OrthoMaM v12: a database of curated single-copy ortholog alignments and trees to study mammalian evolutionary genomics.

To date, the databases built to gather information on gene orthology do not provide end-users with descriptors of the molecular evolution information and phylogenetic pattern of these orthologues. In this context, we developed OrthoMaM, a database of ORTHOlogous MAmmalian Markers describing the evolutionary dynamics of coding sequences in mammalian genomes. OrthoMaM version 12 includes 15,868 alignments of orthologous coding sequences (CDS) from the 190 complete mammalian genomes currently available. All annotations and 1-to-1 orthology assignments are based on NCBI. Orthologous CDS can be mined for potential informative markers at the different taxonomic levels of the mammalian tree. To this end, several evolutionary descriptors of DNA sequences are provided for querying purposes (e.g. base composition and relative substitution rate). The graphical web interface allows the user to easily browse and sort the results of combined queries. The corresponding multiple sequence alignments and ML trees, inferred using state-of-the art approaches, are available for download both at the nucleotide and amino acid levels. OrthoMaM v12 can be used by researchers interested either in reconstructing the phylogenetic relationships of mammalian taxa or in understanding the evolutionary dynamics of coding sequences in their genomes. OrthoMaM is available for browsing, querying and complete or filtered download at https://orthomam.mbb.cnrs.fr/.

Phylogeny, Ecology, and Gene Families Covariation Shaped the Olfactory Subgenome of Rodents.

Olfactory receptor (OR) genes represent the largest multigenic family in mammalian genomes and encode proteins that bind environmental odorant molecules. The OR repertoire is extremely variable among species and is subject to many gene duplications and losses, which have been linked to ecological adaptations in mammals. Although they have been studied on a broad taxonomic scale (i.e., placental), finer sampling has rarely been explored in order to better capture the mechanisms that drove the evolution of the OR repertoire. Among placental mammals, rodents are well-suited for this task, as they exhibit diverse life history traits, and genomic data are available for most major families and a diverse array of lifestyles. In this study, 53 rodent published genomes were mined for their OR subgenomes. We retrieved more than 85,000 functional and pseudogene OR sequences that were subsequently classified into phylogenetic clusters. Copy number variation among rodents is similar to that of other mammals. Using our OR counts along with comparative phylogenetic approaches, we demonstrated that ecological niches such as diet, period of activity, and a fossorial lifestyle strongly impacted the proportion of OR pseudogenes. Within the OR subgenome, phylogenetic inertia was the main factor explaining the relative variations of the 13 OR gene families. However, a striking exception was a convergent 10-fold expansion of the OR family 14 among the phylogenetically divergent subterranean mole-rat lineages belonging to Bathyergidae and Spalacidae families. This study illustrates how the diversity of the OR repertoire has evolved among rodents, both shaped by selective forces stemming from species life history traits and neutral evolution along the rodent phylogeny.

Revisiting gene typing and phylogeny of Trypanosoma cruzi reference strains: Comparison of the relevance of mitochondrial DNA, single-copy nuclear DNA, and the intergenic region of mini-exon gene.

Chagas disease is a widespread neglected disease in Latin America. Trypanosoma cruzi, the causative agent of the disease, is currently subdivided into six DTUs (discrete typing units) named TcI-TcVI, and although no clear association has been found between parasite genetics and different clinical outcomes of the disease or different transmission cycles, genetic characterization of T. cruzi strains remains crucial for integrated epidemiological studies. Numerous markers have been used for this purpose, although without consensus. These include mitochondrial genes, single or multiple-copy nuclear genes, ribosomal RNA genes, and the intergenic region of the repeated mini-exon gene. To increase our knowledge of these gene sequences and their usefulness for strain typing, we sequenced fragments of three mitochondrial genes, nine single-copy nuclear genes, and the repeated intergenic part of the mini-exon gene by Next Generation Sequencing (NGS) on a sample constituted of 16 strains representative of T. cruzi genetic diversity, to which we added the corresponding genetic data of the 38 T. cruzi genomes fully sequenced until 2022. Our results show that single-copy nuclear genes remain the gold standard for characterizing T. cruzi strains; the phylogenetic tree from concatenated genes (3959 bp) confirms the six DTUs previously recognized and provides additional information about the alleles present in the hybrid strains. In the tree built from the three mitochondrial concatenated genes (1274 bp), three main clusters are identified, including one with TcIII, TcIV, TcV, and TcVI DTUs which are not separated. Nevertheless, mitochondrial markers remain necessary for detecting introgression and heteroplasmy. The phylogenetic tree built from the sequence alignment of the repeated mini-exon gene fragment (327 bp) displayed six clusters, but only TcI was associated with a single cluster. The sequences obtained from strains belonging to the other DTUs were scattered into different clusters. Therefore, while the mini-exon marker may bring, for some biological samples, some advantages in terms of sensibility due to its repeated nature, mini-exon sequences must be used with caution and, when possible, avoided for T. cruzi typing and phylogenetic studies.

