Pharmacological activities of some Argyranthemum species growing in the Canary Islands.

Methanolic extracts of eight subspecies of genus Argyranthemum were evaluated against brine shrimps, human cancer cell lines, malarial parasites and microorganisms under in vitro conditions. In the shrimp assay, samples of A. adauctum ssp. adauctum, A. adauctum ssp. erythrocarpon and A. frutetescens ssp. succulentum were active with ED50 values in the range of around 300 to 360 microg/ml. In the Caco-2, HepG2 and MCF-7 cell lines, the samples A. adauctum ssp. jacobaeifolium and A. adauctum ssp. palmensis were active with LC50 values ranging between 80-90 microg/ml. The secondary assay results of antimalarial activity of samples, A. adauctum ssp. adauctum, A. adauctum ssp. dugourii, A. adauctum ssp. erythrocarpon and A. adauctum ssp. jacobaeifolium have IC50 values <50 microg/ml. The samples demonstrated broad specific antimicrobial activity against five different microorganisms.

Plant genetic diversity in the Canary Islands: a conservation perspective.

The Canary Islands are an Atlantic volcanic archipelago with a rich flora of ∼570 endemic species. The endemics represent ∼40% of the native flora of the islands, and ∼20% of the endemics are in the E (endangered) category of the International Union for Conservation of Nature. A review of allozyme variation in 69 endemic species belonging to 18 genera and eight families is presented. The average species-level genetic diversity (H(T)) at allozyme loci is 0.186, which is twice as high as the mean reported for endemics of Pacific archipelagos. Possible factors contributing to this higher diversity are discussed, but the reasons remain obscure. An average of 28% of the allozyme diversity within species resides among populations, indicating a high level of interpopulational differentiation. Studies of reproductive biology indicate that many of the endemic species are outcrossers. The high total diversity within species, the relatively high differentiation among populations, and the outcrossing breeding systems have implications for species conservation. Decreased population sizes in outcrossing species would promote biparental inbreeding and increase inbreeding depression. The relatively high proportion of allozyme diversity among populations indicates that the most effective strategy for preserving genetic variation in species is to conserve as many populations as possible. The genetic diversity in many Canary Island endemics is endangered by: (1) overgrazing by introduced animals, such as barbary sheep, goats, mouflons, rabbits, and sheep; (2) interspecific hybridization following habitat disturbance or planting of endemics along roadsides or in public gardens; (3) competition with alien plant species; and (4) decline of population size because of urban development and farming.

The complete external transcribed spacer of 18S-26S rDNA: amplification and phylogenetic utility at low taxonomic levels in asteraceae and closely allied families.

For molecular phylogenetic reconstruction of some intrageneric groups of plants, a DNA region is needed that evolves more rapidly than the internal transcribed spacer (ITS) of the 18S-26S nuclear ribosomal DNA (nrDNA) repeat. If the region identified is nuclear, it would also be desirable for it to undergo rapid concerted evolution to eliminate problems with coalescence. The external transcribed spacer (ETS) of the nrDNA repeat has shown promise for intrageneric phylogenetic reconstruction, but only the 3' end of the region has been utilized for phylogenetic reconstruction and "universal" primers for PCR amplification have been elusive. We present a method for reliably amplifying and sequencing the entire ETS throughout Asteraceae and some closely allied families. We also show that the ETS is more variable and phylogenetically informative than the ITS in three disparate genera of Asteraceae-Argyranthemum (tribe Anthemideae), Asteriscus (tribe Inuleae), and Helianthus (tribe Heliantheae). The full ETS was amplified using a primer (ETS1f) within the intergenic spacer in combination with a primer (18S-2L) in the 5' end of the highly conserved 18S gene. ETS1f was designed to correspond to a highly conserved region found in Helianthus and Crepis, which are in separate subfamilies of Asteraceae. ETS1f/18S-2L primed in all of the tribes of Asteraceae as well as exemplar taxa from Campanulaceae, Goodeniaceae, and Calyceraceae. For both Argyranthemum and Asteriscus, we were able to directly sequence the ETS PCR products when a single band was produced. When multiple bands were produced, we gel-purified and occasionally cloned the band of interest before sequencing. Although PCR produced single bands for Helianthus species, it was necessary to clone Helianthus amplifications prior to sequencing due to multiple intragenomic ETS repeat types. Alignment of ETS sequences for Argyranthemum and Asteriscus was straightforward and unambiguous despite some subrepeat structure in the 5' end. For Helianthus, different numbers of large tandem subrepeats in different species required analysis of the orthology of the subrepeats prior to alignment. In all three genera, the ETS provided more informative variation for phylogenetic reconstruction and allowed better resolution of relationships than the ITS. Although cloned sequences from Helianthus differed, intragenomic clones consistently formed clades. This result indicated that concerted evolution was proceeding rapidly enough in ETS that species-specific phylogenetic signal was retained. It should be now be possible to use the entire ETS for phylogenetic reconstruction of recently diverged lineages in Asteraceae and at least three other families (approximately 26,000 species or about 8% of all angiosperms).

Molecular evidence for multiple origins of woodiness and a new world biogeographic connection of the Macaronesian island endemic Pericallis (Asteraceae: senecioneae).

