose et al., 2016). The independent evolution of sex chromosomes and dosage compensation within the Culicidae (Biedler and Tu, 2016), and the rapid evolution of the M-locus in the An. gambiae species complex (Hall et al., 2016), left open the question whether dosage compensation is disrupted in An. gambiae complex hybrids. Our analysis of median X:A and 2:3 gene expression ratios showed that dosage compensation is not disrupted in An. coluzzi - An. arabiensis or An. coluzzii - An. quadriannulatus hybrids, irrespective of the direction of the cross. Median X:A gene expression ratios did not differ between males and females of these hybrids; all were near 1.0, indicating complete compensation. These results demonstrate that genes on the male X chromosome are hyper-expressed to match (on average) the expression of autosomal genes. Median male to female expression ratios for X-linked and autosomal loci differ significantly within all samples in this study (parental species and hybrids) but both of these ratios are near one. This finding is in agreement with prior work on dosage compensation in An. gambiae s.s. and An. stephensi (Rose et al., 2016, Jiang et al., 2015) which showed female-biased expression of X linked loci. In all species and hybrids in the present study, median M:F expression ratios are lower (female-biased) for X-linked genes compared to autosomal loci. This pattern could result from a number of evolutionary pressures that we discuss below. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Rose et al.'s (2016) analysis of dosage compensation in An. gambiae s.s. (M form) larvae and pupae found evidence for complete dosage compensation in both developmental stages. Median X:A expression ratios were found to be close to one in male and female larvae and pupae. While they note differential abundances of genes between the X chromosome and autosomes that are sex-biased in their expression, autosomal genes in this study were approximately equally expressed in both sexes, and X-linked genes were slightly female biased. Female-biased expression of X-linked genes was strongest in larvae compared to the pupae. Female-biased expression of X-linked genes could result from an absence of dosage compensation in male testes (Rose et al., 2016, Baker and Russell, 2011), an under- representation of male-biased genes on the X chromosome (Magnusson et al., 2012), X chromosome inactivation in the male germline (Magnusson et al., 2012), tissue-specific dosage effects in males (Baker and Russell, 2011), or a combination of these factors. In An. gambiae s.s. genes with testes-specific expression are under-represented on the X chromosome, while genes with ovary-specific expression are not (Baker and Russell, 2011). Furthermore, male-biased, somatically expressed genes are under-represented on the X chromosome (Baker et al., 2011, Magnusson et al., 2012). Spermatogenesis occurs primarily during the 4th instar larvae and pupal stages in Anopheles mosquitoes (Clements 1992, Krzywinska and Krzywinski, 2009) and no genes that show male-biased expression during these developmental stages are X-linked (Magnusson et al., 2011). An analysis of gene expression in the An. gambiae male pupae germline performed by Rose et al (2016) suggests that, like Drosophila and other Diptera (Meiklejohn and Presgraves, 2012, Vicoso and Bachtrog, 2015), the X chromosome is not hyper-expressed in the testes, and thus does not show dosage compensation in this tissue (Rose et al., 2016, Baker and Russell, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 2011). Sterile F1 male hybrids in the An. gambiae complex exhibit atrophied testes, malformed sperm, or a total lack of sperm due to the disruption of meiosis (Slotman et al., 2004). However, their somatic tissues appear to develop normally. The goal of our analysis was to confirm that dosage compensation takes place in the closely related members of the An. gambiae species complex, and to discern whether or not dosage compensation is mis-regulated or otherwise disrupted in F1 hybrid males. Our results show female-biased expression of X-linked loci in both hybrids and pure species males, and no significant differences in X:A and 2:3 median expression levels between hybrid and pure species males. These results indicate that dosage compensation behaves normally in hybrid males: non- sex-biased X-linked genes are hyper expressed in somatic tissues and testes have 1X expression of non-sex biased, X-linked genes. The rapid radiation of species in the An. gambiae species complex provides a unique study system for investigating fundamental questions in evolutionary biology and genetics. Despite ongoing introgression and low levels of genetic differentiation between members of the An. gambiae complex (Fontaine et al., 2015), reproductive and ecological isolation has allowed for local adaptation, behavioral divergence, and structural reorganization of their genomes (Neafsey et al., 2015). This study demonstrates that despite the genetic and ecological divergence between An. gambiae complex member species, dosage compensation operates in pure species and hybrid males to balance X-linked and autosomal gene expression. Acknowledgements This study was funded by a National Science Foundation Doctoral Dissertation Improvement Grant (award # 1601675) to K.C. Deitz and M.A. Slotman and a Texas A&M University Genomics Seed Grant to K.C. Deitz and M.A. Slotman. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 References Bachtrog D. 2013. 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Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Tables Table 1 Median X:A expression ratios, and their 95% confidence intervals (CI), for each sample of the An. coluzzii - An. arabiensis species comparison and the An. coluzzii - An. quadriannulatus species comparison at RPKM > 10. The results of the between sample ANOVA (F-value and probability > F) and Kruskal-Wallis test ( X and p-value) are reported. Species / F1 Biological Species / F1 Biological Sex Median 95% CI Sex Median 95% CI Hybrid Replicate Hybrid Replicate QUAD Female 1 0.96 (0.89,1.01) ARAB Female 1 1.13 (0.93,1.35) QUAD Female 2 0.98 (0.92,1.04) ARAB Female 2 0.97 (0.79,1.13) QUAD Male 1 0.99 (0.92,1.04) ARAB Male 1 0.99 (0.79,1.14) QUAD Male 2 0.93 (0.86,1) ARAB Male 2 0.96 (0.75,1.13) COLZ Female 1 0.98 (0.93,1.03) COLZ Female 1 1.00 (0.77,1.19) COLZ Female 2 1.00 (0.94,1.07) COLZ Female 2 1.07 (0.87,1.33) COLZ Male 1 0.97 (0.9,1.03) COLZ Male 1 1.05 (0.86,1.3) COLZ Male 2 0.99 (0.94,1.03) COLZ Male 2 0.95 (0.7,1.13) QUAD x COLZ Female 1 1.02 (0.97,1.09) ARAB x COLZ Female 1 1.03 (0.87,1.19) QUAD x COLZ Female 2 1.02 (0.96,1.06) ARAB x COLZ Female 2 1.04 (0.9,1.18) QUAD x COLZ Male 1 0.99 (0.94,1.04) ARAB x