> Thesis




Doctoral Thesis


A case study in the genus Limonium (Plumbaginaceae)

  Was presented in Valencia in December1997 in front of the following jury members:

Andrés Moya Simarro

Vernor Heywood

Marcelino Pérez de la Vega

Josep A. Roselló i Picornell

Julio Rozas Uras

And supervised by:

Fernando González Candelas

"Clasification makes organic diversity accesible to the other biological disciplines. Without it, most of them would be unable to give meaning to their findings."
Mayr & Ashlock
"Todas las plantas deberían de considerarse recursos fitogenéticos...Y quienes trabajamos con plantas en peligro, es bueno que lo hagamos con la conciencia de que nuestra actividad no es algo romántico o quijotesco, sino que con ella está directamente imbricado el futuro de la Humanidad".
César Gómez Campo


    The main aim of my thesis was the study of natural populations from an ecological and evolutionary point of view with a final application to biodiversity management and its conservation. The integration of ecology and evolutionary tools has allowed the use of molecular markers for phylogenetics and population genetics analyses of variability, which is essential for the conservation of biodiversity. Some authorities in this matter now recognize that most of the questions currently resolved in this context could have not been answered without the use of molecular markers. As model organism we chose the genus Limonium (sea lavander). This genus is particularly interesting because it presents all the main mechanisms of rapid speciation known in plants, i.e. geographic and reproductive population isolation due to the patchy distribution of the natural ecosystems its species inhabit as well as the high frequency of hybridization and poliploidy, and the possibility of reproduction through apomixis. These special features and the close morphological relationship exhibited by the species of this genus interfere enormously in its taxonomic study specially when using morphological characters. On theoretical grounds molecular analysis could bypass 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, I analyzed RFLP in the chloroplast DNA using a genomic library that I developed previously for Limonium narbonense. Secondly, I performed ITS region sequence analysis of the nuclear rDNA cistron, which includes the ITS1, the ITS2, and the 5.8 subunit, was performed. Most of the Limonium species studied belong to section Limonium, this is the section of the genus with the larger number of species and the one in which most Mediterranean endemisms are classified. Our conclusions from this study could only be discussed based on the unique, morphological classification of the genus that existed so far. We used several methods for the molecular data analyses. All the phylogenies derived from both, the nuclear and the chloroplast genomes are in disagreement with this previous morphological classification. 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 level of divergence among these later species with both molecular data is consistent with a recent origin of this group.
The fragility of natural ecosystems where Limonium species inhabit has lead to the nearly extinction of several endemisms, 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 (now known as L. perplexum) We analysed 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 on each population.


Phylogenetic studies:

-plasmidic and phagic genomic libraries


-ITS sequencing

Population studies:




Phylogenetic reconstruction methods

-parsimony analysis

-distance analysis

Population variability and structure methods

-minimum spanning network and multivariant analysis

-nucleon diversity analysis

-AMOVA and HOMOVA analysis


    Carmen Palacios, Josep A. Rosselló & Fernando González-Candelas

A comparative phylogenetic analysis of the genus Limonium (Plumbaginaceae) using RFLP cpDNA and ITS sequencing.

Molecular Phylogenetics and Evolution 14, 232-249 (2000)

    Carmen Palacios & Fernando González-Candelas

Lack of genetic Variability in the rare and endangered plant species Limoniumcavanillesii using RAPD markers

Molecular Ecology 6, 671-675 (1997)

    Carmen Palacios & Fernando González-Candelas

AFLP analysis of the critically endangered plant Limonium cavanillesii and comparison with RAPD analysis

Journal of Heredity 90(4), 485-489 (1999)

    Carmen Palacios y Fernando González-Candelas

Analysis of population genetic structure and variability in the endemic and endangered plant species Limoniumdufourii (Plumbaginaceae)

Molecular Ecology 6, 1107-1122 (1997)


    The results obtained through the study of intra and interspecific variability in the different Limonium species by using empirical and conceptual tools from the Population Genetics and Phylogenetics, has allowed us to draw several conclusions, not only from micro and macroevolutionary patterns and processes of these species, but also for the management of the populations of the most endangered species in the Comunidad Valenciana, Limonium cavanillesii and L. dufourii.

