Juan I. Montoya-Burgos

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland

Recent genome-wide analyses have revealed patterns of positive selection acting on protein-coding genes in humans and mammals. To assess whether the conclusions drawn from these analyses are valid for other vertebrates and to identify mammalian specificities, I have investigated the selective pressure acting on protein-coding genes of the puffer fishes Tetraodon and Takifugu. My results indicate that the strength of purifying selection in puffer fishes is similar to previous reports for murids but stronger in hominids, which have a smaller population size. Gene ontology analyses show that more than half of the biological processes targeted by positive selection in mammals are also targeted in puffer fishes, highlighting general patterns for vertebrates. Biological processes enriched with positively selected genes that are shared between mammals and fishes include immune and defense responses, signal transduction, regulation of transcription and several of their descendent terms. Mammalian-specific processes displaying an excess of positively selected genes are related to sensory perception and neurological processes. The comparative analyses also revealed that, for both mammals and fishes, genes encoding extracellular proteins are preferentially targeted by positive selection, indicating that adaptive evolution occurs more often in the extra-cellular environment rather than inside the cell. Moreover, I present here the first genome-wide characterization of neutrally-evolving regions of protein-coding genes. This analysis revealed an unexpectedly high proportion of genes containing both positively selected motifs and neutrally-evolving regions, uncovering a strong link between neutral evolution and positive selection. I speculate that neutrally-evolving regions are a major source of novelties screened by natural selection.

Montoya-Burgos J I

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Recent genome-wide analyses have revealed patterns of positive selection acting on protein-coding genes in humans and mammals. To assess whether the conclusions drawn from these analyses are valid for other vertebrates and to identify mammalian specificities, I have investigated the selective pressure acting on protein-coding genes of the puffer fishes Tetraodon and Takifugu. My results indicate that the strength of purifying selection in puffer fishes is similar to previous reports for murids but stronger in hominids, which have a smaller population size. Gene ontology analyses show that more than half of the biological processes targeted by positive selection in mammals are also targeted in puffer fishes, highlighting general patterns for vertebrates. Biological processes enriched with positively selected genes that are shared between mammals and fishes include immune and defense responses, signal transduction, regulation of transcription and several of their descendent terms. Mammalian-specific processes displaying an excess of positively selected genes are related to sensory perception and neurological processes. The comparative analyses also revealed that, for both mammals and fishes, genes encoding extracellular proteins are preferentially targeted by positive selection, indicating that adaptive evolution occurs more often in the extra-cellular environment rather than inside the cell. Moreover, I present here the first genome-wide characterization of neutrally-evolving regions of protein-coding genes. This analysis revealed an unexpectedly high proportion of genes containing both positively selected motifs and neutrally-evolving regions, uncovering a strong link between neutral evolution and positive selection. I speculate that neutrally-evolving regions are a major source of novelties screened by natural selection.

Surget-Groba Y, Montoya-Burgos J I

Department of Zoology and Animal Biology, University of Geneva, 1211 Geneva 4, Switzerland.

Transcriptome analysis has important applications in many biological fields. However, assembling a transcriptome without a known reference remains a challenging task requiring algorithmic improvements. We present two methods for substantially improving transcriptome de novo assembly. The first method relies on the observation that the use of a single k-mer length by current de novo assemblers is suboptimal to assemble transcriptomes where the sequence coverage of transcripts is highly heterogeneous. We present the Multiple-k method in which various k-mer lengths are used for de novo transcriptome assembly. We demonstrate its good performance by assembling de novo a published next-generation transcriptome sequence data set of Aedes aegypti, using the existing genome to check the accuracy of our method. The second method relies on the use of a reference proteome to improve the de novo assembly. We developed the Scaffolding using Translation Mapping (STM) method that uses mapping against the closest available reference proteome for scaffolding contigs that map onto the same protein. In a controlled experiment using simulated data, we show that the STM method considerably improves the assembly, with few errors. We applied these two methods to assemble the transcriptome of the non-model catfish Loricaria gr. cataphracta. Using the Multiple-k and STM methods, the assembly increases in contiguity and in gene identification, showing that our methods clearly improve quality and can be widely used. The new methods were used to assemble successfully the transcripts of the core set of genes regulating tooth development in vertebrates, while classic de novo assembly failed.

Di-Poi N, Montoya-Burgos J I, Miller H, Pourquie O, Milinkovitch M C, Duboule D

National Research Center Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.

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.

