Hemagglutinins (HA) from different influenza A virus subtypes share as little as ∼40% amino acid identity, yet their protein structure and cell entry function are highly conserved. Here we examine the extent that sequence constraints on HA differ across three subtypes. To do this, we first use pseudovirus deep mutational scanning to measure how all amino-acid mutations to an H7 HA affect its cell entry function. We then compare these new measurements to previously described measurements of how all mutations to H3 and H5 HAs affect cell entry function. We find that ∼50% of HA sites display substantially diverged preferences for different amino acids across the HA subtypes. The sites with the most divergent amino-acid preferences tend to be buried and have biochemically distinct wildtype amino acids in the different HA subtypes. We provide an example of how rewiring the interactions among contacting residues has dramatically shifted which amino acids are tolerated at specific sites. Overall, our results show how proteins with the same structure and function can become subject to very different site-specific evolutionary constraints as their sequences diverge. ### Competing Interest Statement JDB consults for Apriori Bio, Invivyd, Pfizer, GSK, and the Vaccine Company. JDB and BD are inventors on Fred Hutch licensed patents related to the deep mutational scanning of viral proteins. National Institute of Allergy and Infectious Diseases, R01AI165821, 75N93021C00015 U.S. National Science Foundation, DGE-2140004 Howard Hughes Medical Institute, https://ror.org/006w34k90

SpaceBar is a cellular barcoding strategy for simultaneous analysis of cell clonal and spatial identities.

Nature Methods - SpaceBar is a cellular barcoding strategy for simultaneous analysis of cell clonal and spatial identities.

The extent to which adaptation can be predicted, particularly for traits with complex genetic bases, is unknown. Here, we leveraged a model complex trait, model species, and high-powered longitudinal ...

For over a century, evolutionary biologists have been motivated to understand the mechanisms through which organisms adapt to their environments. Coloration and pigmentation are remarkably variable wi...

The nature of standing genetic variation remains a central debate in population genetics, with differing perspectives on whether common variants are almost always neutral as suggested by neutral and n...

A new version of nanorate DNA sequencing, with an error rate lower than five errors per billion base pairs and compatible with whole-exome and targeted capture, enables epidemiological-scale studies of somatic mutation and selection and the generation of high-resolution selection maps across coding and non-coding sites for many genes.

Nature - Cancer evolutionary dynamics are quantitatively inferred using a method, EVOFLUx, applied to fluctuating DNA methylation.

In simple microbial populations, the fitness effects of most selected mutations are generally taken to be constant, independent of genotype frequency. This assumption underpins predictions about evolutionary dynamics, epistatic interactions, and the maintenance of genetic diversity in populations. Here, we systematically test this assumption using beneficial mutations from early generations of the Escherichia coli Long-Term Evolution Experiment (LTEE). Using flow cytometry-based competition assays, we find that frequency-dependent fitness effects are the norm rather than the exception, occurring in approximately 80\% of strain pairs tested. Most competitions exhibit negative frequency-dependence, where fitness advantages decline as mutant frequency increases. Furthermore, we demonstrate that the strength of frequency-dependence is predictable from invasion fitness measurements, with invasion fitness explaining approximately half of the biological variation in frequency-dependent slopes. Additionally, we observe violations of fitness transitivity in several strain combinations, indicating that competitive relationships cannot always be predicted from fitness relative to a single reference strain alone. Through high-resolution measurements of within-growth cycle dynamics, we show that simple resource competition explains a substantial portion of the frequency-dependence: when faster-growing genotypes dominate populations, they deplete shared resources more rapidly, reducing the time available for fitness differences to accumulate. Our results demonstrate that even in a simple model system designed to minimize ecological complexity, subtle ecological interactions between closely related genotypes create frequency-dependent selection that can fundamentally alter evolutionary dynamics. ### Competing Interest Statement The authors have declared no competing interest.

Cells require highly polymorphic receptors to perform accurate self/non-self recognition. In the amoeba Dicytostelium discoideum, polymorphic TgrB1 & TgrC1 proteins are used to bind sister cells and e...

Evolution is expected to involve mutations that are small and modular in effect, but recent findings suggest that mutations early in an adaptive process can have strong and pleiotropic effects. This s...

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Embryonic development typically requires precise coordination among cells to achieve reproducible outcomes, leading to the assumption that cellular heterogeneity must be minimized or buffered against....

Gastruloids are three-dimensional stem-cell-based models that recapitulate key aspects of mammalian gastrulation, including formation of an anterior-posterior (AP) axis. However, we do not have detail...

The constraints that govern the evolution of gene expression patterns across development remain unclear. Single-cell RNA sequencing can detail these constraints by systematically profiling homologous ...

DNA methylation is emerging as a highly sensitive and specific marker of cancer initiation and progression. How these cancer-specific methylation changes are established in the decades before cancer, ...