Full Mitochondrial Genome Research Articles

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus.

ABSTRACTObjectivesArchaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis.Materials and MethodsExtracted DNA from six individuals at the 700‐year‐old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in‐solution capture techniques, followed by Illumina high‐throughput sequencing.ResultsFull mitogenomes (7–34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth.DiscussionDental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220–228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

Evolutionary Relationships among Extinct and Extant Sloths: The Evidence of Mitogenomes and Retroviruses.

Macroevolutionary trends exhibited by retroviruses are complex and not entirely understood. The sloth endogenized foamy-like retrovirus (SloEFV), which demonstrates incongruence in virus–host evolution among extant sloths (Order Folivora), has not been investigated heretofore in any extinct sloth lineages and its premodern history within folivorans is therefore unknown. Determining retroviral coevolutionary trends requires a robust phylogeny of the viral host, but the highly reduced modern sloth fauna (6 species in 2 genera) does not adequately represent what was once a highly diversified clade (∼100 genera) of placental mammals. At present, the amount of molecular data available for extinct sloth taxa is limited, and analytical results based on these data tend to conflict with phylogenetic inferences made on the basis of morphological studies. To augment the molecular data set, we applied hybridization capture and next-generation Illumina sequencing to two extinct and three extant sloth species to retrieve full mitochondrial genomes (mitogenomes) from the hosts and the polymerase gene of SloEFV. The results produced a fully resolved and well-supported phylogeny that supports dividing crown families into two major clades: 1) The three-toed sloth, Bradypus, and Nothrotheriidae and 2) Megalonychidae, including the two-toed sloth, Choloepus, and Mylodontidae. Our calibrated time tree indicates that the Miocene epoch (23.5 Ma), particularly its earlier part, was an important interval for folivoran diversification. Both extant and extinct sloths demonstrate multiple complex invasions of SloEFV into the ancestral sloth germline followed by subsequent introgressions across different sloth lineages. Thus, sloth mitogenome and SloEFV evolution occurred separately and in parallel among sloths.

Full Mitochondrial Genome Sequence of the Sugar Beet Wireworm Limonius californicus (Coleoptera: Elateridae), a Common Agricultural Pest

We report here the full mitochondrial genome sequence of Limonius californicus, a species of click beetle that is an agricultural pest in its larval form. The circular genome is 16.5 kb and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes.

Mitochondrial genome of an Atlantic white shark (Carcharodon carcharias)

Here we report the first full mitochondrial genome sequence for a white shark caught in the Atlantic Ocean. The mitochondrial genome is 16,745 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding control region. The base composition of this mtDNA lineage is A: 30.7%, C: 26.9%, G: 13.8%, and T: 28.6%. In concordance with previous population genetic studies, the Atlantic caught individual forms a separate lineage from individuals caught on either side of the Pacific Ocean.

Mitogenomics of ‘Old World Acraea’ butterflies reveals a highly divergent ‘Bematistes’

Afrotropical Acraeini butterflies provide a fascinating potential model system to contrast with the Neotropical Heliconiini, yet their phylogeny remains largely unexplored by molecular methods and their generic level nomenclature is still contentious. To test the potential of mitogenomes in a simultaneous analysis of the radiation, we sequenced the full mitochondrial genomes of 19 African species. Analyses show the potential of mitogenomic phylogeny reconstruction in this group. Inferred relationships are largely congruent with a previous multilocus study. We confirm a monophyletic Telchinia to include the Asiatic Pareba with a complicated paraphylum, traditional (sub)genus Acraea, toward the base. The results suggest that several proposed subgenera and some species groups within Telchinia are not monophyletic, while two other (sub)genera could possibly be combined. Telchinia was recovered without strong support as sister to the potentially interesting system of distasteful model butterflies known as Bematistes, a name that is suppressed in some treatments. Surprisingly, we find that this taxon has remarkably divergent mitogenomes and unexpected synapomorphic tRNA rearrangements. These gene order changes, combined with evidence for deviating dN/dS ratios and evidence for episodal diversifying selection, suggest that the ancestral Bematistes mitogenome has had a turbulent past. Our study adds genetic support for treating this clade as a distinct genus, while the alternative option, adopted by some authors, of Acraea being equivalent to Acraeini merely promotes redundancy. We pave the way for more detailed mitogenomic and multi-locus molecular analyses which can determine how many genera are needed (possibly at least six) to divide Acraeini into monophyletic groups that also facilitate communication about their biology.

