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Comparative single-nucleus transcriptomics reveals asymmetric evolution of the Drosophila male and female germlines [1]

['Imtiyaz E. Hariyani', 'Department Of Ecology', 'Evolutionary Biology', 'University Of California Irvine', 'Irvine', 'California', 'United States Of America', 'Sudeshna Das', 'Department Of Neurobiology', 'Behavior']

Date: 2026-07 

Reproductive organs vary widely across species yet share conserved cell types that produce gametes, sustaining species’ perpetuation. However, tissue-level comparisons mask critical differences among cell types, obscuring where evolutionary divergence occurs even between closely related species. We quantified expression divergence at cell-type resolution between two sibling species, Drosophila melanogaster and D. simulans, while disentangling adaptive and nonadaptive evolutionary mechanisms. We built a comparative single-nucleus transcriptomic atlas of over 100,000 nuclei from testes and ovaries of both species. Our analysis revealed sharply heterogeneous divergence across testis cell types, contrasting with a broader conservation across ovary cell types. Notably, in both organs, ~40% of genes showing interspecific differences did so in only one cell type. In the testis, spermatogonia were largely conserved, whereas divergence peaked in primary spermatocytes with extensive rewiring of coexpression modules linked to microtubule and mitochondrial functions. In the ovary, expression was largely conserved, except in early germline and late follicle cells, which showed shifts in oogenesis and cell-cycle-related coexpression modules. Divergent cell types in both tissues were enriched for evolutionarily young genes with narrow expression breadth and faster protein evolution rates. Additionally, the ovary exhibited a faster-X effect consistent with adaptive evolution. These findings reveal a fundamental asymmetry in how male and female germlines evolve, with functional constraints relaxed in specific testis cell types but broadly maintained across the ovary. Our work provides an evolutionary framework explaining how core reproductive functions are safeguarded during species diversification while identifying germline cells that drive evolutionary change.

Funding: This work was supported by a National Science Foundation ( https://www.nsf.gov/ ) Award to J.M.R (MCB-2129845), and a National Institutes of Health ( https://www.nih.gov/ ) grant to V.S. (5R01AG071683-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Raw sequencing data, demultiplexed by sample for each sublibrary sequenced, were deposited as part of the NCBI BioProject PRJNA1273697. Further results are available in the supplementary files ( Supporting information ), and the processed expression data are browsable in a web app at https://ranzlab-server.bio.uci.edu/Drosophila_snRNA-seq_Gene_Expression_Browser_Testis/ and https://ranzlab-server.bio.uci.edu/Drosophila_snRNA-seq_Gene_Expression_Browser_Ovary/ . The custom scripts used for this study are available at Zenodo [ 147 ] ( https://doi.org/10.5281/zenodo.16622709 ).

Copyright: © 2026 Hariyani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To address these questions, we used snRNA-seq to compare the testis and ovary transcriptomes of three strains spanning two closely related species, D. melanogaster and D. simulans, which diverged ∼1.4 mya [ 42 ]. We characterized expression divergence across cell types by integrating two complementary analyses: differential expression at the level of individual genes and network divergence at a systems level. We also examined how differentiation trajectories in spermatogenesis and oogenesis relate to pleiotropic constraint, using proxies such as expression breadth and phylogenetic gene age. Our results reveal a dichotomy in the tempo and mode of transcriptome evolution between the male and female germline. Testis transcriptomes evolve rapidly and in a highly cell-type-specific manner, with divergence in meiotic cells driven by a combination of adaptive and nonadaptive mechanisms. In contrast, the ovary exhibits more conserved expression properties, consistent with stronger pleiotropic constraint—except in a subset of germline and late-stage follicle cells, which show pronounced divergence. We further show that the cell types accumulating divergent genes also tend to have narrow expression breadth and younger phylogenetic age, especially in the testis, and that divergent genes are more likely to be on the X chromosome in the ovary. Together, these findings uncover how distinct evolutionary mechanisms shape gene expression programs across cell types in the male and female germlines.

