1992-1998 Ph.D and Associate Professor contract
During my PhD (1992-1997) and Associate
Professor contract (1997-1998), I applied population genetics
and evolutionary biology concepts to the conservation of endangered
plant species of the cosmopolitan genus Limonium (sea lavender)
using different molecular markers. Resolving the phylogenetic
relationships among the species of the complex section Limonium,
which is mostly comprised of polyploid and asexual species,
was the second axis of my thesis project. Two students of
my supervisor carried out analyses using microsatellites and
quantitative characters in a second phase of my thesis project
and during my contract as Associate Professor. I helped these
students in their fieldwork, trained them in molecular biology
and helped to set up the new molecular methodologies in our
lab.
My training included several courses and workshops that broad
my knowledge in Genetics and Evolutionary Ecology. A post-grade
course in the use of databases and networks for molecular
biology and the use of Linux and related operating systems
and shells for some of my analysis have proven a great benefit
for my research career.
Comparative phylogenetic analysis of the genus
Limonium (Plumbaginaceae) using RFLP cpDNA and ITS sequencing.
Palacios et al. Molecular Phylogenetics and Evolution 14,
232-249 (2000)
The genus Limonium presents all
the main mechanisms of rapid speciation known in plants, i.e.
geographic and reproductive population isolation due to the
patchy distribution of species, as well as, the high frequency
of hybridization and polyploidy, and the possibility of reproduction
through apomixis. The special features of the genus and the
close morphology relationships exhibited by its species interfere
enormously in its taxonomic study specially when using morphological
characters. On theoretical grounds, molecular analysis could
overcome this drawback and offer robust hypothesis on the
evolution of Limonium species. Due to the reticular evolution
of the genus, both organular and nuclear molecular markers
were employed for this purpose. First, we analyzed RFLP in
the cpDNA using a genomic library that I previously developed
for Limonium narbonense. Second, I performed sequence analysis
of the nuclear ribosomal DNA cistron, which includes the ITS1,
the ITS2, and the 5.8 rRNA subunit. Most of the Limonium species
studied belong to section Limonium, which is the section of
the genus with the largest number of species and the one in
which most Mediterranean endemic species are classified. Our
conclusions from this study can only be discussed based on
the unique, non-phylogenetic classification of the genus.
We used distance, maximum-likelihood and parsimony analyses
to determine the phylogenetic affiliations of the different
species. All the phylogenies derived from both, the nuclear
and the chloroplast genomes are in disagreement with previous
classifications based on morphological characteristics. For
instance, two species, L. narbonense and L. vulgare, classified
within section Limonium show levels of divergence similar
to species from other sections in which the genus is subdivided.
The rest of the species are monophyletic. The low levels of
divergence among them using both markers is consistent with
a recent origin for this group in congruence with the high
speciation rates expected for this genus. Nevertheless the
endangered populations we were interested in (see below) rendered
a clear species status appearing differentiated from other
taxa as expected.
Population genetics studies in the endangered species Limonium
doufourii and Limonium cavanillesii
Palacios et al. Molecular Ecology 6(7): 671-675 (1997)
Palacios et al. Molecular Ecology 6(12): 1107-1122 (1997)
Palacios et al. Journal of Heredity 90(4): 485-489 (1999)
Palacios et al. Molecular Ecology 8(4): 654-657 (1999)
Palop et al. Conservation Genetics 0: 1-3
Rodriguez et al. Conservation Genetics 4: 386-391 (2003)
The fragility of natural ecosystems where Limonium species
inhabit has lead to the nearly extinction of several endemic
species, especially those situated in the Mediterranean basin
where tourism and agriculture activities have damaged these
habitats enormously. Two of the most endangered species of
the genus are L. dufourii and L. cavanillesii. We analyzed
their intraspecific variability and population genetic structure
using two DNA fingerprinting PCR based markers: AFLP and RAPDs.
Limonium cavanillesii is an extremely endangered
species from which only one natural population remains on
our Mediterranean coasts. It is triploid and apomictic. The
analysis of genetic variation using RAPDs revealed no polymorphic
markers. As an additional effort to find variability we used
AFLP technique. Only 11, very closely related, phenotypes
were found. The low variability levels in this species confirm
that the population has suffered from a recent and severe
bottleneck. However, its distribution of pairwise differences
was typical of a population in equilibrium in the past. Future
management measures taken on the species should consider the
variability encountered, for instance for the establishment
of new self-sustained natural populations and in reintroduction
experiments.
