staff

Michel C Milinkovitch

Full Professor in LANE

  • T: +41 22 379 33 38
  • office 4024b (Sciences III)
  • Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan. Elife 2016 ;5():. 10.7554/eLife.16087. PMC4969037.

    Guerreiro I, Gitto S, Novoa A, Codourey J, Nguyen Huynh TH, Gonzalez F, Milinkovitch MC, Mallo M, Duboule D

    abstract

    Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

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  • Amelanism in the corn snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene. Sci Rep 2015 ;5():17118. srep17118. 10.1038/srep17118. PMC4657000.

    Saenko SV, Lamichhaney S, Martinez Barrio A, Rafati N, Andersson L, Milinkovitch MC

    abstract

    The corn snake (Pantherophis guttatus) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of corn snakes, transcriptomic and genomic next-generation sequencing, exome assembly, and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11(th) intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in corn snakes. As amelanistic snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using corn snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages.

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  • Reptilian Transcriptomes v2.0: An Extensive Resource for Sauropsida Genomics and Transcriptomics. Genome Biol Evol 2015 Jun;7(6):1827-41. evv106. 10.1093/gbe/evv106. PMC4494049.

    Tzika AC, Ullate-Agote A, Grbic D, Milinkovitch MC

    abstract

    Despite the availability of deep-sequencing techniques, genomic and transcriptomic data remain unevenly distributed across phylogenetic groups. For example, reptiles are poorly represented in sequence databases, hindering functional evolutionary and developmental studies in these lineages substantially more diverse than mammals. In addition, different studies use different assembly and annotation protocols, inhibiting meaningful comparisons. Here, we present the "Reptilian Transcriptomes Database 2.0," which provides extensive annotation of transcriptomes and genomes from species covering the major reptilian lineages. To this end, we sequenced normalized complementary DNA libraries of multiple adult tissues and various embryonic stages of the leopard gecko and the corn snake and gathered published reptilian sequence data sets from representatives of the four extant orders of reptiles: Squamata (snakes and lizards), the tuatara, crocodiles, and turtles. The LANE runner 2.0 software was implemented to annotate all assemblies within a single integrated pipeline. We show that this approach increases the annotation completeness of the assembled transcriptomes/genomes. We then built large concatenated protein alignments of single-copy genes and inferred phylogenetic trees that support the positions of turtles and the tuatara as sister groups of Archosauria and Squamata, respectively. The Reptilian Transcriptomes Database 2.0 resource will be updated to include selected new data sets as they become available, thus making it a reference for differential expression studies, comparative genomics and transcriptomics, linkage mapping, molecular ecology, and phylogenomic analyses involving reptiles. The database is available at www.reptilian-transcriptomes.org and can be enquired using a wwwblast server installed at the University of Geneva.

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  • R(2)OBBIE-3D, a Fast Robotic High-Resolution System for Quantitative Phenotyping of Surface Geometry and Colour-Texture. PLoS ONE 2015 ;10(6):e0126740. 10.1371/journal.pone.0126740. PONE-D-14-42512. PMC4454658.

    Martins AF, Bessant M, Manukyan L, Milinkovitch MC

    abstract

    While recent imaging techniques provide insights into biological processes from the molecular to the cellular scale, phenotypes at larger scales remain poorly amenable to quantitative analyses. For example, investigations of the biophysical mechanisms generating skin morphological complexity and diversity would greatly benefit from 3D geometry and colour-texture reconstructions. Here, we report on R(2)OBBIE-3D, an integrated system that combines a robotic arm, a high-resolution digital colour camera, an illumination basket of high-intensity light-emitting diodes and state-of-the-art 3D-reconstruction approaches. We demonstrate that R(2)OBBIE generates accurate 3D models of biological objects between 1 and 100 cm, makes multiview photometric stereo scanning possible in practical processing times, and enables the capture of colour-texture and geometric resolutions better than 15 μm without the use of magnifying lenses. R(2)OBBIE has the potential to greatly improve quantitative analyses of phenotypes in addition to providing multiple new applications in, e.g., biomedical science.

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  • Phylogeography and support vector machine classification of colour variation in panther chameleons. Mol. Ecol. 2015 Jul;24(13):3455-66. 10.1111/mec.13241. PMC4755148.

    Grbic D, Saenko SV, Randriamoria TM, Debry A, Raselimanana AP, Milinkovitch MC

    abstract

    Lizards and snakes exhibit colour variation of adaptive value for thermoregulation, camouflage, predator avoidance, sexual selection and speciation. Furcifer pardalis, the panther chameleon, is one of the most spectacular reptilian endemic species in Madagascar, with pronounced sexual dimorphism and exceptionally large intraspecific variation in male coloration. We perform here an integrative analysis of molecular phylogeography and colour variation after collecting high-resolution colour photographs and blood samples from 324 F. pardalis individuals in locations spanning the whole species distribution. First, mitochondrial and nuclear DNA sequence analyses uncover strong genetic structure among geographically restricted haplogroups, revealing limited gene flow among populations. Bayesian coalescent modelling suggests that most of the mitochondrial haplogroups could be considered as separate species. Second, using a supervised multiclass support vector machine approach on five anatomical components, we identify patterns in 3D colour space that efficiently predict assignment of male individuals to mitochondrial haplogroups. We converted the results of this analysis into a simple visual classification key that can assist trade managers to avoid local population overharvesting.

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  • Photonic crystals cause active colour change in chameleons. Nat Commun 2015 ;6():6368. ncomms7368. 10.1038/ncomms7368. PMC4366488.

    Teyssier J, Saenko SV, van der Marel D, Milinkovitch MC

    abstract

    Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.

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  • Two waves of anisotropic growth generate enlarged follicles in the spiny mouse. Evodevo 2014 ;5():33. 10.1186/2041-9139-5-33. 2041-9139-5-33. PMC4335386.

    Montandon SA, Tzika AC, Martins AF, Chopard B, Milinkovitch MC

    abstract

    Mammals exhibit a remarkable variety of phenotypes and comparative studies using novel model species are needed to uncover the evolutionary developmental mechanisms generating this diversity. Here, we undertake a developmental biology and numerical modeling approach to investigate the development of skin appendages in the spiny mouse, Acomys dimidiatus.

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  • Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs. Proc. Natl. Acad. Sci. U.S.A. 2014 Oct;111(41):E4332-41. 1412268111. 10.1073/pnas.1412268111. PMC4205661.

    Cornelis G, Vernochet C, Malicorne S, Souquere S, Tzika AC, Goodman SM, Catzeflis F, Robinson TJ, Milinkovitch MC, Pierron G, Heidmann O, Dupressoir A, Heidmann T

    abstract

    Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Syncytins have been identified in Euarchontoglires (primates, rodents, Leporidae) and Laurasiatheria (Carnivora, ruminants) placental mammals. Here, we searched for similar genes in species that retained characteristic features of primitive mammals, namely the Malagasy and mainland African Tenrecidae. They belong to the superorder Afrotheria, an early lineage that diverged from Euarchotonglires and Laurasiatheria 100 Mya, during the Cretaceous terrestrial revolution. An in silico search for env genes with full coding capacity within a Tenrecidae genome identified several candidates, with one displaying placenta-specific expression as revealed by RT-PCR analysis of a large panel of Setifer setosus tissues. Cloning of this endogenous retroviral env gene demonstrated fusogenicity in an ex vivo cell-cell fusion assay on a panel of mammalian cells. Refined analysis of placental architecture and ultrastructure combined with in situ hybridization demonstrated specific expression of the gene in multinucleate cellular masses and layers at the materno-fetal interface, consistent with a role in syncytium formation. This gene, which we named "syncytin-Ten1," is conserved among Tenrecidae, with evidence of purifying selection and conservation of fusogenic activity. To our knowledge, it is the first syncytin identified to date within the ancestrally diverged Afrotheria superorder.