COLZ Male 1 0.94 (0.67,1.17) QUAD x COLZ Male 2 1.01 (0.96,1.05) ARAB x COLZ Male 2 0.92 (0.77,1.06) COLZ x QUAD Female 1 1.00 (0.95,1.05) COLZ x ARAB Female 1 0.98 (0.84,1.1) COLZ x QUAD Female 2 0.99 (0.94,1.06) COLZ x ARAB Female 2 1.00 (0.83,1.15) COLZ x QUAD Male 1 0.99 (0.93,1.04) COLZ x ARAB Male 1 0.99 (0.82,1.16) COLZ x QUAD Male 2 1.01 (0.96,1.07) COLZ x ARAB Male 2 0.97 (0.8,1.15) ANOVA F-value 1.83 ANOVA F-value 0.38 ANOVA Pr(>F) 0.03 ANOVA Pr(>F) 0.984 2 2 Kruskal-Wallis X 31.55 Kruskal-Wallis X 5.34 Kruskal-Wallis p-value 0.01 Kruskal-Wallis p-value 0.989 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Table 2 Comparison of M:F expression ratio distributions, separated by X-linked and autosomal genes. Genes are only included if they have an expression level greater than 10.0 RPKM in all samples within a comparison (An. coluzzii - An. arabiensis or An. coluzzii - An. quadriannulatus). F-values and p-values (Pr(>F)) are reported for an ANOVA, which was used to test for significant differences between the means of the X-linked and autosomal M:F expression ratio distributions. X and p-values are reported for the Dunn's test, which was used to test for significant differences between the medians of the X-linked and autosomal M:F expression ratio distributions. X-linked Autosomal ANOVA Dunn's Test Species / F1 Hybrid Median 95% CI Median 95% CI F-value Pr(>F) X p-value COLZ 0.99 (0.97,1) 1.05 (1.05,1.06) 0.09 0.904 47.29 0.000 ARAB 0.91 (0.87,0.95) 0.91 (0.9,0.92) 0.05 0.997 6.79 0.005 COLZ x ARAB 0.95 (0.93,0.97) 0.98 (0.97,0.98) 0.04 0.999 7.45 0.003 ARAB x COLZ 0.90 (0.84,0.95) 1.00 (0.99,1.02) 0.07 0.968 6.59 0.005 COLZ 0.99 (0.97,1) 1.04 (1.04,1.05) 0.08 0.993 35.16 0.000 QUAD 0.89 (0.83,0.94) 0.96 (0.95,0.97) 0.05 0.999 25.54 0.000 COLZ x QUAD 1.07 (1.03,1.11) 1.11 (1.1,1.12) 0.10 0.976 10.09 0.001 QUAD x COLZ 0.87 (0.85,0.89) 0.90 (0.89,0.9) 0.05 0.999 13.00 0.000 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Hybrid Parental Hybrid Parental Figures female male female male 1.2 1.2 ARAB_female1 COLZ_female1 ARAB_female2 1.0 COLZ_female2 1.0 ARAB_male1 COLZ_male1 ARAB_male2 COLZ_male2 ARABxCOLZ_female1 0.8 COLZxQUAD_female1 0.8 ARABxCOLZ_female2 COLZxQUAD_female2 ARABxCOLZ_male1 COLZxQUAD_male1 0.6 ARABxCOLZ_male2 0.6 COLZxQUAD_male2 COLZ_female1 QUAD_female1 COLZ_female2 QUAD_female2 1.2 COLZ_male1 QUAD_male1 1.2 COLZ_male2 QUAD_male2 COLZxARAB_female1 QUADxCOLZ_female1 1.0 1.0 COLZxARAB_female2 QUADxCOLZ_female2 COLZxARAB_male1 QUADxCOLZ_male1 COLZxARAB_male2 QUADxCOLZ_male2 0.8 0.8 0.6 0.6 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 RPKM Threshold RPKM Threshold Figure 1 Scatter plot of X:A (squares) and 2:3 (circles) expression ratios at increasing minimum RPKM thresholds for the An. coluzzii - An. arabiensis species comparisons (left) and the An. coluzzii - An. quadriannulatus species comparisons. Panels separate male and female (x-axis), and parental and hybrid samples (y-axis). Lines represent the 95% confidence interval of the median for each distribution. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Chromosomal Ratio Chromosomal Ratio COLZ COLZ ARAB QUAD X−linked X−linked Autosomal Autosomal COLZxARAB COLZxQUAD ARABxCOLZ QUADxCOLZ 0 1 2 3 4 0 1 2 3 4 Male to Female Median Expression Ratio Male to Female Median Expression Ratio Figure 2 Box plot of M:F expression ratios distributions for the An. coluzzii - An. arabiensis species comparisons (left) and the the An. coluzzii - An. quadriannulatus species comparisons (right), separated by X-linked (pink) and autosomal genes (blue). Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology and Evolution Oxford University Press

The Effect of Hybridization on Dosage Compensation in Member Species of the Anopheles gambiae Species Complex

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Abstract

Dosage compensation has evolved in concert with Y-chromosome degeneration in many taxa that exhibit heterogametic sex chromosomes. Dosage compensation overcomes the biological challenge of a "half dose" of X chromosome gene transcripts in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes and interact with genes expressed on the autosomes. Sex determination and heterogametic sex chromosomes have evolved multiple times in Diptera, and in each case the genetic control of dosage compensation is tightly linked to sex determination. In the Anopheles gambiae species complex (Culicidae), maleness is conferred by the Y-chromosome gene Yob, which despite its conserved role between species is polymorphic in its copy number between them. Previous work demonstrated that male An. gambiae s.s. males exhibit complete dosage compensation in pupal and adult stages. In the present study we have extended this analysis to three sister species in the An. gambiae complex: An. coluzzii, An. arabiensis, and An. quadriannulatus. In addition, we analyzed dosage compensation in bi-directional F1 hybrids © The Author(s) 2018. . Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 between these species to determine if hybridization results in the mis-regulation and disruption of dosage compensation. Our results confirm that dosage compensation operates in the An. gambiae species complex through the hyper-transcription of the male X chromosome. Additionally, dosage compensation in hybrid males does not differ from parental males, indicating that hybridization does not result in the mis-regulation of dosage compensation. Keywords: Anopheles, Dosage Compensation, Hybridization, RNAseq Introduction Sex chromosomes evolved independently from autosomes multiple times within Diptera (Vicoso and Bachtrog, 2015, Bachtrog et al., 2014). While X chromosomes largely retain their original gene content (Vicoso and Charlesworth, 2006), sexually antagonistic mutations that occur on nascent Y-chromosomes suppress recombination with the X (Rice 1996). This leads to Y-chromosome degeneration as male-biased genes move from the X to the Y chromosome or the autosomes or are lost (Parisi et al., 2003), and eventually the formation of heterogametic sex chromosomes (Bachtrog 2013). Old Y-chromosomes harbor few protein coding genes and are repeat-rich, hampering, until recently, their sequencing and characterization (Mahajan and Bachtrog, 2017). As neo-Y chromosomes degenerate, X-linked genes that are not female-biased in their expression must be equalized or compensated for in the heterogametic sex. The evolution of dosage compensation (DC) has occurred in concert with Y- chromosome degeneration in many taxa that exhibit heterogametic sex determination (Disteche 2012, Graves 2015). DC overcomes the biological challenge of a "half dose" of X chromosome gene transcripts (or Z chromosome Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 