The complete mitochondrial genome of Zamia integrifolia uncovers distinct patterns of genomic turnover and repeat expansion in Zamiaceae

Abstract

Gymnosperm mitochondrial genomes exhibit substantial variation in size and structure, yet the evolutionary dynamics of Cycadales remain insufficiently explored beyond the genus Cycas. We assembled the complete mitochondrial genome of Zamia integrifolia using Oxford Nanopore long-read and MGI short-read sequencing. Genome assembly, polishing, and annotation were performed through an integrated bioinformatics pipeline. Repetitive elements and mitochondrial plastid DNA transfers (MTPTs) were characterized. Whole-genome synteny and structural rearrangements were analyzed. Phylogenomic relationships and topological discordance (gene and site concordance factors, gCF/sCF) were evaluated with gene. Lineage-specific substitution rates ( dN , dS , and ω ) were estimated under a branch-specific model. The genome (487,460 bp) reveals a ~74 kb expansion relative to Cycas, driven primarily by the accumulation of non-redundant repeats (107,836 bp) and extensive plastid DNA integration (63,674 bp). Quantitative metrics indicate a process of active turnover where ancestral spacers are purged and replaced by lineage-specific elements. Despite this structural flux, the gene content and local order remain conserved under purifying selection. Our phylogenetic reconstruction positions Cycadales as the sister group to all other extant gymnosperms, although gene-tree support for this node is low (gCF = 34.8%; sCF = 44.0%),and this topology is discordant with the Cycadales–Ginkgoales clade often recovered in nuclear datasets. We discuss how the exceptionally low substitution rates in cycad mitogenomes may preserve ancestral polymorphisms, leading to incomplete lineage sorting (ILS) that confounds deep node resolution, and we also consider long-branch attraction (LBA) and other sources of systematic error as alternative explanations for the observed discordance. These findings highlight a distinct evolutionary mode in Zamiaceae involving structural fluidity amidst sequence stasis and underscore the complexity of resolving gymnosperm phylogeny using organellar markers.