Abstract
Lungs are essential respiratory organs in terrestrial vertebrates, present in most bony fishes but absent in cartilaginous fishes, making them an ideal model for studying organ evolution. Here we analysed single-cell RNA sequencing data from adult and developing lungs across vertebrate species, revealing significant similarities in cell composition, developmental trajectories and gene expression patterns. Surprisingly, a large proportion of lung-related genes, coexpression patterns and many lung enhancers are present in cartilaginous fishes despite their lack of lungs, suggesting that a substantial genetic foundation for lung development existed in the last common ancestor of jawed vertebrates. In addition, the 1,040 enhancers that emerged since the last common ancestor of bony fishes probably contain lung-specific elements that led to the development of lungs. We further identified alveolar type 1 cells as a mammal-specific alveolar cell type, along with several mammal-specific genes, including ager and sfta2, that are highly expressed in lungs. Functional validation showed that deletion of sfta2 in mice leads to severe respiratory defects, highlighting its critical role in mammalian lung features. Our study provides comprehensive insights into the evolution of vertebrate lungs, demonstrating how both regulatory network modifications and the emergence of new genes have shaped lung development and specialization across species.
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Data availability
All sequencing data and genome assemble have been deposited in the NCBI database (PRJNA1026724).
Code availability
The code is available via GitHub at https://github.com/YeLi0909/vertebrate-lung (ref. 116).
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Acknowledgements
The project was supported by the National Natural Science Foundation of China (32122021, 32370452, 82200040, 32225009 and 32100367), the National Key R&D Program of China (2022YFC3400300), the New Cornerstone Investigator Program to W.W., the 1000 Talent Project of Shaanxi Province to K.W. and Q.Q., and the Fundamental Research Funds for the Central Universities.
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K.W., Q.Q., Y.L. and W.W. designed this project and research aspects. C.F., B.W. and M.H. performed sample collection. T.X., F.Z., Y.L. and J.H. contributed to sequencing library construction for CUT&Tag. M.H. performed the scRNA-seq and bulk RNA-seq data analysis for white-spotted bamboo shark. J.Z. conducted the search for CNE. Y.L. conducted the remaining data analysis. C.Z., W.X., Z.L., L.Z. and P.X. provided valuable suggestions for the study. Y.Z. and Z.Z. contributed to the experimental components of this project. Y.L. and K.W. contributed to figure design. Y.L., K.W., Q.Q. and W.W. wrote the manuscript. K.W. and W.W. amended it.
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Extended data
Extended Data Fig. 1 Comparative analysis of cell types and gene expressions across vertebrate species using single-cell RNA sequencing data.
This figure displays a phylogenetic tree of various species (from Senegal bichir to rat), with corresponding cell counts and UMAP plots showing cell type clustering. Each species’ plot is color-coded to represent different cell types, belonging to immune, stromal, endothelial and epithelial cells. Adjacent to the UMAP plots, a dot plot heatmap illustrates gene expression patterns across cell types, with dot size indicating the percentage of cells expressing each gene and color intensity showing expression levels. The UMAP plots for pig, human, mouse, and rat are provided at the bottom. The light color block highlights the data generated for this study.
Extended Data Fig. 2 Cross-species integration of lung cells from nine vertebrate species.
a, UMAP plots showcasing lung cell distributions from each species, derived from merged SAMAP coordinates. Species range from Senegal bichir to rat, illustrating evolutionary diversity. b, Stacked bar chart quantifying the proportional composition of cell types across species. This visualization highlights interspecies variations in lung cellular makeup. c, Comparative UMAP plots demonstrating the integration efficacy of six different computational methods (CCA, RPCA, MNN, scANVI, scVI, and LIGER) on lung cells from all nine species. The upper row is color-coded by species origin, while the lower row is color-coded by cell types as annotated from each species.
Extended Data Fig. 3 Conservation of cellular communication, transcription factor networks, and gene expression in vertebrate lungs.
a, Co-expression network of 23 conserved lung transcription factors and their partial target genes. This network illustrates the complex regulatory relationships governing lung cell identity and function across species. b, Comparative VEGF signaling networks in Senegal bichir, African lungfish, and mouse lungs, derived from CellChat analysis. Circle sizes indicate cell type proportions, while edge widths represent intercellular communication probabilities. This visualization highlights conserved signaling patterns across evolutionarily distant species. c, Dotplot depicting the expression patterns of lung-specific genes across various mouse tissue cell populations. Rows represent individual genes, columns represent cell types, and color intensity indicates scaled average expression levels.
Extended Data Fig. 4 The shared and diverged gene expression pattern between mouse and chicken.
a, UMAP clustering diagram of mouse lung development. The left plot shows the overall cell type distribution, with major populations such as stromal cells, epithelial cells, endothelial cells, and specialized cell types (AT1, AT2, secretory, and ciliated cells) clearly demarcated. The right series of plots demonstrate the temporal progression of lung cell populations from embryonic day 12 (E12) through postnatal day 14 (P14), showcasing the dynamic changes in cellular composition during development. b, Similar and diverged gene expression patterns in epithelial cells between mouse (top row) and chicken (bottom row) during lung development. The left four genes (nkx2-1, sftpc, lpcatl1, fgfr2) exhibit similar expression patterns across both species, indicating conserved developmental processes. In contrast, the right two genes (etv5, shh) show divergent expression patterns, suggesting species-specific adaptations in lung development. c, Part of genes with mammalian-specific expression patterns during lung development. The dot plot compares expression levels across various species, from chicken to rat, emphasizing genes with higher expression or prevalence in mammalian lungs. d, The dynamic proportions of major cell types during lung development in both chicken (top) and mouse (bottom). Notably, the proportion of stromal cells decreases over time in both species, indicating a conserved developmental trend. e, Heatmap of 32 shared signaling pathways in stromal cells between mouse and chicken. f, The expression of key genes in chicken stromal cells that are known to be highly expressed in mouse lung stromal cells during development. g, Heatmaps displaying genes with progressively increasing expression levels in chicken endothelial cells (left) and mouse endothelial cells (right). h, Bubble plot showing enriched GO terms in endothelial (385 genes). i, Venn diagram showing the number of lung development genes and the lung adult gene set (the upper part is the gene set conserved expressed in the adult lungs, and the lower part is the gene set specifically expressed in the lungs).
