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 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'.
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.