Limonium dufourii is also triploid and apomictic.
The remaining six populations from this species are distributed
along the Valencia and Castellón provinces. Most of
these populations are at the verge of extinction. To establish
the relationships among the different AFLP and RAPD patterns,
multivariate statistical analyses and minimum spanning networks
were employed. All of them demonstrated that these relationships
are not coincident with the actual population subdivision.
To explain this result, the analysis of nucleon diversity
allowed us to infer the historical demographic pattern of
each population and to establish a hypothesis on the evolution
of the species. Patterns of population genetic structure determined
from AMOVA and HOMOVA analyses were extremely useful for the
establishment of ex situ and in situ conservation management
measures necessary for on each population.
A study of several quantitative traits in L. dufourii
lead us to determine that the two markedly different genetic
haplotypes defined by RAPD and AFLP markers above were significantly
correlated with morphometric variation. The study of quantitative
genetic traits is of great importance in conservation as it
can be directly related to the fitness and survival of the
population. Consequently, more informed decisions on the management
of this species could be adopted.
DNA fingerprinting techniques provide us with genetic markers
that have a dominant nature. The drawbacks in the analysis
treatment of this kind of markers lead us to develop microsatellites
in the genus Limonium as an alternative codominant molecular
marker. We have demonstrated the validity of these microsatellite
markers already developed across Limonium species and their
use as heterologous SSRs. Comparison of results from both
types of markers will help to determine if the conclusions
extracted from DNA fingerprinting methods are biased due to
their dominant nature. In this case, the known advantages
of these markers might be counter-acted by the lower precision
of their results.
Postdoctoral training
During the first two postdoctoral research
contracts I acquired knowledge in the coevolution of organisms
associations (host-symbionts), comparative genome evolution,
as well as, patterns and processes in protein evolution. Through
these studies I became aware of the importance of informatics
to analyze the enormous amount of information that new molecular
technologies can now provide us with. Consequently, during
my postdoctoral contract at the Josephine Bay Paul Center
for Comparative Molecular Biology and Evolution (JBPC) of
the Marine Biological Laboratory (MBL, Woods Hole, MA, USA),
I learned to program in Perl and participated at the Workshop
in Molecular Biology and Evolution as well as other relevant
biocomputational analysis sessions on both, DNA and protein
expression results. Improving my skills in bioinformatics
along with deepening my knowledge in the analysis of nucleic
acids and protein sequences was greatly facilitated through
the excellent bioinformatics platform at the JBPC. I further
developed experience in high-throughput sequencing analysis
through the Tinto River NASA Astrobiology project, an ecosystem
model I have been working on since then. The integration of
my evolutionary ecological knowledge to an in-depth study
of bacterial community composition in parallel with environmental
variables from the Tinto River has demonstrated a tremendous
potential for understanding the evolutionary processes behind
the patterns of biodiversity encountered in this natural populations.
During my postdoctoral positions at the Max Planck Institute
of Marine Biology in Bremen (Germany) and at the Observatoire
Océanologique de Banyuls I continue studying microbial
populations diversity and functions at another extreme environment,
sunken woods in marine waters. During this postdoctoral research
I gained experience in methods to fingerprint microbial communities,
and improved my skills on 16S rRNA gene (rDNA) high-throughput
sequencing analysis and phylogenetics and on linking biodiversity
to ecosystem functioning through multivariate analysis methods.
Finally, my research at the UMR 5244 CNRS-University of Perpignan
Via Domitia-EPHE (UPVD) aims at the study of the causes of
schistosomiasis sickness re-emergence where it was eradicated.
Schistosomiasis is caused by the parasite Schistosoma
mansoni. We are studying the biodiversity of natural
populations of this species from different hosts populations
to unravel the factors underneath the population genetic structures
observed, which are key to understanding from which hosts
populations the sickness is reemerging. Through this research
I want to broad my knowledge in population genetics and ecological
analysis combining molecular and history trait markers.
