DNA barcoding and DNA taxonomy
DNA barcoding and DNA taxonomy approach is the topic I like the most, and in which I have been more involved in recent months. In fact, biodiversity profiling of large communities using a barcoding and DNA taxonomy approaches will be the topic selected for my future application to the Ramon y Cajal Fellowships. The results of two small projects on beetles, developed in the Natural History Museum working with Dr Alfried Vogler, have been already submitted: one on species delimitation using bardoces alone in an endemic Australian radiation of the genus Rivacindela, and another on biodiversity assessment of Copelatus radiation from Fiji. Recently, our group has been granted with three large projects on seven insect orders of Madagascar, the water beetles from Europe, and Scarabaeidae from South America involving the sequencing of more than 20000 specimens.
Why a barcoding approach?
At the beginning of 21st century the biodiversity of many areas of the Earth is still known inadequately. The high levels of ecosystem endangerment and destruction, particularly in the 42 biodiversity hot spots catalogued by the UNESCO, has prompted urgent calls for new improved systems of inventorying biodiversity and disseminating the information to conservation planners. In recent years a new taxonomic approach called 'DNA barcoding' has been proposed to aiding determining species. This method uses a short DNA sequence, generally the mitochondrial cox1 gene, rather than morphological characters. The early successes of DNA ‘barcoding’ (Hebert et al., 2003a, b; Hebert et al., 2004) suggest that large-scale surveys of DNA variation would accelerate studies on ecology, biodiversity and conservation planning of poorly studied ecosystems and groups of organisms. Recently, several museums, herbaria, universities, biodiversity inventory agencies, and commercial experts have created the international consortium Barcode of Life (CBOL). The use of DNA sequence difference for species recognition, assessment, and taxonomic description is not novel. Instead, the aim of this consortium is (i) promotion of community wide standards and protocols regarding choice of gene(s), electronic databasing, specimen collection data and vouchering and (ii) its explicit use as a system for rapid species recognition and identification in applied contexts outside of species inventory and taxonomy. These include taxonomic accuracy, low cost, ease of application in diverse contexts (including by non-specialists), portability, routine and immediate access to information, and utility across a broad phylogenetic and taxonomic spectrum of organisms, including many species new to science. Its core or essential function is the ability to correctly assign a given sample to individual named species, or to identify the sample as belonging to an unnamed (new) species. DNA barcoding is not intended to provide precise data regarding phylogenetic (evolutionary) relationships, although barcoding efforts are compatible with and ideally will complement phylogenetic studies. However, many scientists have raised concerns about taxonomy based on DNA data alone, viz.: (i) whether DNA variation in the targeted taxa is sufficient to discriminate closely related species, (ii) whether genetic groups can be directly translated into formal species because potentially complicated evolutionary history of gene trees may obscure species history (DNA taxonomy), and (iii) how to incorporate the taxonomic knowledgement built in the last three centuries in the new framework. I am particularly interested in study whether genetic variation of short mitochondrial sequences can delimit species boundaries in close related species.
Phylogenetics: developing new nuclear markers and intron evolution.
At the present, I am working in Dr. Alfried Vogler’s laboratory in the NaturalHistoryMuseum. The main goal of our project is to generate new phylogenetic markers based on ESTs libraries to build the world-wide phylogeny of the Cicindela (Coleoptera: Cicindelidae). The dating of the nodes will be used to estimate and compare the speciation rate in different Cicindela lineages. As result of this project we have submitted a manuscript about the world-wide phylogeny of Cicindela s. l. group (Coleoptera: Cicindelidae) based on a new phylogenetic marker, the muscle specific protein 20 (homologous to Drosophila melanogaster Mp20 protein).
