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/.

PhylteR: Efficient Identification of Outlier Sequences in Phylogenomic Datasets.

In phylogenomics, incongruences between gene trees, resulting from both artifactual and biological reasons, can decrease the signal-to-noise ratio and complicate species tree inference. The amount of data handled today in classical phylogenomic analyses precludes manual error detection and removal. However, a simple and efficient way to automate the identification of outliers from a collection of gene trees is still missing. Here, we present PhylteR, a method that allows rapid and accurate detection of outlier sequences in phylogenomic datasets, i.e. species from individual gene trees that do not follow the general trend. PhylteR relies on DISTATIS, an extension of multidimensional scaling to 3 dimensions to compare multiple distance matrices at once. In PhylteR, these distance matrices extracted from individual gene phylogenies represent evolutionary distances between species according to each gene. On simulated datasets, we show that PhylteR identifies outliers with more sensitivity and precision than a comparable existing method. We also show that PhylteR is not sensitive to ILS-induced incongruences, which is a desirable feature. On a biological dataset of 14,463 genes for 53 species previously assembled for Carnivora phylogenomics, we show (i) that PhylteR identifies as outliers sequences that can be considered as such by other means, and (ii) that the removal of these sequences improves the concordance between the gene trees and the species tree. Thanks to the generation of numerous graphical outputs, PhylteR also allows for the rapid and easy visual characterization of the dataset at hand, thus aiding in the precise identification of errors. PhylteR is distributed as an R package on CRAN and as containerized versions (docker and singularity).

Pangolin Genomes Offer Key Insights and Resources for the World's Most Trafficked Wild Mammals.

Pangolins form a group of scaly mammals that are trafficked at record numbers for their meat and purported medicinal properties. Despite their conservation concern, knowledge of their evolution is limited by a paucity of genomic data. We aim to produce exhaustive genomic resources that include 3,238 orthologous genes and whole-genome polymorphisms to assess the evolution of all eight extant pangolin species. Robust orthologous gene-based phylogenies recovered the monophyly of the three genera and highlighted the existence of an undescribed species closely related to Southeast Asian pangolins. Signatures of middle Miocene admixture between an extinct, possibly European, lineage and the ancestor of Southeast Asian pangolins, provide new insights into the early evolutionary history of the group. Demographic trajectories and genome-wide heterozygosity estimates revealed contrasts between continental versus island populations and species lineages, suggesting that conservation planning should consider intraspecific patterns. With the expected loss of genomic diversity from recent, extensive trafficking not yet realized in pangolins, we recommend that populations be genetically surveyed to anticipate any deleterious impact of the illegal trade. Finally, we produce a complete set of genomic resources that will be integral for future conservation management and forensic endeavors for pangolins, including tracing their illegal trade. These comprise the completion of whole-genomes for pangolins through the hybrid assembly of the first reference genome for the giant pangolin (Smutsia gigantea) and new draft genomes (∼43x-77x) for four additional species, as well as a database of orthologous genes with over 3.4 million polymorphic sites.

Reconciling fossils with phylogenies reveals the origin and macroevolutionary processes explaining the global cycad biodiversity.

The determinants of biodiversity patterns can be understood using macroevolutionary analyses. The integration of fossils into phylogenies offers a deeper understanding of processes underlying biodiversity patterns in deep time. Cycadales are considered a relict of a once more diverse and globally distributed group but are restricted to low latitudes today. We still know little about their origin and geographic range evolution. Combining molecular data for extant species and leaf morphological data for extant and fossil species, we study the origin of cycad global biodiversity patterns through Bayesian total-evidence dating analyses. We assess the ancestral geographic origin and trace the historical biogeography of cycads with a time-stratified process-based model. Cycads originated in the Carboniferous on the Laurasian landmass and expanded in Gondwana in the Jurassic. Through now-vanished continental connections, Antarctica and Greenland were crucial biogeographic crossroads for cycad biogeography. Vicariance is an essential speciation mode in the deep and recent past. Their latitudinal span increased in the Jurassic and restrained toward subtropical latitudes in the Neogene in line with biogeographic inferences of high-latitude extirpations. We show the benefits of integrating fossils into phylogenies to estimate ancestral areas of origin and to study evolutionary processes explaining the global distribution of present-day relict groups.

A comprehensive phylogeny and revised taxonomy illuminate the origin and diversification of the global radiation of Papilio (Lepidoptera: Papilionidae).