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  • Two waves of anisotropic growth generate enlarged follicles in the spiny mouse EvoDevo 2014, 5:33

    Sophie A Montandon, Athanasia C Tzika, António F Martins, Bastien Chopard and Michel C Milinkovitch

    abstract

    Background Mammals exhibit a remarkable variety of phenotypes and comparative studies using novel model species are needed to uncover the evolutionary developmental mechanisms generating this diversity. Here, we undertake a developmental biology and numerical modeling approach to investigate the development of skin appendages in the spiny mouse, Acomys dimidiatus. Results We demonstrate that Acomys spines, possibly involved in display and protection, are enlarged awl hairs with a concave morphology. The Acomys spines originate from enlarged placodes that are characterized by a rapid downwards growth which results in voluminous follicles. The dermal condensation (dermal papilla) at the core of the follicle is very large and exhibits a curved geometry. Given its off-centered position, the dermal papilla generates two waves of anisotropic proliferation, first of the posterior matrix, then of the anterior inner root sheath (IRS). Higher in the follicle, the posterior and anterior cortex cross-section areas substantially decrease due to cortex cell elongation and accumulation of keratin intermediate filaments. Milder keratinization in the medulla gives rise to a foamy material that eventually collapses under the combined compression of the anterior IRS and elongation of the cortex cells. Simulations, using linear elasticity theory and the finite-element method, indicate that these processes are sufficient to replicate the time evolution of the Acomys spine layers and the final shape of the emerging spine shaft. Conclusions Our analyses reveal how hair follicle morphogenesis has been altered during the evolution of the Acomys lineage, resulting in a shift from ancestral awl follicles to enlarged asymmetrical spines. This study contributes to a better understanding of the evolutionary developmental mechanisms that generated the great diversity of skin appendage phenotypes observed in mammals.

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  • The genome sequence of the corn snake (Pantherophis guttatus), a valuable resource for EvoDevo studies in squamates. Int. J. Dev. Biol. 2014 ;58(10-12):881-8. 150060at. 10.1387/ijdb.150060at.

    Ullate-Agote A, Milinkovitch MC, Tzika AC

    abstract

    Squamates (snakes and lizards) exhibit a striking variety of phenotypes, with little known on their generative mechanisms. Studies aiming to understand the genetic basis of this wide diversity in morphology, physiology and ecology will greatly benefit from whole genome sequencing initiatives, as they provide the foundation for comparative analyses and improve our understanding of the evolution, development and diversification of traits. Here, we present the first draft genome of the corn snake Pantherophis guttatus, an oviparous snake that we promote as a particularly appropriate model species for evolutionary developmental studies in squamates. We sequenced 100-base paired-end reads from multiple individuals of a single family (parents and offspring) that produced a genome assembly of 1.53 gigabases (Gb), roughly covering 75% of the expected total genome size, and 297,768 scaffolds >1 Kb. We were able to fully retrieve 86, and partially another 106, of the 248 CEGMA core genes, indicating that a high genome completeness was achieved, even though the assembly is fragmented. Using MAKER2, we annotated 10,917 genes with high confidence (Annotation Edit Distance (AED)<1) and an additional 5,263 predicted genes matched with the species' transcriptome. Numerous colour and colour pattern morphs exist in P. guttatus, making it an ideal model to study the genetic determinism, development, and evolution of adaptive colour traits in reptiles. Using our draft genome and a Single-Nucleotide Polymorphism (SNP) calling approach, we confirmed the interval with the causative mutation for the amelanistic phenotype, a result supported by a parallel exome-based study.

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  • Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards. BMC Biol. 2013 ;11():105. 1741-7007-11-105. 10.1186/1741-7007-11-105. PMC4021644.

    Saenko SV, Teyssier J, van der Marel D, Milinkovitch MC

    abstract

    Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma.

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  • Crocodylians evolved scattered multi-sensory micro-organs. Evodevo 2013 ;4(1):19. 2041-9139-4-19. 10.1186/2041-9139-4-19. PMC3711810.

    Di-Poï N, Milinkovitch MC

    abstract

    During their evolution towards a complete life cycle on land, stem reptiles developed both an impermeable multi-layered keratinized epidermis and skin appendages (scales) providing mechanical, thermal, and chemical protection. Previous studies have demonstrated that, despite the presence of a particularly armored skin, crocodylians have exquisite mechanosensory abilities thanks to the presence of small integumentary sensory organs (ISOs) distributed on postcranial and/or cranial scales.

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  • Patterns of ossification in southern versus northern placental mammals. Evolution 2013 Jul;67(7):1994-2010. 10.1111/evo.12071.

    Hautier L, Bennett NC, Viljoen H, Howard L, Milinkovitch MC, Tzika AC, Goswami A, Asher RJ

    abstract

    Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in "northern" (Laurasiatheria and Euarchontoglires) versus "southern" (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence-ANOVA (analysis of variance) and event-paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).

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  • Recovery of a nearly extinct Galápagos tortoise despite minimal genetic variation. Evol Appl 2013 Feb;6(2):377-83. 10.1111/eva.12014. PMC3586625.

    Milinkovitch MC, Kanitz R, Tiedemann R, Tapia W, Llerena F, Caccone A, Gibbs JP, Powell JR

    abstract

    A species of Galápagos tortoise endemic to Española Island was reduced to just 12 females and three males that have been bred in captivity since 1971 and have produced over 1700 offspring now repatriated to the island. Our molecular genetic analyses of juveniles repatriated to and surviving on the island indicate that none of the tortoises sampled in 1994 had hatched on the island versus 3% in 2004 and 24% in 2007, which demonstrates substantial and increasing reproduction in situ once again. This recovery occurred despite the parental population having an estimated effective population size <8 due to a combination of unequal reproductive success of the breeders and nonrandom mating in captivity. These results provide guidelines for adapting breeding regimes in the parental captive population and decreasing inbreeding in the repatriated population. Using simple morphological data scored on the sampled animals, we also show that a strongly heterogeneous distribution of tortoise sizes on Española Island observed today is due to a large variance in the number of animals included in yearly repatriation events performed in the last 40 years. Our study reveals that, at least in the short run, some endangered species can recover dramatically despite a lack of genetic variation and irregular repatriation efforts.

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  • Contrasted evolution of the vomeronasal receptor repertoires in mammals and squamate reptiles. Genome Biol Evol 2013 ;5(2):389-401. evt013. 10.1093/gbe/evt013. PMC3590772.

    Brykczynska U, Tzika AC, Rodriguez I, Milinkovitch MC

    abstract

    The vomeronasal organ (VNO) is an olfactory structure that detects pheromones and environmental cues. It consists of sensory neurons that express evolutionary unrelated groups of transmembrane chemoreceptors. The predominant V1R and V2R receptor repertoires are believed to detect airborne and water-soluble molecules, respectively. It has been suggested that the shift in habitat of early tetrapods from water to land is reflected by an increase in the ratio of V1R/V2R genes. Snakes, which have a very large VNO associated with a sophisticated tongue delivery system, are missing from this analysis. Here, we use RNA-seq and RNA in situ hybridization to study the diversity, evolution, and expression pattern of the corn snake vomeronasal receptor repertoires. Our analyses indicate that snakes and lizards retain an extremely limited number of V1R genes but exhibit a large number of V2R genes, including multiple lineages of reptile-specific and snake-specific expansions. We finally show that the peculiar bigenic pattern of V2R vomeronasal receptor gene transcription observed in mammals is conserved in squamate reptiles, hinting at an important but unknown functional role played by this expression strategy. Our results do not support the hypothesis that the shift to a vomeronasal receptor repertoire dominated by V1Rs in mammals reflects the evolutionary transition of early tetrapods from water to land. This study sheds light on the evolutionary dynamics of the vomeronasal receptor families in vertebrates and reveals how mammals and squamates differentially adapted the same ancestral vomeronasal repertoire to succeed in a terrestrial environment.