The genus Cicindela (Coleoptera: Cicindelidae) is a species-rich cosmopolitan group of tiger beetles used as model for inter-continental comparisons of lineage diversification. Basal relationships of continental groups are important for this analysis but have been difficult to resolve with standard mtDNA markers. Here we used the mp20 gene, a single copy nuclear marker coding for a muscle associated protein in insects, for phylogenetics of basal groups in Cicindela. Nearly-full length sequences were obtained for 51 cicindelids, including major taxonomic groups from all continents, and resulted in sequences of minimally 1.2 kb and spanning three introns. Phylogenetic signal of exon and intron sequences was compared with that from four gene regions (2.4 kb total) of mtDNA. Mp20 sequences contributed two thirds of the total support of the combined analysis, with most signal from the introns. However, sequence alignment in introns presented a challenge, and minor variation of parameter values introduced great changes in tree topology. We conclude that basal clades of Cicindela are largely coincident with major biogeographic regions, with major clades confined, respectively, to Australasia, the Holarctic, the Indian subcontinent and Africa, and to South and Central America. Mp20 exon and intron sequences also contributed greatly to estimating divergence times based on a molecular clock. The inferred dates suggested that basal splits of continental lineages are younger than would be expected from continental break-up, with most of the species diversity in Cicindela accumulating only over the past 20-30 mya.
In late 2001, I moved to the NaturalHistoryMuseum in London with the main goal to generate new molecular markers to resolve phylogenetic relationships in beetles at genus and family level in which classical mitochondrial and nuclear markers generally show little resolution. We constructed several ESTs libraries from different beetle families to design primers to amplify the selected genes. As result of this project we are finishing a manuscript about the world-wide phylogeny of Cicindela sensu lato group (Coleoptera: Cicindelidae) based on a new molecular marker, the muscle specific protein 20 (homologous to Drosophila melanogaster Mp20 protein).
I am still working on island radiation (and continental as well) but recently my research interests shifted to genes involved in speciation, and the use of short mitochondrial DNA sequences ('barcodes') to delimited species boundaries. For further details about a project about genes involved in speciation please see the attached file entitled 'Genes&Speciation.doc'.
REstos
Repetitive DNA: Heterochromatin and satellite DNA evolution
My research has focused on the repetitive DNA (junk DNA for many people), and particularly on satellite DNA. This highly repetitive DNA, unique to eukaryotic organisms, is organized in long tandem repeats in the constitutive heterochromatin (telomeric and pericentromeric regions of chromosomes). I am especially attracted of the mechanisms explaining the evolutionary dynamics and the function of this fraction of the genome.
I started studying the heterochromatin of Misolampus goudoti (Coleoptera: Tenebrionidae) during my MSc thesis in the laboratory of Genetics of the University of Balearic Islands (Spain). In this species the heterochromatin constitutes a large block in the pericentromeric regions of the chromosomes and small blocks in the telomers. Molecular and cytogenetics studies of those regions revealed that the main components of the centromeric blocks are two satellite DNAs, with repeat units of 190 bp and 1.2 Kb. In contrast, telomeric blocks are composed of the 1.2 Kb-long repeats only.
During my PhD in the same laboratory, I studied the satellite DNA from 35 taxa of the genus Pimelia (Coleoptera: Tenebrionidae) All taxa have large heterochromatic blocks in the pericentromeric regions of all chromosomes except Y chromosome. Digestion of genomic DNA with Eco RI or Hind III revealed the characteristic ladder of oligomers of satellite DNAs. Satellite DNA fraction makes between 25 and 45% of those genomes. FISH of cloned repeats showed the same chromosomal location than the heterochromatin. 26 taxa have a single major satellite composed of tandem repeats of about 357 bp in length and 69 % A-T rich (PIM357). Phylogenetic analysis based on sequence divergence clusters PIM357 repeats into three groups mostly in accordance with the geographic origin of the species: Iberian-Balearic, Moroccan, and Canary Islands groups. These groups are in accordance with the mitochondrial phylogeny (Juan et al., 1995, Pons et al, 2002a). Iberian-Balearic and African Pimelia species show a high homogenisation of the sequences and a gradual fixation of species-specific DNA sequences. This gradual evolution of stDNA sequences has been postulated in the molecular drive model (Dover, 1982, 1986), but few cases have been experimentally demonstrated (Strachan et al., 1985; Bachmann, & Sperlich; 1993; Mantovani et al., 1998). On the other hand, Canarian species show low homogenisation and fixation of their repetitive sequences and spreading of divergent stDNA subfamilies. These subfamilies seems to be randomly distributed in the heterochromatin in P. sparsa sparsa but in P. radula ascendens are compartmentalised in different chromosomes and organised in higher-order repeats (Pons et al., 2002b). Evidence of gene conversion in some Canarian Pimelia (Pons et al., 2002a) suggests that recombination is involved in concerted evolution of the repeats. In addition, the other 9 species of this genus have major satellites belonging to families with larger monomer lengths (500-700 bp). Altogether, within the genus Pimelia there !