Complete mitochondrial genome of the speckled dace Rhinichthys osculus, a widely distributed cyprinid minnow of western North America

The speckled dace Rhinichthys osculus (order Cypriniformes), also known as the carpita pinta, is a small cyprinid minnow native to western North America. Here, we report the sequencing of the full mitochondrial genome (mitogenome) of R. osculus from a male fish collected from the Amargosa River Canyon in eastern California, USA. The assembled mitogenome is 16 658 base pair (bp) nucleotides, and encodes 13 protein-coding genes, and includes both a 12S and a 16S rRNA, 22 tRNAs, and a 985 bp D-loop control region. Mitogenome synteny reflects that of other Ostariophysian fishes with the majority of genes and RNAs encoded on the heavy strand (H-strand) except nd6, tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro. The availability of this R. osculus mitochondrial genome – the first complete mitogenome within the lineage of Rhinichthys riffle daces – provides a foundation for resolving evolutionary relationships among morphologically differentiated populations of R. osculus.

Mitochondrial Genome Sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoidea: Diphyllobothriidae).

The present study was performed to compare the mitochondrial genomes between 2 Spirometra tapeworms, Spirometra erinaceieuropaei and Spirometra decipiens (Cestoidea: Diphyllobothriidae), which larval stages are important etiological agents of sparganosis in humans. For each species, the full mitochondrial genome was amplified in 8 overlapping fragments using total genomic DNA purified from a single worm as the template. The mitochondrial genomes were 13,643 bp (S. erinaceieuropaei) and 13,641 bp (S. decipiens) in length and contained 36 genes; 12 protein-coding genes, 2 ribosomal RNA (rRNA, small and large subunits), and 22 transfer RNAs (tRNAs). The 12 protein-coding genes constituted 10,083 bp (S. erinaceieuropaei) and 10,086 bp (S. decipiens) of their respective mitochondrial genomes. The tRNA genes, ranging in length from 56 to 70 bp, were identified based on putative secondary structures such as the typical cloverleaf shape. A total of 23 intergenic sequences, varying from 1 to 204 bp in size, were interspersed in S. erinaceieuropaei (total, 504 bp) and S. decipiens (total, 496 bp) mtDNA. The 12 protein-coding genes of S. erinaceieuropaei and S. decipiens differed by 12.4%, whereas the overall difference in mtDNA sequence between S. erinaceieuropaei and S. decipiens was 12.9%. Thus, from the standpoint of the mitochondrial genome, S. decipiens represents a valid species that can be distinguished from S. erinaceieuropaei.

Complete mitochondrial genome of Sinergasilus polycolpus (Copepoda:Poecilostomatoida)

The full mitochondrial (mt) genome of Sinergasilus polycolpus is 14 000 bp in length, and it contains 13 protein-coding genes, 22 transfer RNA genes, 2 rRNA genes and 1 non-coding region. The genome organization, nucleotide composition and codon usage are different compared to other Copepodas, such as Lernaea cyprinacea. The full mt genome of S. polycolpus provides useful information for further studies on epidemiology and phylogenetics.

Rapid assembly of taxonomically validated mitochondrial genomes from historical insect collections

Polymerase chain reaction approaches for DNA sequencing from dried collection specimens of insects usually suffer from contamination, short fragment length, and limited phylogenetic power. As an alternative, we use shotgun metagenomic sequencing from mixed pools of DNA, from which mitochondrial sequences are filtered in silico. We extracted DNA from a single leg of 35 species of British butterflies that had been stored in the Natural History Museum for between 9 and 35 years. Illumina MiSeq (1/3 of flow cell) produced mitogenome sequences of > 10 kb for ten species, of which six were (almost) complete. In addition, full or partial barcode sequences for 31 species were recovered, which, in almost all cases, showed perfect (100%) identity to sequences available in the BOLD database under the same taxon name. The mean length of sequence reads was 167 bp (putative mitochondrial reads) and 153 bp (all reads). Assembly of contigs > 10 kb from these short reads was apparently error free, except in the case of two closely-related species of Pieris that formed chimeras. Contiguous sequences are difficult to obtain from degraded DNA with Sanger technology but the Illumina short reads are a natural fit for dry-collection specimens. The obvious possibility for scaling up this protocol provides exciting prospects for the large-scale indexing of natural history collections using partial or full mitochondrial genomes.

Mitochondrial DNA heteroplasmy in the emerging field of massively parallel sequencing

Long an important and useful tool in forensic genetic investigations, mitochondrial DNA (mtDNA) typing continues to mature. Research in the last few years has demonstrated both that data from the entire molecule will have practical benefits in forensic DNA casework, and that massively parallel sequencing (MPS) methods will make full mitochondrial genome (mtGenome) sequencing of forensic specimens feasible and cost-effective. A spate of recent studies has employed these new technologies to assess intraindividual mtDNA variation. However, in several instances, contamination and other sources of mixed mtDNA data have been erroneously identified as heteroplasmy. Well vetted mtGenome datasets based on both Sanger and MPS sequences have found authentic point heteroplasmy in approximately 25% of individuals when minor component detection thresholds are in the range of 10–20%, along with positional distribution patterns in the coding region that differ from patterns of point heteroplasmy in the well-studied control region. A few recent studies that examined very low-level heteroplasmy are concordant with these observations when the data are examined at a common level of resolution. In this review we provide an overview of considerations related to the use of MPS technologies to detect mtDNA heteroplasmy. In addition, we examine published reports on point heteroplasmy to characterize features of the data that will assist in the evaluation of future mtGenome data developed by any typing method.

