A comprehensive phylogenomic study unveils evolutionary patterns and challenges in the mitochondrial genomes of Carcharhiniformes: A focus on Triakidae.

The complex evolutionary patterns in the mitochondrial genome (mitogenome) of the most species-rich shark order, the Carcharhiniformes (ground sharks) has led to challenges in the phylogenomic reconstruction of the families and genera belonging to the order, particularly the family Triakidae (houndsharks). The current state of Triakidae phylogeny remains controversial, with arguments for both monophyly and paraphyly within the family. We hypothesize that this variability is triggered by the selection of different a priori partitioning schemes to account for site and gene heterogeneity within the mitogenome. Here we used an extensive statistical framework to select the a priori partitioning scheme for inference of the mitochondrial phylogenomic relationships within Carcharhiniformes, tested site heterogeneous CAT + GTR + G4 models and incorporated the multi-species coalescent model (MSCM) into our analyses to account for the influence of gene tree discordance on species tree inference. We included five newly assembled houndshark mitogenomes to increase resolution of Triakidae. During the assembly procedure, we uncovered a 714 bp-duplication in the mitogenome of Galeorhinus galeus. Phylogenetic reconstruction confirmed monophyly within Triakidae and the existence of two distinct clades of the expanded Mustelus genus. The latter alludes to potential evolutionary reversal of reproductive mode from placental to aplacental, suggesting that reproductive mode has played a role in the trajectory of adaptive divergence. These new sequences have the potential to contribute to population genomic investigations, species phylogeography delineation, environmental DNA metabarcoding databases and, ultimately, improved conservation strategies for these ecologically and economically important species.

The Non-Dereliction in Evolution: Trophic Specialisation Drives Convergence in the Radiation of Red Devil Spiders (Araneae: Dysderidae) in the Canary Islands.

Natural selection plays a key role in deterministic evolution, as clearly illustrated by the multiple cases of repeated evolution of ecomorphological characters observed in adaptive radiations. Unlike most spiders, Dysdera species display a high variability of cheliceral morphologies, which has been suggested to reflect different levels of specialization to feed on isopods. In this study, we integrate geometric morphometrics and experimental trials with a fully resolved phylogeny of the highly diverse endemic species from the Canary Islands to 1) quantitatively delimit the different cheliceral morphotypes present in the archipelago, 2) test their association with trophic specialization, as reported for continental species, 3) reconstruct the evolution of these ecomorphs throughout the diversification of the group, 4) test the hypothesis of convergent evolution of the different morphotypes, and 5) examine whether specialization constitutes a case of evolutionary irreversibility in this group. We show the existence of 9 cheliceral morphotypes and uncovered their significance for trophic ecology. Further, we demonstrate that similar ecomorphs evolved multiple times in the archipelago, providing a novel study system to explain how convergent evolution and irreversibility due to specialization may be combined to shape phenotypic diversification in adaptive radiations.

Comparative mitogenomics of Brachiopods reveals conservatism in articulate species and unusualness in inarticulate species.

BACKGROUND: Brachiopods are a phylum of marine invertebrates with over 10,000 fossil species. Today, there are fewer than 500 extant species assigned to the class Articulata or Inarticulata and for which knowledge of evolutionary genetics and genomics is still poor. Until now, complete mitogenome sequences of two inarticulate species and four articulate species were available. METHODS AND RESULTS: The complete mitogenome of the inarticulate brachiopod species Lingula reevii (20,778 bp) was obtained by using next generation sequencing. It contains 12 protein-coding genes (the annotation of atp8 is unsure), two ribosomal RNA genes, 26 transfer RNA genes, and one supernumerary ORF that is also conserved in the inarticulate species Lingula anatina. It is hypothesized that this ORF could represent a Lingula-specific mtORFan gene (without obvious homology to other genes). Comparative mitogenomics indicate the mitochondrial gene order of L. reevii is unique among brachiopods, and that compared to articulate species, inarticulate species exhibit massive mitogenome rearrangements, deviant ATP8 protein sequences and supernumerary ORFs, possibly representing species- or lineage-specific mtORFan genes. CONCLUSION: The results of this study enrich genetics knowledge of extant brachiopods, which may eventually help to test hypotheses about their decline.

