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Early structure-informed approaches improved interpretability by mapping single-site metrics onto protein structures, and later methods introduced three-dimensional (3D) sliding windows to capture spatially clustered signals missed by linear window approaches. These frameworks, however, are restricted to predefined statistics and narrowly defined 3D window types, limiting the scope of questions that can be addressed. We developed an R package, evo3D, as a new framework for structure-informed evolutionary analysis that supports a wide range of downstream statistics and scales from simple to complex structures. evo3D extracts structure-informed multiple sequence alignment subsets (spatial haplotypes), making the structure-informed unit of analysis directly available to users. The framework supports fixed-count and fixed-distance spatial windows, introduces residue and codon analysis modes, and extends to multimers, interfaces, and multiple structural models through a single wrapper, run_evo3d(). We demonstrate evo3D's utility by performing an epitope-level diversity scan of hepatitis C virus E1\/E2 complex, identifying conserved spatial neighborhoods missed by linear sliding windows, and by evaluating evo3D's scalability on the octameric Chikungunya virus E1\/E2 assembly. Importantly, evo3D formalizes the core components of structure-informed analysis of molecular evolution and removes technical barriers. As a result, the framework streamlines the evaluation of evolutionary patterns directly within 3D structural contexts, and we anticipate its wide application in molecular evolution studies. The package is available at github.com\/bbroyle\/evo3D.<\/jats:p>","DOI":"10.1093\/molbev\/msag111","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T19:25:06Z","timestamp":1777577106000},"source":"Crossref","is-referenced-by-count":0,"title":["evo3D R package: a spatial haplotype framework for structure-informed analysis of molecular evolution"],"prefix":"10.1093","volume":"43","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3608-4491","authenticated-orcid":false,"given":"Bradley K","family":"Broyles","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, Purdue University , West Lafayette, IN ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1696-8203","authenticated-orcid":false,"given":"Qixin","family":"He","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Purdue University , West Lafayette, IN ,","place":["USA"]},{"name":"Purdue Institute of Inflammation, Immunology and Infectious Disease , West Lafayette, IN ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"2026052006030447200_msag111-B1","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"2026052006030447200_msag111-B2","doi-asserted-by":"publisher","first-page":"12245","DOI":"10.1128\/JVI.02070-15","article-title":"Monoclonal antibodies directed toward the hepatitis C virus glycoprotein E2 detect antigenic differences modulated by the N-terminal hypervariable region 1 (HVR1), HVR2, and intergenotypic variable region","volume":"89","author":"Alhammad","year":"2015","journal-title":"J Virol."},{"key":"2026052006030447200_msag111-B3","doi-asserted-by":"publisher","first-page":"447","DOI":"10.1006\/jmbi.2000.4474","article-title":"ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information1","volume":"307","author":"Armon","year":"2001","journal-title":"J Mol Biol."},{"key":"2026052006030447200_msag111-B4","doi-asserted-by":"publisher","first-page":"704","DOI":"10.1038\/s41586-024-07783-5","article-title":"The hepatitis C virus envelope protein complex is a dimer of heterodimers","volume":"633","author":"Augestad","year":"2024","journal-title":"Nature"},{"key":"2026052006030447200_msag111-B5","doi-asserted-by":"publisher","first-page":"418","DOI":"10.1053\/j.gastro.2018.08.060","article-title":"Approaches, progress, and challenges to hepatitis C vaccine development","volume":"156","author":"Bailey","year":"2019","journal-title":"Gastroenterology"},{"key":"2026052006030447200_msag111-B6","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the false discovery rate: a practical and powerful approach to multiple testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J R Stat Soc Ser B Methodol"},{"key":"2026052006030447200_msag111-B7","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1007\/s00239-004-0223-4","article-title":"Tertiary windowing to detect positive diversifying selection","volume":"60","author":"Berglund","year":"2005","journal-title":"J Mol Evol"},{"key":"2026052006030447200_msag111-B8","doi-asserted-by":"publisher","first-page":"3997","DOI":"10.1093\/bioinformatics\/btv494","article-title":"Msa: an R package for multiple sequence alignment","volume":"31","author":"Bodenhofer","year":"2015","journal-title":"Bioinformatics"},{"key":"2026052006030447200_msag111-B9","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1186\/1471-2105-10-421","article-title":"BLAST+: architecture and applications","volume":"10","author":"Camacho","year":"2009","journal-title":"BMC Bioinformatics"},{"key":"2026052006030447200_msag111-B10","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1007\/978-3-540-35306-5_10","volume-title":"Structural approaches to sequence evolution: molecules, networks, populations. 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