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes.

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.

Complete mitochondrial genome of Military Macaw (Ara militaris): its comparison with mitogenomes of two other Ara species

ABSTRACTThe Military Macaw is one of the eight species of the genus Ara. The genus is one of six genera, which form morphologically diverse group termed as Macaws. Parrots of this group differ in body size on demand of the genus and species. Six of Ara species are classified as large Macaws. Based on morphological similarities and differences, these species can be segregated into three pairs according to their plumage coloration. Representative mitochondrial genomes were sequenced only for A. glaucogularis (blue and yellow coloration) and A. macao (predominantly red/scarlet). Ara militaris is one of two predominantly green species and full mitochondrial genome of considered species was sequenced in this study.It’s comparison with A. glaucogularis and A. macao mitogenomes revealed higher degree of identity between militaris and macao sequences than between militaris and glaucogularis mtDNAs. Ara militaris mitogenome will be indispensable to refine the phylogenetic relationships within Macaw group.

Analysis of Whole Mitogenomes from Ancient Samples.

Ancient mitochondrial DNA has been used in a wide variety of paleontological and archeological studies, ranging from population dynamics of extinct species to patterns of domestication. Most of these studies have traditionally been based on the analysis of short fragments from the mitochondrial control region, analyzed using PCR coupled with Sanger sequencing. With the introduction of high-throughput sequencing, as well as new enrichment technologies, the recovery of full mitochondrial genomes (mitogenomes) from ancient specimens has become significantly less complicated. Here we present a protocol to build ancient extracts into Illumina high-throughput sequencing libraries, and subsequent Agilent array-based capture to enrich for the desired mitogenome. Both are based on previously published protocols, with the introduction of several improvements aimed to increase the recovery of short DNA fragments, while keeping the cost and effort requirements low. This protocol was designed for enrichment of mitochondrial DNA in ancient or other degraded samples. However, the protocols can be easily adapted for using for building libraries for shotgun-sequencing of whole genomes, or enrichment of other genomic regions.

Octocoral mitochondrial genomes provide insights into the phylogenetic history of gene order rearrangements, order reversals, and cnidarian phylogenetics.

We use full mitochondrial genomes to test the robustness of the phylogeny of the Octocorallia, to determine the evolutionary pathway for the five known mitochondrial gene rearrangements in octocorals, and to test the suitability of using mitochondrial genomes for higher taxonomic-level phylogenetic reconstructions. Our phylogeny supports three major divisions within the Octocorallia and show that Paragorgiidae is paraphyletic, with Sibogagorgia forming a sister branch to the Coralliidae. Furthermore, Sibogagorgia cauliflora has what is presumed to be the ancestral gene order in octocorals, but the presence of a pair of inverted repeat sequences suggest that this gene order was not conserved but rather evolved back to this apparent ancestral state. Based on this we recommend the resurrection of the family Sibogagorgiidae to fix the paraphyly of the Paragorgiidae.This is the first study to show that in the Octocorallia, mitochondrial gene orders have evolved back to an ancestral state after going through a gene rearrangement, with at least one of the gene orders evolving independently in different lineages. A number of studies have used gene boundaries to determine the type of mitochondrial gene arrangement present. However, our findings suggest that this method known as gene junction screening may miss evolutionary reversals.Additionally, substitution saturation analysis demonstrates that while whole mitochondrial genomes can be used effectively for phylogenetic analyses within Octocorallia, their utility at higher taxonomic levels within Cnidaria is inadequate. Therefore for phylogenetic reconstruction at taxonomic levels higher than subclass within the Cnidaria, nuclear genes will be required, even when whole mitochondrial genomes are available.

Complete mitochondrial genome of Blue-headed Macaw (Primolius couloni): its comparison with mitogenome of Blue-throated Macaw (Ara glaucogularis)

Primolius is a genus of midsized Macaws comprising three species. Blue-headed Macaws (Primolius couloni) are native to eastern Peru, extreme western Brazil and north-western Bolivia. In this study, full mitochondrial genome of considered species was sequenced. It is 16,995 bp long and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. Its comparison with published Blue-throated Macaw (Ara glaucogularis) mitogenome revealed their high degree of identity. Primolius couloni mitogenome is the first complete genomic sequence of this genus. It will be indispensable to refine the phylogenetic relationships within the tribe Arini and will enrich the resource of markers for systematic, phylogenetic and population genetic studies.

