Assistant professor @ Faculty of Sciences University of Lisbon Faculdade de Ciencias da Universidade de Lisboa Departamento de Biologia Animal/Centro de Biologia Animal Computational Biology & Population Genomics Group Lisbon Lisbon, 1749-016 Portugal Website
Author(s): Nunes, VL, Mendes R, Quartau JA, Seabra SG, Paulo OS, Simões PC
The genus Tettigettalna is composed of nine morphologically similar cicada species, being seven of these endemic to the southern Iberian Peninsula. Males produce calling songs to attract females for pair formation and reproduction. These acoustic signals are thought to be species-specific mate recognition systems with an important role in the maintenance of reproductive isolation among cicadas. Because some Tettigettalna species are often found in sympatry, we investigated the patterns of intra- and interspecific variation in male calling songs of both allopatric and sympatric populations of two sibling species, T. argentata and T. mariae. Morphometric and genetic analyses were also performed. The results confirmed the distinction of species-specific calling songs based on time variables, but the calling songs of both species recorded in sympatry were downshifted in sound frequency. Interspecific variation in morphology is low but both species revealed a significant increase in general body size in sympatric populations. Sequence analyses with one mitochondrial gene (cytochrome oxidase I) and two nuclear genes (calmodulin and elongation factor) revealed that T. argentata is geographically structured, forming two distinct clades (North and South) and that specimens of T. mariae cannot be entirely distinguished from those of T. argentata (clade South). The genetic diversity and the distribution range are much smaller for T. mariae than for T. argentata and most mitochondrial haplotypes detected in T. mariae are shared with T. argentata (clade South), which is usually found in sympatry and parapatry with T. mariae. The genetic overlap between this pair of sibling species might have been caused by incomplete lineage sorting or recent introgression. It remains unclear whether the differences in species-specific male songs are enough to fully prevent hybridization when these species are in sympatry.
Author(s): Ribeiro, CAM, Rodrigues A, Almeida HM, Glushkova M, Batista D, Paulo OS
Cork oak is the most abundant tree in Portugal, which together with its economical and social importance, has granted it the status of National Tree. It is the only species that produces renewable commercial cork, occurring across a vast range of climatic conditions in its natural range of distribution, the Mediterranean region, where it exists for over 60 Ma. So processes of local adaptation are likely to have occurred. Identifying candidate genes underlying genetic differences for adaptive traits can help to understand how species have adapted to their environment and to predict how they will respond to future climatic changes, which is a special concern in regions such as the Mediterranean Basin, where a substantial decrease in precipitation and a pronounced warming is expected in the near future. Here, we intend to assess cork oak potential capacity for adaptation by analysing population differentiation and diversity in candidate genes related with adaptive traits and test for associations with environmental variables. Five candidate genes for bud burst and osmotic stress were analyzed (in total, 1890 bp) in 25 populations from its distribution range. From the 19SNPs analyzed, several showed differences between populations, which might be involved in adaptive responses of cork oak. Our analysis, showed the presence of common haplotypes among various populations but some differences in the frequency of alleles between populations.
A correlation study of allele frequency/genotype with phenotypic and environmental conditions, revealed some significant correlations between specific heterozygous genotypes and characteristics such as plant height, altitude and rainfall. We expect that further exploitation of these data will bring some insights on the molecular basis controlling local adaptation. Funded by FCT, grant SFRH/BD/68670/2010, projects PTDC/AGRGPL/104966/2008 and PTDC/AGR-AAM/104364/2008.
Author(s): Seabra, SG, Rodrigues AS, Silva SE, Silva J, Marabuto E, Pina-Martins F, Gharbi K, Blaxter M, Borges PAV, Jiggins C, Quartau JA, Paulo OS
Philaenus spumarius (Insecta, Hemiptera, Aphrophoridae) has for long been a subject of interest of evolutionary biologists due to its heritable colour polymorphism that shows evidence of balancing selection and of clinal variation in the colour mophs frequencies. We are studying the adaptive significance of this polymorphism, particularly to understand if the melanic morphs (e.g. “marginellus” morph) have any advantage/disadvantage in terms of survival and reproductive success, efficiency of egg maturation and resistance to desiccation compared to non-melanic morphs (“typicus” and “trilineatus”). Results so far indicate a higher survival, higher number of eggs clutches and higher number of eggs laid by the “trilineatus” females than “typicus” or “marginellus” females. We are also taking a genomic approach for a) the identification of genetic basis of the colour polymorphism and b) for detecting signatures of balancing and directional selection in the genome of P. spumarius. For this purpose we are applying RAD sequencing in a) a set of samples from the three different morphs referred above, using a high frequency cutter enzyme (PstI) and in b) another set of samples from 8 populations across the distribution range of the species representing the main mitochondrial haplogroups, using a lower frequency cutter enzyme (SbfI). We are also assembling a draft of the genome that will aid in the identification of homologous regions to available references, although the very large genome size of this insect constitutes an extra challenge.