Digging for the spiny rat and hutia phylogeny using a gene capture approach, with the description of a new mammal subfamily.

Next generation sequencing (NGS) and genomic database mining allow biologists to gather and select large molecular datasets well suited to address phylogenomics and molecular evolution questions. Here we applied this approach to a mammal family, the Echimyidae, for which generic relationships have been difficult to recover and often referred to as a star phylogeny. These South-American spiny rats represent a family of caviomorph rodents exhibiting a striking diversity of species and life history traits. Using a NGS exon capture protocol, we isolated and sequenced ca. 500 nuclear DNA exons for 35 species belonging to all major echimyid and capromyid clades. Exons were carefully selected to encompass as much diversity as possible in terms of rate of evolution, heterogeneity in the distribution of site-variation and nucleotide composition. Supermatrix inferences and coalescence-based approaches were subsequently applied to infer this family's phylogeny. The inferred topologies were the same for both approaches, and support was maximal for each node, entirely resolving the ambiguous relationships of previous analyses. Fast-evolving nuclear exons tended to yield more reliable phylogenies, as slower-evolving sequences were not informative enough to disentangle the short branches of the Echimyidae radiation. Based on this resolved phylogeny and on molecular and morphological evidence, we confirm the rank of the Caribbean hutias - formerly placed in the Capromyidae family - as Capromyinae, a clade nested within Echimyidae. We also name and define Carterodontinae, a new subfamily of Echimyidae, comprising the extant monotypic genus Carterodon from Brazil, which is the closest living relative of West Indies Capromyinae.

OrthoMaM v10: Scaling-Up Orthologous Coding Sequence and Exon Alignments with More than One Hundred Mammalian Genomes.

We present version 10 of OrthoMaM, a database of orthologous mammalian markers. OrthoMaM is already 11 years old and since the outset it has kept on improving, providing alignments and phylogenetic trees of high-quality computed with state-of-the-art methods on up-to-date data. The main contribution of this version is the increase in the number of taxa: 116 mammalian genomes for 14,509 one-to-one orthologous genes. This has been made possible by the combination of genomic data deposited in Ensembl complemented by additional good-quality genomes only available in NCBI. Version 10 users will benefit from pipeline improvements and a completely redesigned web-interface.

MACSE v2: Toolkit for the Alignment of Coding Sequences Accounting for Frameshifts and Stop Codons.

Multiple sequence alignment is a prerequisite for many evolutionary analyses. Multiple Alignment of Coding Sequences (MACSE) is a multiple sequence alignment program that explicitly accounts for the underlying codon structure of protein-coding nucleotide sequences. Its unique characteristic allows building reliable codon alignments even in the presence of frameshifts. This facilitates downstream analyses such as selection pressure estimation based on the ratio of nonsynonymous to synonymous substitutions. Here, we present MACSE v2, a major update with an improved version of the initial algorithm enriched with a complete toolkit to handle multiple alignments of protein-coding sequences. A graphical interface now provides user-friendly access to the different subprograms.

Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?

We examined specimens of the macrostigma trout Salmo macrostigma, which refers to big black spots on the flanks, to assess whether it is an example of taxonomic inflation within the brown trout Salmo trutta complex. Using new specimens, publicly available data and a mitogenomic protocol to amplify the control and cytochrome b regions of the mitochondrial genome from degraded museum samples, including one syntype specimen, the present study shows that the macrostigma trout is not a valid species. Our results suggest the occurrence of a distinct evolutionary lineage of S. trutta in North Africa and Sicily. The name of the North African lineage is proposed for this lineage, which was found to be sister to the Atlantic lineage of brown trout, S. trutta.

