Marie-Ka Tilak

OrthoMaM: a database of orthologous genomic markers for placental mammal phylogenetics.

BackgroundMolecular sequence data have become the standard in modern day phylogenetics. In particular, several long-standing questions of mammalian evolutionary history have been recently resolved thanks to the use of molecular characters. Yet, most studies have focused on only a handful of standard markers. The availability of an ever increasing number of whole genome sequences is a golden mine for modern systematics. Genomic data now provide the opportunity to select new markers that are potentially relevant for further resolving branches of the mammalian phylogenetic tree at various taxonomic levels.DescriptionThe EnsEMBL database was used to determine a set of orthologous genes from 12 available complete mammalian genomes. As targets for possible amplification and sequencing in additional taxa, more than 3,000 exons of length > 400 bp have been selected, among which 118, 368, 608, and 674 are respectively retrieved for 12, 11, 10, and 9 species. A bioinformatic pipeline has been developed to provide evolutionary descriptors for these candidate markers in order to assess their potential phylogenetic utility. The resulting OrthoMaM (Orthologous Mammalian Markers) database can be queried and alignments can be downloaded through a dedicated web interface http://kimura.univ-montp2.fr/orthomam.ConclusionThe importance of marker choice in phylogenetic studies has long been stressed. Our database centered on complete genome information now makes possible to select promising markers to a given phylogenetic question or a systematic framework by querying a number of evolutionary descriptors. The usefulness of the database is illustrated with two biological examples. First, two potentially useful markers were identified for rodent systematics based on relevant evolutionary parameters and sequenced in additional species. Second, a complete, gapless 94 kb supermatrix of 118 orthologous exons was assembled for 12 mammals. Phylogenetic analyses using probabilistic methods unambiguously supported the new placental phylogeny by retrieving the monophyly of Glires, Euarchontoglires, Laurasiatheria, and Boreoeutheria. Muroid rodents thus do not represent a basal placental lineage as it was mistakenly reasserted in some recent phylogenomic analyses based on fewer taxa. We expect the OrthoMaM database to be useful for further resolving the phylogenetic tree of placental mammals and for better understanding the evolutionary dynamics of their genomes, i.e., the forces that shaped coding sequences in terms of selective constraints.

The evolutionary radiation of Arvicolinae rodents (voles and lemmings): relative contribution of nuclear and mitochondrial DNA phylogenies

BackgroundMitochondrial and nuclear genes have generally been employed for different purposes in molecular systematics, the former to resolve relationships within recently evolved groups and the latter to investigate phylogenies at a deeper level. In the case of rapid and recent evolutionary radiations, mitochondrial genes like cytochrome b (CYB) are often inefficient for resolving phylogenetic relationships. One of the best examples is illustrated by Arvicolinae rodents (Rodentia; Muridae), the most impressive mammalian radiation of the Northern Hemisphere which produced voles, lemmings and muskrats. Here, we compare the relative contribution of a nuclear marker – the exon 10 of the growth hormone receptor (GHR) gene – to the one of the mitochondrial CYB for inferring phylogenetic relationships among the major lineages of arvicoline rodents.ResultsThe analysis of GHR sequences improves the overall resolution of the Arvicolinae phylogeny. Our results show that the Caucasian long-clawed vole (Prometheomys schaposnikowi) is one of the basalmost arvicolines, and confirm that true lemmings (Lemmus) and collared lemmings (Dicrostonyx) are not closely related as suggested by morphology. Red-backed voles (Myodini) are found as the sister-group of a clade encompassing water vole (Arvicola), snow vole (Chionomys), and meadow voles (Microtus and allies). Within the latter, no support is recovered for the generic recognition of Blanfordimys, Lasiopodomys, Neodon, and Phaiomys as suggested by morphology. Comparisons of parameter estimates for branch lengths, base composition, among sites rate heterogeneity, and GTR relative substitution rates indicate that CYB sequences consistently exhibit more heterogeneity among codon positions than GHR. By analyzing the contribution of each codon position to node resolution, we show that the apparent higher efficiency of GHR is due to their third positions. Although we focus on speciation events spanning the last 10 million years (Myr), CYB sequences display highly saturated codon positions contrary to the nuclear exon. Lastly, variable length bootstrap predicts a significant increase in resolution of arvicoline phylogeny through the sequencing of nuclear data in an order of magnitude three to five times greater than the size of GHR exon 10.ConclusionOur survey provides a first resolved gene tree for Arvicolinae. The comparison of CYB and GHR phylogenetic efficiency supports recent assertions that nuclear genes are useful for resolving relationships of recently evolved animals. The superiority of nuclear exons may reside both in (i) less heterogeneity among sites, and (ii) the presence of highly informative sites in third codon positions, that evolve rapidly enough to accumulate synapomorphies, but slow enough to avoid substitutional saturation.

Molecular phylogenetics unveils the ancient evolutionary origins of the enigmatic fairy armadillos.