Phylogenetic assessment within a species complex of a subterranean rodent (Geomys bursarius) with conservation implications for isolated subspecies.

Range contraction and expansion from glaciation have led to genetic divergence that may be particularly pronounced in fossorial species with low dispersal. The plains pocket gopher (Geomys bursarius) is a fossorial species that ranges widely across North America but has a poorly understood phylogeny. We used mitogenomes (14,996 base pairs) from 56 individuals across seven subspecies, plus two outgroup species, to assess genetic divergence from minimum spanning trees, measure genetic distances, and infer phylogenetic trees using BEAST. We found G. b. wisconsinensis was monophyletic with recent divergence. Further assessment is needed for G. b. major because it was paraphyletic and exhibited inconsistent groupings with other clades. Importantly, we identified G. b. illinoensis as being genetically distinct and monophyletic likely due to a unique colonization event eastward across the Mississippi River. Because G. b. illinoensis faces continued pressures from niche reduction and habitat loss, we recommend that G. b. illinoensis be considered an evolutionary significant unit warranting conservation actions to promote connectivity and restore suitable habitat. Such conservation efforts should benefit other grassland species including those originating from clades west of the Mississippi River that may also be evolutionary significant units.

Comparative and phylogenetic analyses of mitochondrial genomes in Elateridae (Coleoptera: Elateroidea).

The click-beetles (Elateridae) are a species-rich beetle family that is easily recognizable. They are distributed in all zoogeographical regions with over 11,000 species. Comparative studies of the structural characteristics of mitochondrial genomes (mitogenomes), as well as phylogenetic relationships of click-beetles, can improve our understanding of mitogenomic evolution. In this study, we determined four mitogenomes from Elateridae by next-generation sequencing. The four mitogenomes were 16,005 to 16,930 bp in length with 37 typical genes and a control region (A + T-rich region). Combined with previously reported elaterid mitogenomes, all PCGs initiate with either the standard start codon of ATN or TTG. According to the nonsynonymous/synonymous mutation ratio (Ka/Ks) of all PCGs, the highest and the lowest evolutionary rates were found for atp8 and cox1, respectively. Among the control regions of the four mitogenomes, several different patterns and numbers of tandem repeats were identified, which was the primary cause of the length variation in control regions. Phylogenetic analyses were conducted based on 13 protein-coding genes and two ribosomal RNA genes from 33 species of Elateridae and two outgroups. The Bayesian inference and maximum likelihood trees had an identical topological structure. The monophyly of Cardiophorinae, Agrypninae and Elaterinae was recovered with high support in all topologies, and the Tetralobinae was placed as the earliest branch in the Elateridae. Expanding the availability of mitogenomic and genomic data from a broader range of click-beetles could provide more clarity on the disputed relationships among subfamilies within Elateridae.

Comparative analysis of the two suborders of Thysanoptera and characterization of the complete mitochondrial genome of Thrips parvispinus.

Thrips parvispinus is a serious sucking pest on a number of economically important crops in the oriental region. It has gained importance recently for its drastic range extension distribution as an invasive pest. Here, the complete mitochondrial genome (15,067 bp) of Thrips parvispinus was sequenced and characterized. It possesses 37 genes and the putative noncoding region is duplicated. Comparative analyses of nucleotide diversity, skewness, codon usage bias, and selection pressure in mitochondrial protein-coding genes of the available 31 thrips mitogenomes (24 Terebrantia + 7 Tubulifera) were performed. Phylogenetic analysis showed a sister relationship of T. parvispinus to the clade (T. florum + T. hawaiiensis). Phylogenetic analyses formed the monophyly of subfamilies Phlaeothripinae and Idolothripinae within the family Phlaeothripidae (Suborder Tubulifera). Low nucleotide diversity was indicative of reversal of strand asymmetry in the Tubulifera. Neutrality analysis showed that directional mutation plays a major role in shaping codon usage bias in both suborders. Principal component analysis indicated distinct codon usage patterns in each suborder. Our data suggested weaker selection constrains on Terebrantia than in the Tubulifera. More tubuliferan mitogenomes are required to resolve previous classification hypotheses and elucidate genome evolution in these two suborders.