transcripts in ZZ/ZW systems) in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes. In Drosophila and Anopheles the regulation of DC is closely tied to the sex-determination pathway despite striking differences in sex determination between these genera. In Drosophila females, a double dose of X chromosome-linked signal elements (XSE) initiates female-specific pre-mRNA splicing of sex lethal (Sxl) transcripts. Sxl in turn regulates sex-specific splicing of transformer (tra), which, along with its non-sex-specific co-factor transformer2 (tra2), promotes female-specific splicing of doublesex (dsx) and fruitless (fru) pre-mRNAs. Male and female isoforms of dsx and fru modulate the expression of genes involved in sexually dimorphic morphologies and physiologies. A single dose of XSE fails to initiate the cascade of female- specific splicing and male transcripts are produced (Penalva and Sanchez, 2003). While male and female isoforms of DSX share the same DNA-binding domain, they differ in their C-terminal domains that confer sex-specific gene regulation (Clough et al., 2014). Dsx and fru are highly conserved in insects despite their variety of sex-determination mechanisms (Herpin and Schartl, 2015, Salz and Erickson, 2010, Wilkins 1995). In mosquitoes (Diptera: Culicidae), sex determination is controlled by a M-factor located on the Y chromosome in the subfamily Anophelinae, which has heterogametic sex chromosomes (Krzywinska et al., 2016), or on the homomorphic sex-determining chromosome of the subfamily Culicinae (Hall et al., 2016). Transcription of the M-factor during early embryo development activates a cascade of sex-specific gene splicing that controls male development through sex-specific splicing of dsx and fru. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 In the Anopheles gambiae species complex the M-factor Yob is located within the male- determining locus of the Y chromosome. Yob expression is male-specific, begins within 2.5 hours of embryo oviposition, and occurs prior to dsx splicing by up to six hours (Krzywinska et al., 2016). This indicates that Yob is either a direct or indirect upstream regulator of dsx splicing. Yob is the only protein-coding gene on the Y chromosome that is shared by all members of the An. gambiae species complex (Hall et al., 2016). Yob is polymorphic in copy number between An. gambiae complex species and within An. gambiae s.s. (Hall et al. 2016). An. stephensi, which diverged from the An. gambiae species complex ~30 million years ago, lacks a Yob homolog, indicating a recent origin of Yob in the An. gambiae complex lineage (Hall et al. 2016). Bernardini et al. (2017) were able introgress the An. gambiae Y chromosome into an An. arabiensis genetic background using an F1 x F0 crossing strategy, which allowed them to overcome a severe bottleneck associated with this cross. They found that when isolated, An. gambiae and An. arabiensis Y-chromosomes are interchangeable; the An. gambiae Y chromosome has no effect on An. arabiensis gene expression and its introgression does not cause male sterility. Thus, by itself a heterospecific Y-chromosome does not appear to disrupt dosage compensation in this cross. However, we do not know how dosage compensation is affected in F1 hybrids when the genetic background of the autosomes is not homozygous. DC of the hemizygous X in male Drosophila occurs through the binding of the male- specific lethal (MSL) complex to the X. The MSL complex is comprised of four proteins encoded by the male-specific lethal (msl) genes 1-3 and maleless (mle), all of which cause male inviability when mutated (Penalva and Sanchez, 2003). Sxl mutations, or genes impacting its regulation, result in over-expression of genes on the X chromosome. This results in female lethality during embryogenesis (Erickson and Quintero, 2007, Lucchesi and Skripsky, 1981, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Gergen 1987, Hilfiker and Amrien, 1995) likely due to the mis-regulation of DC (Biedler and Tu, 2016, Cline 1978). Like Drosophila, An. stephensi and An. gambiae s.s exhibit complete DC through the hyper-transcription of the X chromosome (Jing et al., 2015, Rose et al., 2016). Ectopic injections of Yob mRNA into An. gambiae embryos causes female lethality due to abnormal X chromosome transcription, but has no effect on males. Additionally, knockdown of Yob in male An. gambiae embryos results in 100% mortality (Krzywinska et al., 2016). Krzywinska et al (2016) suggested that these results implicate Yob as an upstream regulator of DC in Anopheles gambiae due to the interconnected roles of sex-determination genes and DC regulatory pathways in other insects. Orthologs of the five Drosophila genes that encode the MSL complex are present in the An. gambiae transcriptome. However, Anopheles msl-1 and msl-2 are highly diverged from their Drosophila orthologs (Rose et al., 2016), which may indicate that they may have different functions in Anopheles. Studies in Drosophila initially implicated defects in DC regulation in hybrid male lethality (Chatterjee et al., 2007, Rodriguez et al., 2007, Bachtrog 2008) under a model where MSL proteins and their binding sites evolved on the D. melanogaster lineage in a way that a heterospecific mix of MSL complex proteins in F1 D. melanogaster - D. simulans hybrid males did not effectively regulate DC. Barbash (2010) subsequently refuted this hypothesis through a genetic screen of mle and msl mutant lines. The An. gambiae species complex harbors the most important vectors of human malaria and has recently emerged as a model system for the study of speciation genetics (Neafsey et al., 2015). However, little is known about dosage compensation regulation in Anopheles: sex determination differs drastically between Drosophila and Anopheles, our knowledge of the link Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 between Yob expression and DC regulation is incomplete, and msl-1 and msl-2 are highly diverged in sequence, and perhaps function, between Drosophila and Anopheles. In the present study, we explored whether DC regulation has diverged between members of An. gambiae species complex, and is mis-regulated in their hybrids, through an analysis of genome-wide gene expression in three sister species: An. coluzzii, An. arabiensis, and An. quadriannulatus, as well as reciprocal F1 hybrids derived from An. coluzzii x An. arabiensis and An. coluzzi x An. quadriannulatus crosses. Materials and Methods Mosquito Rearing Mosquitoes were derived from the SUA2La (An. coluzzii), SANGQUA (aka SANGWE, An. quadriannulatus), and DONGOLA (An. arabiensis) strains, or from bi-directional crosses between An. coluzzii and An. arabiensis, as well as An. coluzzii and An. quadriannulatus. Cross