Fairy armadillos or pichiciegos (Xenarthra, Dasypodidae) are among the most elusive mammals. Due to their subterranean and nocturnal lifestyle, their basic biology and evolutionary history remain virtually unknown. Two distinct species with allopatric distributions are recognized: Chlamyphorus truncatus is restricted to central Argentina, while Calyptophractus retusus occurs in the Gran Chaco of Argentina, Paraguay, and Bolivia. To test their monophyly and resolve their phylogenetic affinities within armadillos, we obtained sequence data from modern and museum specimens for two mitochondrial genes (12S RNA [MT-RNR1] and NADH dehydrogenase 1 [MT-ND1]) and two nuclear exons (breast cancer 1 early onset exon 11 [BRCA1] and von Willebrand factor exon 28 [VWF]). Phylogenetic analyses provided a reference phylogeny and timescale for living xenarthran genera. Our results reveal monophyletic pichiciegos as members of a major armadillo subfamily (Chlamyphorinae). Their strictly fossorial lifestyle probably evolved as a response to the Oligocene aridification that occurred in South America after their divergence from Tolypeutinae around 32 million years ago (Mya). The ancient divergence date (∼17Mya) for separation between the two species supports their taxonomic classification into distinct genera. The synchronicity with Middle Miocene marine incursions along the Paraná river basin suggests a vicariant origin for pichiciegos by the disruption of their ancestral range. Their phylogenetic distinctiveness and rarity in the wild argue in favor of high conservation priority.

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

Leaf-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.

Diversification of South American spiny rats (Echimyidae): a multigene phylogenetic approach

Fabre, P.‐H., Galewski, T., Tilak, M.‐k. & Douzery, E.J.P. (2012) Diversification of South American spiny rats (Echimyidae): a multigene phylogenetic approach. —Zoologica Scripta, 00, 000–000.

Is leprosy spreading among nine-banded armadillos in the southeastern United States?

In the United States, nine-banded armadillo (Dasypus novemcinctus) populations are derived from two sources: (1) a continuous range expansion from Mexico led to western populations, some of which, particularly along the western Gulf Coast and west side of the Mississippi River delta, exhibit persistently high rates of leprosy infection, and (2) a small group of animals released from captivity in Florida gave rise to eastern populations that were all considered leprosy free. Given that western and eastern populations have now merged, an important question becomes, to what extent is leprosy spreading into formerly uninfected populations? To answer this question, we sampled 500 animals from populations in Mississippi, Alabama, and Georgia. Analyses of nuclear microsatellite DNA markers confirmed the historic link between source populations from Texas and Florida, but did not permit resolution of the extent to which these intermediate populations represented eastern versus western gene pools. Prevalence of leprosy was determined by screening blood samples for the presence of antibodies against Mycobacterium leprae and via polymerase chain reaction amplification of armadillo tissues to detect M. leprae DNA. The proportion of infected individuals within each population varied from 0% to 10%. Although rare, a number of positive individuals were identified in eastern sites previously considered uninfected. This indicates leprosy may be spreading eastward and calls into question hypotheses proposing leprosy infection is confined because of ecologic constraints to areas west of the Mississippi River.

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.

Systematics of hairy armadillos and the taxonomic status of the Andean hairy armadillo (Chaetophractus nationi)

Hairy armadillos constitute an ecologically homogeneous and morphologically similar group with currently 5 species classified in the subfamily Euphractinae. Among them, the Andean hairy armadillo Chaetophractus nationi (Xenarthra, Cingulata, Dasypodidae) is a small, endangered armadillo that has long been suspected to represent a high-altitude variant of Chaetophractus vellerosus. Here, we report the 1st phylogenetic systematics assessment of hairy armadillos using morphological and molecular analyses of all described species with focus on the status of the Andean hairy armadillo. Multivariate analyses of shape variation based on 3-dimensional landmark coordinates of skulls allowed a clear differentiation of each species with the exception of C. vellerosus and C. nationi, within which only a latitudinal and/or altitudinal gradient in size was apparent. Moreover, analyses of mitochondrial DNA control region (D-loop) revealed a single C. nationi haplotype that appeared to be identical with a C. vellerosus haplotype from Argentina. Identical sequences in C. vellerosus and C. nationi were also observed for 3 of the 5 non-coding nuclear markers investigated. Based on these data, we propose that C. nationi should be considered as a synonym of C. vellerosus. However, this taxonomic change should not preclude the protection of the high-altitude Bolivian populations that are steadily declining because of their overexploitation for traditional purposes. Finally, phylogenetic analyses of euphractine armadillos based on a combination of 6 non-coding nuclear markers and 2 nuclear exons suggest the paraphyly of the genus Chaetophractus, with C. vellerosus being more closely related to Zaedyus pichiy than to C. villosus.

A cost-effective straightforward protocol for shotgun Illumina libraries designed to assemble complete mitogenomes from non-model species

The mitogenome is an inescapable tool in conservation biology studies. Yet, its routine sequencing may remain tricky despite next-generation sequencing technologies. An enrichment step is often necessary but not always straightforward depending on the initial DNA quality or quantity. Furthermore, the availability of close mitochondrial DNA reference sequences for non-model species limits the primer design for long-range PCR or bait synthesis. Here we propose an easy and cost-effective protocol without enrichment step for building and sequencing multiplexed Illumina libraries from small quantities of either high-quality or degraded genomic DNA. We validated the approach through the successful assembly of the complete mitogenome of 60 bats and 7 tunicates. Our protocol allows the sequencing and assembly of mitochondrial genomes from non-model species with sufficient coverage for applications in conservation genetics.

A phylogenomic framework and timescale for comparative studies of tunicates

BackgroundTunicates 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.ResultsPhylogenetic 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.ConclusionsOur 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.