Background: Mitochondrial gene transfer/loss is common in land plants, and therefore the fate of missing mitochondrial genes has attracted more and more attention. The gene content of gymnosperm mitochondria varies greatly, supplying a system for studying the evolutionary fate of missing mitochondrial genes.

Results: Here we studied the tempo and pattern of mitochondrial gene loss/transfer in gymnosperms represented by all 13 families, using high-throughput sequencing of both DNA and cDNA. All 41 mitochondrial protein-coding genes were found in cycads, Ginkgo and Pinaceae, whereas multiple mitochondrial genes were absent in Conifer II and Gnetales. In Conifer II, gene transfer from mitochondria to the nucleus followed by loss of the mitochondrial copy was common, but complete loss of a gene in both mitochondrial and nuclear genomes was rare. In contrast, both gene transfer and loss were commonly found in Gnetales. Notably, in Conifer II and Gnetales, the same five mitochondrial genes were transferred to the nuclear genome, and these gene transfer events occurred, respectively, in ancestors of the two lineages. A two-step transfer mechanism (retroprocessing and subsequent DNA-mediated gene transfer) may be responsible for mitochondrial gene transfer in Conifer II and Gnetales. Moreover, the mitochondrial gene content variation is correlated with gene length, GC content, hydrophobicity, and nucleotide substitution rates in land plants.

Conclusions: This study reveals a complete evolutionary scenario for mitochondrial genes of gymnosperms and the factors responsible for mitochondrial gene content variation in land plants.

To avoid the influence of RNA editing sites, cDNA sequences were used in the phylogenetic analysis. Two basal angiosperms with all 41 mitochondrial genes, i.e., Amborella trichopoda and Liriodendron tulipifera, were selected to represent angiosperms, and two ferns, i.e., Ophioglossum californicum and Psilotum nudum, were selected as outgroups. All mitochondrial genes were concatenated directly. If one gene was proven to be transferred to the nucleus in a species, we first reconstructed the single-gene trees so that we could infer when and how many times this gene was transferred to the nuclear genome. The GTRGAMMA and PROTGAMMAAUTO models were used in the nucleotide and AA matrices, respectively, and RaxML v. 8.2.12 was used to reconstruct the phylogenetic relationships with 100 bootstrap replicates.