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  • Crocodile head scales are not developmental units but emerge from physical cracking. Science 2013 Jan;339(6115):78-81. science.1226265. 10.1126/science.1226265.

    Milinkovitch MC, Manukyan L, Debry A, Di-Poï N, Martin S, Singh D, Lambert D, Zwicker M

    abstract

    Various lineages of amniotes display keratinized skin appendages (feathers, hairs, and scales) that differentiate in the embryo from genetically controlled developmental units whose spatial organization is patterned by reaction-diffusion mechanisms (RDMs). We show that, contrary to skin appendages in other amniotes (as well as body scales in crocodiles), face and jaws scales of crocodiles are random polygonal domains of highly keratinized skin, rather than genetically controlled elements, and emerge from a physical self-organizing stochastic process distinct from RDMs: cracking of the developing skin in a stress field. We suggest that the rapid growth of the crocodile embryonic facial and jaw skeleton, combined with the development of a very keratinized skin, generates the mechanical stress that causes cracking.

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  • Development and embryonic staging in non-model organisms: the case of an afrotherian mammal. J. Anat. 2013 Jan;222(1):2-18. 10.1111/j.1469-7580.2012.01509.x. PMC3552411.

    Werneburg I, Tzika AC, Hautier L, Asher RJ, Milinkovitch MC, Sánchez-Villagra MR

    abstract

    Studies of evolutionary developmental biology commonly use 'model organisms' such as fruit flies or mice, and questions are often functional or epigenetic. Phylogenetic investigations, in contrast, typically use species that are less common and mostly deal with broad scale analyses in the tree of life. However, important evolutionary transformations have taken place at all taxonomic levels, resulting in such diverse forms as elephants and shrews. To understand the mechanisms underlying morphological diversification, broader sampling and comparative approaches are paramount. Using a simple, standardized protocol, we describe for the first time the development of soft tissues and some parts of the skeleton, using μCT-imaging of developmental series of Echinops telfairi and Tenrec ecaudatus, two tenrecid afrotherian mammals. The developmental timing of soft tissue and skeletal characters described for the tenrecids is briefly compared with that of other mammals, including mouse, echidna, and the opossum. We found relatively few heterochronic differences in development in the armadillo vs. tenrec, consistent with a close relationship of Xenarthra and Afrotheria. Ossification in T. ecaudatus continues well into the second half of overall gestation, resembling the pattern seen in other small mammals and differing markedly from the advanced state of ossification evident early in the gestation of elephants, sheep, and humans.

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  • Reptilian-transcriptome v1.0, a glimpse in the brain transcriptome of five divergent Sauropsida lineages and the phylogenetic position of turtles. Evodevo 2011 ;2(1):19. 2041-9139-2-19. 10.1186/2041-9139-2-19. PMC3192992.

    Tzika AC, Helaers R, Schramm G, Milinkovitch MC

    abstract

    Reptiles are largely under-represented in comparative genomics despite the fact that they are substantially more diverse in many respects than mammals. Given the high divergence of reptiles from classical model species, next-generation sequencing of their transcriptomes is an approach of choice for gene identification and annotation.

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  • Alien eggs in duck nests: brood parasitism or a help from Grandma? Mol. Ecol. 2011 Aug;20(15):3237-50. 10.1111/j.1365-294X.2011.05158.x.

    Tiedemann R, Paulus KB, Havenstein K, Thorstensen S, Petersen A, Lyngs P, Milinkovitch MC

    abstract

    Intraspecific brood parasitism (IBP) is a remarkable phenomenon by which parasitic females can increase their reproductive output by laying eggs in conspecific females' nests in addition to incubating eggs in their own nest. Kin selection could explain the tolerance, or even the selective advantage, of IBP, but different models of IBP based on game theory yield contradicting predictions. Our analyses of seven polymorphic autosomal microsatellites in two eider duck colonies indicate that relatedness between host and parasitizing females is significantly higher than the background relatedness within the colony. This result is unlikely to be a by-product of relatives nesting in close vicinity, as nest distance and genetic identity are not correlated. For eider females that had been ring-marked during the decades prior to our study, our analyses indicate that (i) the average age of parasitized females is higher than the age of nonparasitized females, (ii) the percentage of nests with alien eggs increases with the age of nesting females, (iii) the level of IBP increases with the host females' age, and (iv) the number of own eggs in the nest of parasitized females significantly decreases with age. IBP may allow those older females unable to produce as many eggs as they can incubate to gain indirect fitness without impairing their direct fitness: genetically related females specialize in their energy allocation, with young females producing more eggs than they can incubate and entrusting these to their older relatives. Intraspecific brood parasitism in ducks may constitute cooperation among generations of closely related females.

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  • Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci. Mol Ecol Resour 2011 May;11(3):550-6. 10.1111/j.1755-0998.2011.02986.x.

    Ciofi C, Tzika AC, Natali C, Watts PC, Sulandari S, Zein MS, Milinkovitch MC

    abstract

    Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions.

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  • MetaPIGA v2.0: maximum likelihood large phylogeny estimation using the metapopulation genetic algorithm and other stochastic heuristics. BMC Bioinformatics 2010 ;11():379. 1471-2105-11-379. 10.1186/1471-2105-11-379. PMC2912891.

    Helaers R, Milinkovitch MC

    abstract

    The development, in the last decade, of stochastic heuristics implemented in robust application softwares has made large phylogeny inference a key step in most comparative studies involving molecular sequences. Still, the choice of a phylogeny inference software is often dictated by a combination of parameters not related to the raw performance of the implemented algorithm(s) but rather by practical issues such as ergonomics and/or the availability of specific functionalities.

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  • Historical constraints on vertebrate genome evolution. Genome Biol Evol 2010 ;2():13-8. 10.1093/gbe/evp052. PMC2839353.

    Milinkovitch MC, Helaers R, Tzika AC

    abstract

    Recent analyses indicated that genes with larger effect of knockout or mutation and with larger probability to revert to single copy after whole genome duplication are expressed earlier in development. Here, we further investigate whether tissue specificity of gene expression is constrained by the age of origin of the corresponding genes. We use 38 metazoan genomes and a comparative genomic application system to integrate inference of gene duplication with expression data from 17,503 human genes into a strictly phylogenetic framework. We show that the number of anatomical systems in which genes are expressed decreases steadily with decreased age of the genes' first appearance in the phylogeny: the oldest genes are expressed, on average, in twice as many anatomical systems than the genes gained recently in evolution. These results are robust to different sources of expression data, to different levels of the anatomical system hierarchy, and to the use of gene families rather than duplication events. Finally, we show that the rate of increase in gene tissue specificity correlates with the relative rate of increase in the maximum number of cell types in the corresponding taxa. Although subfunctionalization and increase in cell type number throughout evolution could constitute, respectively, the proximal and ultimate causes of this correlation, the two phenomena are intermingled. Our analyses identify a striking historical constraint in gene expression: the number of cell types in existence at the time of a gene appearance (through duplication or de novo origination) tends to determine its level of tissue specificity for tens or hundreds of millions of years.