Taxonomic Status and Phylogenetic Relationship of the Charadriidae Family Based on Complete Mitogenomes.

Background: The Charadriiformes provide a good source for researching evolution owing to their diverse distribution, behavior, morphology, and ecology. However, in the Charadrii, family-level relationships remain understudied, and the monophyly of Charadriidae is also a subject of controversy. Methods: In the present study, we generated complete mitogenomes for two species, Charadrius leschenaultii and Charadrius mongolus, which were found to be 16,905 bp and 16,844 bp in length, respectively. Among the 13 protein codon genes, we observed variation in the rate of non-synonymous substitution rates, with the slowest rate found in COI and the fastest rate observed in ATP8. The Ka/Ks ratio for all Charadriidae species was significantly lower than one, which inferred that the protein-coding genes underwent purifying selection. Results: Phylogenetic analysis based on the genes of Cyt b, 12S and ND2 revealed that the genus Pluvialis is the sister group of three families (Haematopodidae, Ibidorhynchidae, Recurvirostridae). However, the phylogenetic analysis based on complete mitogenomes indicated that the genus Pluvialis is within the Charadriidae family. Conclusion: This study highlights the importance of carefully selecting the number of genes used to obtain accurate estimates of the species tree. It also suggests that relying on partial mtDNA genes with fast-evolving rates may lead to misleading results when resolving the Pluvialis sister group. Future research should focus on sequencing more mitogenomes at different taxonomic levels to gain a better understanding of the features and phylogenetic relationships within the Charadriiformes order.

Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae).

BACKGROUND: Mitochondrial genomes are the most sequenced genomes after bacterial and fungal genomic DNA. However, little information on mitogenomes is available for multiple metazoan taxa, such as Culicoides, a globally distributed, megadiverse genus containing 1,347 species. AIM:  Generating novel mitogenomic information from single Culicoides sonorensis and C. biguttatus specimens, comparing available mitogenome mapping and de novo assembly tools, and identifying the best performing strategy and tools for Culicoides species. RESULTS: We present two novel and fully annotated mitochondrial haplotypes for two Culicoides species, C. sonorensis and C. biguttatus. We also annotated or re-annotated the only available reference mitogenome for C. sonorensis and C. arakawae. All species present a high similarity in mitogenome organization. The general gene arrangement for all Culicoides species was identical to the ancestral insect mitochondrial genome. Only short spacers were found in C. sonorensis (up to 30 bp), contrary to C. biguttatus (up to 114 bp). The mitochondrial genes ATP8, NAD2, NAD6, and LSU rRNA exhibited the highest nucleotide diversity and pairwise interspecific p genetic distance, suggesting that these genes might be suitable and complementary molecular barcodes for Culicoides identification in addition to the commonly utilized COI gene. We observed performance differences between the compared mitogenome generation strategies. The mapping strategy outperformed the de novo assembly strategy, but mapping results were partially biased in the absence of species-specific reference mitogenome. Among the utilized tools, BWA performed best for C. sonorensis while SPAdes, MEGAHIT, and MitoZ were among the best for C. biguttatus. The best-performing mitogenome annotator was MITOS2. Additionally, we were able to recover exogenous mitochondrial DNA from Bos taurus (biting midges host) from a C. biguttatus blood meal sample. CONCLUSIONS: Two novel annotated mitogenome haplotypes for C. sonorensis and C. biguttatus using High-Throughput Sequencing are presented. Current results are useful as the baseline for mitogenome reconstruction of the remaining Culicoides species from single specimens to HTS and genome annotation. Mapping to a species-specific reference mitogenome generated better results for Culicoides mitochondrial genome reconstruction than de novo assembly, while de novo assembly resulted better in the absence of a closely related reference mitogenome. These results have direct implications for molecular-based identification of these vectors of human and zoonotic diseases, setting the basis for using the whole mitochondrial genome as a marker in Culicoides identification.

A global molecular phylogeny yields insights into the dispersal and invasion history of Junonia, a butterfly genus with remarkable dispersal abilities.