Author(s): Modesto, IS, Miguel C, Pina-Martins F, Glushkova M, Veloso M, Batista D, Paulo OS
Cork oak (Quercus suber L.) is an evergreen tree species holding a great economical and social relevance within its distribution range, the western Mediterranean Basin, associated with cork production. This tree is exploited mainly in orchards known as montado, which comprise great biodiversity. Despite its importance, cork oak stands have been facing a significant decline and climate changes expected to occur during this century may aggravate this decay. In this scenario, the assessment of adaptive genetic variation is essential to understand how cork oak may cope with these threats and to delineate management strategies of its genetic resources. In this work, we started by validating single nucleotide polymorphisms (SNPs) detected through the analysis of cork oak pyrosequenced transcriptome, for a set of putative functional genes. Six fragments were then selected to be analysed with the purpose of finding signatures of natural selection. Samples from populations representing the species’ entire distribution range were sequenced and several neutrality tests were performed as well as environmental association tests. Two gene fragments showed signals of balancing selection: the Arabidopsis thaliana orthologs non-expresser of pathogenesis related 1 (NPR1), involved in plant defence response against pathogens, and auxin response factor 16 (ARF16), a gene previously identified as a candidate gene for drought resistance. Additionally, in a class I heat shock protein (HSP), one amino acid position was detected as possibly being under positive selection and associated with several precipitation variables, revealing its potential relevance in adaptation to local climatic conditions. In this study the first steps were taken to start unveiling important information on cork oak adaptation to biotic and abiotic environmental conditions.
Funded by Fundação para a Ciência e Tecnologia (FCT): projects PTDC/AGR-GPL/104966/2008 and EST/036/2009.
The Mediterranean area is well known for retaining and generating biodiversity during climate and habitat shifts taking place since the end of the Miocene. This important biodiversity hotspot is both the source and the result of a complex process of differentiation and interaction between evolving lineages and their changing environment. However, regarding stationary Mediterranean specialists, which are expected to benefit and expand northwards with climate amelioration, little is still known.
Using the butterfly Euchloe tagis, an Atlanto-Mediterranean ecological specialist with a fragmented and restricted distribution, this study attempts at answering pertinent biogeographic aspects on the species and its interaction with the changing ecosystem. We sequenced a 599bp barcode region fragment of the COI gene from all major populations to better understand current its genetic patterns and evolutionary history.
Main results suggest an origin in North Africa, from where the sister species Euchloe pechi is endemic. While one of the Moroccan populations diverged earlier from the remaining stock, all remaining are more closely related despite the existence of differentiated major mitochondrial lineages. It seems that Iberia has played a pivotal role in the generation of diversity in this species: two consistent genetic clusters are partly sympatric whereas French and Italian populations are considerably different. The second Moroccan population from the Rif Mountains shows little segregation from French samples and this issue is discussed on a phylogeographic framework supported by molecular-clock analysis.
This pattern suggests this species experienced polycentric refugia during climate cycles, leading to such genetic structure and a putative re-colonization of Africa. Moreover, the neutral genetic pattern reported in this study challenges current taxonomy based on morphological differentiation and suggests a more complex evolution of this species.
Author(s): Rodrigues, AS, Silva SE, Marabuto E, Silva DN, Silva J, Wilson MR, Thompson V, Yurtsever S, Halkka A, Gharbi K, Blaxter M, Borges PAV, Quartau JA, Jiggins C, Paulo OS, Seabra SG
We investigated the evolutionary history of the meadow spittlebug Philaenus spumarius, a widespread and polyphagous insect species of the Holarctic region, limited in its range by the presence of enough humidity in the earlier life stages, and that has a remarkable dorsal colour/pattern polymorphism of the adults. The mitochondrial gene cytochrome c oxidase subunit I (COI) revealed three main haplogroups in Europe: the “western”, the “eastern-Mediterranean” and the “north-eastern” haplogroups, which overlapped in some regions. Evidence of recent divergence events at less than one million years ago in southern Mediterranean peninsulas followed by northward population expansions were found. Recent gene-flow events between the main southern peninsulas and between the Iberian Peninsula and North Africa were also detected. A probable British origin for the insular populations of the Azores and New Zealand was revealed, as well as multiple geographic origins for North American populations from western and northern Europe.
