Mitogenomics of Hesperelaea, an extinct genus of Oleaceae

Abstract

The recent developments in high-throughput DNA sequencing allowed major advances in organelle genomics. Assembly of mitochondrial genomes (hereafter mitogenomes) in higher plants however remains a challenge due to their large size and the presence of plastid-derived regions and repetitive sequences. In this study, we reconstructed the first mitogenome of Oleaceae using a herbarium specimen of the extinct genus Hesperelaea collected in 1875. Paired-end reads produced with the HiSeq technology (shotgun) in a previous study were re-used. With an approach combining reference-guided and de novo assembly, we obtained a circular molecule of 658,522 bp with a mean coverage depth of 35 ×. We found one large repeat (ca. 8 kb) and annotated 46 protein-coding genes, 3 rRNA genes and 19 tRNA genes. A phylogeny of Lamiales mitogenomes confirms Oleaceae as sister to a group comprising Lamiaceae, Phyrmaceae and Gesneriaceae. The Hesperelaea mitogenome has lower rates of synonymous and non-synonymous substitution compared to Nicotiana tabacum than other available mitogenomes of Lamiales. To conclude, we show that mitogenome reconstruction in higher plants is possible with shotgun data, even from poorly preserved DNA extracted from old specimens. This approach offers new perspectives to reconstruct plant phylogenies from mitochondrial markers, and to develop functional mitogenomics in Oleaceae.

Introduction

Recent advances in high-throughput DNA sequencing, so-called Next Generation Sequencing (NGS), have revolutionized phylogenetics by generating massive genomic resources (Harrison and Kidner, 2011). In particular, the genome skimming approach (i.e. low coverage shotgun sequencing) allowed the assembly of the most represented genomic regions such as the nuclear ribosomal cluster and cytoplasmic genomes (e.g. Cronn et al., 2008, Straub et al., 2012, Besnard et al., 2013, Malé et al., 2014). In plants, genome skimming has been mainly used to generate chloroplast genomes (hereafter plastomes) and nearly complete nuclear ribosomal sequences (Straub et al., 2012), but the method can also be used to assemble sequences from the mitochondrial genome (hereafter mitogenome) (e.g. Ma et al., 2012, Bock et al., 2014, Malé et al., 2014). In addition, the genome skimming method was successfully applied to low-quality DNA extracted from herbarium specimens, which are generally not amenable to classical PCR (e.g. Staats et al., 2013, Besnard et al., 2014, Bakker et al., 2016, Zedane et al., 2016). This approach thus offers the possibility to include in phylogenetic analyses rare and extinct species from natural history collections.

Both plastomes and mitogenomes are maternally inherited in numerous plant groups, in particular in angiosperms, but there are a few exceptions (e.g. Corriveau and Coleman, 1988, Birky, 1995). Polymorphism of the plastome has been extensively used in phylogeographic studies, in contrast with the mitogenome (e.g. Ruhlman and Jansen, 2014). The general lack of interest in plant mitogenomes for phylogenetic analyses mainly stems from their supposed low sequence variation (Wolfe et al., 1987, Smith and Keeling, 2015). Consequently, the assembly of plant mitochondrial sequences from shotgun data was reported only in a few studies (e.g. Ma et al., 2012, Bock et al., 2014, Malé et al., 2014). In addition, reconstructing full mitogenome sequences is challenging due to the presence of both long duplications and plastid-derived regions (Mower et al., 2012b, Smith and Keeling, 2015). These characteristics are lineage-specific and make the assembly of plant mitogenome from short sequence reads very complex, with no standard procedure (as proposed for reconstructing plastomes; Straub et al., 2012, Bakker et al., 2016). Yet, mitogenomic variation in plants may be valuable in investigating not only phylogenetic relationships among species, but also in studying patterns of substitution (e.g. Zhu et al., 2014) or the genetic determinism of maternally inherited traits such as the cytoplasmic male sterility (e.g. Liu et al., 2011, Mower et al., 2012a, Hiroshi et al., 2014, Touzet and Meyer, 2014).

Here, we describe the assembly of the first mitogenome of Oleaceae (Lamiales). We applied our approach to degraded DNA extracted from an old herbarium specimen collected 141 years ago on Guadalupe Island, Mexico. This specimen represents the only known remain of the genus Hesperelaea, which is now considered extinct (Watson, 1876, Zedane et al., 2016). Our study demonstrates that shotgun sequencing data, even from poorly preserved DNA, can be used to generate mitochondrial DNA datasets. Molecular analysis of available mitogenomes of Lamiales reveals deep structural reorganizations and heterogeneous evolutionary rates between mitochondrial genes. Finally, we discuss the implication of our results for further studies on the Oleaceae functional mitogenomics and phylogenetics.