DOI: 10.5061/dryad.f4qrfj78h
title: Data from: High-resolution single-cell analyses reveal evolutionary constraints and evolvability of sexual circuits in Drosophila

description: To understand how the cellular and molecular composition of neural
 circuits evolves to generate species-specific behaviors, we performed
 single-cell transcriptomic profiling of sexual circuits labeled by the sex
 determination gene doublesex (dsx) in adult males of four Drosophila
 species: D. melanogaster, D. yakuba, D. santomea, and D. teissieri. Using
 this dataset, we compared molecularly defined dsx⁺ cell types across
 species, quantified transcriptomic divergence within each cell type, and
 analyzed differences in gene expression. In addition, we characterized sex
 differences in dsx⁺ neurons by generating a single-cell transcriptomic
 dataset from adult females of D. melanogaster, enabling us to examine
 species divergence in the context of sexual dimorphism. These datasets are
 shared here for public access. 
description: # Data from: High-resolution single-cell analyses reveal evolutionary
 constraints and evolvability of sexual circuits in Drosophila Dataset DOI:
 [10.5061/dryad.f4qrfj78h](https://doi.org/10.5061/dryad.f4qrfj78h) ##
 Description of the data and file structure We generated flies with
 genetically labeled *doublesex+* (*dsx*) neurons and dissected the brains
 and ventral nerve cords of ~100 flies per sample, dissociated the tissue
 into single cells, and used fluorescence-activated cell sorting to
 collect *dsx*+ neurons. Next, we followed standard 10X genomics protocols
 to generate single-cell libraries before using CellRanger to generate
 expression matrices. We used Seurat for clustering analyses to explore the
 evolution of *dsx*+ neurons across *Drosophila* species and between male
 and female *D. melanogaster.* In our clustering analyses, to explore the
 heterogeneity of cell types, we identified and separated out cells from
 each *dsx*+ parental clusters and performed additional clustering
 analyses. We included three replicates of *D. melanogaster* males, three
 replicates of *D. yakuba* males, one replicate of *D. santomea* males, one
 replicate of *D. teissieri* males, and one replicate of *D.
 melanogaster* females. We only included genes with orthologs across all
 four species for data analyses.  ### Files and variables #### File:
 FourSpecies_RdsFiles.zip **Description:** Rds files including clustering,
 metadata, and expression data for males from *D. melanogaster, D. yakuba,
 D. santomea, and D. teissieri*. File names indicate the specific cell
 populations included in the clustering analyses. Gene expression also can
 be directly visualized on UMAP space through a Shiny app
 ([https://apps.yenchungchen.com/dsx_neurons/](https://apps.yenchungchen.com/dsx_neurons/)). Parameters used for Seurat clustering for the four-species male dataset are as follows: | Parental cluster | # PCs included in clustering | Clustering resolution | | :---------------- | :--------------------------- | :-------------------- | | All | 15 | 0.5 | | aDN | 3 | 0.05 | | pC1 | 16 | 0.11 | | pC2l | 12 | 0.05 | | pC2m | 8 | 0.05 | | pCd-1 | 4 | 0.1 | | pCd-2 | 3 | 0.05 | | TN1 | 7 | 0.1 | | TN2 | 7 | 0.05 | | ANm | 44 | 0.18 | 
 
 
 #### File: MaleFemale_RdsFiles.zip **Description:** Rds files including
 clustering, metadata, and expression data for *D. melanogaster* males and
 females. Male data were downsampled to match the cellular coverage of
 female data prior to data analyses. File names indicate which cells are
 included for the clustering. Gene expression also can be directly
 visualized on UMAP space through a Shiny app
 ([https://apps.yenchungchen.com/dsx_neurons/](https://apps.yenchungchen.com/dsx_neurons/)). Parameters used for Seurat clustering for D. melanogaster male/female dataset are as follows: | Parental cluster | # PCs included in clustering | Clustering resolution | | :---------------- | :--------------------------- | :-------------------- | | All | 22 | 0.5 | | aDN | 3 | 0.1 | | pC1 | 13 | 0.2 | | pC2l | 12 | 0.12 | | pC2m | 9 | 0.1 | | pCd-1 | 3 | 0.15 | | pCd-2 | 3 | 0.5 | | TN1 | 5 | 0.15 | | TN2 | 3 | 0.7 | | ANm | 46 | 1.7 | 
 
 #### File: dsx_neurons_yakuba_male_rawdata.zip **Description:** Barcode,
 feature, and matrix files for each of the three male *D. yakuba*
 replicates. The barcode file includes the cell-associated barcode
 sequences, the features file contains metadata for each gene (gene ID and
 name), and the matrix file contains a sparse matrix of UMI counts linking
 each gene to each barcode.  #### File:
 dsx_neurons_melanogaster_male_rawdata.zip **Description:** Barcode,
 feature, and matrix files for each of the three male *D. melanogaster*
 replicates. The barcode file includes the cell-associated barcode
 sequences, the features file contains metadata for each gene (gene ID and
 name), and the matrix file contains a sparse matrix of UMI counts linking
 each gene to each barcode.  #### File:
 dsx_neurons_teissieri_male_rawdata.zip **Description:** Barcode, feature,
 and matrix files for the male *D. teissieri* data. The barcode file
 includes the cell-associated barcode sequences, the features file contains
 metadata for each gene (gene ID and name), and the matrix file contains a
 sparse matrix of UMI counts linking each gene to each barcode.  #### File:
 dsx_neurons_melanogaster_female_rawdata.zip **Description:** Barcode,
 feature, and matrix files for the female *D. melanogaster* data. The
 barcode file includes the cell-associated barcode sequences, the features
 file contains metadata for each gene (gene ID and name), and the matrix
 file contains a sparse matrix of UMI counts linking each gene to each
 barcode.  #### File: dsx_neurons_santomea_male_rawdata.zip
 **Description:** Barcode, feature, and matrix files for the male *D.
 santomea* data. The barcode file includes the cell-associated barcode
 sequences, the features file contains metadata for each gene (gene ID and
 name), and the matrix file contains a sparse matrix of UMI counts linking
 each gene to each barcode.  ## Access information Other publicly
 accessible locations of the data: * The first *D. melanogaster* and *D.
 yakuba* replicates (MelSample1 and YakSample1) are also available from
 NCBI's GEO: GSE262732.