Restriction site associated DNA (RAD) sequencing was also applied in a set of 8 populations from the different mitochondrial haplogroups and the differentiation patterns obtained from a set of over 15,000 SNPs were concordant with COI results and with a better resolved tree.
The widespread use of Next Generation Sequencing (NGS) techniques and its application to non model organisms provided biologists with large amounts of genetic data and the ability to address problems which were untreatable just a few years ago.
However, the frequent lack of reference genomes on non-model organisms usually creates difficulties to researchers when trying to answer specific genetic questions, such as finding candidate genes or looking for intra and inter-population variation.
For instance, mining SNPs in NGS datasets of anonymous pooled individuals that cannot be compared to a reference is still not a simple task.
Here is described 4Pipe4, a NGS data analysis pipeline, optimized for SNP mining in the aforementioned datasets, particularly on Roche 454 data.
In order to assess its efficiency, a dataset of anonymous pooled individuals of Quercus suber (Cork Oak), which does not have a reference genome available, was analysed with 4Pipe4, and a subset of tenths of SNPs detected by the pipeline was randomly selected and sequenced in an array for validation.
The results of the genotyping array were explored to: a) provide insights on population structure and gene flow patterns and b) make an association study with environmental factors such as temperature, precipitation or drought periods.
This combined approach of 454/genotyping array with the 4Pipe4 pipeline proved to be a very efficient and cost effective way to obtain validated and mapped SNPs from orthologous regions, for population genomics studies.
Recent studies have been focusing on the phylogenetic relationships among the endemic Columba from the Macaronesia – C. palumbus azorica (Azores Islands), C. trocaz (Madeira Island), C. bollii and C. junoniae (both from Canary Islands) – through several approaches, namely mtDNA and microsatellites, with somewhat incongruent results. In the present study, the origin of the endemic subspecies C. p. azorica was inferred based on five nuclear fragments. Overall, C. p. azorica shows higher haplotype diversity and nucleotide diversity values than the mainland subspecies C. p. palumbus and the Madeiran C. trocaz for the most introns analysed, which was not expected since the insular populations frequently have less genetic diversity than the mainland populations. Our results also support a previous study in which it is described that C. trocaz is phylogenetically closer to C. bollii than to C. p. palumbus (Dourado et al., in press). Surprisingly, for the Azorean subspecies, while some samples cluster with the mainland C. p. palumbus, others share the same group with C. trocaz. The most plausible hypothesis to explain these results is a colonisation of the Azores by an ancestral of C. trocaz and C. bollii, that then diverged due to geographic isolation. More recently, the mainland subspecies C. p. palumbus reached those islands, conducing to admixture between both groups. Hybridization may have conducted to introgression of mtDNA from C. p. palumbus into the other individuals that inhabited Azores Islands, and mtDNA from C. p. palumbus prevailed. Therefore, instead of a simple isolated woodpigeon population, C. p. azorica is a much more complex and interesting event in pigeon evolution than formerly alleged.
Author(s): Brás, PG, Martins R, Martins J, Rebelo MT, Franco JC, Mateus C, Paulo OS, Figueiredo E, Seabra SG
Adults of the tiger-fly, Coenosia attenuata Stein, are the only known predators of adult stages of important greenhouse crop pests such as whiteflies and leafminers. C. attenuata has been recognized as a potential biological control agent in greenhouses but no studies to date have been done to characterize the genetic variation and differentiation across the distribution range of the species. The phylogeographic study on samples from Europe, North and South America and Western Asia based on cytochrome oxidase I mitochondrial gene reveals generally high haplotype diversity but low nucleotide diversity, suggesting a recent expansion, and shows evidences of a recent colonization of North and South America from the Palearctic. The probable origin of the species within the Paleotropical region are discussed, as well as the dispersal patterns of the species and the dates of divergence of populations.