directions are indicated by female x male. Mosquitoes were reared in 12 hr light / 12 hr dark cycles at 25 ˚C and 70-80% relative humidity with access to 5% sucrose solution. Colonies were blood fed via an artificial membrane with warmed, defibrinated sheep blood (Hemostat Laboratories, Dixon, CA). Adults were provided wetted filter paper during the period of 48-72 hr post-feeding for oviposition. Larvae were fed Tetra brand TetraColor Tropical Crisps (fish food) ad libitum until pupation. Mosquitoes were collected 12 hr post-pupation and sexed prior to RNA extraction. Bi-directional crosses were performed by sexing 50-100 male and female pupae from the respective parental colonies prior to mixing them in adult cages. Rearing conditions of F1 larvae and collection of F1 pupae were performed as described above. RNA Isolation and Sequencing Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Mosquitoes were sexed 12 hr post-pupation. Total RNA extractions were performed on five pupae per replicate using a Qiagen RNeasy Mini kit (Qiagen, Redwood City, CA). RNA samples were quantified using a NanoDrop spectrophotometer and their quality was assessed using an Agilent Bioanalyzer. Equimolar amounts of two to four extraction products (10 or 20 total mosquito pupae) were combined to form a single biological replicate. Pooled RNA samples were again run on the Agilent Bioanalyzer and NanoDrop spectrophotometer prior to library prep and sequencing. Library prep was performed using an Illumina TruSeq RNA library prep kit (Illumina Inc., San Diego, CA). Two biological replicates for each strain, cross, and sex (28 libraries total) were sequenced on seven lanes of Illumina HiSeq 2500 machine using 125 bp single-end chemistry. Raw RNA sequencing reads are available on the NCBI Sequence Read Archive at project accession number PRJNAXXXXXX. Pseudo-Genome Construction The first step of our analysis was to construct an An. gambiae-based pseudo-genome for each parental species. By incorporating SNPs from each species into the AgamP4 genome, we were able to use the AgamP4 coordinates and gene set as a common framework to quantify transcript abundance. FastQC package version 0.11.4 (Babraham Bioinformatics) was used to examine read quality. Illumina TruSeq adapters were removed using Trimmomatic version 0.30 (Bolger et al., 2014), while simultaneously soft-clipping the reads from both 5' and 3' ends to an average phred quality score of 20, with no single bp in a four bp window below a phred quality score of 10. Only reads ≥ 50 bp were retained for alignment and subsequent analyses. Trimmed RNAseq data from the three species were initially aligned to their respective, species-specific genomes, which were obtained from VectorBase.org (AcolM1 for An. coluzzii, AaraD1 for An. arabiensis, and AquaS1 for An. quadriannulatus, Giraldo-Calderon et al., 2015). Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Initial alignments were performed using the splice-aware alignment software STAR version 2.4.2a using default parameters with the exception of the option "-- outFilterMismatchNoverLmax 0.1" (Dobin et al., 2013). Next, an index was created for each alignment, which annotated all splice junctions found during the first alignment round. This index informed the second alignment with STAR (as above), which re-aligned the reads around splice junctions. Picard tools version 2.8.1 (http://broadinstitute.github.io/picard/) was used to mark and remove PCR duplicates. BAM alignments from each of the three species (two biological replicates of males and females) were then merged using Picard MergeBamAlignment, and SAMtools/BCFtools version 1.3 (Li et al., 2009) was used to call variants within each merged alignment using the BCFtools "call" tool. This process was informed by a prior mutation rate of 0.02. Next, GATK was used to filter variant call files for only bi-allelic SNPs. The vcfutils.pl varFilter function, a component of the BCFtools package, was used to filter SNPs using default parameters with the exception of not filtering for strand bias, as this can be expected in RNAseq data. Filtered SNPs were converted to the genomic coordinates of the An. gambiae s.s. PEST strain AgamP4 genome (Holt et al., 2002) using the Picard Tools (github.com/broadinstitute/picard ) LiftoverVCF function and a chain file to match base-pair coordinates between genomes. Chain files were created from pair-wise multiple-alignment files between the An. coluzzii, An. arabiensis, and An. quadriannulatus genomes and the An. gambiae s.s. PEST reference originally published by Fontaine et al. (2015), which were downloaded from VectorBase.org. Once SNPs were converted to AgamP4 coordinates, they were incorporated into the AgamP4 genome using the GATK FastaAlternateReferenceMaker to create a pseudo-genome for each species using the AgamP4 backbone/coordinates. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Next, we again performed a STAR 2-pass alignment (as above) for each parental library using the species-specific, AgamP4-baesed pseudo-genome as a reference. SNPs were called using the methods described above, and were then incorporated into the previous pseudo-genome reference to further populate the AgamP4-based pseudo-genome of each species with variation from that strain. Subsequently, the AgamP4-based pseudo-genomes were used as a reference to create a cDNA pseudo-genome for each species using the generate_transcripts function of rSeq version 0.2.2 (Jiang et al., 2009, Salzman et al., 2011). Gene transcript coordinates were derived from the AgamP4.4 gene set (VectroBase.org). Quantification of Gene Expression Parental species libraries (An. coluzzii, An. arabiensis, and An. quadriannulatus) were aligned to their respective cDNA pseudo-genomes using the --very-sensitive mode of Bowtie 2 version 2.2.9 with default parameters (Langmead and Salzberg, 2012). Only uniquely mapped reads were retained to calculate transcript abundances. RNAseq reads from F1 hybrids were aligned to diploid pseudo-genomes comprised of the AgamP4.4 based cDNA pseudo-genomes of each parental species. rSeq version 0.2.2 (Jiang et al., 2009, Salzman et al., 2011) was used to create cDNA genomes from the AgamP4 based pseudo-genomes of each parental species (as above). The maternal and paternal cDNA genomes for each hybrid were then merged into a diploid genome using the ASE-TIGAR program (Nariai et al., 2016, http://nagasakilab.csml.org/ase-tigar/), which concatenates the cDNA genomes of the maternal and paternal strains into one genome file. RNAseq reads from F1 hybrids were assessed for quality and trimmed using FastQC and Trimmomatic as described above. Reads were mapped to the diploid, bi-parental cDNA pseudo-genomes for the respective