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  • Changes in Hox genes' structure and function during the evolution of the squamate body plan. Nature 2010 Mar;464(7285):99-103. nature08789. 10.1038/nature08789.

    Di-Poï N, Montoya-Burgos JI, Miller H, Pourquié O, Milinkovitch MC, Duboule D

    abstract

    Hox genes are central to the specification of structures along the anterior-posterior body axis, and modifications in their expression have paralleled the emergence of diversity in vertebrate body plans. Here we describe the genomic organization of Hox clusters in different reptiles and show that squamates have accumulated unusually large numbers of transposable elements at these loci, reflecting extensive genomic rearrangements of coding and non-coding regulatory regions. Comparative expression analyses between two species showing different axial skeletons, the corn snake and the whiptail lizard, revealed major alterations in Hox13 and Hox10 expression features during snake somitogenesis, in line with the expansion of both caudal and thoracic regions. Variations in both protein sequences and regulatory modalities of posterior Hox genes suggest how this genetic system has dealt with its intrinsic collinear constraint to accompany the substantial morphological radiation observed in this group.

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  • 2x genomes--depth does matter. Genome Biol. 2010 ;11(2):R16. gb-2010-11-2-r16. 10.1186/gb-2010-11-2-r16. PMC2872876.

    Milinkovitch MC, Helaers R, Depiereux E, Tzika AC, Gabaldón T

    abstract

    Given the availability of full genome sequences, mapping gene gains, duplications, and losses during evolution should theoretically be straightforward. However, this endeavor suffers from overemphasis on detecting conserved genome features, which in turn has led to sequencing multiple eutherian genomes with low coverage rather than fewer genomes with high-coverage and more even distribution in the phylogeny. Although limitations associated with analysis of low coverage genomes are recognized, they have not been quantified.

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  • Phylogenomics of unusual histone H2A Variants in Bdelloid rotifers. PLoS Genet. 2009 Mar;5(3):e1000401. 10.1371/journal.pgen.1000401. PMC2642717.

    Van Doninck K, Mandigo ML, Hur JH, Wang P, Guglielmini J, Milinkovitch MC, Lane WS, Meselson M

    abstract

    Rotifers of Class Bdelloidea are remarkable in having evolved for millions of years, apparently without males and meiosis. In addition, they are unusually resistant to desiccation and ionizing radiation and are able to repair hundreds of radiation-induced DNA double-strand breaks per genome with little effect on viability or reproduction. Because specific histone H2A variants are involved in DSB repair and certain meiotic processes in other eukaryotes, we investigated the histone H2A genes and proteins of two bdelloid species. Genomic libraries were built and probed to identify histone H2A genes in Adineta vaga and Philodina roseola, species representing two different bdelloid families. The expressed H2A proteins were visualized on SDS-PAGE gels and identified by tandem mass spectrometry. We find that neither the core histone H2A, present in nearly all other eukaryotes, nor the H2AX variant, a ubiquitous component of the eukaryotic DSB repair machinery, are present in bdelloid rotifers. Instead, they are replaced by unusual histone H2A variants of higher mass. In contrast, a species of rotifer belonging to the facultatively sexual, desiccation- and radiation-intolerant sister class of bdelloid rotifers, the monogononts, contains a canonical core histone H2A and appears to lack the bdelloid H2A variant genes. Applying phylogenetic tools, we demonstrate that the bdelloid-specific H2A variants arose as distinct lineages from canonical H2A separate from those leading to the H2AX and H2AZ variants. The replacement of core H2A and H2AX in bdelloid rotifers by previously uncharacterized H2A variants with extended carboxy-terminal tails is further evidence for evolutionary diversity within this class of histone H2A genes and may represent adaptation to unusual features specific to bdelloid rotifers.

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  • Population genetics of Galápagos land iguana (genus Conolophus) remnant populations. Mol. Ecol. 2008 Dec;17(23):4943-52. MEC3967. 10.1111/j.1365-294X.2008.03967.x.

    Tzika AC, Rosa SF, Fabiani A, Snell HL, Snell HM, Marquez C, Tapia W, Rassmann K, Gentile G, Milinkovitch MC

    abstract

    The Galápagos land iguanas (genus Conolophus) have faced significant anthropogenic disturbances since the 17th century, leading to severe reduction of some populations and the extinction of others. Conservation activities, including the repatriation of captive-bred animals to depleted areas, have been ongoing since the late 1970s, but genetic information has not been extensively incorporated. Here we use nine species-specific microsatellite loci of 703 land iguanas from the six islands where the species occur today to characterize the genetic diversity within, and the levels of genetic differentiation among, current populations as well as test previous hypotheses about accidental translocations associated with early conservation efforts. Our analyses indicate that (i) five populations of iguanas represent distinct conservation units (one of them being the recently discovered rosada form) and could warrant species status, (ii) some individuals from North Seymour previously assumed to be from the natural Baltra population appear related to both Isabela and Santa Cruz populations, and (iii) the five different management units exhibit considerably different levels of intrapopulation genetic diversity, with the Plaza Sur and Santa Fe populations particularly low. Although the initial captive breeding programmes, coupled with intensive efforts to eradicate introduced species, saved several land iguana populations from extinction, our molecular results provide objective data for improving continuing in situ species survival plans and population management for this spectacular and emblematic reptile.

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  • Development of 10 highly-polymorphic microsatellite markers in the vulnerable Galápagos land iguanas (genus Conolophus). Mol Ecol Resour 2009 Jan;9(1):376-9. 10.1111/j.1755-0998.2008.02457.x.

    Rosa SF, Monteyne D, Milinkovitch MC

    abstract

    The two species of Galápagos land iguanas (Conolophus subcristatus and C. pallidus) are listed as 'vulnerable' species by the International Union for the Conservation of Nature (IUCN Red List; http://www.iucnredlist.org). Here, we report on the isolation and characterization of 10 microsatellite markers using 562 individuals sampled on all Galápagos islands where Conolophus species occur today. We show that these 10 loci are highly polymorphic and display diagnostic alleles for five out of the six island populations. These markers will be useful for Conolophus population genetic analyses as well as for guiding ongoing captive breeding programmes.

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  • Characterization of microsatellite loci in the European pond turtle Emys orbicularis. Mol Ecol Resour 2009 Jan;9(1):189-91. 10.1111/j.1755-0998.2008.02205.x.

    Ciofi C, Tzika AC, Natali C, Chelazzi G, Naziridis T, Milinkovitch MC

    abstract

    A set of eight highly polymorphic microsatellite markers was isolated and characterized from a genomic library enriched for dinucleotide repeats in the European pond turtle, Emys orbicularis. The markers were tested for polymorphism in a total of 33 turtles sampled in two natural ponds in the nature reserve of Kerkini, northern Greece. Number of alleles varied from 10 to 18, and expected heterozygosity ranged between 0.738 and 0.921. This novel set of loci will be particularly useful to assess fine-scale population structure and for parentage analysis in E. orbicularis.

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  • Controlling population evolution in the laboratory to evaluate methods of historical inference. PLoS ONE 2008 ;3(8):e2960. 10.1371/journal.pone.0002960. PMC2491900.

