Deep Sequencing of Mixed Total DNA without Barcodes Allows Efficient Assembly of Highly Plastic Ascidian Mitochondrial Genomes

Ascidians or sea squirts form a diverse group within chordates, which includes a few thousand members of marine sessile filter-feeding animals. Their mitochondrial genomes are characterized by particularly high evolutionary rates and rampant gene rearrangements. This extreme variability complicates standard polymerase chain reaction (PCR) based techniques for molecular characterization studies, and consequently only a few complete Ascidian mitochondrial genome sequences are available. Using the standard PCR and Sanger sequencing approach, we produced the mitochondrial genome of Ascidiella aspersa only after a great effort. In contrast, we produced five additional mitogenomes (Botrylloides aff. leachii, Halocynthia spinosa, Polycarpa mytiligera, Pyura gangelion, and Rhodosoma turcicum) with a novel strategy, consisting in sequencing the pooled total DNA samples of these five species using one Illumina HiSeq 2000 flow cell lane. Each mitogenome was efficiently assembled in a single contig using de novo transcriptome assembly, as de novo genome assembly generally performed poorly for this task. Each of the new six mitogenomes presents a different and novel gene order, showing that no syntenic block has been conserved at the ordinal level (in Stolidobranchia and in Phlebobranchia). Phylogenetic analyses support the paraphyly of both Ascidiacea and Phlebobranchia, with Thaliacea nested inside Phlebobranchia, although the deepest nodes of the Phlebobranchia–Thaliacea clade are not well resolved. The strategy described here thus provides a cost-effective approach to obtain complete mitogenomes characterized by a highly plastic gene order and a fast nucleotide/amino acid substitution rate

Data from: New DNA data from a Transthyretin nuclear intron suggest an Oligocene to Miocene diversification of living South America opossums (Marsupialia: Didelphidae).

Phylogenetic relationships of 19 species of didelphid marsupials were studied using two nuclear markers, the non-coding transthyretin intron 1 (TTR) and the coding interphotoreceptor retinoid binding protein exon 1 (IRBP), and two mitochondrial genes, the protein-coding cytochrome b (cyt-b) and the structural 12S ribosomal DNA (12S rDNA). Evolutionary dynamics of these four markers were compared to each other, revealing the appropriate properties presented by TTR intron 1 together with its well supported and resolved phylogenetic signal. Nuclear markers supported the monophyly of medium and large-sized opossums Metachirus+(Chironectes, Lutreolina, Didelphis, Philander), and the paraphyly of mouse-sized opossums, with the genera Gracilinanus, Thylamys, and Marmosops as a sister group to medium and large-sized didelphids. Conflicting branching patterns between mitochondrial and nuclear data involved the phylogenetic position of Marmosa-Micoureus-Monodelphis relative to other mouse-sized opossums. Nuclear phylogenetic inferences among genera were confirmed by the presence of synapomorphic indels observed in TTR intron 1. A Bayesian relaxed molecular clock dating of didelphid evolution using nuclear markers estimated their origin in the Middle Eocene (39.8 million years ago), with subsequent diversification during the Oligocene (Deseadan) and Miocene

Contrasting GC-content dynamics across 33 mammalian genomes: Relationship with life-history traits and chromosome sizes

The origin, evolution, and functional relevance of genomic variations in GC content are a long-debated topic, especially in mammals. Most of the existing literature, however, has focused on a small number of model species and/or limited sequence data sets. We analyzed more than 1000 orthologous genes in 33 fully sequenced mammalian genomes, reconstructed their ancestral isochore organization in the maximum likelihood framework, and explored the evolution of third-codon position GC content in representatives of 16 orders and 27 families. We showed that the previously reported erosion of GC-rich isochores is not a general trend. Several species (e.g., shrew, microbat, tenrec, rabbit) have independently undergone a marked increase in GC content, with a widening gap between the GC-poorest and GC-richest classes of genes. The intensively studied apes and (especially) murids do not reflect the general placental pattern. We correlated GC-content evolution with species life-history traits and cytology. Significant effects of body mass and genome size were detected, with each being consistent with the GC-biased gene conversion model

SDM: Une Méthode de Distance Rapide pour les Etudes de Phylogénomique

National audienceLes études de phylogénomique se proposent de reconstruire la phylogénie d’un ensemble de taxons en utilisant un grand nombre de gènes homologues. Les données, de tailles “génomiques”, imposent des méthodes rapides. Dans un tel contexte, les méthodes de distance constituent une approche de choix, qu’il s’agisse de réaliser des études exploratoires, ou bien de construire un premier arbre qui sera raffiné en- suite par une approche plus lourde de type maximum de vraisemblance (ML). Néanmoins, une distance évolutive estimée directement à partir des gènes concaténés induit généralement un signal topologique perturbé. Nous proposons ici une nouvelle méthode, nommée Super Distance Matrix (SDM), consistant à combiner une collection de matrices de distances évolutives obtenues à partir de chaque gène en une seule supermatrice de distance. Cette supermatrice est ensuite utilisée pour reconstruire un arbre à l’aide d’une méthode de distance classique. Le principe consiste à déformer les matrices sources sans modi- fier leur message topologique, de manière à minimiser leur éloignement réciproque au sens de l’écart quadratique. Une difficulté est que les matrices sources correspondent à des ensembles de taxons qui ne sont que partiellement recouvrants. Nous montrons que ce problème s’exprime comme la minimisation d’un critère quadratique sous contraintes linéaires, ce qui revient à résoudre un système linéaire. La résolution de ce système creux a une complexité pratique de l’ordre de , où représente le nombre de taxons, le nombre de matrices et , ce qui permet d’obtenir très rapidement la supermatrice de distance souhaitée. Nous étudions les performances de SDM à l’aide de simulations. Plusieurs utili- sations de SDM sont envisagées, de l’étude exploratoire rapide à des approches plus lourdes en temps calculs. Nous montrons que SDM constitue une alternative pertinente à la méthode standard “Matrix Representation with Parsimony” (MRP), en particulier lorsque les matrices sont peu recouvrantes. Nous montrons également que SDM construit un excellent arbre de départ pour une approche basée sur le critère ML, qui permet à la fois de réduire les temps calculs et de gagner en précision. Nous analysons à l’aide de SDM le jeu de données moléculaires de Gatesy et al. [14] composé de quarante-huit gènes et soixante-quinze mammifères. Les résultats inférés par SDM indiquent une très forte hétérogénéité des vitesses d’évolution dans cette collection de gènes et confirment les résultats théoriques obtenus par simulations