Patterns and processes of genome evolution in Buchnera aphidicola,
the obligate endosymbiont of aphids
Van Ham R. et al. PNAS 100(2): 581-586 (2003)
Palacios et al. Mol. Biol. Evol. 19(9): 1575-1584 (2002)
From 1998 to 2000, I joined the just created Centro de Astrobiología
(CAB, INTA-CSIC, Madrid, Spain) to sequence the genome of
the endosymbiont of aphids, Buchnera sp. The aim of this project
was to gain insights on the minimal organization necessary
for life. We sequenced the genome of the intracellular symbiont
Buchnera aphidicola from the aphid Baizongia pistacea. This
strain diverged 80–150 million years ago from the common
ancestor of two previously sequenced Buchnera strains. As
a consequence of using a field-collected, nonclonal sample
of insects as source material our genome assembly unveiled
1,200 polymorphic sites demonstrating intrapopulational variation
in this species. Comparison of this genome with two other
Buchnera genomes revealed nearly perfect gene-order conservation,
indicating that the onset of genomic stasis coincided closely
with establishment of the symbiosis with aphids 200 million
years ago. Extensive genome reduction predates the coevolution
of Buchnera and its host. However, although at a slower rate,
gene loss continues among the extant Buchnera lineages indicating
degeneration rather than adaptation patterns of genome evolution.
Nevertheless, through the comparative analysis of genome-wide
patterns of amino-acid usage of this obligate endosymbiont
with the close free-living relative Escherichia coli, we were
able to detect that selection differentially affects amino
acid content of proteins with different expression and hydropathy
levels. Thus, I brought my knowledge in evolutionary ecology
and my expertise in multivariate statistical methods to better
understand the evolution of the genome of the Buchnera species
already sequenced when I joined the JBPC in my first postdoctoral
position at the USA.
Development and application of a new method for high-throughput
analysis of microbial community composition, Serial Analysis
of V6 Ribosomal Sequence Tags (SARST-V6)
Selected publications:
Kysela et al. Environmental Microbiology 7: 356-364 (2005)
Ward et al. International Journal of Astrobiology Supplement
1:77 (2004)
Palacios et al. BSH/ VLIZ Special Publication No. 37 pp. 145-150
(2007)
Palacios et al. (submitted)
Amaral-Zettler et al. (in preparation)
In a second phase of my postdoctoral
research I aimed to reorient my career towards ecological
diversity and evolution at both the DNA and protein levels.
I got introduced into the Sogin's Lab by helping to build
a microarray of the organism Microcoleus chthonoplastes that
served to understand how genes are regulated in this prevalent
component of hypersaline microbial mats. In this side project
I gained some knowledge in protein annotation, database construction
and bioinformatics. But my main initial project was to explore
new technologies for unraveling the microbial population structure
of natural environments. Our efforts resulted in a novel DNA
sequence-based technique named Serial Analysis of Ribosomal
Sequence Tags (SARST-V6). The method exploits the same principle
of SAGE (Serial Analysis of Gene Expression) but through the
concatemerization of the small (50-150bp), yet phylogenetically
informative, V6, hypervariable region of the rRNA gene. The
application of this technology to sediments of the Guaymas
basin hydrothermal vents revealed a microbial community composition
that resemble results from full-length sequences except for
some differences that could be attributed to primer biased.
Our results lead us to conclude that SARST-V6 allows for efficient
and exhaustive sampling of the biodiversity of natural microbial
communities. From 2003 until date, I have been studying the
ecological evolution of the extreme Tinto River (Figure 1).
An in depth analysis of bacterial community composition of
this ecological model using SARST-V6 in concomitance with
appropriate ecological sampling, physico-chemical parameters
measurements and multivariate statistical analysis tools has
revealed new patterns of bacterial population structure previously
hidden to microbiologists probably due to technical limitations.
These patterns allows us to infer the importance of evolutionary
processes like mutation and dispersion in microbial community
dynamics and biogeography, which conform to the hypothesis
of global distribution of ecotypes.
Until now, molecular surveys of the Tinto River have been
restricted to domain-level investigations of prokaryotes or
eukaryotes but combined studies have not been undertaken.
SARST-V6 in combination with traditional full-length rRNA
gene sequencing of novel taxa is being used to explore the
river’s diversity at the three-domain level of life.