Complete mitochondrial genome of Red-bellied Macaw (Orthopsittaca manilata): its comparison with mitogenome of Blue-throated Macaw (Ara glaucogularis)

The Red-bellied Macaw (Orthopsittaca manilata) is a species of the monotypic genus Orthopsittaca. The genus is one of the six genera, which form morphologically diverse group termed as Macaws. Individuals of Orthopsittaca manilata species are found in extremely large Amazonian area of South America. In this study, full mitochondrial genome of considered species was sequenced. It is 16,985 bp long and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. Its comparison with published Blue-throated Macaw (Ara glaucogularis) mitogenome revealed their high degree of identity.Presented Orthopsittaca manilata mitogenome is the first complete genomic sequence of this genus. It will enrich the resource of molecular markers for future examination of evolutionary diversification of Macaws. It will be also indispensable to refine the phylogenetic relationships within the tribe Arini.

The complete mitochondrial genome of the invasive house crow Corvus splendens (Passeriformes: Corvidae)

The complete mitochondrial genome of the invasive house crow (Corvus splendens) was sequenced (GenBank accession number: KJ766304) using the MiSeq Personal Sequencer (Illumina, San Diego, CA). The mitochondrial genome is 16,962 bp in length, comprising 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal subunit genes and a non-coding control region. The mitogenome structural organization is identical to that of the other Corvus species and related genera. The overall base composition of C. splendens is 30.65% for A, 29.71% for C, 14.84% for G and 24.80% for T, with an AT content of 55.45%. We propose to use full mitochondrial genome to address taxonomic issues and to study the population genetics of crows.

Mitochondrial Sequence Changes in Keratoconus Patients

We investigated whether a group of patients with keratoconus (KTCN) harbor mutations in the mitochondrial genome. We sequenced the full mitochondrial genome in a group of Saudi patients with KTCN (n = 26) and 100 ethnically matched controls who had no KTCN by examination. A total of 10 KTCN patients (38.5%) had potentially pathogenic nonsynonymous mtDNA mutations. Of the nonsynonymous sequence changes detected, 4 (40%) were in Complex I, one was in the tRNA(Glutamine), one was in tRNA(Tryptophan), one was in tRNA(Asparagine), one was in tRNA(Histidine), and two were in the tRNA(Leucine2). One nonsynonymous sequence change was heteroplasmic, whereas all the remaining 9 were homoplasmic. These sequence changes were not detected in controls of similar ethnicity. Four sequence changes were novel (were not reported previously) and 5 were reported previously. Additionally, we detected 54 synonymous (does not result in an amino acid change) sequence changes with no pathologic significance. If our results are confirmed in a larger cohort and multiple ethnicities, then mtDNA mutation may be considered as a genetic risk factor contributing indirectly through the oxidative stress mechanism to the development and/or progression of KTCN.

Complete mitochondrial genome of Critically Endangered Blue-throated Macaw (Ara glaucogularis): its comparison with partial mitogenome of Scarlet Macaw (Ara macao)

Blue-throated Macaw (Ara glaucogularis) is Critically Endangered species of parrot endemic to small Bolivian area. In this study, full mitochondrial genome of considered species was sequenced. It is 16,983 bp long and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. It`s comparison with published Ara macao mitogenome revealed their high degree of identity. On the other hand, analysis of both the genome compositions showed incompleteness of Ara macao CYTB gene. Hence, mitogenome of Ara macao species occurred only partial sequence. In consequence, Ara glaucogularis mitogenome is the first complete Macaw sequence, which will be indispensable to refine the phylogenetic relationships within the tribe Arini and will enrich the resource of markers for systematic, phylogenetic and population genetic studies.

A Protocol for mtGenome Analysis on Large Sample Numbers.

The mitochondrial genome is widely studied in a variety of fields, such as population, forensic, and human and medical genetics. Most studies have been limited to a small portion of the sequence that, although highly diverse, does not describe the total variability. The arrival of modern high-throughput sequencing technologies has made it possible to investigate larger sequences in a shorter amount of time as well as in a more affordable fashion. This work aims to describe a protocol for sequencing and analyzing the complete mitochondrial genome with the Ion PGM™ platform. To evaluate the protocol, the mitochondrial genome was sequenced to approximately 210 Mbp, with high-quality sequences distributed between 12 samples that had an average coverage of 1023× per sample. Several variant callers were compared to improve the protocol outcome. The results suggest that it is possible to run up to 120 samples per run without any loss of any significant quality. Therefore, this protocol is an efficient and accurate tool for full mitochondrial genome analysis.