Overview of Peromyscus hypothalamus single-cell atlas.

A) Images of Peromyscus maniculatus and Peromyscus polionotus and summary of their social behavioral differences. Image credit: ©Joel Sartore/ Photo Ark. B) Schematic of the medial preoptic area of the hypothalamus. Dotted boxes indicate the area dissected. C) Schematic of pooling and sample assignment strategy used for snRNA-seq. D) UMAP visualization of all 52,121 neuronal nuclei collected across 6 males and 6 females from each of the P. maniculatus and P. polionotus species, colored by cell cluster. E) UMAP visualization of all neuronal nuclei, colored by the assigned Mus cell type label. Only Peromyscus clusters for which a homologous Mus cell type label could be found are colored; remaining nuclei are shown in gray. F) Heatmap of the proportion of each Peromyscus cell cluster (rows) that are assigned to each Mus cell type label (columns). Only Peromyscus clusters for which a homologous Mus cell type label could be found are shown.

Differential abundance of cell types across species and sex.

A) Schematic of procedure used to test for differential abundance of cell clusters. B) Barplots of log2(fold change) across species (P. pol / P. man, left) and across sex (male / female, right) for each cell cluster. Bars are colored by - log10(FDR). C) Boxplots of cell abundances (y-axis) of four cell types differentially abundant across species and D) of two cell types differentially abundant across sex. Cell abundances across samples are normalized using TMM normalization (Methods).

Immunostaining and cell counts of regional populations of differentially abundant cell types.

A) Representative images of immunoreactive (ir) staining of AVP+ neurons in the PVN (top) and SON (bottom) of P. maniculatus and P. polionotus. Scale bar represents 200μm. B) Boxplots of AVP+ neuron number counts in the PVN (left) and SON (right) of females and males of P. maniculatus (P. man) and P. polionotus (P.pol): ***: p<0.001 (one-sided Mann Whitney test). C and D) Same as A and B, but of OXT+ neurons. E) Representative images of ir staining of CALB1+ neurons in the BNST (top) and SDN-POA (bottom) of a female and male P. maniculatus and P. polionotus. Scale bar represents 200μm. F) Boxplots of the area of CALB1+ in the BNST (left) or CALB1+ neuron number counts in the SDN-POA (right) of females and males of P. maniculatus and P. polionotus: **: p<0.01; *: p<0.05; ns: not significant (one-sided Mann Whitney test).

Neuropeptides conserved in sex-bias in same cell types across P. maniculatus and P. polionotus

Differential gene expression across species.

A) Schematic of procedure used to pseudobulk gene expression counts and test for differential gene expression across species. B) Barplot of the number of DE genes for each cell cluster (bars) colored by P. maniculatus or P. polionotus bias. C) Scatter plot of cell cluster abundance (x-axis) and number of differentially expressed genes (y-axis) identified in each cell cluster (dots). Line of best fit (gray line) and 95% confidence interval (gray shading) are shown. D) Left: Volcano plot of log2(fold change) (x-axis) and log10(FDR) (y-axis) of DE genes across species. Negative values of log2(fold change) indicate P. maniculatus bias and positive values indicate P. polionotus bias. For genes DE in more than one cell type, the cell type with the highest expression level is shown. Genes are colored by gene categories listed on the right. Select outlier genes are labeled. Right: Barplot of enrichment scores (Methods) for each gene category. Dotted line at 1.0 indicates no enrichment or depletion. ***: FDR<0.001; **: FDR<0.01; *: FDR<0.05; ns: not significant.

Differential gene expression across sex.

A) Barplots of the number of sex-biased genes for each cell cluster (bars) in P. maniculatus (top) and P. polionotus (bottom), colored by female- or male-bias. B) Barplot of the number of female- and male-biased genes aggregated across all cell clusters in P. maniculatus (orange), P. polionotus (blue), or shared across both species (gray). C) Scatter plots of cell cluster abundance (x-axis) and number of DE genes (y-axis) identified in each cell cluster (dots) for P. maniculatus (left) and P. polionotus (right). Line of best fit (gray line) and 95% confidence interval (gray shading) are shown in each scatter plot.

Enrichment of neuropeptides in sex-biased genes.

A) Left: Volcano plots of log2(fold change) (x-axis) and log10(FDR) (y-axis) of sex-biased genes in P. maniculatus. Negative values of log2(fold change) indicate female bias and positive values indicate male bias. For genes sex-biased in more than one cell type, the cell type with the highest expression level is shown. Genes are colored by gene categories listed on the right. Select outlier genes are labeled. Right: Barplot of enrichment scores for each gene category. Dotted line at 1.0 indicates no enrichment or depletion. ***: FDR<0.001; *: FDR<0.05. B) Same as A but for P. polionotus. C) Venn diagrams of female- (top) and male-biased (bottom) neuropeptides, categorized by whether they are P. maniculatus specific, P. polionotus specific, or shared. Some neuropeptides (e.g. Nts, Tac1, Oxt, Gnrh1) are female-based in some cell types and male-biased in others. Neuropeptides that share sex-bias in the same cell type across species are starred and listed in more detail in Table 1.