cross using the --very-sensitive mode Bowtie 2 version 2.2.9 with default parameters (Langmead and Salzberg, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 2012). F1 hybrid reads were allowed to align up to 100 times across the diploid reference genome. ASE-TIGAR was used to identify the most likely parental genome of origin and location of each transcript. Thus, only the highest probability map position of each transcript was quantified. Total transcript abundance (maternal + paternal expression in F1 hybrids) was used for the subsequent analysis of dosage compensation. Analysis of Dosage Compensation Transcript abundances were standardized by converting them to RPKMs (reads per kilo- base of transcript per million reads) in R (www.cran.r-project.org, R Core Team, 2013). Two methods were used to assess dosage compensation in species and F1 hybrid samples. First, the ratio of median expression of X-linked and autosomal genes in each sample was determined. This was performed by dividing the RPKM value of each X-linked gene by the median RPKM expression level of all autosomal loci. The "boot" package in R (Davison and Hinkley, 1997, Cantly and Ripley, 2015) was used to calculate 95% confidence intervals of the median of this distribution for each sample using 10,000 replicates. X to autosomal (X:A) median gene expression was analyzed at increasing minimum RPKM values between zero and 10. In order to be included in the analysis at a specific RPKM threshold, a gene had to exceed the specified RPKM level in all samples in a species comparison (ex. An. coluzzii, An. arabiensis, and their hybrids). Because of this, the number of genes included in this analysis differs for An. coluzzi between the An. coluzzi - An. arabiensis and the An. coluzzi - An. quadriannulatus species comparisons. An analysis of X:A median gene expression without RPKM cut-off (RPKM ≥ 0.0) would also include transcriptionally inactive genes which would bias the analyses (Kharchenko et al., 2011). Additionally, work in Drosophila has shown that genes with low expression have a lower degree of dosage compensation (McAnally and Yampolsky, 2010). Thus, by analyzing Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 X:A expression ratios at increasing minimum RPKM thresholds (RPKM > 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0) we were able to identify the minimum RPKM level that removes bias during subsequent gene expression analyses. A comparison of median X:A gene expression ratios between males and females shows if genes on the male hemizygous X chromosome is hyper-expressed to match the level of X chromosome expression in females. In females we expect X:A expression ratios to be at or near one. If the X chromosome is hyper-expressed in males, the X:A expression ratio should be similar to that of females. If dosage compensation is absent, the X:A expression ratio of males should be half that of females. X:A expression ratios in males and females were also compared to the expression ratio of the second and third chromosomes (2:3). We expect the 2:3 expression ratio to be equal in both sexes. Thus, 2:3 median expression serves as an internal standard with which to compare X:A median expression. The 2:3 expression ratios were calculated by correcting the RPKM value of each chromosome 2 gene by the median RPKM of all chromosome 3 genes. The 2:3 expression ratios were also analyzed at increasing minimum RPKM thresholds (RPKM > 0.0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0). The 95% confidence intervals of the median of the expression ratio distribution was calculated using the "boot" package in R using 10,000 replicates (Davison and Hinkley, 1997, Cantly and Ripley, 2015). A Kruskal-Wallis test was used to test for significant differences between the median of the X:A expression ratio distribution between each sample (Kruskal and Wallis, 1952). We used an ANOVA to test for significant differences in the mean of the X:A expression ratio distribution within and between each sample in a species comparison. The same tests were performed to analyze significant differences between the distributions of 2:3 expression ratios between samples. Where appropriate, a Tukey's post-hoc test (Tukey 1949) was Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 performed to test for significant differences in the means of pair-wise comparisons, and a Dunn's test (Dunn 1964) was used to test for significant differences in the medians of pair-wise comparisons. Additionally, we calculated male to female (M:F) expression ratios of X-linked and autosomal genes. Genes were only included in this analysis if they exceeded a RPKM of 10.0 across all samples in a comparison (see results). The expectation of this approach is that the M:F ratio of X-linked and the M:F ratio of autosomal genes will not differ if dosage compensation is operating effectively. M:F expression ratios were calculated by first calculating the mean RPKM expression of each gene between the two biological replicates for each sample. Then, the male to female expression ratio was calculated for each gene within a species or F1 hybrid. Medians and 95% confidence intervals were calculated for X-linked and autosomal distributions separately. The 95% confidence intervals of the median of each M:F expression ratio distribution were calculated as described above. An ANOVA was used to test for significant differences between the means of the X-linked, M:F expression ratio distribution and the autosomal M:F expression ratio distribution within each species and F1 hybrid. Similarly, a Kruskal-Wallis test was used to test for significant differences between the medians of the X-linked, M:F expression ratio distribution and the autosomal M:F expression ratio distribution within each species and F1 hybrid. Results RNA Sequencing and Alignment The sequencing effort yielded an average of 67.23 million reads per sample, though this number is skewed by one sample (COLZ x QUAD Female 2), which had 248.18 million reads (Supplementary Tables S1, S2). If this sample is excluded, the mean number of reads per sample Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 is 60.53 million, ranging from 51.73 to 75.01 million. The mean mapping efficiency of parental libraries to their respective, species-specific reference genomes was 85.5% (min. 80.9%, max 88.7%, Supplementary Table S1). The mapping efficiency of parental species libraries increased when aligned to their respective AgamP4-based pseudo-genomes (mean 97.3%, min. 95.2%, max 99.1%, Supplementary Table S1). The mapping efficiency of all libraries to their respective cDNA pseudo-genomes was reduced in comparison to either DNA genome alignment (mean 63.8%, min. 62.3%, max 65.2%, Supplementary Table