In Cold Blood: Compositional Bias and Positive Selection Drive the High Evolutionary Rate of Vampire Bats Mitochondrial Genomes.

Mitochondrial genomes of animals have long been considered to evolve under the action of purifying selection. Nevertheless, there is increasing evidence that they can also undergo episodes of positive selection in response to shifts in physiological or environmental demands. Vampire bats experienced such a shift, as they are the only mammals feeding exclusively on blood and possessing anatomical adaptations to deal with the associated physiological requirements (e.g., ingestion of high amounts of liquid water and iron). We sequenced eight new chiropteran mitogenomes including two species of vampire bats, five representatives of other lineages of phyllostomids and one close outgroup. Conducting detailed comparative mitogenomic analyses, we found evidence for accelerated evolutionary rates at the nucleotide and amino acid levels in vampires. Moreover, the mitogenomes of vampire bats are characterized by an increased cytosine (C) content mirrored by a decrease in thymine (T) compared with other chiropterans. Proteins encoded by the vampire bat mitogenomes also exhibit a significant increase in threonine (Thr) and slight reductions in frequency of the hydrophobic residues isoleucine (Ile), valine (Val), methionine (Met), and phenylalanine (Phe). We show that these peculiar substitution patterns can be explained by the co-occurrence of both neutral (mutational bias) and adaptive (positive selection) processes. We propose that vampire bat mitogenomes may have been impacted by selection on mitochondrial proteins to accommodate the metabolism and nutritional qualities of blood meals.

A phylogenomic framework and timescale for comparative studies of tunicates.

BACKGROUND: Tunicates are the closest relatives of vertebrates and are widely used as models to study the evolutionary developmental biology of chordates. Their phylogeny, however, remains poorly understood, and to date, only the 18S rRNA nuclear gene and mitogenomes have been used to delineate the major groups of tunicates. To resolve their evolutionary relationships and provide a first estimate of their divergence times, we used a transcriptomic approach to build a phylogenomic dataset including all major tunicate lineages, consisting of 258 evolutionarily conserved orthologous genes from representative species. RESULTS: Phylogenetic analyses using site-heterogeneous CAT mixture models of amino acid sequence evolution resulted in a strongly supported tree topology resolving the relationships among four major tunicate clades: (1) Appendicularia, (2) Thaliacea + Phlebobranchia + Aplousobranchia, (3) Molgulidae, and (4) Styelidae + Pyuridae. Notably, the morphologically derived Thaliacea are confirmed as the sister group of the clade uniting Phlebobranchia + Aplousobranchia within which the precise position of the model ascidian genus Ciona remains uncertain. Relaxed molecular clock analyses accommodating the accelerated evolutionary rate of tunicates reveal ancient diversification (~ 450-350 million years ago) among the major groups and allow one to compare their evolutionary age with respect to the major vertebrate model lineages. CONCLUSIONS: Our study represents the most comprehensive phylogenomic dataset for the main tunicate lineages. It offers a reference phylogenetic framework and first tentative timescale for tunicates, allowing a direct comparison with vertebrate model species in comparative genomics and evolutionary developmental biology studies.

Convergent Acquisition of Nonembryonic Development in Styelid Ascidians.

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

Life-History Traits Evolved Jointly with Climatic Niche and Disturbance Regime in the Genus Leucadendron (Proteaceae).

Organisms have evolved a diversity of life-history strategies to cope with variation in their environment. Persistence as adults and/or seeds across recruitment events allows species to dampen the effects of environmental fluctuations. The evolution of life cycles with overlapping generations should thus permit the colonization of environments with uncertain recruitment. We tested this hypothesis in Leucadendron (Proteaceae), a genus with high functional diversity native to fire-prone habitats in the South African fynbos. We analyzed the joint evolution of life-history traits (adult survival and seed-bank strategies) and ecological niches (climate and fire regime), using comparative methods and accounting for various sources of uncertainty. In the fynbos, species with canopy seed banks that are unable to survive fire as adults display nonoverlapping generations. In contrast, resprouters with an underground seed bank may be less threatened by extreme climatic events and fire intervals, given their iteroparity and long-lasting seed bank. Life cycles with nonoverlapping generations indeed jointly evolved with niches with less exposure to frost but not with those with less exposure to drought. Canopy seed banks jointly evolved with niches with more predictable fire return, compared to underground seed banks. The evolution of extraordinary functional diversity among fynbos plants thus reflects, at least in part, the diversity of both climates and fire regimes in this region.

