Marie Pagès

Revisiting the taxonomy of the Rattini tribe: a phylogeny-based delimitation of species boundaries

BackgroundRodents are recognized as hosts for at least 60 zoonotic diseases and may represent a serious threat for human health. In the context of global environmental changes and increasing mobility of humans and animals, contacts between pathogens and potential animal hosts and vectors are modified, amplifying the risk of disease emergence. An accurate identification of each rodent at a specific level is needed in order to understand their implications in the transmission of diseases. Among the Muridae, the Rattini tribe encompasses 167 species inhabiting South East Asia, a hotspot of both biodiversity and emerging and re-emerging diseases. The region faces growing economical development that affects habitats, biodiversity and health. Rat species have been demonstrated as significant hosts of pathogens but are still difficult to recognize at a specific level using morphological criteria. DNA-barcoding methods appear as accurate tools for rat species identification but their use is hampered by the need of reliable identification of reference specimens. In this study, we explore and highlight the limits of the current taxonomy of the Rattini tribe.ResultsWe used the DNA sequence information itself as the primary information source to establish group membership and estimate putative species boundaries. We sequenced two mitochondrial and one nuclear genes from 122 rat samples to perform phylogenetic reconstructions. The method of Pons and colleagues (2006) that determines, with no prior expectations, the locations of ancestral nodes defining putative species was then applied to our dataset. To give an appropriate name to each cluster recognized as a putative species, we reviewed information from the literature and obtained sequences from a museum holotype specimen following the ancient DNA criteria.ConclusionsUsing a recently developed methodology, this study succeeds in refining the taxonomy of one of the most difficult groups of mammals. Most of the species expected within the area were retrieved but new putative species limits were also indicated, in particular within Berylmys and Rattus genera, where future taxonomic studies should be directed. Our study lays the foundations to better investigate rodent-born diseases in South East Asia and illustrates the relevance of evolutionary studies for health and medical sciences.

Next-generation sequencing for rodent barcoding: species identification from fresh, degraded and environmental samples.

Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.

Cytonuclear discordance among Southeast Asian black rats (Rattus rattus complex)

Black rats are major invasive vertebrate pests with severe ecological, economic and health impacts. Remarkably, their evolutionary history has received little attention, and there is no firm agreement on how many species should be recognized within the black rat complex. This species complex is native to India and Southeast Asia. According to current taxonomic classification, there are three taxa living in sympatry in several parts of Thailand, Cambodia and Lao Peoples Democratic Republic, where this study was conducted: two accepted species (Rattus tanezumi, Rattus sakeratensis) and an additional mitochondrial lineage of unclear taxonomic status referred to here as ‘Rattus R3’. We used extensive sampling, morphological data and diverse genetic markers differing in rates of evolution and parental inheritance (two mitochondrial DNA genes, one nuclear gene and eight microsatellite loci) to assess the reproductive isolation of these three taxa. Two close Asian relatives, Rattus argentiventer and Rattus exulans, were also included in the genetic analyses. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial phylogeny studies identified three reciprocally monophyletic clades in the black rat complex. However, studies of the phylogeny of the nuclear exon interphotoreceptor retinoid‐binding protein gene and clustering and assignation analyses with eight microsatellites failed to separate R. tanezumi and R3. Morphometric analyses were consistent with nuclear data. The incongruence between mitochondrial and nuclear (and morphological) data rendered R. tanezumi/R3 paraphyletic for mitochondrial lineages with respect to R. sakeratensis. Various evolutionary processes, such as shared ancestral polymorphism and incomplete lineage sorting or hybridization with massive mitochondrial introgression between species, may account for this unusual genetic pattern in mammals.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

BackgroundIn vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs).ResultsWe sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades.ConclusionsIn consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands.

Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans

We reviewed the associations of immunity-related genes with susceptibility of humans and rodents to hantaviruses, and with severity of hantaviral diseases in humans. Several class I and class II HLA haplotypes were linked with severe or benign hantavirus infections, and these haplotypes varied among localities and hantaviruses. The polymorphism of other immunity-related genes including the C4A gene and a high-producing genotype of TNF gene associated with severe PUUV infection. Additional genes that may contribute to disease or to PUUV infection severity include non-carriage of the interleukin-1 receptor antagonist (IL-1RA) allele 2 and IL-1β (-511) allele 2, polymorphisms of plasminogen activator inhibitor (PAI-1) and platelet GP1a. In addition, immunogenetic studies have been conducted to identify mechanisms that could be linked with the persistence/clearance of hantaviruses in reservoirs. Persistence was associated during experimental infections with an upregulation of anti-inflammatory responses. Using natural rodent population samples, polymorphisms and/or expression levels of several genes have been analyzed. These genes were selected based on the literature of rodent or human/hantavirus interactions (some Mhc class II genes, Tnf promoter, and genes encoding the proteins TLR4, TLR7, Mx2 and β3 integrin). The comparison of genetic differentiation estimated between bank vole populations sampled over Europe, at neutral and candidate genes, has allowed to evidence signatures of selection for Tnf, Mx2 and the Drb Mhc class II genes. Altogether, these results corroborated the hypothesis of an evolution of tolerance strategies in rodents. We finally discuss the importance of these results from the medical and epidemiological perspectives.

