PreprintCited by 19Genomics and Phylogenetic StudiesMachine Learning in BioinformaticsProtein Structure and Dynamics

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Do Protein Language Models Learn Phylogeny?

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Tule, Sanjana, Foley, Gabriel, Bodén, Mikael·2024·Briefings in Bioinformatics

Preprint — published as 10.1093/bib/bbaf047 · Paper page

Abstract

Deep machine learning demonstrates a capacity to uncover evolutionary relationships directly from protein sequences, in effect internalising notions inherent to classical phylogenetic tree inference. We connect these two paradigms by assessing the capacity of protein-based language models (pLMs) to discern phylogenetic relationships without being explicitly trained to do so. We evaluate ESM2, ProtTrans and MSA-Transformer relative to classical phylogenetic methods, while also considering sequence insertions and deletions (indels) across 114 Pfam datasets. The largest ESM2 model tends to outperform other pLMs (including the multimodal ESM3) by recovering phylogenetic relationships among homologous protein sequences in both low- and high-gap settings. pLMs agree with conventional phylogenetic methods in general, but more so for protein families with fewer implied indels, highlighting indels as a key factor differentiating classical phylogenetics from pLMs. We find that pLMs preferentially capture broader as opposed to finer evolutionary relationships within a specific protein family, where ESM2 has a sweet spot for highly divergent sequences, at remote distance. Less than 10% of neurons are sufficient to broadly recapitulate classical phylogenetic distances; when used in isolation the difference between the paradigms is further diminished. We show these neurons are polysemantic, shared among different homologous families but never fully overlapping. We highlight the potential of ESM2 as a complementary tool for phylogenetic analysis, especially when extending to remote homologs that are difficult to align and imply complex histories of insertions and deletions.