The nymphalid butterfly genus Junonia has remarkable dispersal abilities. Occurring on every continent except Europe and Antarctica, Junonia are often among the only butterflies on remote oceanic islands. The biogeography of Junonia has been controversial, plagued by taxonomic disputes, small phylogenetic datasets, incomplete taxon sampling, and shared interspecific mitochondrial haplotypes. Junonia originated in Africa but its route into the New World remains unknown. Presented here is, to our knowledge, the most comprehensive Junonia phylogeny to date, using full mitogenomes and nuclear ribosomal RNA repeats from 40 of 47 described species. Junonia is monophyletic and the genus Salamis is its probable sister clade. Genetic exchange between Indo-Pacific Junonia villida and New World Junonia vestina is evident, suggesting a trans-Pacific route into the New World. However, in both phylogenies, the sister clades to most New World Junonia contain both African and Asian species. Multiple trans-Atlantic or trans-Pacificinvasions could have contributed to New World diversification. Hybridization and lateral transfer of mitogenomes, already well-documented in New World Junonia, also occurs in at least two Old World lineages (Junonia orithya/Junonia hierta and Junonia iphita/Junonia hedonia). Variation associated with reticulate evolution creates challenges for phylogenetic reconstruction, but also may have contributed to patterns of speciation and diversification in this genus.

The longest mitochondrial protein in metazoans is encoded by the male-transmitted mitogenome of the bivalve Scrobicularia plana.

Cytochrome c oxidase subunit II (COX2) is one of the three mitochondrially encoded proteins of the complex IV of the respiratory chain that catalyses the reduction of oxygen to water. The cox2 gene spans about 690 base pairs in most animal species and produces a protein composed of approximately 230 amino acids. We discovered an extreme departure from this pattern in the male-transmitted mitogenome of the bivalve Scrobicularia plana with doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA), which possesses an important in-frame insertion of approximately 4.8 kb in its cox2 gene. This feature-an enlarged male cox2 gene-is found in many species with DUI; the COX2 protein can be up to 420 amino acids long. Through RT-PCRs, immunoassays and comparative genetics, the evolution and functionality of this insertion in S. plana were characterized. The in-frame insertion is conserved among individuals from different populations and bears the signature of purifying selection seemingly indicating maintenance of functionality. Its transcription and translation were confirmed: this gene produces a polypeptide of 1892 amino acids, making it the largest metazoan COX2 protein known to date. We hypothesize that these extreme modifications in the COX2 protein affect the metabolism of mitochondria containing the male-transmitted mtDNA in Scrobicularia plana.

Phylomitogenomics provides new perspectives on the Euphasmatodea radiation (Insecta: Phasmatodea).

Phasmatodea species diversity lies almost entirely within its suborder Euphasmatodea, which exhibits a pantropical distribution and is considered to derive from a recent and rapid evolutionary radiation. To shed light on Euphasmatodea origins and diversification, we assembled the mitogenomes of 17 species from transcriptomic sequencing data and analysed them along with 22 already available Phasmatodea mitogenomes and 33 mitogenomes representing most of the Polyneoptera lineages. Maximum Likelihood and Bayesian Inference approaches retrieved consistent topologies, both showing the widespread conflict between phylogenetic approaches and traditional systematics. We performed a divergence time analysis leveraging ten fossil specimens representative of most polyneopteran lineages: the time tree obtained supports an older radiation of the clade with respect to previous hypotheses. Euphasmatodea diversification is inferred to have started ~ 187 million years ago, suggesting that the Triassic-Jurassic mass extinction and the breakup of Pangea could have contributed to the process. We also investigated Euphasmatodea mitogenomes patterns of dN, dS and dN/dS ratio throughout our time-tree, trying to characterize the selective regime which may have shaped the clade evolution.

Comparative Mitogenomics of Fungal Species in Stachybotryaceae Provides Evolutionary Insights into Hypocreales.