    Mardulyn P, Vaesen MA, Milinkovitch MC

    abstract

    Natural populations of known detailed past demographic history are extremely valuable to evaluate methods of historical inference, yet are extremely rare. As an alternative approach, we have generated multiple replicate microsatellite data sets from laboratory-cultured populations of a gonochoric free-living nematode, Caenorhabditis remanei, that were constrained to pre-defined demographic histories featuring different levels of migration among populations or bottleneck events of different magnitudes. These data sets were then used to evaluate the performances of two recently developed population genetics methods, BayesAss+, that estimates recent migration rates among populations, and Bottleneck, that detects the occurrence of recent bottlenecks. Migration rates inferred by BayesAss+ were generally over-estimates, although these were often included within the confidence interval. Analyses of data sets simulated in-silico, using a model mimicking the laboratory experiments, produced less biased estimates of the migration rates, and showed increased efficiency of the program when the number of loci and sampled genotypes per population was higher. In the replicates for which the pre-bottleneck laboratory-cultured populations did not significantly depart from a mutation/drift equilibrium, an important assumption of the program Bottleneck, only a portion of the bottleneck events were detected. This result was confirmed by in-silico simulations mirroring the laboratory bottleneck experiments. More generally, our study demonstrates the feasibility, and highlights some of the limits, of the approach that consists in generating molecular genetic data sets by controlling the evolution of laboratory-reared nematode populations, for the purpose of validating methods inferring population history.

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  • Automated discovery and phylogenetic analysis of new toxin-antitoxin systems. BMC Microbiol. 2008 ;8():104. 1471-2180-8-104. 10.1186/1471-2180-8-104. PMC2446400.

    Guglielmini J, Szpirer C, Milinkovitch MC

    abstract

    Although often viewed as elements "at the service of" bacteria, plasmids exhibit replication and maintenance mechanisms that make them purely "selfish DNA" candidates. Toxin-antitoxin (TA) systems are a spectacular example of such mechanisms: a gene coding for a cytotoxic stable protein is preceded by a gene coding for an unstable antitoxin. The toxin being more stable than the antitoxin, absence of the operon causes a reduction of the amount of the latter relative to the amount of the former. Thus, a cell exhibiting a TA system on a plasmid is 'condemned' either not to loose it or to die.

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  • Population structure of an endemic vulnerable species, the Jamaican boa (Epicrates subflavus). Mol. Ecol. 2008 Jan;17(2):533-44. MEC3588. 10.1111/j.1365-294X.2007.03588.x.

    Tzika AC, Koenig S, Miller R, Garcia G, Remy C, Milinkovitch MC

    abstract

    The Jamaican boa (Epicrates subflavus; also called Yellow boa) is an endemic species whose natural populations greatly and constantly declined since the late 19th century, mainly because of predation by introduced species, human persecution, and habitat destruction. In-situ conservation of the Jamaican boa is seriously hindered by the lack of information on demographic and ecological parameters as well as by a poor understanding of the population structure and species distribution in the wild. Here, using nine nuclear microsatellite loci and a fragment of the mitochondrial cytochrome b gene from 87 wild-born individuals, we present the first molecular genetic analyses focusing on the diversity and structure of the natural populations of the Jamaican boa. A model-based clustering analysis of multilocus microsatellite genotypes identifies three groups that are also significantly differentiated on the basis of F-statistics. Similarly, haplotypic network reconstruction methods applied on the cytochrome b haplotypes isolated here identify two well-differentiated haplogroups separated by four to six fixed mutations. Bayesian and metaGA analyses of the mitochondrial data set combined with sequences from other Boidae species indicate that rooting of the haplotypic network occurs most likely between the two defined haplogroups. Both analyses (based on nuclear and mitochondrial markers) underline an Eastern vs. (Western + Central) pattern of differentiation in agreement with geological data and patterns of differentiation uncovered in other vertebrate and invertebrate Jamaican species. Our results provide important insights for improving management of ex-situ captive populations and for guiding the development of proper in-situ species survival and habitat management plans for this spectacular, yet poorly known and vulnerable, snake.

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  • MANTIS: a phylogenetic framework for multi-species genome comparisons. Bioinformatics 2008 Jan;24(2):151-7. btm567. 10.1093/bioinformatics/btm567.

    Tzika AC, Helaers R, Van de Peer Y, Milinkovitch MC

    abstract

    Practitioners of comparative genomics face huge analytical challenges as whole genome sequences and functional/expression data accumulate. Furthermore, the field would greatly benefit from a better integration of this wealth of data with evolutionary concepts.

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  • An ant colony optimization algorithm for phylogenetic estimation under the minimum evolution principle. BMC Evol. Biol. 2007 ;7():228. 1471-2148-7-228. 10.1186/1471-2148-7-228. PMC2211314.

    Catanzaro D, Pesenti R, Milinkovitch MC

    abstract

    Distance matrix methods constitute a major family of phylogenetic estimation methods, and the minimum evolution (ME) principle (aiming at recovering the phylogeny with shortest length) is one of the most commonly used optimality criteria for estimating phylogenetic trees. The major difficulty for its application is that the number of possible phylogenies grows exponentially with the number of taxa analyzed and the minimum evolution principle is known to belong to the NRho- hard class of problems.

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  • Conceptual bases for quantifying the role of the environment on gene evolution: the participation of positive selection and neutral evolution. Biol Rev Camb Philos Soc 2007 Nov;82(4):551-72. BRV24. 10.1111/j.1469-185X.2007.00024.x.

    Levasseur A, Orlando L, Bailly X, Milinkovitch MC, Danchin EG, Pontarotti P

    abstract

    To demonstrate that a given change in the environment has contributed to the emergence of a given genotypic and phenotypic shift during the course of evolution, one should ask to what extent such shifts would have occurred without environmental change. Of course, such tests are rarely practical but phenotypic novelties can still be correlated to genomic shifts in response to environmental changes if enough information is available. We surveyed and re-evaluated the published data in order to estimate the role of environmental changes on the course of species and genomic evolution. Only a few published examples clearly demonstrate a causal link between a given environmental change and the fixation of a genomic variant resulting in functional modification (gain, loss or alteration of function). Many others suggested a link between a given phenotypic shift and a given environmental change but failed to identify the underlying genomic determinant(s) and/or the associated functional consequence(s). The proportion of genotypic and phenotypic variation that is fixed concomitantly with environmental changes is often considered adaptive and hence, the result of positive selection, even though alternative causes, such as genetic drift, are rarely investigated. Therefore, the second aim herein is to review evidence for the mechanisms leading to fixation.

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  • Escaping the mouse trap: the selection of new Evo-Devo model species. J. Exp. Zool. B Mol. Dev. Evol. 2007 Jul;308(4):337-46. 10.1002/jez.b.21180.

    Milinkovitch MC, Tzika A

    abstract

    Among the many, sometimes contradictory, criteria that have been used for promoting model species, the most prominent has probably been their relevance for understanding human biology. Recently however, the debate has partly shifted from the search for evolutionary conservation (medicine-driven models) to a better understanding of the generative mechanisms underlying biological diversity (Evo-Devo-driven models). Integration of multiple disciplines, beyond developmental genetics and evolutionary molecular genetics, as well as of innovative technologies will help biologists to open the massive realm of living species to genome manipulation and phenotypic investigation. However, a consensual list of model species must still be reached for optimizing the interplay between in silico analyses and in vivo experiments, and we claim that the Evo-Devo community should play a more energetic role in this endeavor. We discuss here a few criteria and limitations of major relevance to the choice of model species for Evo-Devo studies, and promote the use of a pragmatic approach. Finally, given the difficulties related to manipulating and breeding model species, we suggest the development of Evo-Devo virtual zoos maintaining breeding colonies of a selected set of species and from which eggs or staged embryos are available on order.