Diversity of ectomycorrhizal fungi naturally established on containerised Pinus seedlings in nursery conditions

International audienceThe study examined the diversity of ectomycorrhizal fungi, naturally established on roots of containerised Pinus seedlings in a nursery, using PCR-RFLP and sequencing of the nuclear ribosomal. internal transcribed spacer. Seventy-two samples, including ectomycorrhizae and fruit bodies, were examined. Molecular typing assigned the fungal symbionts to four ectomycorrhizal Boletates: Rhizopogon rubescens, Suillus bovinus, S. variegatus, and R. luteolus. R. rubescens was abundant (37.5%), while Suillus and R. luteolus species were moderately established (25-26%) and rare (2.8%), respectively. In addition, Rhizopogon species colonised P nigra ssp. salzmannii seedlings, whereas Suillus species were identified on Pinus nigra ssp. nigra seedlings. The diversity and the ability of these naturally established symbionts under artificial nursery conditions were discussed. The molecular survey investigated here should contribute to successful monitoring of mycorrhizal. application under both nursery and plantation conditions

Accelerated evolutionary rate of housekeeping genes in tunicates.

International audiencePhylogenomics has recently revealed that tunicates represent the sister-group of vertebrates in the newly defined clade Olfactores. However, phylogenomic and comparative genomic studies have also suggested that tunicates are characterized by an elevated rate of molecular evolution and a high degree of genomic divergence. Despite the recurrent interest in the group, the picture of tunicate peculiar evolutionary dynamics is still fragmentary, as it mainly lies in studies focusing on only a few model species. In order to expand the available genomic data for the group, we used the high-throughput 454 technology to sequence the partial transcriptome of a previously unsampled tunicate, Microcosmus squamiger. This allowed us to get further insights into tunicate-accelerated evolution through a comparative analysis based on pertinent phylogenetic markers, i.e., a core of 35 housekeeping genes conserved across bilaterians. Our results showed that tunicates evolved on average about two times faster than the other chordates, yet the degree of this acceleration varied extensively upon genes and upon lineages. Appendicularia and Aplousobranchia were detected as the most divergent groups which were also characterized by highly heterogeneous substitution rates across genes. Finally, an estimation of the d (N)/d (S) ratio in three pairs of closely related taxa within Olfactores did not reveal strong differences between the tunicate and vertebrate lineages suggesting that for this set of housekeeping genes, the accelerated evolution of tunicates is plausibly due to an elevated mutation rate rather than to particular selective effects

Data from: 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

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

International audienceNext 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

Next-generation sequencing and phylogenetic signal of complete mitochondrial genomes for resolving the evolutionary history of leaf-nosed bats (Phyllostomidae)

International audienceLeaf-nosed bats (Phyllostomidae) are one of the most studied groups within the order Chiroptera mainly because of their outstanding species richness and diversity in morphological and ecological traits. Rapid diversification and multiple homoplasies have made the phylogeny of the family difficult to solve using morphological characters. Molecular data have contributed to shed light on the evolutionary history of phyllostomid bats, yet several relationships remain unresolved at the intra-familial level. Complete mitochondrial genomes have proven useful to deal with this kind of situation in other groups of mammals by providing access to a large number of molecular characters. At present, there are only two mitogenomes available for phyllostomid bats hinting at the need for further exploration of the mitogenomic approach in this group. We used both standard Sanger sequencing of PCR products and next-generation sequencing (NGS) of shotgun genomic DNA to obtain new complete mitochondrial genomes from 10 species of phyllostomid bats, including representatives of major subfamilies, plus one outgroup belonging to the closely-related mormoopids. We then evaluated the contribution of mitogenomics to the resolution of the phylogeny of leaf-nosed bats and compared the results to those based on mitochondrial genes and the RAG2 and VWF nuclear makers. Our results demonstrate the advantages of the Illumina NGS approach to efficiently obtain mitogenomes of phyllostomid bats. The phylogenetic signal provided by entire mitogenomes is highly comparable to the one of a concatenation of individual mitochondrial and nuclear markers, and allows increasing both resolution and statistical support for several clades. This enhanced phylogenetic signal is the result of combining markers with heterogeneous evolutionary rates representing a large number of nucleotide sites. Our results illustrate the potential of the NGS mitogenomic approach for resolving the evolutionary history of phyllostomid bats based on a denser species sampling