Despite evolutionary divergence, gametogenesis in Drosophila and mammals shares conserved developmental features, including similar differentiation stages and core molecular pathways [ 36 – 38 ]. In mammals, transcriptome divergence is not uniform across the testis: it peaks in meiotic and post-meiotic germ cells of the testis, while early germline cells remain highly conserved—likely due to strong functional constraints tied to their pleiotropic roles [ 9 , 39 , 40 ]. In the mammalian ovary, the theca cells—endocrine somatic cells that surround the follicle—accumulate greater expression divergence between species compared to other ovarian somatic cell types [ 41 ]. Whether Drosophila exhibits similar patterns of stage- or cell-type-specific divergence in either gonadal tissue remains unknown. Comparative single-cell analyses of closely related Drosophila species remain scarce, making it difficult to discern whether previously reported expression divergence reflects true biological differences or methodological artifacts such as confounding or compensatory effects [ 13 , 16 – 18 ]. If substantial interspecific expression differences exist in Drosophila, it is unclear whether they are broadly distributed across cell types or concentrated in terminally differentiated stages, similar to patterns observed in the mammalian testis.

The gonads are central to understanding expression evolution, as they harbor most sex-biased genes [ 12 ] and exhibit distinct patterns of gene expression [ 13 – 15 ]. Drosophila melanogaster has served as a premier model organism for studying gametogenesis, primarily using bulk transcriptomic data, often derived from whole-body samples [ 16 – 21 ]. More recently, single-cell studies in Drosophila have revealed stage- and cell-type-specific expression patterns across both testis and ovary [ 7 , 22 – 30 ]. For example, DNA damage response genes are enriched in early germline stages of both gonadal tissues [ 22 , 27 ], and late spermatogenesis is marked by an enrichment of de novo genes [ 22 ]. However, reliance on single-strain datasets has limited our ability to capture the intraspecific genetic diversity of D. melanogaster [ 31 – 33 ], which is crucial for understanding functional constraints and identifying expression changes that truly contribute to species divergence [ 34 , 35 ]. Key questions remain unresolved: which gonadal cell types drive interspecific divergence? Do the testis and ovary follow parallel evolutionary trajectories, or do their constraints and modes of evolution fundamentally differ? What coexpression networks and functional pathways underlie this divergence? And do chromosomal location or genomic features explain putative cell-type-specific patterns?

Gene expression changes are a key driver of species adaptation and phenotypic diversification [ 1 – 4 ]. While early comparative transcriptomic studies using microarrays or bulk RNA-sequencing helped characterize large-scale expression differences at the tissue-level, they lacked cell type resolution and were confounded by differences in tissue composition between species [ 5 , 6 ]. Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq, respectively) overcome these limitations by enabling direct comparisons of homologous cell types at unprecedented granularity within complex tissues [ 7 , 8 ]. These approaches have led to more precise evolutionary inferences—for example, in mammalian tissues such as the testis [ 5 , 9 ] and various regions of the brain [ 10 , 11 ]—by clarifying which cell types are most conserved versus those contributing disproportionately to expression divergence.

Results

Combinatorial barcoding enables cell-type-resolved gonadal transcriptomes across Drosophila species To characterize gene expression divergence between closely related Drosophila species at cell-type resolution, we performed single-nucleus RNA-sequencing (Parse Biosciences Evercode v2 [43]) on gonadal tissues from two strains of Drosophila melanogaster (A4, ISO1) and one strain of D. simulans (w501), selected to capture intra- and interspecific divergence within the D. melanogaster species subgroup. We profiled testes from 1 to 2-day-old naïve males and ovaries from 3–day-old virgin females using split-pool combinatorial barcoding [43]. After quality control and doublet removal, we retained 106,328 high-quality nuclei–40,274 nuclei from testis and 66,054 nuclei from ovary–across 12 biological samples (3 strains × 2 tissues × 2 biological replicates). To minimize mapping artifacts due to nucleotide differences, we aligned sequencing reads to strain-specific genome assemblies. We then integrated cross-strain data for each tissue using Harmony [44], which best preserved cell-type and species structure among the tools tested (Methods). With an average of 44,742 reads per nucleus (S1 Table), our dataset is comparable to or exceeds those of previous studies (S2 and S3 Tables), enabling high-resolution comparisons of conserved and divergent expression patterns within and between species.