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  • Mitochondrial genome and nuclear sequence data support myzostomida as part of the annelid radiation. Mol. Biol. Evol. 2007 Aug;24(8):1690-701. msm086. 10.1093/molbev/msm086.

    Bleidorn C, Eeckhaut I, Podsiadlowski L, Schult N, McHugh D, Halanych KM, Milinkovitch MC, Tiedemann R

    abstract

    The echinoderm symbionts Myzostomida are marine worms that show an enigmatic lophotrochozoan body plan. Historically, their phylogenetic origins were obscured due to disagreement about which morphological features are evolutionarily conserved, but now most morphological evidence points to annelid origins. In contrast, recent phylogenetic analyses using different molecular markers produced variable results regarding the position of myzostomids, but all suggested these worms are not derived annelids. To reexamine this issue, we analyzed data from nuclear genes (18S rDNA, 28S rDNA, Myosin II, and Elongation Factor-1alpha), and a nearly complete myzostomid mitochondrial genome. Here, we show that the molecular data are in agreement with the morphological evidence that myzostomids are part of the annelid radiation. This result is robustly supported by mitochondrial (gene order and sequence data) and nuclear data, as well as by recent ultrastructural investigations. Using Bayes factor comparison, alternative hypotheses are shown to lack support. Thus, myzostomids probably evolved from a segmented ancestor and gained a derived anatomy during their long evolutionary history as echinoderm symbionts.

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  • Giant Galápagos tortoises; molecular genetic analyses identify a trans-island hybrid in a repatriation program of an endangered taxon. BMC Ecol. 2007 ;7():2. 1472-6785-7-2. 10.1186/1472-6785-7-2. PMC1820773.

    Milinkovitch MC, Monteyne D, Russello M, Gibbs JP, Snell HL, Tapia W, Marquez C, Caccone A, Powell JR

    abstract

    Giant Galápagos tortoises on the island of Española have been the focus of an intensive captive breeding-repatriation programme for over 35 years that saved the taxon from extinction. However, analysis of 118 samples from released individuals indicated that the bias sex ratio and large variance in reproductive success among the 15 breeders has severely reduced the effective population size (Ne).

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  • Phylogeny and biogeography of a cosmopolitan frog radiation: Late cretaceous diversification resulted in continent-scale endemism in the family ranidae. Syst. Biol. 2006 Aug;55(4):579-94. R5051H424T28H706. 10.1080/10635150600812551.

    Bossuyt F, Brown RM, Hillis DM, Cannatella DC, Milinkovitch MC

    abstract

    Ranidae is a large anuran group with a nearly cosmopolitan distribution. We investigated the phylogenetic relationships and early biogeographic history of ranid frogs, using 104 representatives of all subfamilies and families, sampled from throughout their distribution. Analyses of approximately 1570 bp of nuclear gene fragments (Rag-1, rhod, Tyr) and approximately 2100 bp of the mitochondrial genome (12S rRNA, tRNAVAL, 16S rRNA) indicate that the monophyly of several taxa can be rejected with high confidence. Our tree is characterized by a clear historical association of each major clade with one Gondwanan plate. This prevalence of continent-scale endemism suggests that plate tectonics has played a major role in the distribution of ranid frogs. We performed dispersal-vicariance analyses, as well as analyses constrained by paleogeographic data, to estimate ancestral distributions during early ranid diversification. Additionally, we used molecular clock analyses to evaluate whether these scenarios fit the temporal framework of continental breakup. Our analyses suggest that a scenario in which the ancestors of several clades (Rhacophorinae, Dicroglossinae, Raninae) reached Eurasia via the Indian subcontinent, and the ancestor of Ceratobatrachinae entered via the Australia-New Guinea plate, best fits the paleogeographic models and requires the fewest number of dispersal/vicariance events. However, several alternatives, in which part of the ranid fauna colonized Laurasia from Africa, are not significantly worse. Most importantly, all hypotheses make clear predictions as to where to expect key fossils and where to sample other living ranids, and thus constitute a strong basis for further research.

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  • Molecular phylogenetic analyses indicate multiple independent emergences of parasitism in Myzostomida (Protostomia). Syst. Biol. 2006 Apr;55(2):208-27. T127351LPM06186Q. 10.1080/10635150500481317.

    Lanterbecq D, Rouse GW, Milinkovitch MC, Eeckhaut I

    abstract

    The fossil record indicates that Myzostomida, an enigmatic group of marine worms, traditionally considered as annelids, have exhibited a symbiotic relationship with echinoderms, especially crinoids, for nearly 350 million years. All known extant myzostomids are associated with echinoderms and infest their integument, gonads, celom, or digestive system. Using nuclear (18S rDNA) and mitochondrial (16S and COI) DNA sequence data from 37 myzostomid species representing nine genera, we report here the first molecular phylogeny of the Myzostomida and investigate the evolution of their various symbiotic associations. Our analyses indicate that the two orders Proboscidea and Pharyngidea do not constitute natural groupings. Character reconstruction analyses strongly suggest that (1) the ancestor of all extant myzostomids was an ectocommensal that first infested crinoids, and then asteroids and ophiuroids, and (2) parasitism in myzostomids emerged multiple times independently.

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  • A non-linear optimization procedure to estimate distances and instantaneous substitution rate matrices under the GTR model. Bioinformatics 2006 Mar;22(6):708-15. btk001. 10.1093/bioinformatics/btk001.

    Catanzaro D, Pesenti R, Milinkovitch MC

    abstract

    The general-time-reversible (GTR) model is one of the most popular models of nucleotide substitution because it constitutes a good trade-off between mathematical tractability and biological reality. However, when it is applied for inferring evolutionary distances and/or instantaneous rate matrices, the GTR model seems more prone to inapplicability than more restrictive time-reversible models. Although it has been previously noted that the causes for intractability are caused by the impossibility of computing the logarithm of a matrix characterised by negative eigenvalues, the issue has not been investigated further.

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  • Assessing the applicability of the GTR nucleotide substitution model through simulations. Evol. Bioinform. Online 2006 ;2():145-55. PMC2674669.

    Gatto L, Catanzaro D, Milinkovitch MC

    abstract

    The General Time Reversible (GTR) model of nucleotide substitution is at the core of many distance-based and character-based phylogeny inference methods. The procedure described by Waddell and Steel (1997), for estimating distances and instantaneous substitution rate matrices, R, under the GTR model, is known to be inapplicable under some conditions, ie, it leads to the inapplicability of the GTR model. Here, we simulate the evolution of DNA sequences along 12 trees characterized by different combinations of tree length, (non-)homogeneity of the substitution rate matrix R, and sequence length. We then evaluate both the frequency of the GTR model inapplicability for estimating distances and the accuracy of inferred alignments. Our results indicate that, inapplicability of the Waddel and Steel's procedure can be considered a real practical issue, and illustrate that the probability of this inapplicability is a function of substitution rates and sequence length.We also discuss the implications of our results on the current implementations of maximum likelihood and Bayesian methods.

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  • OligoFaktory: a visual tool for interactive oligonucleotide design. Bioinformatics 2006 Jan;22(1):115-6. bti728. 10.1093/bioinformatics/bti728.

    Schretter C, Milinkovitch MC

    abstract

    The OligoFaktory is a set of tools for the design, on an arbitrary number of target sequences, of high-quality long oligonucleotide for micro-array, of primer pair for PCR, of siRNA and more. The user-centered interface exists in two flavours: a web portal and a standalone software for Mac OS X Tiger. A unified presentation of results provides overviews with distribution charts and relative location bar graphs, as well as detailed features for each oligonucleotide. Input and output files conform to a common XML interchange file format to allow both automatic generation of input data, archiving, and post-processing of results. The design pipeline can use BLAST servers to evaluate specificity of selected oligonucleotides.