The prevalence of woody species in oceanic islands has attracted the attention of evolutionary biologists for more than a century. We used a phylogeny based on sequences of the internal-transcribed spacer region of nuclear ribosomal DNA to trace the evolution of woodiness in Pericallis (Asteraceae: Senecioneae), a genus endemic to the Macaronesian archipelagos of the Azores, Madeira, and Canaries. Our results show that woodiness in Pericallis originated independently at least twice in these islands, further weakening some previous hypotheses concerning the value of this character for tracing the continental ancestry of island endemics. The same data suggest that the origin of woodiness is correlated with ecological shifts from open to species-rich habitats and that the ancestor of Pericallis was an herbaceous species adapted to marginal habitats of the laurel forest. Our results also support Pericallis as closely related to New World genera of the tribe Senecioneae.

Internal transcribed spacer sequence phylogeny of Crambe L. (Brassicaceae): molecular data reveal two Old World disjunctions.

Crambe L. (Brassicaceae) is an Old World genus with a disjunct distribution among four major centers of species diversity. A phylogenetic analysis of nucleotide sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal repeat was conducted with 27 species of Crambe and 18 related genera. Cladistic analyses using weighted and unweighted parsimony support Crambe as a monophyletic genus with three major lineages. The first comprises those taxa endemic to the Macaronesian archipelagos. Taxa with a predominant Mediterranean distribution form the second assemblage, and a disjunction between east Africa (C. abyssinica) and the Mediterranean (C. hispanica) occurs in this clade. The third lineage includes all Eurosiberian-Asian taxa and C. kilimandscharica, a species from the highlands of east Africa. A basal biogeographic split between east Africa and Eurasia is present in the third clade. The patterns of relationships in the ITS tree are concordant with known climatic events in northern Africa and southwestern Asia since the middle Miocene. The ITS trees are congruent with the current sectional classification except for a few members of sections Crambe, Leptocrambe, and Orientecrambe (C. cordifolia, C. endentula, C. kilimandscharica, and C. kotschyana). Low levels of support in the basal branches do not allow resolution of which genera of the subtribes Raphaniae or Brassicinae are sister to Crambe. Both subtribes appear to be highly polyphyletic in the ITS trees.

Molecular evidence for a Mediterranean origin of the Macaronesian endemic genus Argyranthemum (Asteraceae).

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.

A common origin for woody Sonchus and five related genera in the Macaronesian islands: molecular evidence for extensive radiation.

Woody Sonchus and five related genera (Babcockia, Taeckholmia, Sventenia, Lactucosonchus, and Prenanthes) of the Macaronesian islands have been regarded as an outstanding example of adaptive radiation in angiosperms. Internal transcribed spacer region of the nuclear rDNA (ITS) sequences were used to demonstrate that, despite the extensive morphological and ecological diversity of the plants, the entire alliance in insular Macaronesia has a common origin. The sequence data place Lactucosonchus as sister group to the remainder of the alliance and also indicate that four related genera are in turn sister groups to subg. Dendrosonchus and Taeckholmia. This implies that the woody members of Sonchus were derived from an ancestor similar to allied genera now present on the Canary Islands. It is also evident that the alliance probably occurred in the Canary Islands during the late Miocene or early Pliocene. A rapid radiation of major lineages in the alliance is consistent with an unresolved polytomy near the base and low ITS sequence divergence. Increase of woodiness is concordant with other insular endemics and refutes the relictural nature of woody Sonchus in the Macaronesian islands.

Chloroplast DNA evidence of colonization, adaptive radiation, and hybridization in the evolution of the Macaronesian flora.

Most evolutionary studies of oceanic islands have focused on the Pacific Ocean. There are very few examples from the Atlantic archipelagos, especially Macaronesia, despite their unusual combination of features, including a close proximity to the continent, a broad range of geological ages, and a biota linked to a source area that existed in the Mediterranean basin before the late Tertiary. A chloroplast DNA (cpDNA) restriction site analysis of Argyranthemum (Asteraceae: Anthemideae), the largest endemic genus of plants of any volcanic archipelago in the Atlantic Ocean, was performed to examine patterns of plant evolution in Macaronesia. cpDNA data indicated that Argyranthemum is a monophyletic group that has speciated recently. The cpDNA tree showed a weak correlation with the current sectional classification and insular distribution. Two major cpDNA lineages were identified. One was restricted to northern archipelagos--e.g., Madeira, Desertas, and Selvagens--and the second comprised taxa endemic to the southern archipelago--e.g., the Canary Islands. The two major radiations identified in the Canaries are correlated with distinct ecological habitats; one is restricted to ecological zones under the influence of the northeastern trade winds and the other to regions that are not affected by these winds. The patterns of phylogenetic relationships in Argyranthemum indicate that interisland colonization between similar ecological zones is the main mechanism for establishing founder populations. This phenomenon, combined with rapid radiation into distinct ecological zones and interspecific hybridization, is the primary explanation for species diversification.

Numerical analyses of RAPD data highlight the origin of cultivated tagasaste (Chamaecytisus proliferus ssp. palmensis) in the Canary Islands.

Random Amplified Polymorphic DNA (RAPD) was used to generate molecular markers to trace the origin of the fodder legume tagasaste (Chamaecytisus proliferus (L. fil.) Link ssp. palmensis (H. Christ) Kunkel) in the Canary Islands. Results from multivariate analyses of data through "Two Way Indicator Species Analysis" (TWINSPAN) and "Detrended Correspondence Analysis" (DECORANA) showed that genotypes collected on the island of La Palma exhibited a wider range of variation than those from the other islands. This supports the existing hypothesis that tagasaste originated on La Palma and emphasizes the importance of conserving and evaluating germ plasm from this island.