Stachybotrys chartarum is one of the world's ten most feared fungi within the family Stachybotryaceae, although to date, not a single mitogenome has been documented for Stachybotryaceae. Herein, six mitogenomes of four different species in Stachybotryaceae are newly reported. The S. chartarum mitogenome was 30.7 kb in length and contained two introns (one each in rnl and cox1). A comparison of the mitogenomes of three different individuals of S. chartarum showed few nucleotide variations and conservation of gene content/order and intron insertion. A comparison of the mitogenomes of four different Stachybotryaceae species (Memnoniella echinata, Myrothecium inundatum, S. chartarum, and S. chlorohalonata), however, revealed variations in intron insertion, gene order/content, and nad2/nad3 joining pattern. Further investigations on all Hypocreales species with available mitogenomes showed greater variabilities in gene order (six patterns) and nad2/nad3 joining pattern (five patterns) although a dominant pattern always existed in each case. Ancestral state estimation showed that in each case the dominant pattern was always more ancestral than those rare patterns. Phylogenetic analyses based on mitochondrion-encoded genes supported the placement of Stachybotryaceae in Hypocreales. The crown age of Stachybotryaceae was estimated to be approximately the Early Cretaceous (141-142 Mya). This study greatly promotes our understanding of the evolution of fungal species in Hypocreales.

Pliocene-Early Pleistocene Geological Events Structure Pacific Martens (Martes caurina).

The complex topography, climate, and geological history of Western North America have shaped contemporary patterns of biodiversity and species distributions in the region. Pacific martens (Martes caurina) are distributed along the northern Pacific Coast of North America with disjunct populations found throughout the Northwestern Forested Mountains and Marine West Coast Forest ecoregions of the West Coast. Martes in this region have been classified into subspecies; however, the subspecific designation has been extensively debated. In this study, we use genomic data to delineate conservation units of Pacific marten in the Sierra-Cascade-Coastal montane belt in the western United States. We analyzed the mitochondrial genome for 94 individuals to evaluate the spatial distribution and divergence times of major lineages. We further genotyped 401 individuals at 13 microsatellite loci to investigate major patterns of population structure. Both nuclear and mitochondrial DNA suggest substantial genetic substructure concordant with historical subspecies designations. Our results revealed that the region contains 2 distinct mitochondrial lineages: a Cascades/Sierra lineage that diverged from the Cascades/coastal lineage 2.23 (1.48-3.14 mya), consistent with orogeny of the Cascade Mountain chain. Interestingly, Pacific Martes share phylogeographic patterns similar with other sympatric taxa, suggesting that the complex geological history has shaped the biota of this region. The information is critical for conservation and management efforts, and further investigation of adaptive diversity is warranted following appropriate revision of conservation management designations.

Characterization of the complete mitochondrial genome of Amblyomma ovale, comparative analyses and phylogenetic considerations.

In this study, we sequenced two complete mitochondrial genomes of Amblyomma ovale, a tick of public health importance. Sequencing two distinct individuals, the resulting mitochondrial genomes were 14,756 and 14,760 bp in length and maintained the same gene order previously reported in Amblyomma. These were combined with RNA-seq derived mitochondrial sequences from three additional species, Amblyomma aureolatum, Amblyomma maculatum, and Amblyomma moreliae, to carry out mitogenome comparative and evolutionary analyses against all previously published tick mitochondrial genomes. We described a derivative genome rearrangement that isolates Ixodes from the remaining Ixodidae and consists of both a reverse translocation as well as an event of Tandem Duplication Random Loss. Genetic distance analyses indicated that cox2, nd1, nd5, and 16S are good candidates for future population studies in A. ovale. The phylogenetic analyses corroborated the utility of complete mitochondrial genomes as phylogenetic markers within the group. This study further supplements the genome information available for Amblyomma and facilitates future evolutionary and population genetic studies within the genus.

Mitochondrial phylogenomics of the Hymenoptera.

The insect order Hymenoptera presents marvelous morphological and ecological diversity. Higher-level hymenopteran relationships remain controversial, even after recent phylogenomic analyses, as their taxon sampling was limited. To shed light on the origin and diversification of Hymenoptera, in particular the poorly studied Parasitica, we undertook phylogenetic analyses of 40 newly and 43 previously sequenced mitochondrial genomes representing all major clades of Hymenoptera. Various Bayesian inferences using different data partitions and phylogenetic methods recovered similar phylogenetic trees with strong statistical support for almost all nodes. Novel findings of the mitogenomic phylogeny mainly affected the three infraorders Ichneumonomorpha, Proctotrupomorpha and Evaniomorpha, the latter of which was split into three clades. Basal relationships of Parasitica recovered Stephanoidea + (Gasteruptiidae + Aulacidae) as the sister group to Ichneumonomorpha + (Trigonalyoidea + Megalyroidea). This entire clade is sister to Proctotrupomorpha, and Ceraphronoidea + Evaniidae is sister to Aculeata (stinging wasps). Our divergence time analysis indicates that major hymenopteran lineages originated in the Mesozoic. The radiation of early apocritans may have been triggered by the Triassic-Jurassic mass extinction; all extant families were present by the Cretaceous.