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  • Evaluating intraspecific "network" construction methods using simulated sequence data: do existing algorithms outperform the global maximum parsimony approach? Syst. Biol. 2005 Jun;54(3):363-72. Q512W89524N36102. 10.1080/10635150590945377.

    Cassens I, Mardulyn P, Milinkovitch MC

    abstract

    In intraspecific studies, reticulated graphs are valuable tools for visualization, within a single figure, of alternative genealogical pathways among haplotypes. As available software packages implementing the global maximum parsimony (MP) approach only give the possibility to merge resulting topologies into less-resolved consensus trees, MP has often been neglected as an alternative approach to purely algorithmic (i.e., methods defined solely on the basis of an algorithm) "network" construction methods. Here, we propose to search tree space using the MP criterion and present a new algorithm for uniting all equally most parsimonious trees into a single (possibly reticulated) graph. Using simulated sequence data, we compare our method with three purely algorithmic and widely used graph construction approaches (minimum-spanning network, statistical parsimony, and median-joining network). We demonstrate that the combination of MP trees into a single graph provides a good estimate of the true genealogy. Moreover, our analyses indicate that, when internal node haplotypes are not sampled, the median-joining and MP methods provide the best estimate of the true genealogy whereas the minimum-spanning algorithm shows very poor performances.

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  • Separate-component-stabilization system for protein and DNA production without the use of antibiotics. BioTechniques 2005 May;38(5):775-81. 05385RR02.

    Szpirer CY, Milinkovitch MC

    abstract

    Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.

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  • Inferring contemporary levels of gene flow and demographic history in a local population of the leaf beetle Gonioctena olivacea from mitochondrial DNA sequence variation. Mol. Ecol. 2005 May;14(6):1641-53. MEC2537. 10.1111/j.1365-294X.2005.02537.x.

    Mardulyn P, Milinkovitch MC

    abstract

    We have studied mitochondrial DNA variation in a local population of the leaf beetle species Gonioctena olivacea, to check whether its apparent low dispersal behaviour affects its pattern of genetic variation at a small geographical scale. We have sampled 10 populations of G. olivacea within a rectangle of 5 x 2 km in the Belgian Ardennes, as well as five populations located approximately along a straight line of 30 km and separated by distances of 3-12 km. For each sampled individual (8-19 per population), a fragment of the mtDNA control region was polymerase chain reaction-amplified and sequenced. Sequence data were analysed to test whether significant genetic differentiation could be detected among populations separated by such relatively short distances. The reconstructed genealogy of the mitochondrial haplotypes was also used to investigate the demographic history of these populations. Computer simulations of the evolution of populations were conducted to assess the minimum amount of gene flow that is necessary to explain the observed pattern of variation in the samples. Results show that migration among populations included in the rectangle of 5 x 2 km is substantial, and probably involves the occurrence of dispersal flights. This appears difficult to reconcile with the results of a previous ecological field study that concluded that most of this species dispersal occurs by walking. While sufficient migration to homogenize genetic diversity occurs among populations separated by distances of a few hundred metres to a few kilometres, distances greater than 5 km results in contrast in strong differentiation among populations, suggesting that migration is drastically reduced on such distances. Finally, the results of coalescent simulations suggest that the star-like genealogy inferred from the mtDNA sequence data is fully compatible with a past demographic expansion. However, a metapopulation structure alone (without the need to invoke a population expansion event) cannot be dismissed as the cause of this star shape.

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  • Evidence for male dispersal along the coasts but no migration in pelagic waters in dusky dolphins (Lagenorhynchus obscurus). Mol. Ecol. 2005 Jan;14(1):107-21. MEC2407. 10.1111/j.1365-294X.2004.02407.x.

    Cassens I, Van Waerebeek K, Best PB, Tzika A, Van Helden AL, Crespo EA, Milinkovitch MC

    abstract

    Using nine nuclear species-specific microsatellite loci and two mitochondrial gene fragments (cytochrome b and control region), we investigated the processes that have shaped the geographical distribution of genetic diversity exhibited by contemporary dusky dolphin (Lagenorhynchus obscurus) populations. A total of 221 individuals from four locations (Peru, Argentina, southern Africa, and New Zealand) were assayed, covering most of the species' distribution range. Although our analyses identify a general demographic decline in the Peruvian dusky dolphin stock (recently affected by high natural and human-induced mortality levels), comparison between the different molecular markers hint at an ancient bottleneck that predates recent El Niño oscillations and human exploitation. Moreover, we find evidence of a difference in dispersal behaviour of dusky dolphins along the South American coast and across the Atlantic. While data in Peruvian and Argentine waters are best explained by male-specific gene flow between these two populations, our analyses suggest that dusky dolphins from Argentina and southern Africa recently separated from an ancestral Atlantic population and, since then, diverged without considerable gene flow. The inclusion of a few New Zealand samples further confirms the low levels of genetic differentiation among most dusky dolphin populations. Only the Peruvian dusky dolphin stock is highly differentiated, especially at mitochondrial loci, suggesting that major fluctuations in its population size have led to an increased rate of genetic drift.

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  • Local endemism within the Western Ghats-sri Lanka biodiversity hotspot. Science 2004 Oct;306(5695):479-81. 306/5695/479. 10.1126/science.1100167.

    Bossuyt F, Meegaskumbura M, Beenaerts N, Gower DJ, Pethiyagoda R, Roelants K, Mannaert A, Wilkinson M, Bahir MM, Manamendra-Arachchi K, Ng PK, Schneider CJ, Oommen OV, Milinkovitch MC

    abstract

    The apparent biotic affinities between the mainland and the island in the Western Ghats-Sri Lanka biodiversity hotspot have been interpreted as the result of frequent migrations during recent periods of low sea level. We show, using molecular phylogenies of two invertebrate and four vertebrate groups, that biotic interchange between these areas has been much more limited than hitherto assumed. Despite several extended periods of land connection during the past 500,000 years, Sri Lanka has maintained a fauna that is largely distinct from that of the Indian mainland. Future conservation programs for the subcontinent should take into account such patterns of local endemism at the finest scale at which they may occur.

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  • Genetic analysis of a successful repatriation programme: giant Galápagos tortoises. Proc. Biol. Sci. 2004 Feb;271(1537):341-5. 10.1098/rspb.2003.2607. PMC1691607.

    Milinkovitch MC, Monteyne D, Gibbs JP, Fritts TH, Tapia W, Snell HL, Tiedemann R, Caccone A, Powell JR

    abstract

    As natural populations of endangered species dwindle to precarious levels, remaining members are sometimes brought into captivity, allowed to breed and their offspring returned to the natural habitat. One goal of such repatriation programmes is to retain as much of the genetic variation of the species as possible. A taxon of giant Galápagos tortoises on the island of Española has been the subject of a captive breeding-repatriation programme for 33 years. Core breeders, consisting of 12 females and three males, have produced more than 1200 offspring that have been released on Española where in situ reproduction has recently been observed. Using microsatellite DNA markers, we have determined the maternity and paternity of 132 repatriated offspring. Contributions of the breeders are highly skewed. This has led to a further loss of genetic variation that is detrimental to the long-term survival of the population. Modifications to the breeding programme could alleviate this problem.

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  • Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J. Virol. 2004 Mar;78(5):2537-44. PMC369207.

