Forensic mitochondrial DNA (mtDNA) interpretation depends on stable and well-curated reference phylogenies to ensure consistent haplogroup assignment and reporting. The mitoLEAF project was established as a curated successor to PhyloTree, providing a structured backbone of 6,409 haplogroup motifs designed for transparency, interpretability, and controlled nomenclature. Although this backbone supports forensic reliability, the continuous growth of publicly available mitogenomes challenges traditional models in which phylogenies are updated manually and periodically.

Here, we describe the next development stage of mitoLEAF, integrating explicit phylogenetic inference into the update process while preserving backbone stability. Newly retrieved and quality-controlled sequences are incorporated using backbone-constrained tree inference, in which the established haplogroup structure remains fixed as new sequences are integrated, allowing topology-guided identification of emerging subclades and refinement of lineage definitions. This approach introduces reproducible and transparent criteria for structural updates, complementing motif-based curation without destabilizing established forensic nomenclature.

Embedding phylogeny-driven structural revision within a defined analytical workflow also enables systematic evaluation of mtDNA nomenclature complexity and inconsistency. By linking lineage naming directly to reproducible phylogenetic evidence, haplogroup designations can be assessed and stabilized in a transparent manner. A metadata‑driven workflow architecture allows parallel representation of alternative lineage descriptors, creating opportunities to explore more coherent and scalable nomenclature strategies whilst remaining compatible with forensic conventions.

Moving beyond static and manually revised trees, we aim to transition toward guideline‑based phylogenetic pipelines capable of periodic and semi-automated, reproducible updates. Although expert oversight remains essential, this model reduces dependence on individual curators and promotes long‑term sustainability. By combining backbone stability with structured, phylogeny-guided updating, mitoLEAF strengthens haplogroup resolution, enhances interpretive consistency, and supports future-proof forensic mtDNA analysis.

Authors

• Nicole Huber (Institute of Legal Medicine, Medical University of Innsbruck, Austria)
• Arne Dür (Institute of Mathematics, University of Innsbruck, Austria)
• Walther Parson (Institute of Legal Medicine, Medical University of Innsbruck, Austria)