Cetartiodactyla: Updating a time-calibrated molecular phylogeny.

Cetartiodactyla comprises one of the most diverse mammal radiations. Currently, 23 families, 131 genera and more than 330 species are recognized. Several studies have been trying to resolve its phylogenetic relationships. The most comprehensive dated phylogenetic hypothesis available includes only 55% of the extant species, precluding a clear understanding of ecological and evolutionary patterns in Cetartiodactyla. Here, we gathered all mitochondrial genetic data available in GenBank to build a robust Cetartiodactyla calibrated phylogenetic tree using 21 fossil calibration points. We found mitogenomic data for 225 species and included other 93 species from which there was at least one mitochondrial gene available. Using a Bayesian approach, we generated a dated tree comprising 90% of the extant Cetartiodactyla species (n = 318). The major lineages showed robust support and families divergence times are congruent with the available fossil evidence and with previously published phylogenetic hypotheses. By making available a dated phylogeny with extensively sampled clades, we expect to foster future studies on the origin, tempo and mode of Cetartiodactyla diversification.

The age and diversification of metacrangonyctid subterranean amphipod crustaceans revisited.

The Metacrangonyctidae are a small family of amphipod crustaceans of marine origin found only in subterranean continental waters. They display a broad but punctuated distribution between the Caribbean and the Arabian Peninsula, with major disjunctions either due to vicariance by plate tectonics or to occurrence of recent episodes of long-distance transoceanic dispersal. We re-examine the phylogeny of the family and the time frame for its diversification using mitochondrial genomes in the light of two key taxa recently discovered, from Oman (Arabian Peninsula) and the Rif area of Morocco, respectively. We also use a novel fossil calibration scheme of the mitogenome phylogeny. Results of previous analyses based on palaeogeographic calibrations are not contradicted by the new approach, with vicariance by plate tectonics remaining as the main explanatory factor for the amphi-Atlantic distribution displayed by this ancient group of subterranean amphipods.

Complete mitogenome of the entomopathogenic fungus Sporothrix insectorum RCEF 264 and comparative mitogenomics in Ophiostomatales.

The fungal order Ophiostomatales contains numerous species important in medical fields, agriculture, and forestry, and several species have had available mitogenome information. The nuclear genome of the entomopathogenic fungus Sporothrix insectorum has been reported, while its mitogenome remains unknown. Herein, we firstly described the mitogenome of S. insectorum RCEF 264 and then compared Ophiostomatales mitogenomes from both interspecific and intraspecific perspectives. The mitogenome of S. insectorum RCEF 264 was 31,454 bp in length, containing typical fungal mitochondrial genes plus rnpB. Four group I introns interrupted rnl and cox1. Phylogenetic analyses confirmed the placement of S. insectorum RCEF 264 in Ophiostomatales. Comparison of mitogenomes among seven Ophiostomatales species revealed conserved gene contents and a high synteny, although there were also some differences among them. Their mitogenomes showed more than two-fold variations (26.6-65.1 kb) in size, with a total of 37 intron insertional loci from 11 genes (1-25 introns per species). The sole intron shared by all species was an rps3-encoding intron in rnl (mL2450), and this intron-based phylogeny was highly consistent with those constructed using mitochondrial/nuclear genes, suggesting convergent evolution of this intron with Ophiostomatales species. The dendrogram based on presence/absence patterns at all intron loci was quite different from those based on mitochondrial/nuclear genes. Comparison of mitogenomes among two to three intraspecific individuals in Ophiostoma novo-ulmi subsp. novo-ulmi and Sporothrix schenckii revealed mitogenome size variations due to single-nucleotide polymorphisms (SNPs) and indels but without fluctuation of intron numbers for each species. This study greatly enhanced our understanding of mitogenome evolution in Ophiostomatales.

