Temporal and palaeoclimatic context of the evolution of insular woodiness in the Canary Islands.

Insular woodiness (IW), referring to the evolutionary transition from herbaceousness toward woodiness on islands, has arisen more than 30 times on the Canary Islands (Atlantic Ocean). One of the IW hypotheses suggests that drought has been a major driver of wood formation, but we do not know in which palaeoclimatic conditions the insular woody lineages originated. Therefore, we provided an updated review on the presence of IW on the Canaries, reviewed the palaeoclimate, and estimated the timing of origin of woodiness of 24 insular woody lineages that represent a large majority of the insular woody species diversity on the Canaries. Our single, broad-scale dating analysis shows that woodiness in 60%-65% of the insular woody lineages studied originated within the last 3.2 Myr, during which Mediterranean seasonality (yearly summer droughts) became established on the Canaries. Consequently, our results are consistent with palaeoclimatic aridification as a potential driver of woodiness in a considerable proportion of the insular woody Canary Island lineages. However, the observed pattern between insular woodiness and palaeodrought during the last couple of million years could potentially have emerged as a result of the typically young age of the native insular flora, characterized by a high turnover.

Molecular phylogenetics of Lotus (Leguminosae) with emphasis in the tempo and patterns of colonization in the Macaronesian region.

With a wide distribution range including Europe and Asia, Lotus (Leguminosae) represents the largest genus within Loteae. It is particularly diverse in the Mediterreanean region and in the five archipelagos of Macaronesia (Atlantic Ocean). However, little is known about the relationships among the 14 sections currently recognized within Lotus and about the timing and patterns of its colonization in the Macaronesian region. In this investigation, we use four DNA regions (nuclear ribosomal ITS plus three plastid regions) in the most comprehensive sampling of Lotus species to date (some endemic species within the Canary Islands were poorly represented in previous phylogenetic analyses) to infer relationships within this genus and to establish patterns of colonization in Macaronesia. Divergence time estimates and habitat reconstruction analyses indicate that Lotus likely diverged about 7.86 Ma from its sister group, but all colonization events to Macaronesia occurred more recently (ranging from the last 0.23 to 2.70 Ma). The diversification of Lotus in Macaronesia involved between four and six independent colonization events from four sections currently distributed in Africa and Europe. A major aspect shaping the current distribution of taxa involved intra-island colonization of mainly new habitats and inter-island colonization of mostly similar habitats, with Gran Canaria and Tenerife as the major sources of diversification and of further colonization events. Section Pedrosia is the most diverse in terms of colonization events, number of species, and habitat heterogeneity, including a back-colonization event to the continent. Subsections within Pedrosia radiated into diverse habitat types recently (late Pleistocene, ca 0.23-0.29 Ma) and additional molecular markers and sampling would be necessary to understand the most recent dispersal events of this group within the Canary Islands and Cape Verde.

Fostering Conservation via an Integrated Use of Conventional Approaches and High-Throughput SPET Genotyping: A Case Study Using the Endangered Canarian Endemics Solanum lidii and S. vespertilio (Solanaceae).

Islands provide unique opportunities to integrated research approaches to study evolution and conservation because boundaries are circumscribed, geological ages are often precise, and many taxa are greatly imperiled. We combined morphological and hybridization studies with high-throughput genotyping platforms to streamline relationships in the endangered monophyletic and highly diverse lineage of Solanum in the Canarian archipelago, where three endemic taxa are currently recognized. Inter-taxa hybridizations were performed, and morphological expression was assessed with a common-garden approach. Using the eggplant Single Primer Enrichment Technology (SPET) platform with 5,093 probes, 74 individuals of three endemic taxa (Solanum lidii, S. vespertilio subsp. vespertilio, and S. vespertilio subsp. doramae) were sampled for SNPs. While morphological and breeding studies showed clear distinctions and some continuous variation, inter-taxon hybrids were fertile and heterotic for vigor traits. SPET genotyping revealed 1,421 high-quality SNPs and supported four, not three, distinct taxonomic entities associated with post-emergence geological, ecological and geographic factors of the islands. Given the lack of barriers to hybridization among all the taxa and their molecular differences, great care must be taken in population management. Conservation strategies must take account of the sexual and breeding systems and genotypic distribution among populations to successfully conserve and restore threatened/endangered island taxa, as exemplified by Solanum on the Canary Islands.