Cellular composition of Drosophila gonads is conserved across species We identified conserved germline and somatic cell populations in both testis and ovary, whose proportions and marker expression were highly conserved across all three strains. Using uniform manifold approximation and projection (UMAP) and graph-based clustering, we found 10 testis and 17 ovary cell types and annotated them using known marker genes (S4 Table). In the testis, we identified cells undergoing mitosis (Mi; GSC/early spermatogonia (ES) and late spermatogonia (LS)) and meiosis (Me; early spermatocytes (ESp), mid spermatocytes (MSp), late spermatocytes (LSp), maturing primary spermatocytes (MPSp), and post-meiotic spermatids (S)), along with three somatic populations (S; epithelial (EC), cyst (CC), and hub (HC) cells). In the ovary, we distinguished early germline cells in the germarium (G; germline stem cells and germarium region 1 and 2a (GSC/G1-2a), germarium region 2a and 2b (G2a-2b), and germarium region 2b and 3 cells (G2b-3)) from somatic cells in the germarium (GS; follicle stem cells (FSC/pre-FCs), early follicle cells (EFC), and stalk and polar cells (SPC)), and multiple somatic populations in the epithelium (EpS; main body follicle cells (V7, V8, V9-10A, C12, C14), oviduct (O), ovarian sheath muscle (OSM), stretch cells (SC), and terminal corpus luteum cells (TCLC)). These somatic cells play essential roles in stem cell maintenance, germ cell differentiation and survival, and sex-specific germline development [45–47]. UMAP visualization faithfully recapitulated the expected spatial arrangement of these cell types, despite the absence of spatial data (Fig 1A and 1B). We validated these annotations using developmental trajectory reconstruction, which recovered the expected progression of germline differentiation (S1 and S2 Figs). PNG larger image

TIFF original image Download: Fig 1. Single-nucleus transcriptomic landscapes of Drosophila testis and ovary across strains. UMAP visualization of transcriptomic profiles from the (A) testis and (B) ovary, respectively, colored by annotated cell types. Each cluster represents a distinct cell type that corresponds to a particular stage during spermatogenesis and oogenesis, respectively, combined for all three strains (above) and individually in D. melanogaster strains A4 and ISO1, and D. simulans strain w501 (below). Stacked bar plot showing the relative fraction of identified cell types for each strain in the (C) testis and (D) ovary, respectively. Stacked bar plot showing the proportion of all cells annotated for each identified cell type across the three strains in the (E) testis and (F) ovary, respectively. All annotated cell types were included after quality control filtering, regardless of number of nuclei. Testis cell types: EC, epithelial cells; HC, hub cells; CC, cyst cells; ES, germline stem cells and early spermatogonia; LS, late spermatogonia; ESp, early spermatocytes; MSp, mid spermatocytes; LSp, late spermatocytes; MPSp, maturing primary spermatocytes; and S, spermatids. Ovary cell types: GSC/G1-2a, germline stem cells and germarium region 1 and 2a cells; G2a-2b, germarium region 2a and 2b cells; G2b-3, germarium region 2b and 3 cells; SPC, stalk and polar cells; FSC/preFCs, follicle stem cells and pre-follicle cells; EFC, early follicle cells; MFC, mitotic follicle cells stage 1-5; PMFC, post-mitotic follicle cells stage 6; V7, vitellogenic main-body follicle cells (MBFCs) stage 7; V8, vitellogenic MBFCs stage 8; V9-10A, vitellogenic MBFCs stage 9-10A; C12, choriogenic MBFCs stage 12; C14, choriogenic MBFCs stage 14; TCLC, terminal corpus luteum cells; SC, stretch cells; OSM, ovarian sheath muscle; and O, oviduct. Un, unannotated. https://doi.org/10.1371/journal.pbio.3003869.g001 The testis and ovary cell types displayed strong signatures of evolutionary stability. The different strains exhibited similar overall contributions to each cell type (Fig 1C and 1D), with minimal differences in relative cell type composition (Figs 1E, 1F, and S3; S5 Table and S1 Text). This resulted in highly correlated cell proportions across strains (S6 Table). Furthermore, marker gene expression remained remarkably consistent across strains for all annotated cell types (S4 and S5 Figs). Principal component analysis revealed that expression profiles clustered by species and by broad cell type category—mitotic, meiotic/post-meiotic, and somatic in the testis, and along a developmental trajectory continuum in the ovary (Fig 2A and 2B). Biological replicates consistently clustered together, and the two strains of D. melanogaster always grouped more closely with each other than with D. simulans, reflecting their phylogenetic relatedness and confirming the validity of our sample preparation and sequencing pipeline. Together, these findings indicate the conserved developmental architecture of gametogenesis, enabling cell-type-level comparisons of gene expression evolution across species. PNG larger image

