Helena Cotrim

Comparative structure of the labellum in Ophrys fusca and O. lutea (Orchidaceae)

The morphology and anatomy of the labellar epidermal cells and the way in which they are arranged are described in an attempt to locate and characterize the osmophore in Ophrys fusca and O. lutea. The micromorphology of the labellum of these two species is similar. Four types of epidermal cells are present on the adaxial surface of the labellum. Long unicellular trichomes with straight tips cover the basal region of the labellum, whereas short unicellular trichomes with polygonal flattened bases form the reflective median speculum. The apical region of the labellum possesses a villous indumentum of long acuminate trichomes with bent or sinuate tips. Large smooth-walled, dome-shaped papillae occur on the margins and on the distal region of the abaxial surface of the labellum. These remarkable papillae have high polarity; the protoplasm at the apex of each cell contains several small vacuoles, while a prominent nucleus surrounded by numerous hypertrophied amyloplasts occurs at the opposite end of the cell. Positive reactions to Vogels staining test and to Sudan black B enabled us to conclude that the osmophores of both species are composed of these peculiar secretory epidermal cells and by two or three subsecretory layers of parenchyma cells.

Genetic diversity of three endangered species of Echium L. (Boraginaceae) endemic to Cape Verde Islands

Echium hypertropicum, E. stenosiphon and E. vulcanorum are the three endemics representative of the genus Echium (Boraginaceae) in Cape Verde archipelago. The aim of this study is to provide a first attempt at estimating genetic diversity among natural populations of these endangered Echium species based on RAPD, so as to provide data available for future appropriate strategies for their conservation. PCO and UPGMA of RAPD analysis suggests a close genetic proximity between the Southern endemic species (E. hypertropicum and E. vulcanorum) and shows that the levels of polymorphism strongly differ between these two Echium species (27 and 29% respectively) and E. stenosiphon (74%), the Northern endemic species. Mantel test also corroborates a close genetic proximity between genetic and geographic data. Population genetic analysis of E. stenosiphon revealed low levels of gene flow between islands (Nm = 0.32) being S. Nicolau the most isolated as evident in PCO. Furthermore the differentiation between groups of individuals belonging to putative subspecies was tested by AMOVA. According to our results there is no genetic basis to consider the two subspecies of E. stenosiphon namely ssp. stenosiphon and ssp. lindbergii. Our results enable us to suggest that E. stenosiphon must be ranked as a Threatened species. Measures aiming at conservation of E. hypertropicum and E. vulcanorum must be implemented at short-term taking into account the small number of existing plants and its low genetic variability.

Silene rothmaleri P. Silva (Caryophyllaceae), a rare, fragmented but genetically diverse species

Silene rothmaleri is an endemic Portuguese species considered extinct until 1992, when it was rediscovered in the wild with a highly fragmented distribution. These rare plants occur along the southwestern Portuguese coast in small populations, which in addition to phenological differences that occur along the north–south gradient could create a pattern of genetic isolation. To evaluate the degree of genetic diversity and estimate the relationship between population fragmentation and genetic variability, we analysed the five known populations of S. rothmaleri using random amplified polymorphic DNA. Degree of polymorphism and Shannon Index of phenotypic diversity revealed high levels of diversity, found mainly within populations. PCo and cluster analysis revealed a distinct north–south cline, which was confirmed by spatial autocorrelation (Mantel) analysis. This indicates the existence of gene flow between small nearby populations and its insufficiency between widely separated populations. Levels of gene flow (Nm) estimated from the Shannon Index reveal a pattern consistent with a larger past distribution that went through a period of contraction and lack of gene flow followed by population differentiation. The central and largest population probably acts as a core of genetic variability inherited as a relict from a larger and more diverse ancestral population.