Mitogenomic Phylogeny, Diversification, and Biogeography of South American Spiny Rats.

Echimyidae is one of the most speciose and ecologically diverse rodent families in the world, occupying a wide range of habitats in the Neotropics. However, a resolved phylogeny at the genus-level is still lacking for these 22 genera of South American spiny rats, including the coypu (Myocastorinae), and 5 genera of West Indian hutias (Capromyidae) relatives. Here, we used Illumina shotgun sequencing to assemble 38 new complete mitogenomes, establishing Echimyidae, and Capromyidae as the first major rodent families to be completely sequenced at the genus-level for their mitochondrial DNA. Combining mitogenomes and nuclear exons, we inferred a robust phylogenetic framework that reveals several newly supported nodes as well as the tempo of the higher level diversification of these rodents. Incorporating the full generic diversity of extant echimyids leads us to propose a new higher level classification of two subfamilies: Euryzygomatomyinae and Echimyinae. Of note, the enigmatic Carterodon displays fast-evolving mitochondrial and nuclear sequences, with a long branch that destabilizes the deepest divergences of the echimyid tree, thereby challenging the sister-group relationship between Capromyidae and Euryzygomatomyinae. Biogeographical analyses involving higher level taxa show that several vicariant and dispersal events impacted the evolutionary history of echimyids. The diversification history of Echimyidae seems to have been influenced by two major historical factors, namely (1) recurrent connections between Atlantic and Amazonian Forests and (2) the Northern uplift of the Andes.

Fast and accurate branch lengths estimation for phylogenomic trees.

BACKGROUND: Branch lengths are an important attribute of phylogenetic trees, providing essential information for many studies in evolutionary biology. Yet, part of the current methodology to reconstruct a phylogeny from genomic information - namely supertree methods - focuses on the topology or structure of the phylogenetic tree, rather than the evolutionary divergences associated to it. Moreover, accurate methods to estimate branch lengths - typically based on probabilistic analysis of a concatenated alignment - are limited by large demands in memory and computing time, and may become impractical when the data sets are too large. RESULTS: Here, we present a novel phylogenomic distance-based method, named ERaBLE (Evolutionary Rates and Branch Length Estimation), to estimate the branch lengths of a given reference topology, and the relative evolutionary rates of the genes employed in the analysis. ERaBLE uses as input data a potentially very large collection of distance matrices, where each matrix is obtained from a different genomic region - either directly from its sequence alignment, or indirectly from a gene tree inferred from the alignment. Our experiments show that ERaBLE is very fast and fairly accurate when compared to other possible approaches for the same tasks. Specifically, it efficiently and accurately deals with large data sets, such as the OrthoMaM v8 database, composed of 6,953 exons from up to 40 mammals. CONCLUSIONS: ERaBLE may be used as a complement to supertree methods - or it may provide an efficient alternative to maximum likelihood analysis of concatenated alignments - to estimate branch lengths from phylogenomic data sets.

Thrice better than once: quality control guidelines to validate new mitogenomes.

Mitogenomic data are increasingly used in evolutionary biology and ecology, stressing the importance for double checking the authenticity of DNA sequences. For example, Szczesniak et al. (2013) recently published the mitochondrial genome of a bat, the Leschenault's rousette (Rousettus leschenaultii). Here we show using straightforward phylogenetic analyses of available chiropteran sequence data that the taxonomic attribution of the reported mitogenome is erroneous. The purportedly-new complete mitochondrial genome likely belongs to the Egyptian fruit bat (R. aegyptiacus) for which a reference sequence already exists. We propose that future articles reporting complete mitochondrial genome sequences should mandatorily include maximum likelihood trees inferred from (i) the standard barcoding marker for the taxon under focus, which would benefit from the massive data available in public databases, and (ii) the available mitogenomes of closely related species. We also strongly advise these trees be presented as phylograms so that all pertinent phylogenetic information is displayed in the form of a topology and its associated branch lengths. Along with compulsory information on the geographical location and origin of the specimen, these new standards should help avoiding the publication of taxonomically misidentified mitogenomes that might end up as reference sequences in public databases and re-used in subsequent meta-analyses.