S1). However, this reduction was uniform across species and may result from an incomplete AgamP4 gene annotation. F1 hybrid RNAseq reads were aligned to the respective bi-parental cDNA pseudo-genome for that cross. After filtering BAM alignments with ASE-Tigar, the average mapping efficiency for F1 hybrid libraries was 64.3% (min. 62.3%, max. 66.7%, Supplementary Table S2). Thus, the mapping efficiency of F1 hybrid and parental libraries to the cDNA pseudo-genomes was equivalent. Dosage compensation in An. coluzzii, An. arabiensis, and An. quadriannulatus The analysis of dosage compensation can be biased when genes with very low expression are included. To overcome this challenge we calculated X:A and chromosome 2:3 gene expression ratios at increasing minimum RPKM thresholds from zero to 10. The number of genes included in the An. coluzzii - An. arabiensis and An. coluzzii - An. quadriannulatus comparison datasets do not differ substantially at any RPKM threshold (Supplementary Table S3). The medians of the X:A and 2:3 distributions were tested for significant differences at each RPKM threshold. X:A median expression ratios approach 1.0 for male and female An. coluzzii, An. arabiensis, and An. quadriannulatus as the RPKM threshold increases (Supplementary Tables Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 S4, S5), and at RPKM > 10, all 95% confidence intervals of the median exression ratios include 1.0 (Figure 1, Table 1). The same is true for 2:3 median expression ratios for males and females of all parental species (Figure 1, Supplementary Tables S8, S9). This result demonstrates that at RPKM > 10, 2:3 median expression ratios serve as an appropriate control with which to compare X:A expression in the effort to determine if dosage compensation is active in males. We tested for significant differences between the medians of the X:A and 2:3 expression ratio distributions in males and females of each parental species and found no significant differences at a minimum RPKM > 10.0 (X p-value > 0.05, Supplementary Tables S8, S9). This result was confirmed through pair-wise tests for differences between of X:A and 2:3 median expression ratios between males and females, with in each parental species. X:A an 2:3 median expression ratios did not differ between any pair-wise comparisons of males and females at RPKM > 2.0 (Supplementary Tables S10, S11). Another way to explore whether dosage compensation is operating is by comparing directly the median male to female expression ratios of X-linked and autosomal genes. Due to the under-representation of male-biased on the X-chromosome (Magnusson et al., 2011, Magnusson et al., 2012), we expect the male to female expression ratios of X-linked genes to be female-biased, but nonetheless close to one. Based on the findings of the chromosome ratio analyses (above), only genes with a minimum RPKM > 10.0 across all samples in a species comparison were included in this analysis. The median M:F expression ratios ranged between 0.89 and 0.99 for X-linked genes in parental species, and between 0.91 and 1.05 for autosomal loci (Table 2, Figure 2). An ANOVA found no significant difference between the means of the X-linked and autosomal distributions, however, a Dunn's test found significant differences between the medians of the distributions Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 (Table 2). Despite these differences, all values are close to 1.0. In each species, the median of the M:F expression ratio distribution was lower for X-linked genes vs. autosomal genes (Figure 2), indicating, as expected, a higher proportion of male-biased genes expressed on the autosomes compared to the X chromosome. An additional explanation for this pattern (though not mutually exclusive) is a higher proportion of female-biased genes on the X chromosome relative to the autosomes. However, previous studies found that genes with female-biased expression in An. gambiae are randomly distributed amongst the X chromosome and autosomes (Magnusson et al., 2012). These results confirm that dosage compensation is acting to balance X chromosome and autosomal gene expression in hemizygous males of An. coluzzi, An. arabiensis, and An. quadriannulatus, and that X chromosome dosage is equivalent between males of these species. Dosage compensation in Anopheles gambiae complex hybrids Like parental species, X:A and 2:3 median expression ratios approach 1.0 for male and female F1 hybrids as the RPKM threshold increases regardless of the parental species or direction of the cross (Supplementary Tables S4, S5). At RPKM > 10 the 95% confidence intervals of the X:A median expression ratios include 1.0 for all hybrids (Figure 1, Table 1). The same is true for 2:3 median expression ratios (Supplementary Tables S6, S7). The X:A median expression ratio of bi-directional hybrids between An. coluzzii and An. arabiensis do not differ significantly from parental species at RPKM > 0.2 (Supplementary Table 4) or at RPKM > 0.5 for 2:3 expression ratios (Supplementary Table 6). A Kruskal-Wallis test found no significant differences between median X:A and 2:3 expression ratios within both males and female hybrids at RPKM > 10 (Supplementary Table S8). Additionally, between sex Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 comparisons of median X:A and 2:3 expression ratios in F1 hybrids found no significant differences at RPKM > 10 (Supplementary Table S10). Similar patterns were found in bi-directional hybrids between An. coluzzi and An. quadriannulatus. The median X:A expression ratios of F1 males and females did not differ from parental species at RPKM > 10 (Supplementary Table S5), and median 2:3 expression ratios did not differ above RPKM > 0.5 (Supplementary Table S7). No significant differences were found between median X:A and 2:3 expression ratios within both males and female hybrids at RPKM > 10 (Supplementary Table S9), with on exception (COLZ x QUAD Female 1, Kruskal-Wallis p- value = 0.036). However, this difference is not significant after Bonferroni correction, and this result has no relevant bearing on our dosage compensation analyses. Finally, between sex comparisons of median X:A and 2:3 expression ratios in F1 hybrids found no significant differences above RPKM > 0.5 (Supplementary Table S11). The median M:F expression ratios of ranged between 0.87 and 1.07 for X-linked genes in hybrids, and between 0.90 and 1.11 for autosomal loci (Table 2, Figure 2). Corresponding to the analysis of parental species, an ANOVA found no significant difference between the means of the X-linked and autosomal distributions, but a Dunn's test found significant differences between the medians (Table 2). These results demonstrate that hybridization between An. coluzzii and