Extended Data Fig. 5 Evolution of lung-related genes in cartilaginous fish and analysis of lung-associated enhancers.
a, Phylogenetic tree depicting the evolutionary history of the cd14 (left), ctss and ctsk (rigth) across various species. Numbers at each node represent posterior probabilities (as percentages), indicating the level of support for each branching event. b, Bar plot showing the proportion of lung-related genes originating from 2R-WGD, which is significantly higher than the background proportion. c, Phylogenetic tree depicting the evolutionary history of the sftpb gene across various species. d, Heatmap showing the expression levels of lung-specific genes in various tissues of the bamboo shark (bulk RNA-seq). e, Comparison of evolutionary rates for lung-related genes (left) and other genes (right) between cartilaginous and bony fish. These plots show the distribution of Ka (nonsynonymous substitution rate), Ks (synonymous substitution rate), and Ka/Ks ratio, all calculation based on the ancestral sequence. f, Bar plots comparing the expression levels (in FPKM) of two key lung-specific genes, sftpb and abca3, across different tissues in three species: African lungfish, Senegal bichir, and bamboo shark. The plots reveal a degree of co-expression of these genes in lungfish and bichir, particularly in lung tissues, while showing no apparent correlation in the bamboo shark. g, UMAP plot showing the distribution of cells co-expressing sftpb and abca3 in the esophagus and stomach from white spotted bamboo shark.
Extended Data Fig. 6 Evolution of lung-related regulatory elements.
a-b, Heatmaps showing the distribution of CUT&Tag signals for histone modifications H3K27ac and H3K4me1 around gene transcription start sites (TSS) in embryonic chicken lungs at 9, 11, and 19 days, and at postnatal day 23(a), as well as in 4-week-old mouse lungs (b). c, Stacked plot depicting the relative genomic positions of selected genes and their nearby CNEs in humans, with different rows representing the evolutionary origins of the CNEs. d, Detailed view of 10 CNEs near the TBX4 gene. Top: human lung Hi-C interactions, TAD (Topologically Associating Domain) distribution, and CNE positions (using hg38 as reference). Middle: H3K27ac signals in chicken and mouse embryonic lungs. Bottom: Sequence conservation across multiple species.
Extended Data Fig. 7 Expression of lung-specific genes in swim bladder.
a, Left: UMAP plot showing integrated cell clustering for lungs of African lungfish, Senegal bichir, and swim bladder of zebrafish. Right: Bar chart showing proportion of each cell population (stromal, immune, epithelial, endothelial) in these three species. b, Dot plot displaying expression of lung-specific genes in different cell populations of the zebrafish swim bladder.
Extended Data Fig. 8 Mammalian-specific adaptations in lung structure and function.
a, UMAP plots illustrating the further specialization of mammalian lung endothelial cells, particularly highlighting a distinct population of lung capillary cells not identified in the five non-mammalian species studied. The upper panel shows mammalian lung endothelial cells, while the lower panel represents non-mammalian species. b, Dot plots demonstrating that capillary marker genes are not prominently expressed in lung endothelial cells of non-mammalian species. Triangles indicate endothelial cells in each species. This plot compares gene expression across different cell types and species. c, RNA in situ hybridization images of ca4 (capillary endothelial cell marker) and vwf (vascular endothelial cell marker) in lung sections from mouse, bearded dragon, and African bullfrog. The results indicate that ca4 expression is not detectable in the lungs of these two non-mammalian species, supporting the findings from (b). d, Clustering relationships among AT1, AT2, and respiratory epithelial cells. The left panel shows a phylogenetic tree constructed using expression data, while the right panel represents a similarity matrix. e, UMAP plot of respiratory epithelial cells from nine vertebrate species, generated using SAMAP. Colors represent cell types (left) and species (right). f, Dot plots illustrating the expression patterns of typical AT2 marker genes and AT1-specific genes (found to be upregulated in mammalian AT1 cells compared to non-mammalian respiratory epithelial cells.) in vertebrate lung epithelial cells. Mammals are shown on the top, and non-mammals on the bottom. The scattered expression patterns in non-mammals suggest the specialization of AT1 cells in mammals.
Extended Data Fig. 9 Mammalian-specific genes may drive cell type specialization.
a, Phylogenetic tree illustrating the origin of the sfta2 gene from a duplication of the rhcg gene. b, Phylogenetic tree illustrating the origin of the scgb3a2 gene. c, Enrichment analysis of genes upregulated in the lungs of sfta2 knockout homozygous mice. The plot shows enriched GO terms, ReactomeDB pathways, and WikiPathways, with dot size indicating gene count and color representing significance.
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Li, Y., Hu, M., Zhang, Z. et al. Origin and stepwise evolution of vertebrate lungs. Nat Ecol Evol 9, 672–691 (2025). https://doi.org/10.1038/s41559-025-02642-6
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DOI: https://doi.org/10.1038/s41559-025-02642-6
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