Author(s): Romeiras, MM, Duarte MC, Santos-Guerra A, Moura M, Batista D, Vieira A, Paulo OS
Understanding the origins and diversification of oceanic island flora has long fascinated evolutionary biologists, ever since Darwin. The Macaronesian Islands, in the North-East Atlantic Region, are considered a hot-spot of biodiversity and contain a large proportion of endemic taxa. Recent molecular studies are providing insights into the patterns of colonization and radiation within the endemic plant species, while the relationships between the wild (i.e. endemics and natives) and the crop species have not been suitably addressed from an evolutionary perspective. The aim of this study is to understand the evolutionary and biogeographic history of the seven native species of Macaronesian Beta-Patellifolia species complex (Subfamily Betoideae, Amaranthaceae), which are ancestral species of the cultivated sugarbeet species (Beta vulgaris subsp. vulgaris). We compare the patterns of diversification identified for Macaronesia with those of the Mediterranean Region to assess to what extent patterns may be generalized across regions. Phylogenetic analyses based on nuclear (ITS) and plastid (rbcL, matK, trnH-psbA, trnL-F) DNA markers produced similar topologies that suggest a recent colonization event in the Macaronesian Region. Despite extensive sampling, low levels of sequence variation are consistently found throughout Beta spp., thus preventing complete resolution among taxa. Origins and diversification of these endemics, natives and crop species within the Macaronesian Region will be discussed in a biogeographic context.
Author(s): Vieira, AC, Silva DN, Varzea V, Silva MC, Paulo OS, Batista D
Ecological speciation through host-shift has been proposed in recent years as a major route for the appearance of novel fungal plant pathogens. Colletotrichum kahawae is an emergent plant pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa at high altitude. This specialist pathogen revealed to be an outstanding model of rapid adaptation and ecological speciation due to its strikingly close proximity (< 2000 yrs) to a non-pathogenic and generalist fungal species. The remarkable genetic proximity of C. kahawae and those Colletotrichum species associated with a qualitatively change in pathogenicity (C. kahawae is able to infect green coffee berries while the closely related species are not) creates a unique opportunity to address fundamental questions behind the evolution of pathogenic fungi. This work aims to use a next generation sequencing approach with Restriction-site Associated DNA (RAD) markers, as well as a broad geographic sampling of C. kahawae and its closely related non-pathogenic sibling, to perform a SNP-based genomic population study and unveil the genomic base putatively responsible for the rapid adaptation and severe virulence of C. kahawae. Revealing, where and how natural selection is acting across the genome could point us into the direction of the underlying genetics that make the patogenicity of C. kahawae unique and specific to green coffee berries. Moreover, understanding the genomic bases behind these specialization processes will allow a more based-informed and effective control not only for C. kahawae populations but also for other plant pathogens with the same speciation mechanism.
Author(s): Fino, J, Figueiredo A, Loureiro A, Silva MC, Batista D, Paulo OS
Coffee berry disease (CBD), caused by the fungus Colletotrichum kahawae, is considered one of the biggest threats to Arabica coffee production in Africa at high altitude. Some coffee genotypes are known to be resistant to CBD, but the molecular genetic basis of coffee resistance is still unknown. With the purpose of gaining some insights on this process, a RNA Illumina sequencing approach was used to characterize the defense response of two coffee genotypes, respectively resistant and susceptible to C. kahawae, during the early stages of the infection process. Three inoculation time-points were selected (24, 48 and 72 hpi) and two biological replicates were collected. Twenty four independent cDNA libraries were sequenced and data is being analyzed to assess differential gene expression when comparing inoculated with control samples. The data was trimmed, and two assemblies were made: one with the control libraries and the other with the inoculated libraries. The first was used in expression quantification for both gene and isoform, by using statistical methods. Particular attention was paid to data normalization, using FPKM algorithm in gene quantification, and a Maximum Likelihood statistical model in isoform quantification. The other assembly is being used to discover C. kahawae genes by codon usage frequency (ECLAT). Although fungal transcripts are weakly represented in the samples, we expect to detect new and unknown genes of C. kahawae. The identification and characterization of expression differences between these contrasting situations will allow us to understand which genes are potentially involved in the resistance response of coffee to C. kahawae. The subsequent identification of the predicted proteins and their location in the metabolic networks could provide new relevant knowledge able to support and improve coffee breeding for resistance to CBD.