Montoya-Burgos J I, Foulon A, Bahechar I

Department of Zoology and Animal Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland. juan.montoya@unige.ch

BACKGROUND: Fast evolving genes are targets of an increasing panel of biological studies, from cancer research to population genetics and species specific adaptations. Yet, their identification and isolation are still laborious, particularly for non-model organisms. We developed a method, named the Inter-Specific Selective Hybridization (ISSH) method, for generating cDNA libraries enriched in fast evolving genes. It utilizes transcripts of homologous tissues of distinct yet related species. Experimental hybridization conditions are monitored in order to discard transcripts that do not find their homologous counterparts in the two species sets as well as transcripts that display a strong complementarity between the two species. Only heteroduplexes that disanneal at low stringency are used for constructing the resulting cDNA library. RESULTS: We demonstrate the efficiency of the ISSH method by generating a brain cDNA library enriched in fast evolving transcripts of a non-model catfish species as well as a control, non-enriched library. Our results indicate that the enriched library contains effectively more fast evolving sequences than the control library. Gene annotation analyses also indicate enrichment in genes with low expression levels and non-ubiquitously expressed genes, both categories encompassing the majority of fast evolving genes. Furthermore, most of the identified transcripts show higher sequence divergence between two closely related catfish species as compared to recognized fast evolving DNA markers. CONCLUSIONS: The ISSH method offers a simple, inexpensive and efficient way to screen the transcriptome for isolating fast evolving genes. This method opens new opportunities in the investigation of biological mechanisms that include fast evolving genes, such as the evolution of lineage specific processes and traits responsible for species adaptation to their environment.

Covain R, Dray S, Fisch-Muller S, Montoya-Burgos J I

Departement d'herpetologie et d'ichtyologie, Museum d'histoire naturelle, 1 route de Malagnou, C.P. 6434, CH-1211 Geneve 6, Switzerland; Departement de zoologie et de biologie animale, Universite d...

Torrico J P, Hubert N, Desmarais E, Duponchelle F, Nunez Rodriguez J, Montoya-Burgos J, Garcia Davila C, Carvajal-Vallejos F M, Grajales A A, Bonhomme F, Renno J F

Facultad de Ciencias Puras y Naturales, IRD UR 175/IBMB, Universidad Mayor San Andres, La Paz, Murillo, Bolivia. jptb_bioevol@yahoo.com

Di-Poi N, Montoya-Burgos J I, Duboule D

National Research Center "Frontiers in Genetics," Department of Zoology and Animal Biology, University of Geneva, 1211 Geneva 4, Switzerland.

Hox genes control many aspects of embryonic development in metazoans. Previous analyses of this gene family revealed a surprising diversity in terms of gene number and organization between various animal species. In vertebrates, Hox genes are grouped into tightly organized clusters, claimed to be devoid of repetitive sequences. Here, we report the genomic organization of the four Hox loci present in the green anole lizard and show that they have massively accumulated retrotransposons, leading to gene clusters larger in size when compared to other vertebrates. In addition, similar repeats are present in many other development-related gene-containing regions, also thought to be refractory to such repetitive elements. Transposable elements are major sources of genetic variations, including alterations of gene expression, and hence this situation, so far unique among vertebrates, may have been associated with the evolution of the spectacular realm of morphological variations in the body plans of Squamata. Finally, sequence alignments highlight some divergent evolution in highly conserved DNA regions between vertebrate Hox clusters, which may coincide with the emergence of mammalian-specific features.

Cardoso Y P, Montoya-Burgos J I

Department of Zoology and Animal Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland.

Abstract Neotropical freshwater fishes have reached an unrivalled diversity, organized into several areas of endemism, yet the underlying processes are still largely unknown. The topographical and ecological characteristics of the Guyanas Region make it an ideal area of endemism in which to investigate the forces that have shaped this great diversity. This region is thought to be inhabited by species descending from Amazonian ancestors, which would have used two documented routes that, however, hardly explain the entrance of species adapted to running waters. Here, we investigate the evolutionary history of Pseudancistrus brevispinis, a catfish endemic to this region and exclusively found in running waters, thus making it an ideal model for investigating colonization routes and dispersal in such habitats. Our analyses, based on mitochondrial and nuclear markers, revealed an unexpected diversity consisting of six monophyletic lineages within P. brevispinis, showing a disjoint distribution pattern. The lineages endemic to Guyanas coastal rivers form a monophyletic group that originated via an ancestral colonization event from the Amazon basin. Evidence given favours a colonization pathway through river capture between an Amazonian tributary and the Upper Maroni River. Population genetic analyses of the most widespread species indicate that subsequent dispersal among Guyanas coastal rivers occurred principally by temporary connections between adjacent rivers during periods of lower sea level, yet instances of dispersal via interbasin river captures are not excluded. During high sea level intervals, the isolated populations would have diverged leading to the observed allopatric species. This evolutionary process is named the sea level fluctuation (SLF) hypothesis of diversification.

Chiachio M C, Oliveira C, Montoya-Burgos J I

Departamento de Morfologia, Universidade Estadual Paulista Julio de Mesquita Filho, Instituto de Biociencias, Laboratorio de Biologia e Genetica de Peixes, Rubiao Junior 18618000, Botucatu, SP, Bra...