TIFF original image Download: Fig 2. Intra- and interspecific expression variation and correlations across cell types in Drosophila gonads. Principal component analysis (PCA) of cell-type pseudo-bulks. (A) Testis. PC1 separates the mitotic cells from the meiotic and somatic cells. PC2 shows separation between species. (B) Ovary. PC1 shows the separation between species. PC2 shows cell progression along the oogenesis trajectory. Correlation matrices depicting Pearson’s correlation coefficients between the expression levels of genes found expressed in common between any two cell types across the three strains assayed in (C) testis and (D) ovary. A4 and ISO1, D. melanogaster strains; w501, D. simulans strain. In this correlation analysis, only cell types with at least 50 cells per strain and 350 cells across strains were included; as a result, the TCLC and SC ovarian cell types were excluded. Genes were required to be expressed in at least 1% of cells with a minimum average expression of 0.01. The order of cell types along both axes is identical, with broadly defined and precise cell types indicated on the x-axis and y-axis, respectively. Broad categories: somatic (S), mitotic (Mi), and meiotic (Me) categories for testis; and germline (G), germarium somatic (GS), and epithelium somatic (EpS) for ovary. Testis cell types: EC, epithelial cells; HC, hub cells; CC, cyst cells; ES, germline stem cells and early spermatogonia; LS, late spermatogonia; ESp, early spermatocytes; MSp, mid spermatocytes; LSp, late spermatocytes; MPSp, maturing primary spermatocytes; and S, spermatids. Ovary cell types: GSC/G1-2a, germline stem cells and germarium region 1 and 2a cells; G2a-2b, germarium region 2a and 2b cells; G2b-3, germarium region 2b and 3 cells; SPC, stalk and polar cells; FSC/preFCs, follicle stem cells and pre-follicle cells; EFC, early follicle cells; MFC, mitotic follicle cells stage 1-5; PMFC, post-mitotic follicle cells stage 6; V7, vitellogenic main-body follicle cells (MBFCs) stage 7; V8, vitellogenic MBFCs stage 8; V9-10A, vitellogenic MBFCs stage 9-10A; C12, choriogenic MBFCs stage 12; C14, choriogenic MBFCs stage 14; TCLC, terminal corpus luteum cells; SC, stretch cells; OSM, ovarian sheath muscle; and O, oviduct. https://doi.org/10.1371/journal.pbio.3003869.g002

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