Author(s): Silva, DN, Vieira A, Talhinhas P, Azinheira HG, Silva MC, Fernandez D, Duplessis S, Batista D, Paulo OS
The Basidiomycota is the second largest group of fungi, encompassing a remarkably diverse and important array of life forms, from mushrooms to smuts and rusts. They play vital roles in recycling nutrients in the global environment and present serious threats to conservation and economic activities as pathogens and wood rotting species. The importance of these species allied with their relatively small and compact genomes have made them prime candidates for sequencing projects, generating a growing platform of genomic resources. However, due to the recent availability of these resources, there has still been no effort to use genome-scale data to study the evolutionary history of this group of organisms. Here we present the results of an ongoing phylogenomics project that includes not only a representative sampling of the Basidiomycota, but also of the sister phylum, Ascomycota, in order to produce a comprehensive data matrix amenable for testing several hypotheses about the evolution of the Basidiomycota. Using state-of-the-art orthology detection methods as well as established and custom made quality control pipelines, we were able to assemble several data sets that range from 614 putative orthologs across 47 species to 3093 putative orthologs across 114 species, including data from complete genome projects and EST databases. Regarding the species-gene ratio, this represents the most comprehensive data matrix assembled so far for the fungal kingdom, allowing us to produce resolved and highly supported phylogenetic trees even among the most deep relationships. Paradoxically, we have also obtained several incongruities between data sets, which may have a substantial impact on the current understanding of fungal evolution. Our current goals reside in achieving a better understanding of these incongruities and adding a geological time-awareness into the evolutionary history of the Basidiomycota. This work is being funded by project PTDC/AGR-GPL/119943/2010.
The Iberian Peninsula is recognized as one of the most important refugia for temperate species during the cyclic climatic changes of the Plio-Pleistocene glacial periods. Mountainous regions within these refugia areas are also believed to retain high levels of endemism and genetic diversity due to the easily tracked suitable habitats by upward-downward movements. Here we examine population genetic structure, phylogeographic and demographic patterns of Lycaena bleusei, a butterfly species endemic to the Central Iberian Mountain System and contrast this to the patterns found for a congeneric widespread species, also occurring in the Iberian Peninsula, Lycaena tityrus. We use two molecular markers to understand if there is congruency in their evolutionary histories or, conversely, if different evolutionary drivers may have shaped these species differently. We find that the two species, which exhibit similar dispersal abilities and ecological requirements, show different demographic and genetic patterns. We hypothesize that these differences are due to the evolutionary forces acting on species that contracted to southern refugia during the Pleistocene and that, ultimately, drove to population differentiation and speciation. We also find evidence for hybridization events between these two species, with L. bleusei males coming in contact with L. tityrus females on a secondary contact zone described here for the first time.
Author(s): Batista, D, Silva DN, Martins R, Pereira AP, Guimarães L, Talhinhas P, Silva MC, Várzea V, Paulo OS
Coffee leaf rust (CLR) caused by the biotrophic fungus Hemileia vastatrix is the most important disease of Arabica coffee, reaching nowadays a worldwide distribution. Breeding for rust resistance has proven successful, but the highly adaptable nature of the fungus shaped by the dynamic system of host-pathogen co-evolution has been a critical limitation for achieving durable CLR resistance. As a consequence a gradual breakdown of resistance has been observed over the years in many improved varieties in several countries. In the presence of such a serious and constant threat of new evolving pathotypes emerging under a strong selective pressure and becoming epidemically spread on a continental scale, sound knowledge on population genetics can be used to determine the major forces and mechanisms driving evolution of pathotypes, providing ground to understand how host resistance genes are overcome. A project was recently initiated to address these questions using RADseq of H. vastatrix isolates comprising a comprehensive along-time coverage of geographical origins and virulence profiles that provides a unique opportunity for detection of temporal variation over the last 50 years. From the data of thousands of SNPs simultaneously genotyped on those samples, we will be searching for phylogeographical patterns and signatures of selection in the genome. This will allow the identification of putative loci/candidate genes of adaptive significance, and provide insights on the mechanisms of pathogen evolution underlying virulence differentiation. In a parallel conventional population analysis of a virulence-related candidate gene, divergent alleles were detected and signs of adaptive relevant genetic variation are being traced, which could lead us on starting to understand rust adaptation.
Funded by FCT (project PTDC/AGR-GPL/119943/2010)