LINGUINE: a phylogeny-aware, orthogroup-based framework for robust ancestral linkage group inference

Reconstructing the chromosomal architecture of ancestral genomes is a central challenge in comparative genomics, yet existing methods for ancestral linkage group (ALG) inference struggle in clades with rapid genome evolution. Current approaches rely predominantly on single-copy orthologous genes to anchor linkage groups between species, limiting marker density and lacking mechanisms to resolve paralogy, which leads to systematic inflation of inferred ALG counts and propagates structural artifacts in downstream analyses. Here we present LINGUINE (LINkage GroUps INfErence), a phylogeny-aware pipeline for robust ALG reconstruction addressing these limitations. LINGUINE uses orthogroups to maximise marker density, employs a Hidden Markov Model to delineate syntenic blocks while accommodating gene loss, rearrangements, and assembly fragmentation, and incorporates an explicit paralogy resolution step prior to ancestral reconstruction. Ancestral states are inferred through iterative post-order traversal of the species tree, progressively integrating information from all descendants. Using GARLIC (Genome reARrangement simuLator for Inferring Chromosomal landscapes), we characterise the limits of synteny signal detectability and define the conditions under which ALG reconstruction remains reliable. Benchmarking on nematodes demonstrates concordant performance with existing tools while incorporating a substantially larger fraction of the gene repertoire. Applied to clitellate annelids, a clade with highly rearranged genomes, LINGUINE recovers biologically coherent ancestral architectures that existing methods fail to resolve. These results establish LINGUINE as a flexible framework for ancestral genome reconstruction in clades where traditional approaches lose resolution. LINGUINE and GARLIC are open source and available in GitHub (https://github.com/MetazoaPhylogenomicsLab/Vargas-Chavez-Fernandez_2026_LINGUINE, and https://github.com/MetazoaPhylogenomicsLab/Vargas-Chavez-Fernandez_2026_GARLIC).

Vargas-Chavez, C., Fernandez, R.

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