Through the study of temporal and spatial changes in microbial
community diversity along with information on physical and
chemical data we are acquiring further knowledge on how geochemical
forces shape community structure in this ecosystem model.
Microbial ecological diversity and functions of
sunken woods in marine environments
Palacios et al. Cahiers de Biologie Marine 47: 415-420 (2006)
Pailleret et al. C.R. Palevol 6:463-468 (2007)
Palacios et al. (submitted)
Ghiglione et al. (in preparation)
In the marine environment, large
organic falls like wood, kelp and whale falls are very interesting
habitats from an evolutionary point of view. Degradation occurs
by specific microbes and animal communities adapted to the
use of this high carbon content materials as substrates. The
phylogenetic similarity between metazoans inhabiting sunken
woods and whale bones with those of other highly reduced environments
like hydrothermal vents and cold seeps suggests that these
environments could act as stepping-stones for the adaptation,
evolution and dispersion of marine chemosynthetic communities.
However, there are no studies that explore the free-living
microbial component of sunken wood ecosystems. Furthermore,
the occurrence of anaerobic microbial communities thriving
on wood falls remains to be demonstrated. In 2005 I started
to explore the microbiology of these habitats at the postdoctoral
position in the Max Planck Institute for Marine Microbiology
(Bremen, Germany). I started by developing a series of methods
to quantitatively study microbial diversity and degradation
processes in sunken woods, from counting bacteria attached
to wood chips to study their cellulolytic activities and for
microbial in situ detection using FISH and CARD-FISH. At present
I have a Marie Curie postdoctoral fellowship to continue my
research in sunken woods at the Observatoire Oceanologique
de Banyuls (France). We have used culture-independent methods
to study the microbial communities on both, artificially immersed
and natural wood samples, located at geographically isolated
coastal and deep-sea environments. Capillary Electrophoresis
Single Strand Conformation Polymophism (CE-SSCP) of the rDNA
has helped to fingerprint the Archaea and Bacteria communities
living on and within the woods to determine their resemblance
among samples. Cloning and sequencing of the rDNA in target
samples is giving clues on the actual diversity and phylogenetic
resemblance of the main metabolic players responsible of energy
and carbon flows. Our results broad our narrow knowledge on
the microbial biofilms that develop around woods in marine
environments and give insights on the ecological importance
of these organic island deposits for the dispersion of chemosynthetic
communities at the ocean basin. Through this postdocs I increased
my knowledge in state of the art high-throughput sequencing
analysis methods like automatic alignment and Bayesian methods
of phylogenetic analysis, as well as, further developed my
skills in the ecological analysis of populations using multivariate
methods.
Genetic structure of Schistosoma mansoni
natural populations: the Oman Republic as an example of schistosomiasis
reemergence
The main aim of my research at the UPVD is to determine the
causes of reemergence of the human sickness schistosomiasis
caused by the parasite Schistosoma mansoni. The Oman Republic
is a good study ground. The sickness was declared eradicated
in this country 15 years ago but the increasing number of
cases detected after year 2000 clearly points towards its
reemergence. The most plausible hypotheses to explain the
reemergence of schistosomiasis is the existence of reservoir
host populations among rats and humans that are not regularly
screened for having the parasite. Individual parasites from
these reservoir hosts have been found different in their chronobiology
but identical in their genetic diversity when using the cytochrome
oxydase I gene (coxI) as a marker. This indicates that all
parasites are Schistosoma mansoni and not other species in
spite of the difference in the history trait marker. Other
mitochondrial (cox3, nad4, and nad5) and nuclear (microsatellites
already developed for this species) molecular markers more
divergent than coxI exist. We are now studying the variability
of these markers to find out which ones can be used as diagnostic
to differentiate parasites from the different hosts. Once
diagnostic markers are identified we will study their variability
in natural parasite populations from different reservoir hosts.
The genetic structure analyses will serve to understand the
gene flow among these populations and whether or not the genetic
differentiation coincides with host adaptations. Understanding
these ecological and evolutionary demographic factors underneath
the variation observed would help to determine which hosts
populations are responsible of schistosomiasis reemergence
in the Oman Republic. This work could then serve as a model
to test the causes of schistosomiasis reemergence worldwide.
Dr. Carmen Palacios de la Cruz
@ Winter 2008