A tale of two forests: ongoing aridification drives population decline and genetic diversity loss at continental scale in Afro-Macaronesian evergreen-forest archipelago endemics.

Background and Aims: Various studies and conservationist reports have warned about the contraction of the last subtropical Afro-Macaronesian forests. These relict vegetation zones have been restricted to a few oceanic and continental islands around the edges of Africa, due to aridification. Previous studies on relict species have generally focused on glacial effects on narrow endemics; however, little is known about the effects of aridification on the fates of previously widespread subtropical lineages. Methods: Nuclear microsatellites and ecological niche modelling were used to understand observed patterns of genetic diversity in two emblematic species, widely distributed in these ecosystems: Canarina eminii (a palaeoendemic of the eastern Afromontane forests) and Canarina canariensis (a palaeoendemic of the Canarian laurel forests). The software DIYABC was used to test alternative demographic scenarios and an ensemble method was employed to model potential distributions of the selected plants from the end of the deglaciation to the present. Key Results: All the populations assessed experienced a strong and recent population decline, revealing that locally widespread endemisms may also be alarmingly threatened. Conclusions: The detected extinction debt, as well as the extinction spiral to which these populations are subjected, demands urgent conservation measures for the unique, biodiversity-rich ecosystems that they inhabit.

Biogeography of Mediterranean Hotspot Biodiversity: Re-Evaluating the 'Tertiary Relict' Hypothesis of Macaronesian Laurel Forests.

The Macaronesian laurel forests (MLF) are dominated by trees with a laurophyll habit comparable to evergreen humid forests which were scattered across Europe and the Mediterranean in the Paleogene and Neogene. Therefore, MLF are traditionally regarded as an old, 'Tertiary relict' vegetation type. Here we address the question if key taxa of the MLF are relictual. We evaluated the relict hypothesis consulting fossil data and analyses based on molecular phylogenies of 18 representative species. For molecular dating we used the program BEAST, for ancestral trait reconstructions BayesTraits and Lagrange to infer ancestral areas. Our molecular dating showed that the origins of four species date back to the Upper Miocene while 14 originated in the Plio-Pleistocene. This coincides with the decline of fossil laurophyllous elements in Europe since the middle Miocene. Ancestral trait and area reconstructions indicate that MLF evolved partly from pre-adapted taxa from the Mediterranean, Macaronesia and the tropics. According to the fossil record laurophyllous taxa existed in Macaronesia since the Plio- and Pleistocene. MLF are composed of species with a heterogeneous origin. The taxa dated to the Pleistocene are likely not 'Tertiary relicts'. Some species may be interpreted as relictual. In this case, the establishment of most species in the Plio-Pleistocene suggests that there was a massive species turnover before this time. Alternatively, MLF were largely newly assembled through global recruitment rather than surviving as relicts of a once more widespread vegetation. This process may have possibly been triggered by the intensification of the trade winds at the end of the Pliocene as indicated by proxy data.

Molecular taxonomic identification in the absence of a 'barcoding gap': a test with the endemic flora of the Canarian oceanic hotspot.

We use a comprehensive subset of Canarian angiosperms corresponding to 23 families, 35 genera and 60 Canarian endemic taxa to test whether this flora is suitable to taxonomic identification with the two proposed plant DNA barcode sequences and whether these sequences may reveal the existence of cryptic species overlooked by morphology. The rate of discrimination success between the insular congeneric samples using the rbcL+matK combination and a 'character-based' approach (where we use only the combination of nucleotide positions in an alignment that allows unambiguous species identification) is higher (82.29%) than that obtained with the 'distance-based' approach (80.20%) used by the CBOL Plant Working Group in 2009 and also when compared with tests conducted in other floras. This suggests that the molecular identification of the Canarian endemic flora can be achieved as successfully as in other floras where the incidence of radiation is not as relevant. The facts that (i) a distance-based criterion was unable to discriminate between congeneric and conspecific comparisons and (ii) only the character-based discrimination criterion resolved cases that the distance-based criterion did not, further support the use of a character discrimination approach for a more efficient DNA barcoding of floras from oceanic islands like the Canaries. Thus, a barcoding gap seems not to be necessary for the correct molecular characterization of the Canarian flora. DNA barcodes also suggest the possible existence of cryptic taxa to be further investigated by morphology and that the current taxonomic status of some of the taxa analysed may need revision.