    - It has been demonstrated the need of an adequate sampling strategy to detect intraspecific variability in molecular phylogenetic studies, especially when there is evidence of hybridization and/or introgression, and cytoplasmic DNA or repetitive gene families are used as markers.

    - The different rDNA and cpDNA types detected in L. delicatulum, L. interjectum and L. furfuraceum are paraphyletic, with divergence levels similar to those of other species from section Limonium. This result reinforces the introgression and/or hybridization hypotheses for the origin of these species.

    - In all phylogenies obtained L. narbonense and L. vulgare, from subsection Genuinae, form a sister clade (monophyletic group) to the rest of the species from section Limonium, with levels of divergence similar or even higher than those of species from other sections of the genus. Consequently, the actual placement of L. narbonense and L. vulgare within section Limonium is questioned.

    - The remaining species from section Limonium always form a monophyletic group in all the topologies resulting from phylogenetic analyses. The low levels of divergence encountered at this level imply a recent origin of these species. In general, there is a lack of resolution within this clade that could be due to the reticular evolution of these highly related species. This fact is confirmed by the lack of concordance between cpDNA and ITS phylogenies.

    - It has been demonstrated the usefulness of comparing the results obtained from cytoplasmic and nuclear markers. Likewise, the use of different methods of phylogenetic analysis has demonstrated to be very helpful to interpret the results obtained and to extract more reliable conclusions about the evolution of these genomes.

    - Besides, in those species in which a more exhaustive analysis of intraspecific variability has been performed, the use of appropriate analytical methods to determine the intraspecific relationships and the population structure has been essential to extract as much information as possible from the molecular data, in spite of the restrictions imposed by dominant markers. It is noteworthy that dicotomic trees are very strict representations of the evolutionary relationships among the phenotypes or populations studied. Other type of representations, such as MSN (minimum spanning networks), have demonstrated to be more adequate, as it is possible to extract more information from the same data.

    - The low levels of variability encountered in the species L. cavanillesii could be due to its obligate apomictic reproductive system. However, only one population with very low numbers has been discovered in nature, which implies that it has passed through a severe bottleneck, after which there has been no chance for mutation to restore the levels of genetic variability expected in a population that could have been in equilibrium in the past, as pointed by the analysis of nucleon diversity.

    - Conservation of this unique L. cavanillesii population must be an urgent measure, as its low genetic variability makes it very sensible to environmental changes. The best strategy might be the protection of the area where it is currently established, declared as a small reserve, and the re-establishment of new self-sustaining populations in suitable habitats. Micropropagation of a representative sample of the variability encountered would be the most appropriate ex situ conservation measure. Finally, we believe that L. cavanillesii should be catalogued as critically endangered according to the IUCN categories of threat.

    - The study of intraspecific variability in the species L. dufourii has demonstrated that there are two divergent phenotype groups (A and B) within Marjal del Moro populations. Moreover, phenotypes that belong to other populations present a higher similarity to those from group B, which points to a common origin of these phenotypes and its posterior dispersion, colonizing suitable habitats north and southwards. But divergence levels among phenotypes from groups A and B are within those expected for intraspecific studies.

    - The high level of population structuring within L. dufourii makes it advisable to prevent transplantation of individuals or seeds from one population to another, with the possible exception of El Saler population, with only 7 individuals, in which reintroduction of new variability from group B individuals of Marjal del Moro populations is recommendable. In situ measures of conservation for the different populations should be complemented by ex situ measures, which must take into account the diversity and population subdivision encountered in the species. L. dufourii should be catalogued as endangered according to the IUCN categories of threat.

    - Comparative results of detectable variability levels with different molecular markers, as AFLPs and RAPDs, can not be generalized even for evolutively close species. However, when PCR based DNA fingerprinting methods are the best choice in a particular study, AFLP method represents the most advantageous technique, if the necessary equipment and the economic resources are available.