Molecular phylogeny and systematics of Dipodoidea: a test of morphology‐based hypotheses

Lebedev, V.S., Bannikova, A.A., Pagès, M., Pisano, J., Michaux, J.R. & Shenbrot, G.I. (2012). Molecular phylogeny and systematics of Dipodoidea: a test of morphology‐based hypotheses. —Zoologica Scripta, 42, 231–249.

Molecular phylogeny of South-East Asian arboreal murine rodents

Recent phylogenetic studies and taxonomic reviews have led to nearly complete resolution of the phylogenetic divisions within the old world rats and mice (Muridae, Murinae). The Micromys division and Pithecheir division are two notable exceptions where groupings of species into these divisions based on morphology and arboreal lifestyle have not been supported by phylogenetic evidence. Several enigmatic species from these divisions have been missing from molecular studies, preventing a rigorous revision of phylogenetic relationships. In this study, we sequenced for the first time one mitochondrial and three nuclear genes from South‐East Asian keystone species of these two arboreal divisions: Hapalomys delacouri (Micromys division), Lenothrix canus and Pithecheir parvus (Pithecheir division). We also complemented the molecular data already available for the two divisions with new data from Sundaic Chiropodomys, Indian Vandeleuria oleracea and the recently described Sulawesian Margaretamys christinae. Using this new phylogenetic framework and molecular dating methodologies, our study allows some more detailed classification of the former Micromys and Pithecheir divisions, while confirming their polyphyletic status. Specifically, the former Micromys division should now be split into four monotypic divisions: Chiropodomys, Hapalomys, Micromys and Vandeleuria divisions. The former Pithecheir division is likely to be refined and restricted to Pithecheir and probably Pithecheirops, whereas Lenothrix and Margaretamys should now be recognized as representatives of the Dacnomys division. Our findings have profound implications with regard to the systematics of Murinae, as well as to the early evolution of murine morphology and dental characters.

Morphological, chromosomal, and genic differences between sympatric Rattus rattus and Rattus satarae in South India

Abstract Two morphological forms of black rats (Rattus cf. rattus) were found living in sympatry in high-altitude dense forests of the Nilgiri Mountains, South India. The 1st one, often brown- or gray-bellied, also is found commensal in lowland settlements and represents Rattus rattus cf. rufescens (Gray 1837), with a diploid number (2N) of 38 chromosomes. The 2nd form, which has most often a pure white belly, has 2N  =  42 chromosomes and is referable to Rattus r. satarae Hinton, 1918, based on morphological comparison with the holotype. A multidisciplinary study indicates that these 2 forms are characterized by clear-cut differences in biochemistry (electrophoresis of homologous isozymes), molecular sequences (mitochondrial and nuclear DNA), and chromosomes (detailed banding analysis). All these data, coupled to diagnostic morphological characteristics, support the hypothesis that Rattus satarae and Rattus rattus are separate, sympatric species, with no gene flow between them. Their similar external morphology is interpreted as the result of convergence through occupying the same ecological niche.

Progress on research on rodents and rodent-borne zoonoses in South-east Asia

Abstract. This review aims to synthesise knowledge regarding the taxonomy of South-east Asian murine rodents and the challenges associated with the identification of habitat preferences and associated rodent-borne diseases. Recent studies concerning the Rattini tribe have identified unclear species boundaries that would benefit from further investigation. The development of barcoding may allow more accurate identification of rodents, specifically for complex species. However, knowledge on the distribution and habitat specialisations of many common murine rodents is still scarce, particularly regarding the specific habitat preferences of most synanthropic rodent species (Rattus tanezumi or Rattus exulans). Several studies have analysed the prevalence of major rodent-borne diseases in South-east Asia and it appears that the greatest risk of rodent zoonoses are in the lowland rain-fed and irrigated landscapes, generally in and around rice fields.

Phylogenetic position of the Ohiya rat (Srilankamys ohiensis) based on mitochondrial and nuclear gene sequence analysis

Buzan, E. V., Pagès, M., Michaux, J. & Krystufek, B. (2011). Phylogenetic position of the Ohiya rat (Srilankamys ohiensis) based on mitochondrial and nuclear gene sequence analysis. —Zoologica Scripta, 40, 545–553.