The Neotropics possess the greatest freshwater fish diversity of the world, rendering the study of their evolutionary history extremely challenging. Loricariidae catfishes are one of the most diverse components of the Neotropical ichthyofauna and despite a long history of classification, major issues still need elucidation. Based on a nuclear gene, we present a robust phylogeny of two former loricariid subfamilies: Hypoptopomatinae and Neoplecostominae. Our results show that Neoplecostominae is nested within Hypoptopomatinae, and is the sister group to the former Otothyrini tribe. According to our results, supplemented by morphological observations, we erect two new subfamilies, the Otothyrinae and a new Hypoptopomatinae, and modify the Neoplecostominae by including the genus Pseudotocinclus. The uncovered evolutionary relationships allow a detailed analysis of their historical biogeography. We tested two Dispersal-Extinction-Cladogenesis models for inferring the distribution range evolution of the new subfamilies, and show that the model having no constrains performs better than a model constraining long-range dispersal. The Maximum Likelihood reconstructions of ancestral ranges showed a marked division between the Amazonian origin of the Hypoptopomatinae and the eastern coastal Brazil+Upper Parana origin of the Neoplecostominae and Otothyrinae. Markedly few instances of dispersal across the border separating the Amazon basin and the Parana-Paraguay+eastern coastal Brazil+Uruguay were reconstructed. This result is in clear contrast with the historical biogeography of many Neotropical fishes, including other Loricariidae. Part of the dispersal limitation may be explained by divergent ecological specialization: lowland rivers versus mountain streams habitats. Moreover, because most species of the new subfamilies are small, we hypothesize that body size-related effects might limit their dispersal, like predation and energetic cost to migration. Finally, morphological and anatomical features are presented that limit or, to the contrary, enhance dispersal capability in these small and fascinating catfishes.

Covain R, Dray S, Fisch-Muller S, Montoya-Burgos J I

Departement d'herpetologie et d'ichtyologie, Museum d'histoire naturelle, 1 route de Malagnou, C.P. 6434, CH-1211 Geneve 6, Switzerland. Raphael.Covain@ville-ge.ch

With the increase of laboratory facilities, molecular phylogenies are playing a predominant role in evolutionary analyses. However, understanding the evolution of morphological traits remains essential for a comprehensive view of the evolution of a group. Here we present a new approach based on co-inertia analysis for identifying characters which variations are dependent to the phylogeny, a prerequisite for analyzing the evolution of characters. Our approach has the advantage of treating the full data set at once, including qualitative and quantitative variables. It provides a graphical output giving the contribution of each variable to the co-structure, allowing a direct discrimination among phylogenetically dependent and independent variables. We have implemented this approach in deciphering the evolution of morphological traits in a highly specialized group of Neotropical catfishes: the Loricariinae. We have first inferred a molecular phylogeny of this group based on the 12S and 16S mitochondrial genes. The resulting phylogeny indicated that the subtribe Harttiini was restricted to the single genus Harttia, and within the subtribe Loricariini, two sister subtribes were distinguished, Sturisomina (new subtribe), and Loricariina. Among Loricariina, the morphological groups Loricariichthys and Loricaria+Pseudohemiodon were confirmed. The co-inertia analysis highlighted a strong relationship between the morphological and the genetic data sets, and identified three quantitative and eight qualitative variables linked to the phylogeny. The evolution of quantitative variables was assessed using the orthogram method and showed a major punctual event in the evolution of the number of caudal-fin rays, and a more gradual pattern of evolution of the number of teeth along the phylogeny. The evolution of qualitative variables was inferred using ancestral states reconstructions and highlighted parallel patterns of evolution in characters linked to the mouth, suggesting co-evolution of the traits for adapting to divergent substrates.

Nikolaev S I, Montoya-Burgos J I, Popadin K, Parand L, Margulies E H, Antonarakis S E

Department of Genetic Medicine and Development, University of Geneva Medical School, 1 Rue Michel-Servet, 1211 Geneva, Switzerland. sergey.nikolaev@medecine.unige.ch

A comprehensive phylogenetic framework is indispensable for investigating the evolution of genomic features in mammals as a whole, and particularly in humans. Using the ENCODE sequence data, we estimated mammalian neutral evolutionary rates and selective pressures acting on conserved coding and noncoding elements. We show that neutral evolutionary rates can be explained by the generation time (GT) hypothesis. Accordingly, primates (especially humans), having longer GTs than other mammals, display slower rates of neutral evolution. The evolution of constrained elements, particularly of nonsynonymous sites, is in agreement with the expectations of the nearly neutral theory of molecular evolution. We show that rates of nonsynonymous substitutions (dN) depend on the population size of a species. The results are robust to the exclusion of hypermutable CpG prone sites. The average rate of evolution in conserved noncoding sequences (CNCs) is 1.7 times higher than in nonsynonymous sites. Despite this, CNCs evolve at similar or even lower rates than nonsynonymous sites in the majority of basal branches of the eutherian tree. This observation could be the result of an overall gradual or, alternatively, lineage-specific relaxation of CNCs. The latter hypothesis was supported by the finding that 3 of the 20 longest CNCs displayed significant relaxation of individual branches. This observation may explain why the evolution of CNCs fits the expectations of the nearly neutral theory less well than the evolution of nonsynonymous sites.