The swallowtail genus Papilio (Lepidoptera: Papilionidae) is species rich, distributed worldwide, and has broad morphological habits and ecological niches. Because of its elevated species richness, it has been historically difficult to reconstruct a densely sampled phylogeny for this clade. Here we provide a taxonomic working list for the genus, resulting in 235 Papilio species, and assemble a molecular dataset of seven gene fragments representing ca. 80% of the currently described diversity. Phylogenetic analyses reconstructed a robust tree with highly supported relationships within subgenera, although a few nodes in the early history of the Old World Papilio remain unresolved. Contrasting with previous results, we found that Papilio alexanor is sister to all Old World Papilio and that the subgenus Eleppone is no longer monotypic. The latter includes the recently described Fijian Papilio natewa with the Australian Papilio anactus and is sister to subgenus Araminta (formerly included in subgenus Menelaides) occurring in Southeast Asia. Our phylogeny also includes rarely studied (P. antimachus, P. benguetana) or endangered species (P. buddha, P. chikae). Taxonomic changes resulting from this study are elucidated. Molecular dating and biogeographic analyses indicate that Papilio originated ca. 30 million years ago (Oligocene), in a northern region centered on Beringia. A rapid early Miocene radiation in the Paleotropics is revealed within Old World Papilio, potentially explaining their low early branch support. Most subgenera originated in the early to middle Miocene followed by synchronous southward biogeographic dispersals and repeated local extirpations in northern latitudes. This study provides a comprehensive phylogenetic framework for Papilio with clarification of subgeneric systematics and species taxonomic changes enumerated, which will facilitate further studies to address questions on their ecology and evolutionary biology using this model clade.

Whole Genome Shotgun Phylogenomics Resolves the Pattern and Timing of Swallowtail Butterfly Evolution.

Evolutionary relationships have remained unresolved in many well-studied groups, even though advances in next-generation sequencing and analysis, using approaches such as transcriptomics, anchored hybrid enrichment, or ultraconserved elements, have brought systematics to the brink of whole genome phylogenomics. Recently, it has become possible to sequence the entire genomes of numerous nonbiological models in parallel at reasonable cost, particularly with shotgun sequencing. Here, we identify orthologous coding sequences from whole-genome shotgun sequences, which we then use to investigate the relevance and power of phylogenomic relationship inference and time-calibrated tree estimation. We study an iconic group of butterflies-swallowtails of the family Papilionidae-that has remained phylogenetically unresolved, with continued debate about the timing of their diversification. Low-coverage whole genomes were obtained using Illumina shotgun sequencing for all genera. Genome assembly coupled to BLAST-based orthology searches allowed extraction of 6621 orthologous protein-coding genes for 45 Papilionidae species and 16 outgroup species (with 32% missing data after cleaning phases). Supermatrix phylogenomic analyses were performed with both maximum-likelihood (IQ-TREE) and Bayesian mixture models (PhyloBayes) for amino acid sequences, which produced a fully resolved phylogeny providing new insights into controversial relationships. Species tree reconstruction from gene trees was performed with ASTRAL and SuperTriplets and recovered the same phylogeny. We estimated gene site concordant factors to complement traditional node-support measures, which strengthens the robustness of inferred phylogenies. Bayesian estimates of divergence times based on a reduced data set (760 orthologs and 12% missing data) indicate a mid-Cretaceous origin of Papilionoidea around 99.2 Ma (95% credibility interval: 68.6-142.7 Ma) and Papilionidae around 71.4 Ma (49.8-103.6 Ma), with subsequent diversification of modern lineages well after the Cretaceous-Paleogene event. These results show that shotgun sequencing of whole genomes, even when highly fragmented, represents a powerful approach to phylogenomics and molecular dating in a group that has previously been refractory to resolution.

Large Variation in the Ratio of Mitochondrial to Nuclear Mutation Rate across Animals: Implications for Genetic Diversity and the Use of Mitochondrial DNA as a Molecular Marker.