    Radjef N, Gordien E, Ivaniushina V, Gault E, Anaïs P, Drugan T, Trinchet JC, Roulot D, Tamby M, Milinkovitch MC, Dény P

    abstract

    Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and propagation and infects nearly 20 million people worldwide. The HDV genome is a compact circular single-stranded RNA genome with extensive intramolecular complementarity. Despite its different epidemiological and pathological patterns, the variability and geographical distribution of HDV are limited to three genotypes and two subtypes that have been characterized to date. Phylogenetic reconstructions based on the delta antigen gene and full-length genome sequence data show an extensive and probably ancient radiation of African lineages, suggesting that the genetic variability of HDV is much more complex than was previously thought, with evidence of additional clades. These results relate the geographic distribution of HDV more closely to the genetic variability of its helper HBV.

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  • A hidden Markov model for progressive multiple alignment. Bioinformatics 2003 Aug;19(12):1505-13.

    Löytynoja A, Milinkovitch MC

    abstract

    Progressive algorithms are widely used heuristics for the production of alignments among multiple nucleic-acid or protein sequences. Probabilistic approaches providing measures of global and/or local reliability of individual solutions would constitute valuable developments.

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  • Structure and evolution of the mitochondrial control region of leaf beetles (Coleoptera: Chrysomelidae): a hierarchical analysis of nucleotide sequence variation. J. Mol. Evol. 2003 Jan;56(1):38-45. 10.1007/s00239-002-2378-1.

    Mardulyn P, Termonia A, Milinkovitch MC

    abstract

    To assess the levels of variation at different evolutionary scales in the mitochondrial (mt) control region of leaf beetles, we sequenced and compared the full mt control region in two genera ( Chrysomela and Gonioctena), in two species within a genus ( Gonioctena olivacea and G. pallida), in individuals from distant populations of these species in Europe, and in individuals from populations separated by moderate (10- to 100-km) to short (<5-km) distances. In all individuals, a highly repetitive section consisting of the tandem repetition of 12 to 17 imperfect copies of a 107- to 159-bp-long core sequence was observed. This repetitive fragment accounts for roughly 50% of the full control-region length. The sequence variability among repeated elements within the control region of a given individual depends on the species considered: the variability within any G. olivacea individual is much higher than that within G. pallida individuals. Comparisons of the repeated elements, in a phylogenetic framework, within and among individuals of G. olivacea and G. pallida suggests that the repetitive section of the control region experienced recurrent duplications/deletions, leading to some degree of concerted evolution. Comparisons between Chrysomela and Gonioctena control regions revealed virtually no significant sequence similarity, except for two long stretches of A's and several [T(T)A(A)] repeats, all found in the control region of other insect orders. Our analyses allowed us to identify portions of the control region with enough variation for population genetic or phylogeographic studies.

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  • Microsatellite analysis of genetic divergence among populations of giant Galápagos tortoises. Mol. Ecol. 2002 Nov;11(11):2265-83. 1617.

    Ciofi C, Milinkovitch MC, Gibbs JP, Caccone A, Powell JR

    abstract

    Giant Galápagos tortoises represent an interesting model for the study of patterns of genetic divergence and adaptive differentiation related to island colonization events. Recent mitochondrial DNA work elucidated the evolutionary history of the species and helped to clarify aspects of nomenclature. We used 10 microsatellite loci to assess levels of genetic divergence among and within island populations. In particular, we described the genetic structure of tortoises on the island of Isabela, where discrimination of different taxa is still subject of debate. Individual island populations were all genetically distinct. The island of Santa Cruz harboured two distinct populations. On Isabela, populations of Volcan Wolf, Darwin and Alcedo were significantly different from each other. On the other hand, Volcan Wolf showed allelic similarity with the island of Santiago. On Southern Isabela, lower genetic divergence was found between Northeast Sierra Negra and Volcan Alcedo, while patterns of gene flow were recorded among tortoises of Cerro Azul and Southeast Sierra Negra. These tortoises have endured heavy exploitation during the last three centuries and recently attracted much concern due to the current number of stochastic and deterministic threats to extant populations. Our study complements previous investigation based on mtDNA diversity and provides further information that may help devising tortoise management plans.

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  • Investigation of the population genetic structure and mating system in the ant Pheidole pallidula. Mol. Ecol. 2002 Sep;11(9):1805-14. 1573.

    Fournier D, Aron S, Milinkovitch MC

    abstract

    The origin of eusociality in haplo-diploid organisms such as Hymenoptera has been mostly explained by kin selection. However, several studies have uncovered decreased relatedness values within colonies, resulting primarily from multiple queen matings (polyandry) and/or from the presence of more than one functional queen (polygyny). Here, we report on the use of microsatellite data for the investigation of sociogenetic parameters, such as relatedness, and levels of polygyny and polyandry, in the ant Pheidole pallidula. We demonstrate, through analysis of mother-offspring combinations and the use of direct sperm typing, that each queen is inseminated by a single male. The inbreeding coefficient within colonies and the levels of relatedness between the queens and their mate are not significantly different from zero, indicating that matings occur between unrelated individuals. Analyses of worker genotypes demonstrate that 38% of the colonies are polygynous with 2-4 functional queens, and suggest the existence of reproductive skew, i.e. unequal respective contribution of queens to reproduction. Finally, our analyses indicate that colonies are genetically differentiated and form a population exhibiting significant isolation-by-distance, suggesting that some colonies originate through budding.

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  • The metapopulation genetic algorithm: An efficient solution for the problem of large phylogeny estimation. Proc. Natl. Acad. Sci. U.S.A. 2002 Aug;99(16):10516-21. 10.1073/pnas.162224399. 162224399. PMC124960.

    Lemmon AR, Milinkovitch MC

    abstract

    Large phylogeny estimation is a combinatorial optimization problem that no future computer will ever be able to solve exactly in practical computing time. The difficulty of the problem is amplified by the need to use complex evolutionary models and large taxon samplings. Hence, many heuristic approaches have been developed, with varying degrees of success. Here, we report on a heuristic approach, the metapopulation genetic algorithm, involving several populations of trees that are forced to cooperate in the search for the optimal tree. Within each population, trees are subjected to evaluation, selection, and mutation events, which are directed by using inter-population consensus information. The method proves to be both very accurate and vastly faster than existing heuristics, such that data sets comprised of hundreds of taxa can be analyzed in practical computing times under complex models of maximum-likelihood evolution. Branch support values produced by the metapopulation genetic algorithm might closely approximate the posterior probabilities of the corresponding branches.

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  • Dual chemical sequestration: a key mechanism in transitions among ecological specialization. Proc. Biol. Sci. 2002 Jan;269(1486):1-6. 10.1098/rspb.2001.1859. PMC1690864.

    Termonia A, Pasteels JM, Windsor DM, Milinkovitch MC

    abstract

    Platyphora leaf beetles form a vast group of tropical species each feeding on a restricted set of host plants and exhibiting bright coloration warning predators against their chemical protection. These beetles offer an exceptional opportunity for understanding the evolution of phytochemical sequestration. Indeed, qualitative studies of defensive secretions indicate that Platyphora species acquire toxicity via sequestration of plant secondary metabolites. All produce pentacyclic triterpene saponins from sequestered plant amyrins, but our analyses also indicate that many Platyphora species produce a dual chemical defence, that is, they are additionally protected by lycopsamine-type pyrolyzidine alkaloids that they also sequester from their host. This paper reports on the evolution of chemical defence and host affiliation in Platyphora leaf beetles as reconstructed on a molecular phylogeny of mitochondrial and nuclear DNA sequences. The analyses indicate that dual sequestration could be the key mechanistic means by which transitions among ecological specializations (i.e. restricted host-plant affiliations) are made possible.

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