DNA barcodes successfully identified Macaronesian Lotus (Leguminosae) species within early diverged lineages of Cape Verde and mainland Africa.

Plant DNA barcoding currently relies on the application of a two-locus combination, matK + rbcL. Despite the universality of these two gene regions across plants, it is suspected that this combination might not have sufficient variation to discriminate closely related species. In this study, we tested the performance of this two-locus plant barcode along with the additional plastid regions trnH-psbA, rpoC1 and rpoB and the nuclear region internal transcribed spacer (nrITS) in a group of 38 species of Lotus from the Macaronesian region. The group has radiated into the five archipelagos within this region from mid-Miocene to early Pleistocene, and thus provides both early divergent and recent radiations that pose a particularly difficult challenge for barcoding. The group also has 10 species considered under different levels of conservation concern. We found different levels of species discrimination depending on the age of the lineages. We obtained 100 % of the species identification from mainland Africa and Cape Verde when all six regions were combined. These lineages radiated >4.5 Mya; however, in the most recent radiations from the end of the Pliocene to the mid-Pleistocene (3.5-1.5 Mya), only 30 % of the species were identified. Of the regions examined, the intergenic region trnH-psbA was the most variable and had the greatest discriminatory power (18 %) of the plastid regions when analysed alone. The nrITS region was the best region when analysed alone with a discriminatory power of 26 % of the species. Overall, we identified 52 % of the species and 30 % of the endangered or threatened species within this group when all six regions were combined. Our results are consistent with those of other studies that indicate that additional approaches to barcoding will be needed in recently evolved groups, such as the inclusion of faster evolving regions from the nuclear genome.

A local dormancy cline is related to the seed maturation environment, population genetic composition and climate.

BACKGROUND AND AIMS: Seed dormancy varies within species in response to climate, both in the long term (through ecotypes or clines) and in the short term (through the influence of the seed maturation environment). Disentangling both processes is crucial to understand plant adaptation to environmental changes. In this study, the local patterns of seed dormancy were investigated in a narrow endemic species, Centaurium somedanum, in order to determine the influence of the seed maturation environment, population genetic composition and climate. METHODS: Laboratory germination experiments were performed to measure dormancy in (1) seeds collected from different wild populations along a local altitudinal gradient and (2) seeds of a subsequent generation produced in a common garden. The genetic composition of the original populations was characterized using intersimple sequence repeat (ISSR) PCR and principal co-ordinate analysis (PCoA), and its correlation with the dormancy patterns of both generations was analysed. The effect of the local climate on dormancy was also modelled. KEY RESULTS: An altitudinal dormancy cline was found in the wild populations, which was maintained by the plants grown in the common garden. However, seeds from the common garden responded better to stratification, and their release from dormancy was more intense. The patterns of dormancy variation were correlated with genetic composition, whereas lower temperature and summer precipitation at the population sites predicted higher dormancy in the seeds of both generations. CONCLUSIONS: The dormancy cline in C. somedanum is related to a local climatic gradient and also corresponds to genetic differentiation among populations. This cline is further affected by the weather conditions during seed maturation, which influence the receptiveness to dormancy-breaking factors. These results show that dormancy is influenced by both long-and short-term climatic variation. Such processes at such a reduced spatial scale highlight the potential of plants to adapt to fast environmental changes.

Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling.

We used an ITS2 primary and secondary structure and Compensatory Base Changes (CBCs) analyses on new French and Spanish Dunallela salina strains to investigate their phylogenetic position and taxonomic status within the genus Dunaliella. Our analyses show a great diversity within D. salina (with only some clades not statistically supported) and reveal considerable genetic diversity and structure within Dunaliella, although the CBC analysis did not bolster the existence of different biological groups within this taxon. The ITS2 sequences of the new Spanish and French D. salina strains were very similar except for two of them: ITC5105 "Janubio" from Spain and ITC5119 from France. Although the Spanish one had a unique ITS2 sequence profile and the phylogenetic tree indicates that this strain can represent a new species, this hypothesis was not confirmed by CBCs, and clarification of its taxonomic status requires further investigation with new data. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some cases, and the ITS2 region does not contain a geographical signal overall.

The origin of bird pollination in Macaronesian Lotus (Loteae, Leguminosae).

