Conserved Patterns of Sex Chromosome Dosage Compensation in the Lepidoptera (WZ/ZZ): Insights from a Moth Neo-Z Chromosome

Abstract

Where previously described, patterns of sex chromosome dosage compensation in the Lepidoptera (moths and butterflies) have several unusual characteristics. Other female-heterogametic (ZW/ZZ) species exhibit female Z-linked expression that is reduced compared with autosomal expression and male Z expression. In the Lepidoptera, however, Z expression typically appears balanced between sexes but overall reduced relative to autosomal expression, that is Z ≈ ZZ < AA. This pattern is not easily reconciled with theoretical expectations for the evolution of sex chromosome dosage compensation. Moreover, conflicting results linger due to discrepancies in data analyses and tissues sampled among lepidopterans. To address these issues, we performed RNA-seq to analyze sex chromosome dosage compensation in the codling moth, Cydia pomonella, which is a species from the earliest diverging lepidopteran lineage yet examined for dosage compensation and has a neo-Z chromosome resulting from an ancient Z:autosome fusion. While supported by intraspecific analyses, the Z ≈ ZZ < AA pattern was further evidenced by comparative study using autosomal orthologs of C. pomonella neo-Z genes in outgroup species. In contrast, dosage compensation appears to be absent in reproductive tissues. We thus argue that inclusion of reproductive tissues may explain the incongruence from a prior study on another moth species and that patterns of dosage compensation are likely conserved in the Lepidoptera. Notably, this pattern appears convergent with patterns in eutherian mammals (X ≈ XX < AA). Overall, our results contribute to the notion that the Lepidoptera present challenges both to classical theories regarding the evolution of sex chromosome dosage compensation and the emerging view of the association of dosage compensation with sexual heterogamety.

Keywords: Cydia pomonella; Lepidoptera; neo-Z chromosome; sex chromosome dosage compensation; sex-biased gene expression.

Fig. 1.—

Summary of dosage compensation studies in the Lepidoptera. Phylogenetic relationships among insect species mentioned in this study is based on Marec et al. (2010) and Wahlberg et al. (2013).

Fig. 2.—

Sex chromosomes comparison between Cydia pomonella and Bombyx mori. The “ancl-Z” and “neo-Z” chromosomal segments of C. pomonella are homologous to the Z chromosome and autosome 15 of B. mori, respectively. Z chromosomes are effectively hemizygous in lepidopteran females; W chromosomes are heterochromatic and are presumed to contain few or no functional genes in this order and are therefore not drawn.

Fig. 3.—

Male reproductive tissues of Cydia pomonella dissected for RNAseq analyses. Testis were separated from the male reproductive tract. The latter was collected as a single tissue comprising the accessory gland (the predominant component), along with seminal vesicles, and the ejaculatory duct (excluding the ejaculatory bulb).

Fig. 4.—

Dosage compensation analysis in Cydia pomonella using tissue-specific RNAseq. (A) Z∼ZZ balance in C. pomonella head, midgut and gonads assessed by the log₂(female:male) expression ratio. Key reference log₂ ratios are marked: −1 (2-fold male over female expression); 0 (equal expression between sexes) and 1 (2-fold female over male expression). (B) Expression levels (log₂ transformed FPKM) of all expressed genes (FPKM>0) in each tissue. The number of total expressed genes is indicated above each boxplot. Plot boxes represent the median and interquartile range of expression levels. The whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range. The dotted horizontal line across the whole plotting area denotes the mean autosomal expression in the head (F&M) and midgut (F&M). (C) Z(Z):AA ratios of median expression of all expressed genes (FPKM>0) in each tissue (orange: ancl-Z(Z):AA; green: neo-Z(Z):AA). Error bars show 95% confidence intervals estimated by 1,000 bootstrap replicates. The horizontal dotted lines denote the reference ratios: 0.5 indicates Z(Z) expression equal to monosomic autosomal expression (A); 1 indicates Z(Z) expression equal to disomic autosomal expression (AA).

Fig. 5.—

Dosage compensation analysis in Cydia pomonella based on interspecific adult head RNAseq data. (A) Expression ratios of orthologous genes expressed in adult head of C. pomonella (Cp) and reference species (Ms: Manduca sexta, Hm: Heliconius melpomene). Grey: AA: AA, green: neo-Z(Z): neo-ZZ, orange: ancl-Z(Z): ancl-ZZ, where AA represents all autosomal genes of M. sexta or H. melpomene except those orthologous to C. pomonella neo-Z(Z), and neo-ZZ represents autosomal genes of M. sexta or H. melpomene orthologous to C. pomonella neo-Z(Z). (B) Distributions of expression levels of orthologous genes expressed in adult head of C. pomonella and M. sexta. Bonferroni corrected P values from Komolgorov–Smirnov tests are shown below each category.

Fig. 6.—

Genomic distribution of tissue-specific genes in Cydia pomonella. Tissue-specific genes were identified under fold-change (FC) cutoff values from 2 to 128. Statistically significant differences assessed by two tailed Fisher’s exact test are indicated on top of bars: single asterisk (*) P<0.05; double asterisk (**) P<0.01 | black asterisk: FC cutoff=2; red asterisk: FC cutoff=128.