Evidence of two deeply divergent co-existing mitochondrial genomes in the Tuatara reveals an extremely complex genomic organization

J Robert Macey,Paul M Hime,Vanessa L Gonzalez,Ella S Buring,Stefan Prost,Daniel G Mulcahy,Charles G Barbieri,Lara Urban,Stephan Pabinger,Dustin P Demeo,Neil J Gemmell,Aaron N Elliott

doi:10.1038/s42003-020-01639-0

Abstract

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. Here we show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. The two molecules, revealed using a combination of short-read and long-read sequencing technologies, differ by 10.4% nucleotide divergence. A single long-read covers an entire mt-molecule for both strands. Phylogenetic analyses suggest a 7–8 million-year divergence between genomes. Contrary to earlier reports, all 37 genes typical of animal mitochondria, with drastic gene rearrangements, are confirmed for both mt-genomes. Also unique to vertebrates, concerted evolution drives three near-identical putative Control Region non-coding blocks. Evidence of positive selection at sites linked to metabolically important transmembrane regions of encoded proteins suggests these two mt-genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

Highlights

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules
Illumina assembly of the Lady Alice Island (LAI) molecule 1 (M1) is 18,078 bases and molecule 2 (M2) 18,315 bases, each containing all 37 genes typical of animal mt-genomes. Molecular features implicate both genomes as mtDNA and not nuclear DNA. These include strong strand-bias against guanine (13.8% M1 and 14.6% M2; Supplementary Note 1), all proteincoding genes translate with no internal stop codons, tRNA genes encode tRNAs with stable secondary structures containing recognized anti-codons[6], and no sequencing reads, whether short or long-read data, were flanked by nuclear DNA
Filtering out mtDNA reads with assignments to M1 or M2, results in 114 mtDNA reads that map to 100% of the LAI M1 Illumina assembly reference

Summary

Introduction

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. We show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. Divergent co-existing mt-genomes are previously only known among bivalvian mollusks, where one mt-genome is typically maternally inherited and the other paternally inherited, a phenomenon known as doubly uniparental inheritance (DUI)[5]. Two deeply divergent mt-molecules are discovered in the Tuatara (LAI), each containing the three genes previously reported as missing, albeit bounded by a series of repeated Control Region copies. The likely time of divergence among these molecules and the selective pressures that may have led to maintain two deeply divergent, coexisting mitochondrial genomes is explored

Methods

Results

Conclusion