Rodents of the Caribbean: origin and diversification of hutias unravelled by next-generation museomics.

The Capromyidae (hutias) are endemic rodents of the Caribbean and represent a model of dispersal for non-flying mammals in the Greater Antilles. This family has experienced severe extinctions during the Holocene and its phylogenetic affinities with respect to other caviomorph relatives are still debated as morphological and molecular data disagree. We used target enrichment and next-generation sequencing of mitochondrial and nuclear genes to infer the phylogenetic relationships of hutias, estimate their divergence ages, and understand their mode of dispersal in the Greater Antilles.We found that Capromyidae are nested within Echimyidae (spiny rats) and should be considered a subfamily thereof. We estimated that the split between hutias and Atlantic Forest spiny rats occurred 16.5 (14.8­18.2) million years ago (Ma), which is more recent than the GAARlandia land bridge hypothesis (34­35 Ma). This would suggest that during the Early Miocene, an echimyid-like ancestor colonized the Greater Antilles from an eastern South American source population via rafting. The basal divergence of the Hispaniolan Plagiodontia provides further support for a vicariant separation between Hispaniolan and western islands (Bahamas, Cuba, Jamaica) hutias. Recent divergences among these western hutias suggest Plio-Pleistocene dispersal waves associated with glacial cycles.

Convergent and correlated evolution of major life-history traits in the angiosperm genus Leucadendron (Proteaceae).

Natural selection is expected to cause convergence of life histories among taxa as well as correlated evolution of different life-history traits. Here, we quantify the extent of convergence of five key life-history traits (adult fire survival, seed storage, degree of sexual dimorphism, pollination mode, and seed-dispersal mode) and test hypotheses about their correlated evolution in the genus Leucadendron (Proteaceae) from the fire-prone South African fynbos. We reconstructed a new molecular phylogeny of this highly diverse genus that involves more taxa and molecular markers than previously. This reconstruction identifies new clades that were not detected by previous molecular study and morphological classifications. Using this new phylogeny and robust methods that account for phylogenetic uncertainty, we show that the five life-history traits studied were labile during the evolutionary history of the genus. This diversity allowed us to tackle major questions about the correlated evolution of life-history strategies. We found that species with longer seed-dispersal distances tended to evolve lower pollen-dispersal distance, that insect-pollinated species evolved decreased sexual dimorphism, and that species with a persistent soil seed-bank evolved toward reduced fire-survival ability of adults.

OrthoMaM v8: a database of orthologous exons and coding sequences for comparative genomics in mammals.

Comparative genomic studies extensively rely on alignments of orthologous sequences. Yet, selecting, gathering, and aligning orthologous exons and protein-coding sequences (CDS) that are relevant for a given evolutionary analysis can be a difficult and time-consuming task. In this context, we developed OrthoMaM, a database of ORTHOlogous MAmmalian Markers describing the evolutionary dynamics of orthologous genes in mammalian genomes using a phylogenetic framework. Since its first release in 2007, OrthoMaM has regularly evolved, not only to include newly available genomes but also to incorporate up-to-date software in its analytic pipeline. This eighth release integrates the 40 complete mammalian genomes available in Ensembl v73 and provides alignments, phylogenies, evolutionary descriptor information, and functional annotations for 13,404 single-copy orthologous CDS and 6,953 long exons. The graphical interface allows to easily explore OrthoMaM to identify markers with specific characteristics (e.g., taxa availability, alignment size, %G+C, evolutionary rate, chromosome location). It hence provides an efficient solution to sample preprocessed markers adapted to user-specific needs. OrthoMaM has proven to be a valuable resource for researchers interested in mammalian phylogenomics, evolutionary genomics, and has served as a source of benchmark empirical data sets in several methodological studies. OrthoMaM is available for browsing, query and complete or filtered downloads at http://www.orthomam.univ-montp2.fr/.