are seven different satellite DNA families, whose sequence repeats are clearly related and have probably evolved from an ancestor sequence through sequence rearrangements and nucleotide substitutions (Pons et al. submitted). Most of the major species-specific satellites of Pimelia species can be, in some cases, found in low copy number in other Pimelia taxa as predicted in the library hypothesis (Salser et al., 1977, Mestrovic et al., 1998). Low copy number sequences are evolving at same rate than the major stDNA sequences, indicating that sequence divergence and concerted evolution of satellite sequences is not directly related to the copy number in Pimelia beetles (Bruvo et al., submitted). All Pimelia sequence units show curvature due to the presence of higher frequency of phased A or T tracts ? 3 which have been related with the heterochromatin condensation in mouse (Martinez-Balbas et al., 1990).
During my postdoc in Rosie Gillespie’s lab in UC Berkeley my work concentrated on the satellite DNA from the genus Tetragnatha (Aranae: Tetragnathidae). Satellite DNAs of 13 endemic Hawaiian Tetragnatha species are very divergent in nucleotide sequence (up to 45 %) though conserve particular homology in some stretches. On the other hand, the length (190-183 bp) and the nucleotide composition (60-55 % A-T rich) are remarkably conserved. The results suggest a common origin and a selective constraint in the length and nucleotide compositions of these repeats but not in their nucleotide sequence (Pons & Gillespie, submitted). Some authors have suggested that natural selection has an important role on the repeats (Stephan & Cho, 1994). This subtle action does not work on sequence per se, but works on certain trait of the sequence (e.g. sequence length) that could be crucial in their function. Surprisingly, the narrow range of repeat length found for Tetragnatha repeats corresponds closely to the range of nucleosomal unit length. Phylogenetic analysis based on sequence divergence of the repeats clusters the species in accordance with the mitochondrial phylogeny and the geological history of the Hawaiian Islands (Pons et al, unpublished).
At the present, I am working in Dr. Alfried Vogler’s laboratory in the Natural History Museum. The main goal of our project is to generate new phylogenetic markers based on ESTs libraries to build the world-wide phylogeny of the Cicindela (Coleoptera: Cicindelidae). The dating of the nodes will be used to estimate and compare the speciation rate in different Cicindela lineages. As result of this project we have submitted a manuscript about the world-wide phylogeny of Cicindela s. l. group (Coleoptera: Cicindelidae) based on a new phylogenetic marker, the muscle specific protein 20 (homologous to Drosophila melanogaster Mp20 protein).
The genus Cicindela (Coleoptera: Cicindelidae) is a species-rich cosmopolitan group of tiger beetles used as model for inter-continental comparisons of lineage diversification. Basal relationships of continental groups are important for this analysis but have been difficult to resolve with standard mtDNA markers. Here we used the mp20 gene, a single copy nuclear marker coding for a muscle associated protein in insects, for phylogenetics of basal groups in Cicindela. Nearly-full length sequences were obtained for 51 cicindelids, including major taxonomic groups from all continents, and resulted in sequences of minimally 1.2 kb and spanning three introns. Phylogenetic signal of exon and intron sequences was compared with that from four gene regions (2.4 kb total) of mtDNA. Mp20 sequences contributed two thirds of the total support of the combined analysis, with most signal from the introns. However, sequence alignment in introns presented a challenge, and minor variation of parameter values introduced great changes in tree topology. We conclude that basal clades of Cicindela are largely coincident with major biogeographic regions, with major clades confined, respectively, to Australasia, the Holarctic, the Indian subcontinent and Africa, and to South and Central America. Mp20 exon and intron sequences also contributed greatly to estimating divergence times based on a molecular clock. The inferred dates suggested that basal splits of continental lineages are younger than would be expected from continental break-up, with most of the species diversity in Cicindela accumulating only over the past 20-30 mya.