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Figures

Figure 1 Evolutionary similarity score (ESS_\rho; y-axis) between layer-specific pLM matrix (x-axis) and onehot matrix. Boxplots are based on 114 Pfam datasets for ESM2-embed (a) and PT-embed (b). In case of MSA-Transformer (c), boxplots are based on 100 Pfam entries. Line plot (d) compares ESS_{r} (y-axis) and line plot (e) compares ESS_\rho (y-axis) for pLMs based on matrices at different layer depths (x-axis).
Figure 1 Evolutionary similarity score (ESS_\rho; y-axis) between layer-specific pLM matrix (x-axis) and onehot matrix. Boxplots are based on 114 Pfam datasets for ESM2-embed (a) and PT-embed (b). In case of MSA-Transformer (c), boxplots are based on 100 Pfam entries. Line plot (d) compares ESS_{r} (y-axis) and line plot (e) compares ESS_\rho (y-axis) for pLMs based on matrices at different layer depths (x-axis).CC BY · PubMed Central
Figure 2 Violin plots for ESS_\rho and ESS_{r} for different pLM matrices against LG, across all applicable low- (a) and high-gap (b) datasets. Bar plots showing the number of datasets where the type of pLM has the greatest ESS across all applicable low- (c) and high-gap (d) datasets.
Figure 2 Violin plots for ESS_\rho and ESS_{r} for different pLM matrices against LG, across all applicable low- (a) and high-gap (b) datasets. Bar plots showing the number of datasets where the type of pLM has the greatest ESS across all applicable low- (c) and high-gap (d) datasets.CC BY · PubMed Central
Figure 3 Distributions of ESS_{r} of select column attention heads in MSA-Transformer, across all applicable low- and high-gap datasets; the scores for MSA-col2 (layer 3, head 12) negatively mirrors MSA-col1 (layer 1, head 5).
Figure 3 Distributions of ESS_{r} of select column attention heads in MSA-Transformer, across all applicable low- and high-gap datasets; the scores for MSA-col2 (layer 3, head 12) negatively mirrors MSA-col1 (layer 1, head 5).CC BY · PubMed Central
Figure 4 Average ESS for the LG matrix against all column attention heads of MSA-Transformer. x-axis show the different heads and y-axis captures the different layers of the MSA-Transformer. (a) Average ESS_\rho for low-gap datasets; (b) average ESS_\rho for high-gap datasets; (c) average ESS_{r} for low-gap datasets; (d) average ESS_{r} for high-gap datasets.
Figure 4 Average ESS for the LG matrix against all column attention heads of MSA-Transformer. x-axis show the different heads and y-axis captures the different layers of the MSA-Transformer. (a) Average ESS_\rho for low-gap datasets; (b) average ESS_\rho for high-gap datasets; (c) average ESS_{r} for low-gap datasets; (d) average ESS_{r} for high-gap datasets.CC BY · PubMed Central
Figure 5 The percentage of close homologous sequence groups of size K drawn from all datasets, indicating different LHS overlap categories using the y-axis, and pLM embedding using the x-axis. We separate analyses for (a) low- and (b) high-gap datasets.
Figure 5 The percentage of close homologous sequence groups of size K drawn from all datasets, indicating different LHS overlap categories using the y-axis, and pLM embedding using the x-axis. We separate analyses for (a) low- and (b) high-gap datasets.CC BY · PubMed Central
Figure 6 Dot plot representing the ‘fine’ (yellow) and ‘broad’ (purple) ESS_{r} (shown on x-axis), as well as the ESS_{r} including ‘all’ (pairs of) sequences (green), for all Pfam datasets sorted on the y-axis in ascending order (top-to-bottom) by the ‘all’ correlation. We include LG matrix vs. PT-embed (top panel) and LG matrix vs. MSA-col1 (bottom panel). Lines in light grey represent low-gap and dark grey represent high-gap Pfam datasets.
Figure 6 Dot plot representing the ‘fine’ (yellow) and ‘broad’ (purple) ESS_{r} (shown on x-axis), as well as the ESS_{r} including ‘all’ (pairs of) sequences (green), for all Pfam datasets sorted on the y-axis in ascending order (top-to-bottom) by the ‘all’ correlation. We include LG matrix vs. PT-embed (top panel) and LG matrix vs. MSA-col1 (bottom panel). Lines in light grey represent low-gap and dark grey represent high-gap Pfam datasets.CC BY · PubMed Central
Figure 7 Analysis of ESS_{r} for different evolutionary distances for all pLM representations. Density of ESS_{r} for ‘close homologs, not divergent’ (a), ‘close homologs, moderately divergent’ (b) and ‘distant/remote homologs, highly divergent’ (c) relative to mean evolutionary distance in each group (from reference sequence). Left-hand side plots in each group show the caricatures of nominal but common phylogenetic tree-motifs (r_{s} is a reference sequence and s_{1}, s_{2}, s_{3} are members of its group).
Figure 7 Analysis of ESS_{r} for different evolutionary distances for all pLM representations. Density of ESS_{r} for ‘close homologs, not divergent’ (a), ‘close homologs, moderately divergent’ (b) and ‘distant/remote homologs, highly divergent’ (c) relative to mean evolutionary distance in each group (from reference sequence). Left-hand side plots in each group show the caricatures of nominal but common phylogenetic tree-motifs (r_{s} is a reference sequence and s_{1}, s_{2}, s_{3} are members of its group).CC BY · PubMed Central
Figure 8 Probing the saliency of neurons. (a) Distribution of the top salient neurons identified by elastic net regression for each pLM representation. ESM2 model shows a high number of neurons used for capturing evolutionary distances between protein sequences. (b) Distribution of the bottom neurons identified by elastic net regression for each pLM representation.
Figure 8 Probing the saliency of neurons. (a) Distribution of the top salient neurons identified by elastic net regression for each pLM representation. ESM2 model shows a high number of neurons used for capturing evolutionary distances between protein sequences. (b) Distribution of the bottom neurons identified by elastic net regression for each pLM representation.CC BY · PubMed Central
Figure 9 Heatmap showing the number of superimposed neurons in the top 10% of neurons for datasets used in evolutionary probe. x-axis and y-axis represent the different datasets used for evolutionary probe. ESM2-embed show a high overlap of neurons compared to the other pLM representation; this might be a characteristic of the protein datasets used for probe rather than property of that particular pLM.
Figure 9 Heatmap showing the number of superimposed neurons in the top 10% of neurons for datasets used in evolutionary probe. x-axis and y-axis represent the different datasets used for evolutionary probe. ESM2-embed show a high overlap of neurons compared to the other pLM representation; this might be a characteristic of the protein datasets used for probe rather than property of that particular pLM.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Tule, S., Foley, G., & Bodén, M.
Affiliations
The University of Queensland (AU)
Venue
Briefings in Bioinformatics
Publisher
openRxiv
Published
2024-09-25
Volume
26
Issue
1
ISSN
1467-5463 (ISSN-L)
OpenAlex
W4407867672
Cited by
19
References
1
Citations by year
2026: 9 · 2025: 10
FWCI
2.58
Type
journal article
Language
en
Open access
Locations
  • Briefings in Bioinformatics (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Machine Learning in Bioinformatics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
protein language models, phylogenetic inference, ESM-2, indels, phylogenetic distance, remote homologs, protein sequence evolution, Pfam protein families
MeSH terms
Machine Learning, Deep Learning, Humans, Phylogeny, Proteins, Evolution, Molecular, INDEL Mutation

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