Ascidian mitogenomics: comparison of evolutionary rates in closely related taxa provides evidence of ongoing speciation events.

Ascidians are a fascinating group of filter-feeding marine chordates characterized by rapid evolution of both sequences and structure of their nuclear and mitochondrial genomes. Moreover, they include several model organisms used to investigate complex biological processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other available mitogenomes, using a novel approach involving detailed whole-mitogenome comparisons of conspecific and congeneric pairs. The evolutionary rate was quite homogeneous at both intraspecific and congeneric level, and the lowest congeneric rates were found in cryptic (morphologically undistinguishable) and in morphologically very similar species pairs. Moreover, congeneric nonsynonymous rates (dN) were up to two orders of magnitude higher than in intraspecies pairs. Overall, a clear-cut gap sets apart conspecific from congeneric pairs. These evolutionary peculiarities allowed easily identifying an extraordinary intraspecific variability in the model ascidian Botryllus schlosseri, where most pairs show a dN value between that observed at intraspecies and congeneric level, yet consistently lower than that of the Ciona intestinalis cryptic species pair. These data suggest ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably, these ongoing speciation events were undetectable by the cox1 barcode fragment, demonstrating that, at low phylogenetic distances, the whole mitogenome has a higher resolving power than cox1. Our study shows that whole-mitogenome comparative analyses, performed on a suitable sample of congeneric and intraspecies pairs, may allow detecting not only cryptic species but also ongoing speciation events.

Developing nuclear DNA phylogenetic markers in the angiosperm genus Leucadendron (Proteaceae): a next-generation sequencing transcriptomic approach.

Despite the recent advances in generating molecular data, reconstructing species-level phylogenies for non-models groups remains a challenge. The use of a number of independent genes is required to resolve phylogenetic relationships, especially for groups displaying low polymorphism. In such cases, low-copy nuclear exons and non-coding regions, such as 3' untranslated regions (3'-UTRs) or introns, constitute a potentially interesting source of nuclear DNA variation. Here, we present a methodology meant to identify new nuclear orthologous markers using both public-nucleotide databases and transcriptomic data generated for the group of interest by using next generation sequencing technology. To identify PCR primers for a non-model group, the genus Leucadendron (Proteaceae), we adopted a framework aimed at minimizing the probability of paralogy and maximizing polymorphism. We anchored when possible the right-hand primer into the 3'-UTR and the left-hand primer into the coding region. Seven new nuclear markers emerged from this search strategy, three of those included 3'-UTRs. We further compared the phylogenetic potential between our new markers and the ribosomal internal transcribed spacer region (ITS). The sequenced 3'-UTRs yielded higher polymorphism rates than the ITS region did. We did not find strong incongruences with the phylogenetic signal contained in the ITS region and the seven new designed markers but they strongly improved the phylogeny of the genus Leucadendron. Overall, this methodology is efficient in isolating orthologous loci and is valid for any non-model group given the availability of transcriptomic data.

Jumping and gliding rodents: mitogenomic affinities of Pedetidae and Anomaluridae deduced from an RNA-Seq approach.

An RNA-Seq strategy was used to obtain the complete set of protein-coding mitochondrial genes from two rodent taxa. Thanks to the next generation sequencing (NGS) 454 approach, we determined the complete mitochondrial DNA genome from Graphiurus kelleni (Mammalia: Rodentia: Gliridae) and partial mitogenome from Pedetes capensis (Pedetidae), and compared them with published rodent and outgroup mitogenomes. We finished the mitogenome sequencing by a series of amplicons using conserved PCR primers to fill the gaps corresponding to tRNA, rRNA and control regions. Phylogenetic analyses of the mitogenomes suggest a well-supported rodent phylogeny in agreement with nuclear gene trees. Pedetes groups with Anomalurus into the clade Anomaluromorpha, while Graphiurus branches within the squirrel-related clade. Moreover, Pedetes+Anomalurus branch with Castor into the mouse-related clade. Our study demonstrates the utility of NGS for obtaining new mitochondrial genomes as well as the importance of choosing adequate models of sequence evolution to infer the phylogeny of rodents.