> Research career


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