# Phylogenetic Reconstructions Based on Mitogenomes Reveal the Paraphyly of the Subfamily Isotominae of Isotomidae (Collembola: Entomobryomorpha)

## TL;DR

Mitochondrial genome data shows that the subfamily Isotominae within Isotomidae is not a natural group, suggesting a need for taxonomic revision.

## Contribution

This study provides new mitogenome data and reveals the paraphyly of Isotominae using phylogenetic analysis.

## Key findings

- Phylogenetic analysis based on mitogenomes shows that Isotominae and Anurophorinae are paraphyletic.
- Parisotoma sp. forms a lineage closer to derived subfamilies than to core Isotominae.
- Pachyotominae is nested within Anurophorinae, suggesting misclassification.

## Abstract

Background: Isotomidae is one of the most common Collembola families, comprising 1484 species belonging to four subfamilies: Isotominae, Proisotominae, Anurophorinae, and Pachyotominae, while the subfamilial classification remains contentious and lack of molecular phylogenetic evidence. Methods: We sequenced and assembled the mitochondrial genomes (mitogenomes) of three species (Parisotoma sp., Folsomia sp. 1, and Folsomia sp. 2. Combining these with 10 mitogenomes available from GenBank, we reconstructed the phylogeny of Isotomidae based on a dataset of 13 species representing all four subfamilies. Results: These new mitogenomes, with lengths of 15,741 bp, 16,295 bp, and 16,765 bp, respectively, exhibit the typical metazoan gene set (13 PCGs, 22 tRNAs, 2 rRNAs) and show high structural conservation with other Collembola species. However, phylogenetic analyses based on concatenated protein-coding genes revealed significant incongruence with traditional classification. While Isotomidae was recovered as monophyletic, both Isotominae and Anurophorinae were recovered as paraphyletic. Specifically, Parisotoma sp. formed a distinct lineage closer to the derived subfamilies than to the core Isotominae, and the representative of Pachyotominae (Paranurophorus simplex) was recovered nested within Anurophorinae, suggesting potential subfamilial misclassification or paraphyly. Furthermore, Proisotoma minuta was identified as an independent sister lineage to the Anurophorinae + Pachyotominae clade. Conclusions: Our findings suggest that the current subfamily boundaries are not natural and that key diagnostic traits, such as furcal structure, likely reflect symplesiomorphies or various forms of homoplasy-including convergent evolution, parallelism, and evolutionary reversals—rather than unique synapomorphies defining monophyletic groups. This study provides essential genomic resources and highlights the need for an integrative taxonomic revision of Isotomidae that incorporates both molecular and morphological data, with particular emphasis on redefining subfamilies boundaries and reassessing diagnostic morphological traits.