Labellum transcriptome reveals alkene biosynthetic genes involved in orchid sexual deception and pollination-induced senescence

One of the most remarkable pollination strategy in orchids biology is pollination by sexual deception, in which the modified petal labellum lures pollinators by mimicking the chemical (e.g. sex pheromones), visual (e.g. colour and shape/size) and tactile (e.g. labellum trichomes) cues of the receptive female insect species. The present study aimed to characterize the transcriptional changes occurring after pollination in the labellum of a sexually deceptive orchid (Ophrys fusca Link) in order to identify genes involved on signals responsible for pollinator attraction, the major goal of floral tissues. Novel information on alterations in the orchid petal labellum gene expression occurring after pollination demonstrates a reduction in the expression of alkene biosynthetic genes using O. fusca Link as the species under study. Petal labellum transcriptional analysis revealed downregulation of transcripts involved in both pigment machinery and scent compounds, acting as visual and olfactory cues, respectively, important in sexual mimicry. Regulation of petal labellum senescence was revealed by transcripts related to macromolecules breakdown, protein synthesis and remobilization of nutrients.

Isolation and characterization of novel polymorphic nuclear microsatellite markers from Ophrys fusca (Orchidaceae) and cross-species amplification

The present study reports the isolation and characterization of eight new polymorphic microsatellite loci from the sexually deceptive orchid Ophrys fusca. Microsatellites were isolated from two partially enriched genomic libraries using FIASCO (Fast Isolation by AFLP of Sequences COntaining repeats). Seventy-three loci were screened for primer design and primer pairs corresponding to eight different loci were selected for microsatellite characterization of two Portuguese populations. Total number of alleles per locus ranged from 10 to 32. All loci showed a high level of observed heterozygosity (H0) ranging from 0.33 to 1 and were possible to amplify in 16 other species of Ophrys using the same primers.

Marked hybridization and introgression in Ophrys sect. Pseudophrys in the western Iberian Peninsula

PREMISE OF THE STUDY Orchids in the genus Ophrys represent extraordinary cases of tight coevolution between plants and their pollinators, and as a result, they present opportunities for studying hybridization, or a lack thereof, during speciation. However, few studies assess the real effect of hybridization in diversification. The three most representative species of section Pseudophrys in the western Iberian Peninsula-O. dyris, O. fusca, and O. lutea-were chosen to study evolutionary relationships and examine speciation. METHODS Using eight specific nuclear microsatellite loci, 357 individuals from 28 locations were studied; 142 of these samples were also studied with four plastid microsatellite loci. Data were analyzed using Bayesian cluster analysis, a median-joint network, and multivariate analysis. KEY RESULTS Many O. dyris and O. fusca specimens had three or four alleles and were therefore treated as tetraploid. Ophrys dyris is poorly genetically separated from O. fusca, and pure populations are rare. Ophrys fusca and O. lutea are distinct, but hybrids/introgressed individuals were detected in most of the populations and supported by plastid haplotypes. Ophrys fusca is subdivided into three well-delimited genetic lineages with a strict geographic correspondence confirmed by plastid haplotypes. CONCLUSIONS Because postzygotic barriers are weak, leakage in this highly specialized orchid-pollinator system contributes to hybridization and introgression. These leakages may have occurred during periods of past climate change, promoting homogenization and the potential for generations of new biodiversity via production of novel genotypes/phenotypes interacting with pollinators.

Metal hyperaccumulation patterns within Plantago phylogeny (Plantaginaceae)

AimsWe aimed to evaluate the presence of the metal hyperaccumulation trait in the phylogeny of Plantago with emphasis on P. subgen. Coronopus. Considering that P. almogravensis is an Al-hyperaccumulator and that phylogenetic links have been described for other Al-hyperaccumulators, we hypothesized that we would find more Plantago taxa with this accumulation trait. We also enquired if other metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn), follow a similar accumulation pattern as Al.MethodsWe analysed metal accumulation data from direct (analyses) and indirect sources (bibliographic) combined with phylogenetic analysis.ResultsRegarding metal accumulation, our results indicate a high probability of hyperaccumulation in Plantago species (nine out of 15 species), namely for the metals Al (6–8), Cu (1), Zn (2–3) and Pb (1). There are also records of Fe (5) considered abnormally high. For P. subgen. Coronopus, hyperaccumulation was perceived in six out of seven species. In particular for Al-hyperaccumulation, a high frequency and dispersion among Plantago sub-ranks is compatible with a phylogenetic relation.ConclusionsOur work suggests that the Al-hyperaccumulation trait is common in Plantago, especially in P. subgen. Coronopus, and contributes to the scarce knowledge of that uncommon trait, namely in Mediterranean plants.