The four red-flowered, apparently bird-pollinated Lotus species from the Canary Islands have previously been classified in their own genus, Rhyncholotus. Currently, they are considered as a separate section within genus Lotus, distinct from other herbaceous Canarian congeners which are yellow-flowered and bee-pollinated. A combined analysis of four nuclear regions (including ITS and three homologues of CYCLOIDEA) and three plastid regions (CYB6, matK and trnH-psbA) nests the four bird-pollinated species within a single extant species of bee-pollinated Lotus (L. sessilifolius), in a very extreme example of species paraphyly. Therefore, our data compellingly support the hypothesis that the Macaronesian Lotus species with a bird pollination syndrome are recently derived from entomophilous ancestors. Calibration of the phylogenetic trees using geological age estimates of the most recent islands (La Palma and El Hierro) together with oldest ages of Fuerteventura indicates that bird pollination evolved ca. 1.7 Ma in the Canarian Lotus. These four bird-pollinated species share a most recent common ancestor (MRCA) with L. sessilifolius that dates to about 2.2 Ma. Our analyses further suggest that the evolution of the bird pollination syndrome was likely triggered by the availability of new niches in La Palma and Tenerife as a result of recent volcanic activity.

The colonization history of Juniperus brevifolia (Cupressaceae) in the Azores Islands.

BACKGROUND: A central aim of island biogeography is to understand the colonization history of insular species using current distributions, fossil records and genetic diversity. Here, we analyze five plastid DNA regions of the endangered Juniperus brevifolia, which is endemic to the Azores archipelago. METHODOLOGY/PRINCIPAL FINDINGS: The phylogeny of the section Juniperus and the phylogeographic analyses of J. brevifolia based on the coalescence theory of allele (plastid) diversity suggest that: (1) a single introduction event likely occurred from Europe; (2) genetic diversification and inter-island dispersal postdated the emergence of the oldest island (Santa Maria, 8.12 Ma); (3) the genetic differentiation found in populations on the islands with higher age and smaller distance to the continent is significantly higher than that on the younger, more remote ones; (4) the high number of haplotypes observed (16), and the widespread distribution of the most frequent and ancestral ones across the archipelago, are indicating early diversification, demographic expansion, and recurrent dispersal. In contrast, restriction of six of the seven derived haplotypes to single islands is construed as reflecting significant isolation time prior to colonization. CONCLUSIONS/SIGNIFICANCE: Our phylogeographic reconstruction points to the sequence of island emergence as the key factor to explain the distribution of plastid DNA variation. The reproductive traits of this juniper species (anemophily, ornithochory, multi-seeded cones), together with its broad ecological range, appear to be largely responsible for recurrent inter-island colonization of ancestral haplotypes. In contrast, certain delay in colonization of new haplotypes may reflect intraspecific habitat competition on islands where this juniper was already present.

The molecular phylogeny of Matthiola R. Br. (Brassicaceae) inferred from ITS sequences, with special emphasis on the Macaronesian endemics.

Matthiola (Brassicaceae) is a genus that is widespread in the Mediterranean and Irano-Turanian regions and includes two species that are endemic to the archipelagos of Madeira and the Canaries in Macaronesia, which is an insular oceanic hotspot of biodiversity harboring many radiating endemic plant lineages. Sequence analyses of the nuclear ITS-1 and ITS-2 regions in a comprehensive geographical sample of Matthiola, encompassing all the endemic Macaronesian populations known to date, suggest independent Mediterranean and NW African origins of the taxa in Madeira and the Canaries, respectively. These molecular data reveal a complex evolutionary landscape that converges with morphological analyses in the recognition of two new Madeiran species. The data also suggest that the Canarian infra-specific endemic taxa described thus far have high (but non-diagnostic) levels of morphological and genetic diversity, and should be included in the single endemic Matthiola bolleana. In agreement with earlier investigations that revealed a high genetic differentiation between the populations of Matthiola in Fuerteventura and Lanzarote, our phylogeny supports independent founder events from the same mainland congener to either island. The consistently derived position of the Moroccan populations within a mostly Canarian clade suggests a further back-colonization of the continent. Notably, the ITS sequence resolution offered by Matthiola is higher than that found in many of the radiating Canarian endemic lineages for which molecular phylogenetic studies abound. Hence, our research discovers largely unexplored pathways to understand plant diversification in this oceanic insular hotspot through the investigation of non-speciose endemics.