In late 2001, I moved to the Natural History Museum in London with the main goal to generate new molecular markers to resolve phylogenetic relationships in beetles at genus and family level in which classical mitochondrial and nuclear markers generally show little resolution. We constructed several ESTs libraries from different beetle families to design primers to amplify the selected genes. As result of this project we are finishing a manuscript about the world-wide phylogeny of Cicindela sensu lato group (Coleoptera: Cicindelidae) based on a new molecular marker, the muscle specific protein 20 (homologous to Drosophila melanogaster Mp20 protein).
I am still working on island radiation (and continental as well) but recently my research interests shifted to genes involved in speciation, and the use of short mitochondrial DNA sequences ('barcodes') to delimited species boundaries. For further details about a project about genes involved in speciation please see the attached file entitled 'Genes&Speciation.doc'.
DNA barcoding and DNA taxonomy approach is the topic I like the most, and in which I have been more involved in recent months. In fact, biodiversity profiling of large communities using a barcoding and DNA taxonomy approaches will be the topic selected for my future application to the Ramon y Cajal Fellowships. The results of two small projects on beetles, developed in the Natural History Museum working with Dr Alfried Vogler, have been already submitted: one on species delimitation using bardoces alone in an endemic Australian radiation of the genus Rivacindela, and another on biodiversity assessment of Copelatus radiation from Fiji. Recently, our group has been granted with three large projects on seven insect orders of Madagascar, the water beetles from Europe, and Scarabaeidae from South America involving the sequencing of more than 20000 specimens.
Why a barcoding approach?
At the beginning of 21st century the biodiversity of many areas of the Earth is still known inadequately. The high levels of ecosystem endangerment and destruction, particularly in the 42 biodiversity hot spots catalogued by the UNESCO, has prompted urgent calls for new improved systems of inventorying biodiversity and disseminating the information to conservation planners. In recent years a new taxonomic approach called 'DNA barcoding' has been proposed to aiding determining species. This method uses a short DNA sequence, generally the mitochondrial cox1 gene, rather than morphological characters. The early successes of DNA ‘barcoding’ (Hebert et al., 2003a, b; Hebert et al., 2004) suggest that large-scale surveys of DNA variation would accelerate studies on ecology, biodiversity and conservation planning of poorly studied ecosystems and groups of organisms. Recently, several museums, herbaria, universities, biodiversity inventory agencies, and commercial experts have created the international consortium Barcode of Life (CBOL). The use of DNA sequence difference for species recognition, assessment, and taxonomic description is not novel. Instead, the aim of this consortium is (i) promotion of community wide standards and protocols regarding choice of gene(s), electronic databasing, specimen collection data and vouchering and (ii) its explicit use as a system for rapid species recognition and identification in applied contexts outside of species inventory and taxonomy. These include taxonomic accuracy, low cost, ease of application in diverse contexts (including by non-specialists), portability, routine and immediate access to information, and utility across a broad phylogenetic and taxonomic spectrum of organisms, including many species new to science. Its core or essential function is the ability to correctly assign a given sample to individual named species, or to identify the sample as belonging to an unnamed (new) species. DNA barcoding is not intended to provide precise data regarding phylogenetic (evolutionary) relationships, although barcoding efforts are compatible with and ideally will complement phylogenetic studies. However, many scientists have raised concerns about taxonomy based on DNA data alone, viz.: (i) whether DNA variation in the targeted taxa is sufficient to discriminate closely related species, (ii) whether genetic groups can be directly translated into formal species because potentially complicated evolutionary history of gene trees may obscure species history (DNA taxonomy), and (iii) how to incorporate the taxonomic knowledgement built in the last three centuries in the new framework. I am particularly interested in study whether genetic variation of short mitochondrial sequences can delimit species boundaries in close related species.