It is commonly assumed that mitochondrial DNA (mtDNA) evolves at a faster rate than nuclear DNA (nuDNA) in animals. This has contributed to the popularity of mtDNA as a molecular marker in evolutionary studies. Analyzing 121 multilocus data sets and four phylogenomic data sets encompassing 4,676 species of animals, we demonstrate that the ratio of mitochondrial over nuclear mutation rate is highly variable among animal taxa. In nonvertebrates, such as insects and arachnids, the ratio of mtDNA over nuDNA mutation rate varies between 2 and 6, whereas it is above 20, on average, in vertebrates such as scaled reptiles and birds. Interestingly, this variation is sufficient to explain the previous report of a similar level of mitochondrial polymorphism, on average, between vertebrates and nonvertebrates, which was originally interpreted as reflecting the effect of pervasive positive selection. Our analysis rather indicates that the among-phyla homogeneity in within-species mtDNA diversity is due to a negative correlation between mtDNA per-generation mutation rate and effective population size, irrespective of the action of natural selection. Finally, we explore the variation in the absolute per-year mutation rate of both mtDNA and nuDNA using a reduced data set for which fossil calibration is available, and discuss the potential determinants of mutation rate variation across genomes and taxa. This study has important implications regarding DNA-based identification methods in predicting that mtDNA barcoding should be less reliable in nonvertebrates than in vertebrates.

High-quality carnivoran genomes from roadkill samples enable comparative species delineation in aardwolf and bat-eared fox.

In a context of ongoing biodiversity erosion, obtaining genomic resources from wildlife is essential for conservation. The thousands of yearly mammalian roadkill provide a useful source material for genomic surveys. To illustrate the potential of this underexploited resource, we used roadkill samples to study the genomic diversity of the bat-eared fox (Otocyon megalotis) and the aardwolf (Proteles cristatus), both having subspecies with similar disjunct distributions in Eastern and Southern Africa. First, we obtained reference genomes with high contiguity and gene completeness by combining Nanopore long reads and Illumina short reads. Then, we showed that the two subspecies of aardwolf might warrant species status (P. cristatus and P. septentrionalis) by comparing their genome-wide genetic differentiation to pairs of well-defined species across Carnivora with a new Genetic Differentiation index (GDI) based on only a few resequenced individuals. Finally, we obtained a genome-scale Carnivora phylogeny including the new aardwolf species.

Genome-wide macroevolutionary signatures of key innovations in butterflies colonizing new host plants.

The mega-diversity of herbivorous insects is attributed to their co-evolutionary associations with plants. Despite abundant studies on insect-plant interactions, we do not know whether host-plant shifts have impacted both genomic adaptation and species diversification over geological times. We show that the antagonistic insect-plant interaction between swallowtail butterflies and the highly toxic birthworts began 55 million years ago in Beringia, followed by several major ancient host-plant shifts. This evolutionary framework provides a valuable opportunity for repeated tests of genomic signatures of macroevolutionary changes and estimation of diversification rates across their phylogeny. We find that host-plant shifts in butterflies are associated with both genome-wide adaptive molecular evolution (more genes under positive selection) and repeated bursts of speciation rates, contributing to an increase in global diversification through time. Our study links ecological changes, genome-wide adaptations and macroevolutionary consequences, lending support to the importance of ecological interactions as evolutionary drivers over long time periods.

MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics.

Thanks to the development of high-throughput sequencing technologies, target enrichment sequencing of nuclear ultraconserved DNA elements (UCEs) now allows routine inference of phylogenetic relationships from thousands of genomic markers. Recently, it has been shown that mitochondrial DNA (mtDNA) is frequently sequenced alongside the targeted loci in such capture experiments. Despite its broad evolutionary interest, mtDNA is rarely assembled and used in conjunction with nuclear markers in capture-based studies. Here, we developed MitoFinder, a user-friendly bioinformatic pipeline, to efficiently assemble and annotate mitogenomic data from hundreds of UCE libraries. As a case study, we used ants (Formicidae) for which 501 UCE libraries have been sequenced whereas only 29 mitogenomes are available. We compared the efficiency of four different assemblers (IDBA-UD, MEGAHIT, MetaSPAdes, and Trinity) for assembling both UCE and mtDNA loci. Using MitoFinder, we show that metagenomic assemblers, in particular MetaSPAdes, are well suited to assemble both UCEs and mtDNA. Mitogenomic signal was successfully extracted from all 501 UCE libraries, allowing us to confirm species identification using CO1 barcoding. Moreover, our automated procedure retrieved 296 cases in which the mitochondrial genome was assembled in a single contig, thus increasing the number of available ant mitogenomes by an order of magnitude. By utilizing the power of metagenomic assemblers, MitoFinder provides an efficient tool to extract complementary mitogenomic data from UCE libraries, allowing testing for potential mitonuclear discordance. Our approach is potentially applicable to other sequence capture methods, transcriptomic data and whole genome shotgun sequencing in diverse taxa. The MitoFinder software is available from GitHub (https://github.com/RemiAllio/MitoFinder).