Ancient mtDNA sequences from the First Australians revisited.

The publication in 2001 by Adcock et al. [Adcock GJ, et al. (2001) Proc Natl Acad Sci USA 98(2):537-542] in PNAS reported the recovery of short mtDNA sequences from ancient Australians, including the 42,000-y-old Mungo Man [Willandra Lakes Hominid (WLH3)]. This landmark study in human ancient DNA suggested that an early modern human mitochondrial lineage emerged in Asia and that the theory of modern human origins could no longer be considered solely through the lens of the "Out of Africa" model. To evaluate these claims, we used second generation DNA sequencing and capture methods as well as PCR-based and single-primer extension (SPEX) approaches to reexamine the same four Willandra Lakes and Kow Swamp 8 (KS8) remains studied in the work by Adcock et al. Two of the remains sampled contained no identifiable human DNA (WLH15 and WLH55), whereas the Mungo Man (WLH3) sample contained no Aboriginal Australian DNA. KS8 reveals human mitochondrial sequences that differ from the previously inferred sequence. Instead, we recover a total of five modern European contaminants from Mungo Man (WLH3). We show that the remaining sample (WLH4) contains ∼1.4% human DNA, from which we assembled two complete mitochondrial genomes. One of these was a previously unidentified Aboriginal Australian haplotype belonging to haplogroup S2 that we sequenced to a high coverage. The other was a contaminating modern European mitochondrial haplotype. Although none of the sequences that we recovered matched those reported by Adcock et al., except a contaminant, these findings show the feasibility of obtaining important information from ancient Aboriginal Australian remains.

Landscape genomics: natural selection drives the evolution of mitogenome in penguins.

BACKGROUND: Mitochondria play a key role in the balance of energy and heat production, and therefore the mitochondrial genome is under natural selection by environmental temperature and food availability, since starvation can generate more efficient coupling of energy production. However, selection over mitochondrial DNA (mtDNA) genes has usually been evaluated at the population level. We sequenced by NGS 12 mitogenomes and with four published genomes, assessed genetic variation in ten penguin species distributed from the equator to Antarctica. Signatures of selection of 13 mitochondrial protein-coding genes were evaluated by comparing among species within and among genera (Spheniscus, Pygoscelis, Eudyptula, Eudyptes and Aptenodytes). The genetic data were correlated with environmental data obtained through remote sensing (sea surface temperature [SST], chlorophyll levels [Chl] and a combination of SST and Chl [COM]) through the distribution of these species. RESULTS: We identified the complete mtDNA genomes of several penguin species, including ND6 and 8 tRNAs on the light strand and 12 protein coding genes, 14 tRNAs and two rRNAs positioned on the heavy strand. The highest diversity was found in NADH dehydrogenase genes and the lowest in COX genes. The lowest evolutionary divergence among species was between Humboldt (Spheniscus humboldti) and Galapagos (S. mendiculus) penguins (0.004), while the highest was observed between little penguin (Eudyptula minor) and Adélie penguin (Pygoscelis adeliae) (0.097). We identified a signature of purifying selection (Ka/Ks < 1) across the mitochondrial genome, which is consistent with the hypothesis that purifying selection is constraining mitogenome evolution to maintain Oxidative phosphorylation (OXPHOS) proteins and functionality. Pairwise species maximum-likelihood analyses of selection at codon sites suggest positive selection has occurred on ATP8 (Fixed-Effects Likelihood, FEL) and ND4 (Single Likelihood Ancestral Counting, SLAC) in all penguins. In contrast, COX1 had a signature of strong negative selection. ND4 Ka/Ks ratios were highly correlated with SST (Mantel, p-value: 0.0001; GLM, p-value: 0.00001) and thus may be related to climate adaptation throughout penguin speciation. CONCLUSIONS: These results identify mtDNA candidate genes under selection which could be involved in broad-scale adaptations of penguins to their environment. Such knowledge may be particularly useful for developing predictive models of how these species may respond to severe climatic changes in the future.