Full Mitochondrial Genome Sequences Research Articles

Ancient DNA analysis of the extinct North American flat-headed peccary (Platygonus compressus)

The geographical range of extant peccaries extends from the southwestern United States through Central America and into northern Argentina. However, from the Miocene until the Pleistocene now-extinct peccary species inhabited the entirety of North America. Relationships among the living and extinct species have long been contentious. Similarly, how and when peccaries moved from North to South America is unclear. The North American flat-headed peccary (Platygonus compressus) became extinct at the end of the Pleistocene and is one of the most abundant subfossil taxa found in North America, yet despite this extensive fossil record its phylogenetic position has not been resolved. This study is the first to present DNA data from the flat-headed peccary and full mitochondrial genome sequences of all the extant peccary species. We performed a molecular phylogenetic analysis to determine the relationships among ancient and extant peccary species. Our results suggested that the flat-headed peccary is sister-taxon to a clade comprising the extant peccary species. Divergence date estimates from our molecular dating analyses suggest that if extant peccary diversification occurred in South America then their common ancestor must have dispersed from North America to South America well before the establishment of the Isthmus of Panama. We also investigated the genetic diversity of the flat-headed peccary by performing a preliminary population study on specimens from Sheriden Cave, Ohio. Flat-headed peccaries from Sheriden Cave appear to be genetically diverse and show no signature of population decline prior to extinction. Including additional extinct Pleistocene peccary species in future phylogenetic analyses will further clarify peccary evolution.

Mitochondrial and chemical profiles reveal a new genus and species of Neotropical termite with snapping soldiers, Palmitermes impostor (Termitidae : Termitinae)

Since the inception of Linnaean taxonomy, termite species and genus descriptions have been mostly based on the morphology of soldiers, sometimes complemented by alate characters, though these are seldom discriminant. However, narrowly soldier-based descriptions may overemphasise ancestral characters and lead to the establishment of non-monophyletic taxa. In this paper, we used an integrative taxonomic approach that incorporates the morphology of all castes, including workers, as well as molecular and chemical data, to describe Palmitermes impostor Hellemans & Roisin, 2017 (Termitidae:Termitinae), a new termite genus and species from French Guiana. Although the soldiers of P. impostor resemble those of Termes Linnaeus, 1758, the digestive tract and mandibles of workers suggest that Palmitermes is closely related to Cavitermes Emerson, 1925. The sister-group relationship between Palmitermes and Cavitermes was confirmed by a phylogenetic reconstruction based on full mitochondrial genome sequences as well as by the comparison of the profiles of cuticular hydrocarbons of workers with those of related taxa. Our study illustrates the benefits of using an integrative taxonomic approach to describe new taxa and the pitfalls of using soldier morphology as the exclusive set of characters in termite systematics.

The mitochondrial genome of the long-billed plover, Charadrius placidus (Charadriiformes: Charadriidae)

The present study provides the first full mitochondrial genome sequence of a long-billed plover from South Korea. This mitogenome is 16,895 bp in length and consists of 13 protein-coding genes, 2 ribosomal-RNA genes, 22 transfer-RNA genes, and a non-coding control region. Overall, base composition was: A, 31.4%; C, 31.1%; G, 13.8%; and T, 23.8%. A frameshift mutation in the ND3 gene was identified and a tandem repeat (AACA) was observed in the D-loop region. The phylogenetic analysis based on concatenated coding genes indicated Charadriidae’s monophyly. These results contribute to further understanding of long-billed plover phylogenetic relationships and species identification.

Complete mitochondrial genome of the smallmouth hardyhead (Atherinosoma microstoma) and its phylogenetic position among the Atheriniform fishes

In this paper, I report the full mitochondrial genome sequence of the smallmouth hardyhead (Atherinosoma microstoma), an endemic marine fish from the shallow coastal waters of southeastern Australia. The mitogenome is 16,573 bp-long with the standard 37 genes all included, with a genomic structure typical of a vertebrate mitogenome. In order to confirm the phylogenetic position of this species, phylogenetic trees were inferred using a data set including publicly available 28 atherinomorph, nine percomorph, and two outgroup mitogenome sequences. The complete mitogenome data of A. microstoma reported here will be useful for further genetics, phylogeography, and phylogenetics studies involving this species.

The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) hongkongensis Provide Evidence for Presence of Cryptic Species.

BackgroundCutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information.Methods and FindingsComplete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large.ConclusionGenetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. ‘Thailand II’, or a quite divergent population of C. D. hongkongensis.

Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq

Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control region (CR), is routinely employed in forensics in human identification and missing persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome some of the major limitations of STS processing, permitting reasonable paths forward for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput and higher sensitivity capabilities. To establish the accuracy and reproducibility of NGS for the development of mtDNA data, 90 DNA extracts that were previously used to generate forensic quality full mtGenomes using STS were sequenced using Nextera XT library preparation and the Illumina MiSeq. Using the same amplicon product, replicate library sets were generated and sequenced at different laboratories, and analysis was performed in replicate using the CLC Genomics Workbench. Both sequencing sets resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including controls) were multiplexed. Overall, 99.9996% concordance was observed between the NGS data and the STS data for the full mtGenome. The only “discordant” calls involved low level point heteroplasmies, with the differences resulting from stochastic variation and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection of a mixed sample previously not detected with STS. Additionally, variant calls were reproducible between sequencing sets and between software analysis versions with the variant frequency only differing by 0.23% and 0.01%, respectively. Further validation studies and specialized software functionality tailored to forensic practice should facilitate the incorporation of NGS processing into standard casework applications. The data herein comprise the largest, and likely most thoroughly examined, complete mtGenome STS-NGS concordance dataset available.

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.

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.

Increased mitochondrial mutation frequency after an island colonization: positive selection or accumulation of slightly deleterious mutations?

Island colonizations are excellent models for studying early processes of evolution. We found in a previous study on mice that had colonized the sub-Antarctic Kerguelen Archipelago about 200 years ago that they were derived from a single founder lineage and that this showed an unexpectedly large number of new mutations in the mitochondrial D-loop. To assess whether positive selection has played a role in the emergence of these variants, we have obtained 16 full mitochondrial genome sequences from these mice. For comparison, we have compiled 57 mitochondrial genome sequences from laboratory inbred lines that became established about 100 years ago, also starting from a single founder lineage. We find that the island mice and the laboratory lines show very similar mutation frequencies and patterns. None of the patterns in the Kerguelen mice provides evidence for positive selection. We conclude that nearly neutral evolutionary processes that assume the presence of slightly deleterious variants can fully explain the patterns. This supports the notion of time-dependency of molecular evolution and provides a new calibration point. Based on the observed mutation frequency, we calculate an average evolutionary rate of 0.23 substitutions per site per Myr for the earliest time frame of divergence, which is about six times higher than the long-term rate of 0.037 substitutions per site per Myr.

Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination

BackgroundAlthough nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle".ResultsHere we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation.ConclusionsThe homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB is supposed to halt replication, it offers a potential mechanistic explanation for frequent recombination in mitochondrial genomes.

Application of full mitochondrial genome sequencing using 454 GS FLX pyrosequencing

The GS FLX pyrosequencing platform using parallel tagged sequencing was tested on 10 Somali individuals for sequencing of the complete mitochondrial genome. The amplicons were sequenced twice with increasing coverage to establish the minimum of coverage needed to produce reliable sequence reads. The genome sequences were compared to previously obtained control regions sequences with Sanger sequencing and 49 SNPs in coding regions of the mitochondrial genome. No discrepancy was found with the three methods except in a poly-C stretch that was estimated to be 16193.1C by sequencing with terminator chemistry and no insert was observed with pyrosequencing. The sequence lengths of both pyrosequencing runs were between 225 and 235 nucleotides. The coverage for the first pyrosequencing run was between 0 and 159 and 10–577 for the second one.

The mastodon mitochondrial genome: a mammoth accomplishment

The mitochondrial genome of an American mastodon was recently sequenced and used to root a phylogenetic analysis that included full mitochondrial genome sequences from woolly mammoths and the two living elephant genera. The study definitively established that mammoth and Asian elephant mitochondrial DNA lineages are more closely related than either is to African elephants. However, it also suggests that a complex evolutionary picture could ultimately emerge and points to similarities between the early evolution of the Elephantidae and that of the gorilla-human-chimpanzee clade.

Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes

Mitochondrial genome sequences provide useful markers for investigating population genetic structures because of their maternal inheritance and high evolutionary rates. There is, however, a paucity of information on mitochondrial genomes for many parasitic organisms, including nematodes, which appears to relate mainly to technical limitations and (for modestly funded laboratories) the cost associated with full mitochondrial genome sequencing. In this article, we describe a simple, relatively inexpensive long-PCR approach for the amplification (using two sets of primers) of the entire mitochondrial genome from individual parasitic nematodes for subsequent sequencing, which overcomes these limitations. We employed two species of human hookworm (Ancylostoma duodenale and Necator americanus; order Strongylida) to establish the long-PCR conditions, and then extended its use to a number of other species of parasitic nematode of the class Secernentea (orders Strongylida, Ascaridida and Rhabditida). The long-PCR method for the amplification of the entire mitochondrial genome from single nematodes, coupled with direct sequencing of amplicons, provides a useful tool for the comparative analysis of genome organisation and evolution of a range of nematode groups. It also creates a platform for molecular ecological and population genetic studies.