An. arabiensis, and An. coluzzii and An. quadriannulatus, does not result in the mis-regulation and disruption of dosage compensation in F1 hybrid males. Discussion We analyzed dosage compensation in the closely related species within the Anopheles gambiae complex; An. coluzzii, An. arabiensis, and An. quadriannulatus, as well as in their F1 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 hybrids. Dosage compensation was measured by comparing the ratio of male to female gene expression on the X-chromosome and autosomes, and by comparing X-chromosome vs. autosome median gene expression levels in males and females. By analyzing bi-directional crosses between An. coluzzii - An. arabiensis and An. coluzzii - An. quadriannulatu, we tested if the species from which the Y or X chromosome is inherited affects dosage compensation in male hybrids. Specifically, these comparisons tested if the observed variation in the M locus between these closely related species may impact dosage compensation and if trans-acting factors from the heterozygous autosomes of hybrids affected X chromosome transcription differently depending on which parental species the X chromosome was inherited from. Our results correspond with previous studies that compared X:A and M:F expression ratios in An. stephensi and An. gambiae s.s. to demonstrate that dosage compensation acts in males through the hyper-transcription of the hemizygous X (Jiang et al., 2015, Rose et al., 2016). Jiang et al. (2015) reported median X:A expression ratios in An. stephensi males and females between 0.92 and 0.98 at minimum RPKM thresholds between 0.0 and 4.0, with no significant differences between median X and autosomal expression in males or females above a minimum RPKM > 2.0. In addition, these authors compared expression ratios between An. stephensi X- linked genes and their one-to-one orthologs in Ae. aegypti, a species with homomorphic sex chromosomes. The median RPKM ratio of X-linked An. stephensi genes to their Ae. aegypti orthologs was close to one in both sexes after normalization, indicating full dosage compensation of these genes on the hemizygous X chromosome of An. stephensi males. The evolution of sex determination and heteromorphic sex chromosomes evolved independently between Drosophila and Anopheles mosquitoes. Drosophila and Anopheles differ in their mechanisms of sex-determination (dosage vs. the Yob M-locus), and the mechanisms Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 controlling dosage compensation in Anopheles differ from those in Drosophila. Sxl controls sex- determination in Drosophila, however, the Anopheles gambiae homolog does not have sex- specific transcripts (Saccone et al., 2002), and is not involved in sex determination (Hall et al., 2016). Additionally, while the orthologs of Drosophila MSL complex proteins are transcribed in An. gambiae, they are highly divergent, and their role, or lack thereof, in dosage compensation is unknown (Rose et al., 2016). The independent evolution of sex chromosomes and dosage compensation within the Culicidae (Biedler and Tu, 2016), and the rapid evolution of the M-locus in the An. gambiae species complex (Hall et al., 2016), left open the question whether dosage compensation is disrupted in An. gambiae complex hybrids. Our analysis of median X:A and 2:3 gene expression ratios showed that dosage compensation is not disrupted in An. coluzzi - An. arabiensis or An. coluzzii - An. quadriannulatus hybrids, irrespective of the direction of the cross. Median X:A gene expression ratios did not differ between males and females of these hybrids; all were near 1.0, indicating complete compensation. These results demonstrate that genes on the male X chromosome are hyper-expressed to match (on average) the expression of autosomal genes. Median male to female expression ratios for X-linked and autosomal loci differ significantly within all samples in this study (parental species and hybrids) but both of these ratios are near one. This finding is in agreement with prior work on dosage compensation in An. gambiae s.s. and An. stephensi (Rose et al., 2016, Jiang et al., 2015) which showed female-biased expression of X linked loci. In all species and hybrids in the present study, median M:F expression ratios are lower (female-biased) for X-linked genes compared to autosomal loci. This pattern could result from a number of evolutionary pressures that we discuss below. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Rose et al.'s (2016) analysis of dosage compensation in An. gambiae s.s. (M form) larvae and pupae found evidence for complete dosage compensation in both developmental stages. Median X:A expression ratios were found to be close to one in male and female larvae and pupae. While they note differential abundances of genes between the X chromosome and autosomes that are sex-biased in their expression, autosomal genes in this study were approximately equally expressed in both sexes, and X-linked genes were slightly female biased. Female-biased expression of X-linked genes was strongest in larvae compared to the pupae. Female-biased expression of X-linked genes could result from an absence of dosage compensation in male testes (Rose et al., 2016, Baker and Russell, 2011), an under- representation of male-biased genes on the X chromosome (Magnusson et al., 2012), X chromosome inactivation in the male germline (Magnusson et al., 2012), tissue-specific dosage effects in males (Baker and Russell, 2011), or a combination of these factors. In An. gambiae s.s. genes with testes-specific expression are under-represented on the X chromosome, while genes with ovary-specific expression are not (Baker and Russell, 2011). Furthermore, male-biased, somatically expressed genes are under-represented on the X chromosome (Baker et al., 2011, Magnusson et al., 2012). Spermatogenesis occurs primarily during the 4th instar larvae and pupal stages in Anopheles mosquitoes (Clements 1992, Krzywinska and Krzywinski, 2009) and no genes that show male-biased expression during these developmental stages are X-linked (Magnusson et al., 2011). An analysis of gene expression in the An. gambiae male pupae germline performed by Rose et al (2016) suggests that, like Drosophila and other Diptera (Meiklejohn and Presgraves, 2012, Vicoso and Bachtrog, 2015), the X chromosome is not hyper-expressed in the testes, and thus does not show dosage compensation in this tissue (Rose et al., 2016, Baker and Russell, Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 2011). Sterile F1 male hybrids in the An. gambiae complex exhibit atrophied testes, malformed sperm, or a total lack of sperm due to the disruption of meiosis (Slotman et al., 2004). However, their somatic tissues appear to develop normally. The goal of our analysis was to confirm that dosage compensation takes place in the closely related members of the An. gambiae species complex, and to discern whether or not dosage compensation is mis-regulated or otherwise disrupted in F1 hybrid males. Our results show female-biased expression of X-linked loci in both hybrids and pure species males, and no significant differences in X:A and 2:3 median expression levels between hybrid and pure species males. These results indicate that dosage compensation behaves normally in hybrid males: non- sex-biased X-linked genes are hyper expressed in somatic tissues and testes have 1X expression of non-sex biased, X-linked genes. The rapid radiation of species in the An. gambiae species complex provides a unique study system for investigating fundamental questions in evolutionary biology and genetics. Despite ongoing introgression and low levels of genetic differentiation between members of the An. gambiae complex (Fontaine et al., 2015), reproductive and ecological isolation has allowed for local adaptation, behavioral divergence, and structural reorganization of their genomes (Neafsey et al., 2015). This study demonstrates that despite the genetic and ecological divergence between An. gambiae complex member species, dosage compensation operates in pure species and hybrid males to balance X-linked and autosomal gene expression. Acknowledgements This study was funded by a National Science Foundation Doctoral Dissertation Improvement Grant (award # 1601675) to K.C. Deitz and M.A. Slotman and a Texas A&M University Genomics Seed Grant to K.C. Deitz and M.A. Slotman. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 References Bachtrog D. 2013. 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Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Tables Table 1 Median X:A expression ratios, and their 95% confidence intervals (CI), for each sample of the An. coluzzii - An. arabiensis species comparison and the An. coluzzii - An. quadriannulatus species comparison at RPKM > 10. The results of the between sample ANOVA (F-value and probability > F) and Kruskal-Wallis test ( X and p-value) are reported. Species / F1 Biological Species / F1 Biological Sex Median 95% CI Sex Median 95% CI Hybrid Replicate Hybrid Replicate QUAD Female 1 0.96 (0.89,1.01) ARAB Female 1 1.13 (0.93,1.35) QUAD Female 2 0.98 (0.92,1.04) ARAB Female 2 0.97 (0.79,1.13) QUAD Male 1 0.99 (0.92,1.04) ARAB Male 1 0.99 (0.79,1.14) QUAD Male 2 0.93 (0.86,1) ARAB Male 2 0.96 (0.75,1.13) COLZ Female 1 0.98 (0.93,1.03) COLZ Female 1 1.00 (0.77,1.19) COLZ Female 2 1.00 (0.94,1.07) COLZ Female 2 1.07 (0.87,1.33) COLZ Male 1 0.97 (0.9,1.03) COLZ Male 1 1.05 (0.86,1.3) COLZ Male 2 0.99 (0.94,1.03) COLZ Male 2 0.95 (0.7,1.13) QUAD x COLZ Female 1 1.02 (0.97,1.09) ARAB x COLZ Female 1 1.03 (0.87,1.19) QUAD x COLZ Female 2 1.02 (0.96,1.06) ARAB x COLZ Female 2 1.04 (0.9,1.18) QUAD x COLZ Male 1 0.99 (0.94,1.04) ARAB x COLZ Male 1 0.94 (0.67,1.17) QUAD x COLZ Male 2 1.01 (0.96,1.05) ARAB x COLZ Male 2 0.92 (0.77,1.06) COLZ x QUAD Female 1 1.00 (0.95,1.05) COLZ x ARAB Female 1 0.98 (0.84,1.1) COLZ x QUAD Female 2 0.99 (0.94,1.06) COLZ x ARAB Female 2 1.00 (0.83,1.15) COLZ x QUAD Male 1 0.99 (0.93,1.04) COLZ x ARAB Male 1 0.99 (0.82,1.16) COLZ x QUAD Male 2 1.01 (0.96,1.07) COLZ x ARAB Male 2 0.97 (0.8,1.15) ANOVA F-value 1.83 ANOVA F-value 0.38 ANOVA Pr(>F) 0.03 ANOVA Pr(>F) 0.984 2 2 Kruskal-Wallis X 31.55 Kruskal-Wallis X 5.34 Kruskal-Wallis p-value 0.01 Kruskal-Wallis p-value 0.989 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Table 2 Comparison of M:F expression ratio distributions, separated by X-linked and autosomal genes. Genes are only included if they have an expression level greater than 10.0 RPKM in all samples within a comparison (An. coluzzii - An. arabiensis or An. coluzzii - An. quadriannulatus). F-values and p-values (Pr(>F)) are reported for an ANOVA, which was used to test for significant differences between the means of the X-linked and autosomal M:F expression ratio distributions. X and p-values are reported for the Dunn's test, which was used to test for significant differences between the medians of the X-linked and autosomal M:F expression ratio distributions. X-linked Autosomal ANOVA Dunn's Test Species / F1 Hybrid Median 95% CI Median 95% CI F-value Pr(>F) X p-value COLZ 0.99 (0.97,1) 1.05 (1.05,1.06) 0.09 0.904 47.29 0.000 ARAB 0.91 (0.87,0.95) 0.91 (0.9,0.92) 0.05 0.997 6.79 0.005 COLZ x ARAB 0.95 (0.93,0.97) 0.98 (0.97,0.98) 0.04 0.999 7.45 0.003 ARAB x COLZ 0.90 (0.84,0.95) 1.00 (0.99,1.02) 0.07 0.968 6.59 0.005 COLZ 0.99 (0.97,1) 1.04 (1.04,1.05) 0.08 0.993 35.16 0.000 QUAD 0.89 (0.83,0.94) 0.96 (0.95,0.97) 0.05 0.999 25.54 0.000 COLZ x QUAD 1.07 (1.03,1.11) 1.11 (1.1,1.12) 0.10 0.976 10.09 0.001 QUAD x COLZ 0.87 (0.85,0.89) 0.90 (0.89,0.9) 0.05 0.999 13.00 0.000 Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Hybrid Parental Hybrid Parental Figures female male female male 1.2 1.2 ARAB_female1 COLZ_female1 ARAB_female2 1.0 COLZ_female2 1.0 ARAB_male1 COLZ_male1 ARAB_male2 COLZ_male2 ARABxCOLZ_female1 0.8 COLZxQUAD_female1 0.8 ARABxCOLZ_female2 COLZxQUAD_female2 ARABxCOLZ_male1 COLZxQUAD_male1 0.6 ARABxCOLZ_male2 0.6 COLZxQUAD_male2 COLZ_female1 QUAD_female1 COLZ_female2 QUAD_female2 1.2 COLZ_male1 QUAD_male1 1.2 COLZ_male2 QUAD_male2 COLZxARAB_female1 QUADxCOLZ_female1 1.0 1.0 COLZxARAB_female2 QUADxCOLZ_female2 COLZxARAB_male1 QUADxCOLZ_male1 COLZxARAB_male2 QUADxCOLZ_male2 0.8 0.8 0.6 0.6 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 0 0.2 0.5 1 2 5 10 RPKM Threshold RPKM Threshold Figure 1 Scatter plot of X:A (squares) and 2:3 (circles) expression ratios at increasing minimum RPKM thresholds for the An. coluzzii - An. arabiensis species comparisons (left) and the An. coluzzii - An. quadriannulatus species comparisons. Panels separate male and female (x-axis), and parental and hybrid samples (y-axis). Lines represent the 95% confidence interval of the median for each distribution. Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Chromosomal Ratio Chromosomal Ratio COLZ COLZ ARAB QUAD X−linked X−linked Autosomal Autosomal COLZxARAB COLZxQUAD ARABxCOLZ QUADxCOLZ 0 1 2 3 4 0 1 2 3 4 Male to Female Median Expression Ratio Male to Female Median Expression Ratio Figure 2 Box plot of M:F expression ratios distributions for the An. coluzzii - An. arabiensis species comparisons (left) and the the An. coluzzii - An. quadriannulatus species comparisons (right), separated by X-linked (pink) and autosomal genes (blue). Downloaded from https://academic.oup.com/gbe/advance-article-abstract/doi/10.1093/gbe/evy108/5026595 by Ed 'DeepDyve' Gillespie user on 07 June 2018

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